Microbial Metabolism: M U T M
Microbial Metabolism: M U T M
Microbial Metabolism: M U T M
Microbial Metabolism
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This article from the Homer, Alaska, online newsletter, which has a label saying “sponsored” in an upper corner, says
that if we take this particular supplement, we will lose weight and have more energy and improved blood sugar levels. The
supplement contains four enzymes: lipase, protease, trypsin, and amylase.
You will learn in this chapter that enzymes are usually named using the substance they act upon followed by the suffix
“-ase.” So three of these enzymes are described in the article in the following way:
☐☐ Lipase “breaks down fat and ensures you’re not getting fatter from having sugar stores in your body for later use.”
☐☐ Protease breaks down protein, “ensuring you have the proper energy to go on with your day easily.”
☐☐ Amylase: The Latin for starch is amylum. The article states that those with low amylase levels in their saliva “usually
experience poor insulin responses and increased blood sugar levels.” It goes on to say, “This is not something that has
been discovered in the lab. It’s the human body’s science and the actual enzyme. What you should know is that beef
enzymes resemble human enzymes. 80% of the human DNA consists of cow or so-called beef material.”
■■ What is the intended message of the article?
■■ What is your critical reading of the summary of the article provided above? Remember that in this context, “critical
reading” does not necessarily mean What criticism do you have? but asks you to apply your knowledge to interpret
whether the article is factual and whether the facts support the intended message.
■■ How would you interpret the news item for your nonmicrobiologist friends?
■■ What is your overall grade for the news item—taking into account its accuracy and the accuracy of its intended effect?
Media Under The Microscope Wrap-Up appears at the end of the chapter.
250
ANABOLISM Bacterial
Glu cell
Phe
ANABOLISM
Lys
Relative complexity of molecules
Ala Macromolecules
CATABOLISM Val
Glucose ANABOLISM
Proteins
Building
blocks Peptidoglycan
Nutrients
from outside Precursor RNA + DNA
Amino acids
or from molecules
Glycolysis Complex lipids
internal Sugars
pathways Pyruvate
Krebs cycle
Nucleotides
Acetyl CoA
Respiratory
Fatty acids
chain
Glyceraldehyde-3-P Some assembly
reactions occur
Fermentation
spontaneously
Figure 10.1 Simplified model of metabolism. Cellular reactions fall into two major categories. Catabolism (yellow) involves the breakdown
of complex organic molecules to extract energy and form simpler end products. Anabolism (blue) uses the energy to synthesize necessary
macromolecules and cell structures from precursors.
to proceed, is measurable and is called the activation energy (or material. Their discovery has lent support for what is known today
energy of activation). This can be overcome by as the “RNA hypothesis,” which states that RNA was in fact the
first genetic material within ancient cells. In natural systems,
1. increasing thermal energy (heating) to increase the velocity
ribozymes are involved in self-splicing or cutting of RNA mol-
of molecules,
ecules during final processing of the genetic code.
2. increasing the concentration of reactants to increase the rate
of molecular collisions, or Apoenzymes: Specificity and the Active Site
3. adding a catalyst.
Apoenzymes range in size from small polypeptides with about
In most living systems, the first two alternatives are not fea- 100 amino acids and a molecular weight of 12,000 to large
sible because elevating the temperature is potentially harmful and polypeptide conglomerates with thousands of amino acids and a
higher concentrations of reactants are not practical. This leaves molecular weight of over 1 million. Like all proteins, an apoen-
only the action of catalysts, and enzymes fill this need efficiently zyme exhibits levels of molecular complexity called the primary,
and potently. Enzymatic catalysts effectively lower the energy of secondary, tertiary, and—in larger enzymes—quaternary organi-
activation, allowing a reaction to progress at a faster pace and with zation (figure 10.3). As we saw in chapter 2, the first three levels
reduced energy input. of structure arise when a single polypeptide chain undergoes
At the molecular level, an enzyme promotes a reaction by an automatic folding process and achieves stability by forming
serving as a physical site upon which the reactant molecules, disulfide and other types of bonds. The actual site where the
called substrates, can be positioned for various interactions. The substrate binds is a crevice or groove called the active site, or
enzyme is usually much larger in size than its substrate, and it catalytic site, and there can be one or several such sites (as shown
presents a unique active site that accepts only that particular sub- in figure 10.3). The three-dimensional shape of each site is formed
strate. Although an enzyme binds to the substrate and participates by the way the amino acid chain or chains are folded. Each type
directly in changes to the substrate, it does not become a part of of enzyme has a different primary structure (type and sequence of
the products, is not used up by the reaction, and can function over amino acids), variations in folding, and unique active sites.
and over again. Enzyme speed is well documented. For example,
the enzyme catalase converts its substrates at the rate of several Enzyme-Substrate Interactions
million per second. For a reaction to take place, the substrate has to nestle into the
active site (figure 10.4). The fit is so specific that it is often
Enzyme Structure described as a “lock-and-key” fit in which the substrate is inserted
into the active site’s pocket.
Most enzymes are proteins, and they can be classified as simple
Once the enzyme-substrate complex has formed, appropriate
or conjugated. Simple enzymes consist of protein alone, whereas
reactions occur on the substrate, often with the aid of a cofactor,
conjugated enzymes (figure 10.2) contain protein and some other
and a product is formed and released. The enzyme can then attach
nonprotein molecule or molecules. The whole conjugated enzyme,
to another substrate molecule and repeat this action.
sometimes referred to as a holoenzyme, is a combination of the
protein, called the apoenzyme in these cases, and one or more Cofactors: Supporting the Work of Enzymes
cofactors. Cofactors are either organic molecules, called coen-
zymes, or inorganic elements (metal ions). For example, catalase, In chapter 9, you learned that microorganisms require specific
an enzyme that we learned in chapter 9 breaks down hydrogen metal ions called trace elements and certain organic growth
peroxide, requires iron as a metallic cofactor. factors. Here we see why they are needed: to assist enzymes. The
There is another type of enzyme that is not made of protein, metal cofactors, including iron, copper, magnesium, manganese,
but of RNA. Named ribozymes, these molecules are remarkable zinc, cobalt, selenium, and many others, help with precise func-
because they are RNA molecules that catalyze reactions on other tions between the enzyme and its substrate. In general, metals
RNA. Ribozymes are thought to be remnants of the earliest mol- activate enzymes, help bring the active site and substrate close
ecules on earth that could have served as both catalysts and genetic together, and participate directly in chemical reactions with the
enzyme-substrate complex.
Coenzymes are one type of cofactor. The general function
Holoenzyme of a coenzyme is to remove a chemical group from one substrate
(purple + red) Coenzyme Coenzyme molecule and add it to another substrate, thereby serving as a
transient carrier of this group. Soon, we shall see that coenzymes
carry and transfer hydrogen atoms, electrons, carbon dioxide, and
Metallic amino groups. One of the most important kinds of coenzymes is
cofactor vitamins, which explains why vitamins are important to nutrition
and may be required as growth factors for living things. Vitamin
deficiencies prevent the complete holoenzyme from forming.
Metallic
Apoenzyme cofactor
(purple) Naming Enzymes
Figure 10.2 Conjugated enzyme structure. Conjugated A standardized system of nomenclature and classification has
enzymes have an apoenzyme (polypeptide or protein) component and been developed to keep things clear. In general, an enzyme name
one or more cofactors. is composed of two parts: (1) a prefix or stem word derived from a
AS
Quaternary
(b) More complex
enzymes have a AS
quaternary structure
consisting of several
polypeptides bound by
weak forces. Often the
active site is formed by
the junction of two
polypeptides. AS
AS
Remember: This
can take place very
quickly—up to
millions of times
per second! Substrates
–Kelly & Heidi
Products
Figure 10.4 Enzyme-substrate reactions. (a) When the enzyme and substrate come together, the substrate (S) must show the correct fit and
position with respect to the enzyme (E). (b) When the ES complex is formed, it enters a transition state. During this temporary but tight interlocking union, the
enzyme participates directly in breaking or making bonds. (c) Once the reaction is complete, the enzyme releases the products.
certain characteristic—usually the substrate acted upon or the type Each enzyme is also assigned a common name that indicates
of reaction catalyzed, or both—followed by (2) the ending -ase. the specific reaction it catalyzes. With this system, an enzyme
This system classifies the enzyme in one of six classes, on the that digests a carbohydrate substrate is a c arbohydrase; a specific
basis of its general biochemical action: carbohydrase, amylase, acts on starch (amylose is a major com-
ponent of starch). An enzyme that hydrolyzes peptide bonds of a
1. Oxidoreductases transfer electrons from one substrate to
protein is a proteinase, protease, or peptidase, depending on the
another, and dehydrogenases transfer a hydrogen from one
size of the protein substrate. Some fats and other lipids are digested
compound to another.
by lipases. DNA is hydrolyzed by deoxyribonuclease, generally
2. Transferases transfer functional groups from one substrate to
shortened to DNase. A synthetase or polymerase bonds together
another.
many small molecules into large molecules. Other examples of
3. Hydrolases cleave bonds on molecules with the addition of water.
enzymes are presented in table 10.2.
4. Lyases add groups to or remove groups from double-bonded
substrates.
5. Isomerases change a substrate into its isomeric1 form. Transfer Reactions by Enzymes
6. Ligases catalyze the formation of bonds with the input of ATP
Other enzyme-driven processes that involve the simple addition or
and the removal of water.
removal of a functional group are important to the overall work-
1. An isomer is a compound that has the same molecular formula as another ings of the cell. Oxidation-reduction and other transfer activities
compound but differs in arrangement of the atoms. are examples of these types of reactions.
Remember how we said that the flow of electrons is critical to Enzymes are not all produced in equal amounts or at equal
metabolism? Some atoms and compounds readily give or receive rates. Some, called constitutive enzymes ( figure 10.6a), are
electrons and participate in oxidation (the loss of electrons) always present and in relatively constant amounts, regardless of the
or reduction (the gain of electrons). The compound that loses cellular environment. The enzymes involved in using glucose, for
the electrons is oxidized, and the compound that receives the example, are very important in metabolism and, for that reason, are
electrons is reduced. These oxidation-reduction (redox) reactions constitutive. Other enzymes are regulated enzymes (figure 10.6b),
are common in the cell. Oxidoreductases remove electrons from and they are either turned on (induced) or turned off (repressed) in
one substrate and add them to another. Their coenzyme carriers response to changes in concentration of the substrate.
are nicotinamide adenine dinucleotide (NAD) and flavin adenine
dinucleotide (FAD). (Take note: Even if by now your eyes are The Role of Microbial Enzymes in Disease
glazing over at all the terms and details, this paragraph is a
Many pathogens secrete unique exoenzymes that help them avoid
valuable one! If you remember the statements in this paragraph,
host defenses or promote their multiplication in tissues. Because
the rest of metabolism will be a lot easier to understand.)
these enzymes contribute to pathogenicity, they are referred to as
virulence factors. Some of them function as toxins. Streptococ-
Location of Enzyme Action cus pyogenes (a cause of throat and skin infections) produces a
Enzymes perform their tasks either inside or outside of the cell streptokinase that digests blood clots and helps the bacterium
in which they were produced. After they are made inside the cell, invade wounds. Another exoenzyme from this bacterium is called
exoenzymes are transported extracellularly, where they break streptolysin. In mammalian hosts, streptolysin damages blood
down (hydrolyze) large food molecules or harmful chemicals. cells and tissues. It is also responsible for lysing red blood cells
Examples of exoenzymes are cellulase, amylase, and penicillinase. used in blood agar dishes, and this trait is used for identifying the
By contrast, endoenzymes function inside the cell. Most enzymes bacteria growing in culture. Pseudomonas aeruginosa, a respira-
of the metabolic pathways are endoenzymes (figure 10.5). tory and skin pathogen, produces elastase and collagenase, which
digest elastin and collagen, two proteins found in connective tis-
sue. These increase the severity of certain lung diseases and burn
Exoenzymes Endoenzymes infections. Clostridium perfringens, an agent of gas gangrene,
synthesizes lecithinase C, a lipase that profoundly damages cell
Substrates Products membranes and accounts for the tissue death associated with this
disease. Not all microbial enzymes digest tissues; some, such as
Products penicillinase, inactivate penicillin and thereby protect a microbe
Active
enzymes from its effects.
Enzyme
Inactive Disease Connection
enzymes Substrate
In competitive inhibition, enzyme activity is stopped by a mol-
ecule that resembles (mimics) the enzyme’s normal substrate.
There are many drugs that use the principle of competitive inhibi-
tion. Many of the drugs developed to treat HIV/AIDS prevent the
(a) (b)
enzyme HIV protease from doing its job, which is to assemble the
Figure 10.5 Types of enzymes, as described by their capsids of new viruses. Certain antidepressants, diuretics, and
location of action. (a) Exoenzymes are released outside the cell to antibiotics also act as competitive inhibitors.
function. (b) Endoenzymes remain in the cell and function there.
Add more
substrate.
Add more
substrate.
Enzyme is induced.
No change in or
(a) amount of enzyme.
The Sensitivity of Enzymes to Their Environment responds to environmental conditions by using those metabolic
reactions that most favor growth and survival. Basically, the
The activity of an enzyme is highly influenced by the cell’s envi-
regulation of metabolism comes about through the regulation of
ronment. In general, enzymes operate only under the natural tem-
enzymes via an elaborate system of checks and balances.
perature, pH, and osmotic pressure of its organism’s habitat. When
enzymes are subjected to changes in these normal conditions,
they tend to be chemically unstable, or labile. Low temperatures Metabolic Pathways
inhibit catalysis, and high temperatures denature the apoenzyme. Metabolic reactions rarely consist of a single action or step. More
Denaturation is a process by which the weak bonds that collec- often, they happen in a multistep series or pathway, with each step
tively maintain the native shape of the apoenzyme are broken. This catalyzed by a different enzyme. An individual reaction can be shown
disruption causes distortion of the enzyme’s shape and prevents in various ways, depending on the purpose at hand (figure 10.7). The
the substrate from attaching to the active site. Such nonfunctional product of one reaction is often the reactant (substrate) for the next,
enzymes block metabolic reactions and can lead to cell death. Low forming a linear chain of reactions. Many pathways have branches
or high pH or certain chemicals (heavy metals, alcohol) are also that provide alternate methods for nutrient processing. Others take a
denaturing agents. When enzymes are nonfunctional, metabolic cyclic form, in which the starting molecule is regenerated to initiate
reactions fail to happen and cell death can follow. another turn of the cycle. On top of that, pathways generally do not
stand alone; they are interconnected and merge at many sites.
Regulation of Enzymatic Activity
and Metabolic Pathways Direct Controls on the Action of Enzymes
Metabolic reactions proceed in a systematic, highly regulated The bacterial cell has many ways of directly influencing the
manner that maximizes the use of nutrients and energy. The cell activity of its existing enzymes. It can inhibit enzyme activity
Multienzyme Systems
down. This form of inhibition is called competitive inhibition
because the mimic is competing with the substrate for the binding
Linear Cyclic Branched site (figure 10.8).
Divergent Convergent
Another form of inhibition can occur with special types of
A
enzymes that have two binding sites—the active site and another
M A X area called the regulatory site (as shown in figure 10.8). These
U
B enzymes are regulated by the binding of molecules in their
V T input N B Y regulatory sites. Often, the regulatory molecule is the product of
C S product Z the enzymatic reaction itself. This provides a negative feedback
Krebs
Cycle
W O P mechanism that can slow down enzymatic activity once a certain
C Z
D Y concentration of product is produced. This is noncompetitive
X inhibition because the regulator molecule does not bind in the
O1 Q M
E same site as the substrate.
O2 R N
Controls on Whether the Enzyme Gets Made
Example: Example: Example: Controlling enzymes by controlling their synthesis is another
Glycolysis Protein Amino acid
catabolism synthesis effective mechanism because enzymes do not last indefinitely.
Some wear out, some are deliberately degraded, and others are
Figure 10.7 Patterns of metabolism. In general, metabolic diluted with each cell division. For the reactions to continue,
pathways consist of a linked series of individual chemical reactions
enzymes eventually must be replaced. This cycle works into the
that produce intermediary metabolites and lead to a final product. These
pathways occur in several patterns, including linear, cyclic, and branched.
scheme of the cell, where replacement of enzymes can be regu-
Virtually every reaction in a series—represented by an arrow—involves a lated according to cell demand.
specific enzyme. Enzyme repression is a mechanism to stop further syn-
thesis of an enzyme somewhere along its pathway. As the level
by supplying a molecule that resembles the enzyme’s normal of the end product from a given enzymatic reaction has built
substrate. This “mimic” can then occupy the enzyme’s active site, to excess, the genes responsible for replacing these enzymes
preventing the actual substrate from binding there. Because the are automatically suppressed (figure 10.9). The response time
mimic cannot actually be acted on by the enzyme or function in is longer than for the direct controls described above, but its
the way the product would have, the enzyme is effectively shut effects last longer.
Substrate
Normal Competitive
substrate inhibitor with Active site
similar shape
Both molecules
compete for
Enzyme the active site.
Regulatory site
Regulatory
molecule
(product)
Enzyme Enzyme
Products
Reaction is blocked Reaction proceeds. Reaction is blocked because binding of
because competitive regulatory molecule in regulatory site
inhibitor is incapable changes conformation of active site so
Reaction proceeds. of becoming a product. Product that substrate cannot enter.
Figure 10.8 Examples of two common control mechanisms for enzyme activity.
1
DNA transcribed into RNA
2
RNA translated into protein
3
Protein
This is where
ere
on in
the inhibition 6
this scenario
rio Excess product binds to
DNA and shuts down 7
e.
takes place. further enzyme production. DNA cannot be transcribed;
- Kelly & Heidi the protein cannot be made.
4
Folds to form functional
enzyme structure
5
Substrate
= +
Products Substrate Enzyme
Figure 10.9 One type of genetic control of enzyme synthesis: enzyme repression. 1 – 5 : The enzyme is synthesized continuously
via uninhibited transcription and translation until enough product has been made. 6 , 7 : Excess product reacts with a site on DNA that regulates
the enzyme’s synthesis, thereby inhibiting further enzyme production.
The inverse of enzyme repression is enzyme induction. In from wasting energy by making enzymes for which no substrates
this process, enzymes appear (are induced) only when suitable are present.
substrates are present—that is, the synthesis of an enzyme is
induced by its substrate. Both mechanisms are important genetic
control systems in bacteria. 10.1 Learning Outcomes—Assess Your Progress
A classic model of enzyme induction is the case of the 1. Describe the relationship among metabolism, catabolism,
lac operon in Escherichia coli we examined in chapter 6. As a and anabolism.
reminder, if E. coli is inoculated into a medium whose principal 2. Fully discuss the structure and function of enzymes.
carbon source is lactose, it will produce the enzyme lactase
3. Differentiate between an apoenzyme and a holoenzyme.
to hydrolyze it into glucose and galactose. If the bacterium is
subsequently inoculated into a medium containing only sucrose 4. Differentiate between an endoenzyme and an exoenzyme,
as a carbon source, it will cease synthesizing lactase and begin and between constitutive and regulated enzymes.
synthesizing sucrase. This response enables the organism to 5. Diagram the four major patterns of metabolic pathways.
adapt to a variety of nutrients, and it also prevents a microbe 6. Describe how enzymes are controlled.
10.2 Finding and Making Use of Energy To reiterate, cells possess specialized enzyme systems that trap
the energy present in the bonds of nutrients as they are progressively
In order to carry out the work of metabolism, cells require constant broken (figure 10.10). During exergonic reactions, energy released
input of some form of usable energy. The energy can come directly by bonds is stored in certain high-energy phosphate bonds, such as
from sunlight (in photosynthesizers) or from free electrons (in in ATP. ATP stores the energy and releases it when needed for ender-
electricity-harvesting bacteria). In most bacteria we examine in this gonic reactions. Before discussing ATP, we examine redox reactions,
book, the energy comes from organic substances, such as sugars, which provide the electrons that are critical to energy production.
when their bonds are broken, releasing and transferring electrons.
The energy is stored in ATP. A Closer Look at Oxidation and Reduction
Redox reactions always occur in pairs, with an electron donor and
Energy in Cells an electron acceptor, which constitute a redox pair. The reaction
Not all cellular reactions are equal with respect to energy. Some can be represented as follows:
release energy, and others require it. For example, a reaction that
Starting state Ending state
proceeds as follows:
enzyme
X+Y Z + energy
+ –
are called endergonic (en-der-gon′-ik) because they require the Redox reactions occur in all cells and are indispensable to
addition of energy. In cells, exergonic and endergonic reactions are the required energy transformations. Important components of
often coupled, so that released energy is immediately put to use. cellular redox reactions are enzymes called oxidoreductases. They
High
Figure 10.10 A simplified model
ATP
6
used to perform of energy production. Glucose
CH 2OH
cellular work is oxidized as it passes through
C5 O
sequential metabolic pathways,
H
H H resulting in the removal of
C4 C1 hydrogens and their accompanying
HO OH ATP electrons. The energy from the
OH H
Hydrogen ions hydrogens and electrons is used
3C C2
with electrons
H OH to generate ATP. Eventually, all
Energy Level of Chemical Compound
H2O
O C O End products
CO2
Low
P P
Adenosine Triphosphate:
Metabolic Money
Let’s look more closely at the energy molecule ATP. ATP has
been described as “metabolic money” because it can be earned,
banked, saved, spent, and exchanged. As a temporary energy
Figure 10.11 Details of NAD reduction. The coenzyme NAD repository, ATP provides a connection between energy-yielding
contains the vitamin nicotinamide (niacin) and the purine adenine
catabolism and the other cellular activities that require energy.
attached to two ribose phosphate molecules. The principal site of
action is on the nicotinamide (blue-boxed areas). Hydrogens and
Some clues to its energy-storing properties lie in its unique
electrons donated by a substrate interact with a carbon on the top of molecular structure.
the ring. One hydrogen bonds there, carrying two electrons (H:), and
the other hydrogen is carried in solution as H+ (a proton). The Molecular Structure of ATP
ATP is a three-part molecule consisting of a nitrogen base
have coenzyme carriers called nicotinamide adenine dinucleotide (adenine) linked to a 5-carbon sugar (ribose), with a chain of
(NAD) (figure 10.11) and flavin adenine dinucleotide (FAD). three phosphate groups bonded to the ribose (figure 10.12). The
Just look one time at the whole name to appreciate that they are
nucleotides, and concentrate on the blue boxes in figure 10.11.
Adenosine Adenosine
That's where the action is. Triphosphate Diphosphate Adenosine
Oxidation-reduction reactions trade electrons back and forth (ATP) (ADP)
along with the energy they contain. The newly reduced compound
H H
(the one that gains electrons) has more energy than it did in its orig- Adenine
N
inal oxidized state. The energy now present in the electron acceptor
can be captured to p hosphorylate (add an inorganic phosphate) to N
N
ADP or to some other compound. This process stores the energy H
in a high-energy molecule (e.g., ATP). In many cases, the cell H N N
handles electrons not as separate entities but rather as parts of an
atom such as hydrogen (which contains a proton and an electron). OH OH OH
H
For simplicity’s sake, we will continue to use the term electron
transfer, but keep in mind that hydrogens are often involved in the HO P O P O P O H
transfer process. The removal of hydrogens from a compound dur-
O
ing a redox reaction is called dehydrogenation. The job of handling O O O
these protons and electrons falls to one or more carriers, which H H
function as short-term storehouses for the electrons until they can H H
Bond that releases
be transferred. As we will see, dehydrogenations are an essential energy when broken OH OH
supplier of electrons for the respiratory electron transport system. Ribose
ANABOLISM
ANABOLISM
ANABOLISM
substrate-level phosphorylation. In substrate-level phosphorylation, on three basic catabolic
ATP is formed by transfer of a phosphate group from a phos- pathways. Frequently,
phorylated compound (substrate) directly to ADP to yield ATP the nutrient is glucose.
(figure 10.13). We will use this nutrient
Other ATPs are formed through oxidative phosphorylation, as an example, but there are many different nutrients that can be
a series of redox reactions occurring during the final phase of the fed into catabolic pathways. There are several pathways that can be
respiratory pathway. Phototrophic organisms have a system called used to break down glucose, but the most common one is glycoly-
photophosphorylation, in which the ATP is formed through a sis (gly-kol′-ih-sis). After glycolysis, organisms use mainly three
series of sunlight-driven reactions. different pathways for producing the needed precursors and energy
(i.e., catabolism) (figure 10.14).
Aerobic respiration is a series of reactions: glycolysis, the
Krebs cycle, and the respiratory chain. This pathway converts
PO4 + ADP + ATP glucose to CO2 and allows the cell to recover significant amounts
of energy (review figure 10.10). Aerobic respiration relies on
Substrate
free oxygen as the final acceptor for electrons and hydrogens and
Figure 10.13 ATP formation by substrate-level produces a large amount of ATP. Aerobic respiration is character-
phosphorylation. The inorganic phosphate and the substrates form istic of many bacteria, fungi, protozoa, and animals. Facultative
a bond with high potential energy. In a reaction catalyzed enzymatically, and aerotolerant anaerobes may use only the glycolysis portion
the phosphate is transferred to ADP, thereby producing ATP. to incompletely oxidize (ferment) glucose. In this case, oxygen
Glycolysis
Glycolysis
Glycolysis
NAD H NAD H NAD H
Fermentation
Electron Transport System Electron Transport System
Using organic
compounds as
Using O2 as electron acceptor Using non- O2 compound as electron acceptor electron acceptor
(SO42–, NO3–, CO32–)
Yields variable
amount of
energy ATP ATP
Alcohols, acids
is not required, organic compounds are the final electron accep- from them also removes electrons that can be used in energy
tors, and a relatively small amount of ATP is produced. Some transfers. The end products of the conversion of these carbon
anaerobic microorganisms metabolize by means of a naerobic compounds are energy-rich ATP and energy-poor carbon diox-
respiration. This system involves the same three pathways as ide and water. Although in our discussion we use glucose as the
aerobic respiration, but it does not use molecular oxygen as the main starting compound, other hexoses (fructose, galactose) and
final electron acceptor. Instead, NO3−, SO42−, CO32−, and other fatty acid subunits can enter the pathways of aerobic respiration
oxidized compounds are utilized. Aspects of fermentation and as well.
anaerobic respiration are covered in subsequent sections of this
chapter. Glycolysis: How It All Starts
Glycolysis uses several steps to convert glucose into pyruvic acid.
Aerobic Respiration Depending on the organism and the conditions, it may be only the
Aerobic respiration is the metabolic process in which electrons first phase of aerobic respiration, or it may serve as the primary met-
are transferred from fuel molecules such as glucose to oxy- abolic pathway (in the case of fermentation). Glycolysis provides a
gen as a final electron acceptor. This pathway is the principal significant means to synthesize ATP and also to generate pyruvic
energy-yielding scheme for aerobic heterotrophs, and it pro- acid, an essential intermediary metabolite.
vides both ATP and metabolic intermediates for many other Glycolysis proceeds along nine steps, starting with glucose
pathways in the cell, including those of protein, lipid, and and ending with pyruvic acid (pyruvate2). An overview of glyco-
carbohydrate synthesis. lysis will be presented here. Figure 10.15 contains the chemical
structures and a visual representation of the reactions. Each of the
nine reactions is catalyzed by a specific enzyme with a specific
Glucose: The Starting Compound
name (not mentioned here).
Carbohydrates such as glucose are good fuels because they are
readily oxidized. That means that they are excellent hydrogen 2. In biochemistry, the terms used for organic acids appear as either the acid form
and electron donors. The enzymatic withdrawal of hydrogen (pyruvic acid) or its salt (pyruvate).
1
C C C C C C Glucose
ATP
First 1
phosphorylation ADP PO4
1
C C C C C C Glucose-6-phosphate
2
PO4
1
C C C C C C Fructose-6-phosphate
ATP
Second 3 ADP
phosphorylation
PO4 PO4
Fructose-1,6-diphosphate
1
C C C C C C (F-1,6-P)
Glyceraldehyde-3-
phosphate
C C C PO4 C C C PO4
To electron transport
To electron transport
NAD NAD
PO43– PO43–
5
NAD H NAD H
Diphosphoglyceric
PO4 C C C PO4 acid PO4 C C C PO4
3-phosphoglyceric
C C C PO4 C C C PO4
acid
7
PO4 PO4
C C C 2-phosphoglyceric acid C C C
8 H2O H2O
C C C Pyruvic acid C C C
First, glucose is activated by adding a phosphate to it, result- occurred and 2 ATPs have been used. The next step involves con-
ing in glucose-6-phosphate. It is then converted (another reaction, verting one C molecule (DHAP) into the other (glyceraldehyde-
another enzyme) to fructose-6-phosphate, and then another phos- 3-P; G-3-P), resulting in two G-3-Ps.
phate is added. The resulting molecule—fructose diphosphate—is From here to the end, everything that happens in glycolysis
more symmetrical and can be split into two 3-carbon molecules happens twice—once to each of the 3-carbon molecules. First,
(figure 10.15, 4 ). At this point, no oxidation- reduction has the G-3-Ps each receives another phosphate. At the same time,
2 NADs in the vicinity are reduced to NADHs. These NADHs The Krebs Cycle: A Carbon
will be used in the last step of catabolism (the electron transport and Energy Wheel
system) to produce ATP.
Although glycolysis is the main route to pyruvate produc- In glycolysis, the oxidation of glucose yields a comparatively small
tion for most organisms, some microbes lack the enzymes for this amount of energy and produces pyruvic acid. Pyruvic acid is still
pathway. There are alternate biochemical reactions such as the energy-rich, containing a number of extractable hydrogens and
Entner-Doudoroff pathway (used by Pseudomonas and Enterococ- electrons to power ATP synthesis, but this can be achieved only
cus species) and the pentose phosphate pathway (used by some through the work of the second and third phases of respiration, in
photosynthetic microbes). Our aim here is to focus on general which pyruvic acid’s hydrogens are transferred to oxygen, produc-
principles, so we will restrict ourselves to glycolysis. ing CO2 and H2O. In the following section, we examine the next
phase of this process, the Krebs cycle (named after the scientist
who first defined it). This set of reactions takes place in the cyto-
Disease Connection plasm of bacteria and is catalyzed by a group of enzymes (some of
which are associated with the cytoplasmic membrane). In eukary-
Enterococci are bacteria that can live peacefully in the intestinal otic cells, this process takes place in the mitochondrial matrix.
tract or the female reproductive tract, but they can occasionally To connect the glycolysis pathway to the Krebs cycle, for
cause disease in those areas or in surgical wounds. In recent either aerobic or anaerobic respiration, the pyruvic acid is first
years, Enterococci that are resistant to the antibiotic vancomy- converted to a starting compound for that cycle (f igure 10.17).
cin have become problematic, particularly in hospitals. They are Here we have an oxidation-reduction reaction, which also
termed VREs (vancomycin-resistant enterococci). They have an releases the first carbon dioxide molecule. It involves a cluster of
extremely flexible metabolism and can use a huge variety of sub- enzymes and coenzyme A that participate in the dehydrogenation
strates to produce energy. (oxidation) of pyruvic acid, the reduction of NAD to NADH, and
the decarboxylation of pyruvic acid to a 2-carbon acetyl group.
The acetyl group remains attached to coenzyme A, forming
acetyl coenzyme A (acetyl CoA) that feeds into the Krebs cycle.
Pyruvic Acid: A Central Metabolite The NADH formed during this reaction will be shuttled into
Pyruvic acid occupies an important position in several pathways, electron transport and used to generate ATP via oxidative phos-
and different organisms handle it in different ways (figure 10.16). phorylation. Keep in mind that all reactions described actually
In strictly aerobic organisms and some anaerobes, pyruvic acid happen twice for each glucose because of the two pyruvates that
enters the Krebs cycle for further processing and energy release. are formed during glycolysis.
Facultative anaerobes can also use a fermentative metabo- The Krebs cycle as depicted in figure 10.17 always looks
lism, in which pyruvic acid is re-reduced into acids or other intimidating. Think of it as a series of eight reactions catalyzed by
products. eight different enzymes.
ll ll l Acetaldehyde
O O H
Alcohol
Acetone
Acetyl CoA Acids, gas 2,3-butanediol
Krebs
cycle
From glycolysis
O
C O–
Pyruvic acid
C O
Steps in the Krebs Cycle the energy production that will occur in electron transport. Along
the way, the 2-carbon acetyl CoA joins with a 4-carbon compound,
As you learned earlier, a cyclic pathway is one in which the start-
oxaloacetic acid, and then participates in seven additional chemi-
ing compound is regenerated at the end. The Krebs cycle has
cal transformations while “spinning off” the NADH and FADH2.
eight steps, beginning with citric acid formation and ending with
That’s why we called the Krebs cycle the “carbon and energy
oxaloacetic acid. As we take a single spin around the Krebs cycle,
wheel.”
it will be helpful to keep track of
∙∙ the numbers of carbons (#C) in each substrate and product, The Respiratory Chain: Electron Transport
∙∙ reactions where CO2 is generated,
and Oxidative Phosphorylation
∙∙ the involvement of the electron carriers NAD and FAD, and
∙∙ where ATP is made. We now come to the energy chain, which is the final “processing
mill” for electrons and hydrogen ions and the major generator of ATP.
The reactions in the Krebs cycle follow. Overall, the electron transport system (ETS) consists of a chain of
1 Oxaloacetic acid (oxaloacetate; 4C) reacts with the acetyl group special redox carriers (proteins) that receives electrons from reduced
(2C) on acetyl CoA, thereby forming citric acid (citrate; 6C) and carriers (NADH, FADH2) generated by glycolysis and the Krebs
releasing coenzyme A so it can join with another acetyl group. cycle and passes them in a sequential and orderly fashion from one
2 Citric acid is converted to isocitric acid (isocitrate; 6C) to redox molecule to the next. The flow of electrons down this chain
prepare this substrate for the decarboxylation and dehydroge- allows the active transport of hydrogen ions to the outside of the
nation of the next step. membrane where the respiratory chain is located. The step that final-
3 Isocitric acid is acted upon by an enzyme complex including izes the transport process is the acceptance of electrons and hydrogen
NAD or NADP (depending on the organism) in a reaction that by oxygen, producing water. This process consumes oxygen. Some
generates NADH or NADPH, splits off a carbon dioxide, and variability exists from one organism to another, but the principal com-
leaves alpha-ketoglutaric acid (α-ketoglutarate; 5C). pounds that carry out these complex reactions are NADH dehydro-
4 Alpha- ketoglutaric acid serves as a substrate for the last genase, flavoproteins, coenzyme Q (ubiquinone), and cytochromes.
decarboxylation reaction and yet another redox reaction The cytochromes contain a tightly bound metal atom at their center
involving coenzyme A and yielding NADH. The product is that is actively involved in accepting electrons and donating them to
the high-energy compound succinyl CoA (4C). the next carrier in the series. The highly compartmentalized structure
of the respiratory chain is an important factor in its function. Note in
At this point, the cycle has released 3 CO2 molecules that bal- figure 10.18 that the electron transport carriers and enzymes are
ance out the original 3-carbon pyruvic acid that began the Krebs embedded in the cytoplasmic membrane in bacteria. The equivalent
cycle. The remaining steps are needed not only to regenerate the structure for housing them in eukaryotes is the inner mitochondrial
oxaloacetic acid to start the cycle again but also to extract more membranes pictured in figure 10.19. We will describe the electron
energy from the intermediate compounds leading to oxaloacetic acid. transport system in both bacteria and eukaryotes.
5 Succinyl CoA is the source of the one substrate-level phos-
phorylation in the Krebs cycle. In most microbes, it proceeds Elements of Electron Transport:
with the formation of GTP, guanosine triphosphate. GTP is The Energy Cascade
readily converted to ATP. The product of this reaction is suc-
The principal questions about the electron transport system are:
cinic acid (succinate; 4C).
How are the electrons passed from one carrier to another in the
6 Succinic acid next becomes dehydrogenated, but in this case,
series? How does this progression result in ATP synthesis? How is
the electron and H+ acceptor is flavin adenine dinucleotide
oxygen (or another electron acceptor) utilized? Although the bio-
(FAD). The enzyme that catalyzes this reaction, succinyl dehy-
chemical details of this process are rather complicated, the basic
drogenase, is found in the bacterial cytoplasmic membrane and
reactions consist of a number of redox reactions now familiar to us.
mitochondrial cristae of eukaryotic cells. FADH2 then directly
In general, the carrier compounds and their enzymes are arranged
enters the electron transport system. Fumaric acid (fumarate;
in linear sequence and are reduced and then oxidized in turn.
4C) is the product of this reaction.
The sequence of electron carriers in the respiratory chain of
7 The addition of water to fumaric acid (called hydration)
most aerobic organisms is
results in malic acid (malate; 4C). This is one of the few
reactions in respiration that directly incorporate water. 1. NADH dehydrogenase;
8 Malic acid is dehydrogenated (with formation of a final 2. flavin mononucleotide (FMN);
NADH), and oxaloacetic acid is formed. This step brings the 3. coenzyme Q;
cycle back to its original starting position, where oxaloacetic 4. cytochrome b;
acid can react with acetyl coenzyme A. 5. cytochrome c1;
6. cytochrome c; and
The Krebs cycle serves to transfer the energy stored in acetyl
7. cytochromes a and a3, which are complexed together.
CoA to NAD+ and FAD by reducing them (transferring hydrogen
ions to them). For that reason, the main products of the Krebs cycle NADH from glycolysis and from the Krebs cycle enters the
are these reduced molecules (as well as 2 ATPs for each glucose chain at the first carrier. This sets in motion the next six steps.
molecule). The reduced coenzymes NADH and FADH2 are vital to With each redox exchange, the energy level of the reactants
H+
H+
H+
H+
ATP
H+ synthase
Cell wall
H+
H+
H+
H+
ADP ATP
H+
is decreased. The released energy is captured and used by the transport carriers shuttle electrons, they actively pump hydrogen ions
ATP synthase complex, stationed along the membrane at the (protons) into the periplasmic space, or the space between the wall
end of the line of ETS carriers. Each NADH that enters electron and the cytoplasmic membrane, depending on whether the bacterium
transport can give rise to 3 ATPs. This coupling of ATP synthe- is gram-positive or gram-negative. This process sets up a concentra-
sis to electron transport is termed o xidative phosphorylation. tion gradient of hydrogen ions called the proton motive force (PMF).
Because the electrons from FADH2 from the Krebs cycle enter The PMF consists of a difference in charge between the outside of the
the cycle at a later point, there is less energy to release, and only membrane (+) and the inside of the membrane (−) (see f igure 10.18).
2 ATPs are the result.
Intermembrane
The Formation of ATP and Chemiosmosis space
H+ ions
How exactly is electron transport linked to ATP production? We will Cristae
first look at the system in bacteria, which have the components of
electron transport embedded in a precise sequence on the cytoplas-
mic membrane. The process is called chemiosmosis. As the electron
Separating the charge has the effect of a battery, which can tem- The single FADH2 produced during the Krebs cycle results in
porarily store potential energy. This charge is maintained by the fact
that H+ can’t cross the membrane. The only site where H+ can dif- 2 ATPs per electron pair = 2 ATPs
fuse into the cytoplasm is at the ATP synthase complex, which sets and
the stage for the final processing of H+ leading to ATP synthesis. 2 × 2 = 4 ATPs per glucose
ATP synthase is a complex enzyme composed of two large units Figure 10.20 summarizes the total of ATP and other
(see figures 10.18 and 10.19). It is embedded in the membrane, but products for the entire aerobic pathway. These totals are the
part of it rotates like a motor and traps chemical energy. As the H+ ions maximum yields possible but may not be fulfilled by many
flow through the center of the enzyme by diffusion, the other com- organisms.
partments pull in ADP and Pi. Rotation causes a three-dimensional
change in the enzyme that bonds these two molecules, thereby releas-
ing ATP into the cytoplasm (see figure 10.18). The enzyme is then Summary of Aerobic Respiration
rotated back to the start position and will continue the process. Originally, we presented a summary equation for respiration. We
Eukaryotic ATP synthesis occurs by means of the same are now in a position to add up the input and output of this equa-
overall process. However, eukaryotes have the ETS stationed tion at various points in the pathways you can see and sum up the
in mitochondrial membranes, between the inner mitochondrial final ATP. In figure 10.20 you can see several important aspects
matrix and the outer intermembrane space (see figure 10.19). of aerobic respiration:
Potential Yield of ATPs from 1. The total possible production of ATP is 40: 4 from glycolysis,
Oxidative Phosphorylation 2 from the Krebs cycle, and 34 from electron transport.
The total of five NADHs (four from the Krebs cycle and one from However, because 2 ATPs were already spent in early
glycolysis) can be used to synthesize glycolysis, this leaves a maximum of 38 ATPs.
The actual totals may be lower in certain eukaryotic cells
3 ATPs per electron pair = 15 ATPs because energy is spent in transporting the NADH produced
and during glycolysis across the mitochondrial membrane. Certain
15 × 2 = 30 ATPs per glucose aerobic bacteria come closest to achieving the full total of 38
AEROBIC RESPIRATION
Glycolysis
NAD H
ATP Carbon Water and Oxygen
Yields 2 ATPs ATP 1 glucose yields 2 pyruvates 2 H2Os produced
CO2
NAD H Krebs
Cycle 2 CO2
FADH2
Yields 2 ATPs ATP Yields 6 CO2 2 H2Os consumed
(1 per pyruvate) (3 per pyruvate)
Figure 10.20 Theoretic ATP yield from aerobic respiration. To attain the theoretic maximum yield of ATP, we assume a ratio of 3 for the
oxidation of NADH to 2 for FADH2. The actual yield is generally lower and varies between eukaryotes and bacteria and among bacterial species.
because they lack mitochondria and, for that reason, do not nitrate reductase
have to use ATP in the transport of NADH across the outer ↓
mitochondrial membrane. NO3− + NADH → NO2− + H2O + NAD+
nitrate nitrite
2. Six carbon dioxide molecules are generated during the Krebs
cycle. The enzyme nitrate reductase catalyzes the removal of oxygen
3. Six oxygen molecules are consumed during electron transport. from nitrate, leaving nitrite and water as products. A test for
4. Six water molecules are produced in electron transport and 2 this reaction is one of the physiological tests used in identifying
in glycolysis, but because 2 are used in the Krebs cycle, this bacteria.
leaves a net number of 6. It has been found that nitrate-reducing bacteria in the mouth
and gut can contribute to pulmonary and cardiovascular diseases
The Terminal Step in the host due to the overproduction of nitrate, nitrite, and other
forms of nitrogen oxide, which damage blood vessels. This is a
The last step, during which oxygen accepts the electrons, is clear example of the interaction of the host microbiome on health
catalyzed by cytochrome aa3, also called cytochrome oxidase. and disease.
This large enzyme complex is specifically adapted to receive
electrons from another cytochrome, pick up hydrogens from the
solution, and react with oxygen to form a molecule of water.
Fermentation
This reaction, though in actuality more complex, is summarized The third main catabolism pathway is fermentation. The defini-
as follows: tion of fermentation is the incomplete oxidation of glucose or other
carbohydrates in the absence of oxygen. This process uses organic
2H+ + 2e− + ½O2 → H2O compounds—as opposed to O2 or oxygen-containing ions—as
Most eukaryotic aerobes have a fully functioning cytochrome the terminal electron acceptors and yields only a small amount of
system, but bacteria exhibit wide-ranging variations in this part ATP generated in glycolysis, the beginning part of fermentation
of the system. Some species lack one or more of the redox steps. (see figure 10.14).
Others have several alternative electron transport schemes. Over time, the term fermentation has acquired several looser
Because many bacteria lack cytochrome oxidase, this variation connotations. Originally, Pasteur called the microbial action of
can be used to differentiate among certain genera of bacteria. yeast during wine production ferments, and to this day, biochem-
An oxidase detection test can be used to help identify members ists use the term in reference to the production of ethyl alcohol
of the genera Neisseria and Pseudomonas and some species of by yeasts acting on glucose and other carbohydrates. Fermenta-
Bacillus. Another variation in the cytochrome system is evident tion is also what bacteriologists call the formation of acid, gas,
in certain bacteria (Klebsiella, Enterobacter) that can grow and other products by the action of various bacteria on pyruvic
even in the presence of cyanide because they lack cytochrome acid. The process is a common metabolic strategy among bac-
oxidase. Cyanide will cause rapid death in humans and other teria. Industrial processes that produce chemicals on a massive
eukaryotes because it blocks cytochrome oxidase, thereby com- scale through the actions of microbes are also called fermenta-
pletely blocking aerobic respiration, but it is harmless to these tions. Each of these usages is acceptable for one application or
bacteria. another.
A potential side reaction of the respiratory chain in aerobic Without the use of an electron transport chain, it may seem
organisms is the incomplete reduction of oxygen to superoxide that fermentation would yield only meager amounts of energy
ion (O2−) and hydrogen peroxide (H2O2). These toxic oxygen (2 ATPs maximum per glucose) and that would slow down
products can be very damaging to cells. Aerobes have neutral- growth. What actually happens, however, is that many bacteria
izing enzymes to deal with these products, including superoxide can grow as fast as they would in the presence of oxygen because
dismutase and catalase. One exception is the genus Streptococ- they speed up the rate of glycolysis. From another standpoint,
cus, which can grow well in oxygen yet lacks both cytochromes fermentation permits independence from molecular oxygen and
and catalase. The tolerance of these organisms to oxygen can be allows colonization of anaerobic environments. It also enables
explained by the neutralizing effects of a special peroxidase. The microorganisms with a versatile metabolism to adapt to variations
lack of cytochromes, catalase, and peroxidases in anaerobes as a in the availability of oxygen. For them, fermentation provides a
rule limits their ability to process free oxygen and contributes to means to grow even when oxygen levels are too low for aerobic
its toxic effects on them. respiration.
Bacteria that digest cellulose in the rumens of cattle are largely
fermentative. After initially hydrolyzing cellulose to glucose, they
Anaerobic Respiration ferment the glucose to organic acids, which are then absorbed as
Some bacteria have evolved an anaerobic respiratory system that the bovine’s principal energy source. Even human muscle cells
functions like the aerobic cytochrome system except that it utilizes can undergo a form of fermentation that permits short periods of
oxygen-containing ions, rather than free oxygen, as the final elec- activity after the oxygen supply in the muscle has been exhausted.
tron acceptor in electron transport (see figure 10.14). Of these, Muscle cells convert pyruvic acid into lactic acid, which allows
the nitrate (NO3−) and nitrite (NO2−) reduction systems are best anaerobic production of ATP to proceed for a time. But this cannot
known. The reaction in species such as Escherichia coli is repre- go on indefinitely, and after a few minutes, the accumulated lactic
sented as: acid causes muscle fatigue.
NAD+
NAD H
Glycolysis
H O
Acetaldehyde
NAD H NAD H
H H H OH
O
NAD+
H C C OH H C C C
OH
H H H H
Ethyl alcohol Lactic acid
further decompose formic acid completely to carbon dioxide and 10.4 Biosynthesis and the Crossing
hydrogen gases. Because enteric bacteria commonly occupy the
intestine, this fermentative activity accounts for the accumulation Pathways of Metabolism
of some types of gas in the intestine. Our discussion now
turns from catabolism
Catabolism of Noncarbohydrate Compounds and energy extraction
We have given you one version of events for catabolism, using glu- to anabolic functions
CATABOLISM
ANABOLISM
ANABOLISM
ANABOLISM
cose, a carbohydrate, as our example. Other compounds serve as and biosynthesis. In
fuel, as well. The more complex polysaccharides are easily broken this section, we present
down into their component sugars, which can enter glycolysis at aspects of intermediary
various points. Microbes also break down other molecules for metabolism, including
their own use, of course. Two other major sources of energy and amphibolic pathways, the synthesis of simple molecules, and the
building blocks for microbes are lipids (fats) and proteins. Both of synthesis of macromolecules.
these must be broken down to their component parts to produce
precursor metabolites and energy. The Efficiency of the Cell
Recall from chapter 2 that fats are fatty acids joined to
It must be obvious by now that cells have mechanisms for careful
glycerol. Enzymes called lipases break these apart. The glycerol
management of carbon compounds. Rather than being dead ends,
is then converted to dihydroxyacetone phosphate (DHAP), which
most catabolic pathways contain strategic molecular intermedi-
can enter step 4 of glycolysis (see figure 10.15). The fatty
ates (metabolites) that can be diverted into anabolic pathways. In
acid component goes through a process called beta oxidation.
this way, a given molecule can serve multiple purposes, and the
Fatty acids have a variable number of carbons. In beta oxidation,
maximum benefit can be derived from all nutrients and metabo-
2-carbon units are successively transferred to coenzyme A, creat-
lites of the cell pool. The property of a system to integrate cata-
ing acetyl CoA, which enters the Krebs cycle. This process can
bolic and anabolic pathways to improve cell efficiency is termed
yield a large amount of energy. Oxidation of a 6-carbon fatty acid
amphibolism (am-fee-bol′-izm).
yields 50 ATPs, compared with 38 for a 6-carbon sugar.
At this point in the chapter, you can appreciate a more com-
Proteins are chains of amino acids. Enzymes called proteases
plex view of metabolism than that presented at the beginning in
break proteins down to their amino acid components, after which
figure 10.1. Figure 10.23 includes the activities of amphibolism
the amino groups are removed by a reaction called deamination
and so provides a more detailed picture. Cells are efficient. They
(figure 10.22). This leaves a carbon compound, which is easily
can divert intermediate products from glycolysis to synthesize car-
converted to one of several Krebs cycle intermediates.
bohydrates if needed. You can see that catabolism and anabolism
are not always strictly separated the way figure 10.1 implies.
10.3 Learning Outcomes—Assess Your Progress
10. Name the three main catabolic pathways and the Amphibolic Sources of Cellular Building Blocks
estimated ATP yield for each. Glyceraldehyde-3-phosphate can be diverted away from glycoly-
11. Construct a paragraph summarizing glycolysis. sis and converted into precursors for amino acid, carbohydrate,
12. Describe the Krebs cycle and compare the process and triglyceride (fat) synthesis. (A precursor molecule is a com-
between bacteria and eukaryotes. pound that is the source of another compound.) We have already
13. Discuss the significance of the electron transport system. noted the numerous directions that pyruvic acid catabolism can
take. In terms of synthesis, pyruvate also plays a pivotal role
14. State two ways in which anaerobic respiration differs from
in providing intermediates for amino acids. In the event of an
aerobic respiration.
inadequate glucose supply, pyruvate serves as the starting point
15. Summarize the steps of microbial fermentation, and list in glucose synthesis from various metabolic intermediates, a
three useful products it can create. process called gluconeogenesis (gloo′-koh-nee′-oh-gen′-uh-sis).
16. Describe how noncarbohydrate compounds are catabolized. The acetyl group that starts the Krebs cycle is another extremely
versatile metabolite that can be fed into a number of synthetic path-
ways. This 2-carbon fragment can be converted as a single unit into
one of several amino acids, or a number of these fragments can
Amino acid Carbohydrate
be condensed into hydrocarbon chains that are important building
NH2 H H O O H H blocks for fatty acid and lipid synthesis. Note that the reverse is also
+
O
O O NAD true—fats can be degraded to acetyl through beta oxidation and
C C C C C C C C C C NH4+
OH H H H OH OH H H OH thereby enter the Krebs cycle as acetyl coenzyme A.
H2O
Pathways that synthesize the nitrogen bases (purines, pyri-
Glutamic acid α-ketoglutaric acid
midines), which are components of DNA and RNA, originate in
Figure 10.22 Deamination. Removal of an amino group amino acids and so can be dependent on intermediates from the
converts an amino acid to an intermediate of carbohydrate metabolism. Krebs cycle as well. Because the coenzymes NAD, NADP, FAD,
Ammonium is a by-product. and others contain purines and pyrimidines similar to the nucleic
Carbohydrate Biosynthesis
Krebs
Cycle
The role of glucose in metabolism and
CO2
energy utilization is so crucial that its bio-
synthesis is ensured by several alternative
NH3 H2 O pathways. Certain structures in the cell
depend on an adequate supply of glucose as
Figure 10.23 An amphibolic view of metabolism. A simple compound such as well. It is the major component of the cel-
glucose is both broken down into smaller parts (catabolism) and modified and used as a building lulose cell walls of some eukaryotes and of
block in anabolism. certain storage granules (starch, glycogen).
acids, their synthetic pathways are also dependent on amino acids. One of the intermediaries in glycolysis,
During times of carbohydrate deprivation, organisms can likewise glucose-6 -
P , is used to form glycogen. Monosaccharides other
convert amino acids to intermediates of the Krebs cycle by deami- than glucose are important in the synthesis of bacterial cell walls.
nation and thereby derive energy from proteins (see figure 10.22). Peptidoglycan contains a linked polymer of muramic acid and
We’ve been discussing these pathways within individual cells, glucosamine. Fructose- 6 -
P from glycolysis is used to form these
but it is clear that microbes and their metabolic intermediaries and two sugars. Carbohydrates (deoxyribose, ribose) are also essential
products influence other microbes and other organisms in their envi- building blocks in nucleic acids. Polysaccharides are the predomi-
ronment. The notions of symbiosis discussed in chapter 9 encompass nant components of cell surface structures such as capsules and the
the ability of adjacent organisms to use their neighbor’s products. glycocalyx, and they are commonly found in slime layers.
ANABOLISM
ANABOLISM
ANABOLISM
blocks that make up the acids are needed to make these proteins. Although some organ-
various macromolecules isms (E. coli, for example) have pathways that will synthesize all
and organelles of the 20 amino acids, others, including animals, lack some or all of the
cell—come from two pathways for amino acid synthesis and must acquire the essential
ones from their diets. Protein synthesis itself is a complex process into chemical energy through photosynthesis. (Some chemoauto-
that requires a genetic blueprint and the operation of intricate cel- trophs derive their energy and nutrients solely from inorganic sub-
lular machinery, as you saw in chapter 6. strates.) The other major products of photosynthesis are organic
carbon compounds, which are produced from carbon dioxide
Assembly of the Cell through a process called carbon fixation.
The component parts of With some notable exceptions, the energy that drives all life
a bacterial cell are syn- processes comes from the sun, but this source is directly available
thesized on a continuous only to photosynthesizers. On land, green plants are the primary
basis, and catabolism is photosynthesizers. In aquatic ecosystems, algae, green and pur-
CATABOLISM
ANABOLISM
ANABOLISM
ANABOLISM
also taking place as long ple bacteria, and cyanobacteria fill this role. It was also recently
as nutrients are present discovered that bacteriophages that infect marine cyanobacteria
and the cell is in a nondor- provide some of the genes allowing these organisms to carry out
mant state. When anabo- photosynthesis.
lism produces enough macromolecules to serve two cells, and when Photosynthetic organisms use light energy to produce high-
DNA replication produces duplicate copies of the cell’s genetic mate- energy glucose from low-energy CO2 and water or as you see in
rial, the cell undergoes binary fission, which results in two cells from Insight 10.1, from electricity. They do this through a series of
one parent cell. The two cells will need twice as many ribosomes, reactions involving light, pigment, CO2, and water, which is used
twice as many enzymes, and so on. The cell has created these during as a source for electrons.
the initial anabolic phases we have described. Before cell division, the Photosynthesis proceeds in two phases: the light-dependent
membrane(s) and the cell wall will have increased in size to create a reactions, which proceed only in the presence of sunlight, and
cell that is almost twice as big as a “newborn” cell. Once synthesized, the light-independent reactions, which proceed regardless of the
the phospholipid bilayer components of the membranes assemble lighting conditions (light or dark).
themselves spontaneously with no energy input. Other assembly Solar energy is delivered in discrete energy packets called
reactions require the input of energy. Proteins and other components photons (also called quanta) that travel as waves. The wavelengths
must be added to the membranes. Growth of the cell wall, accom- of light operating in photosynthesis occur in the visible spectrum
plished by the addition and coupling of sugars and peptides, requires between 400 (violet) and 700 nanometers (red). As this light strikes
energy input. The energy accumulated during catabolic processes photosynthetic pigments, some wavelengths are absorbed, some
provides all the energy for these complex building reactions. pass through, and some are reflected. The activity that has greatest
impact on photosynthesis is the absorbance of light by photosyn-
thetic pigments. These include the chlorophylls, which are green;
10.4 Learning Outcomes—Assess Your Progress carotenoids, which are yellow, orange, or red; and phycobilins,
which are red or blue-green.3 The most important of these pig-
17. Provide an overview of the anabolic stages of metabolism.
ments are the bacterial chlorophylls. These molecules contain a
18. Define amphibolism. photocenter that consists of a magnesium atom held in the center
of a complex, ringed molecule called a porphyrin. As we will see,
10.5 Photosynthesis: It Starts with Light the chlorophyll molecule harvests the energy of photons and con-
verts it to chemical energy. Other pigments such as carotenes trap
As we mentioned earlier, the ultimate source of chemical energy
in many cells comes from the sun. Most organisms depend either
directly or indirectly on the sunlight’s energy, which is converted 3. The color of the pigment corresponds to the wavelength of light it reflects.
This chapter describes metabolism, and states right up front that of material for building blocks and energy. In fact, they metabolize
metabolism has three goals: and grow using only electrons from electricity. They may run the
TCA cycle in reverse, creating acetyl CoA from carbon dioxide.
∙∙ break down large molecules, usually sugars, to get building
From acetyl CoA they can create all of the molecules they need
blocks;
for a new cell. Then again, the very newest bacteria discovered can
∙∙ build molecules the cell needs; and
live (but not reproduce) with no carbon input at all—not even CO2.
∙∙ harvest energy to do the building work.
Dr. Ken Nealson, at the University of Southern California,
Put another way, electron flow—from an energy source to electron whose lab conducted these studies, says, “This is huge. What it
carriers to the electron transport chain—is necessary for energy to means is there’s a whole part of the microbial world that we don’t
be gained. know about.” In addition, there are a slew of practical applications,
Well, nature continues to surprise us with its many different including waste recycling and creating biological fuel cells.
variations on central themes. Scientists have discovered microbes Dr. Nealson is also guessing that this might be the predominant
that survive without using any sugars, which are the usual source mode of life on other planets.
Flagellum
Nucleus
Cell wall
Chloroplast
Stroma
A B
Granum N N
Mg
N N
d en t L igh t- I D C
e p en nd
g ht-D c tio n s R e a c e p en d Glucose
i tio n en
H 2O L R ea s t
Chlorophyll
Photons
ATP
2H + e–
NADP H
2 e–
2
Ele 2 e– NADP + H+
car ctron
rr i on
P700 5
rier
ca ectr
s
s
er
1
Calvin Cycle
NADPH
El
1/2 O2 Photosystem I
2 e– ADP + P i
O2 Photolysis
H+
3 6
H
+
CO2 P680 2H+ ATP
Chloroplast H+ H+ H +
Photosystem II ATP
Figure 10.24 Overview of photosynthesis. The general H2O Proton
synthase
pump
reactions of photosynthesis, divided into two phases called light-
dependent reactions and light-independent reactions. The dependent
Thylakoid membrane Interior of granum
reactions require light to activate chlorophyll pigment and use the 4
of granum
energy given off during activation to split an H2O molecule into oxygen H+
and hydrogen, producing ATP and NADPH. The independent reactions,
which occur either with or without light, utilize ATP and NADPH The main events of the light reaction shown as an exploded view in
produced during the light reactions to fix CO2 into organic compounds one granum.
such as glucose. 1 When light activates photosystem II, it sets up a chain reaction, in
which electrons are released from chlorophyll.
light energy and shuttle it to chlorophyll, functioning like antennae. 2 These electrons are transported along a chain of carriers to
These light-dependent reactions are catabolic (energy-producing) photosystem I.
reactions, which pave the way for the next set of reactions, the 3 The empty position in photosystem II is replenished by photolysis of
H2O. Other products of photolysis are O2 and H+.
light-independent reactions, which need the produced energy for
4 Pumping of H+ into the interior of the granum produces conditions for
synthesis (anabolism). During this phase, carbon atoms from CO2 ATP to be synthesized.
are added to the carbon backbones of organic molecules. 5 The final electron and H+ acceptor is NADP, which receives these from
The detailed biochemistry of photosynthesis is not neces- photosystem I.
sary for this text, but we will provide an overview of the general 6 Both NADPH and ATP are fed into the stroma for the Calvin cycle.
process as it occurs in green plants, algae, and cyanobacteria
(figure 10.24). Many of the basic activities (electron transport and Figure 10.25 The reactions of photosynthesis. A cell of the
phosphorylation) are similar to certain pathways of respiration. eukaryotic motile alga Chlamydomonas, with a single large chloroplast
(magnified cutaway view). The chloroplast contains membranous
compartments called grana where chlorophyll molecules and the
Light-Dependent Reactions photosystems for the light molecules are located.
The same systems that carry the photosynthetic pigments are
also the sites for the light reactions. They occur in the thylakoid
membranes of compartments called grana (singular, granum) in
chloroplasts (figure 10.25). In bacteria, this occurs in special- simultaneously activated by light, but the reactions in photosystem
ized parts of the cytoplasmic membranes. These systems exist II help drive photosystem I. Together the systems are activated by
as two separate complexes called photosystem I (P700) and pho- light, then transport electrons, pump hydrogen ions, and form ATP
tosystem II (P680).4 Both systems contain chlorophyll and are and NADPH.
When photons enter the photocenter of the P680 system
4. The numbers refer to the wavelength of light to which each system is most sensitive. (PS II), the magnesium atom in chlorophyll becomes excited and
releases 2 electrons. The loss of electrons from the photocenter has CO2
two major effects:
P P Splitting
1. It creates a vacancy in the chlorophyll molecule forceful
6-carbon
enough to split an H2O molecule into hydrogen (H+) (elec- intermediate 3-phosphoglyceric
trons and hydrogen ions) and oxygen (O2). This splitting of acid
water, termed photolysis, is the ultimate source of the O2 gas P P
that is an important product of photosynthesis. The electrons Ribulose-1,5-bisphosphate
ATP × 2
5-carbon P
released from the lysed water regenerate photosystem II for its P
next reaction with light.
ADP Calvin Cycle
2. Electrons generated by this first photoevent are immediately P P ADP
shunted through a series of carriers (cytochromes) to the
P700 system. At this same time, hydrogen ions accumulate in ATP
the internal space of the thylakoid complex, thereby produc-
P P
ing an electrochemical gradient.
1,3-bisphosphoglyceric acid
Series of 7-carbon
The P700 system (PS I) has been activated by light so that it and 5-carbon
P
H P
is ready to accept electrons generated by the PS II. The electrons intermediates NADPH × 2
H
it receives are passed along a second transport chain to a com-
plex that uses electrons and hydrogen ions to reduce NADP to
P NADP+
NADPH. (Recall that reduction in this sense entails the addition P
of electrons and hydrogens to a substrate.) Glyceraldehyde-3-
phosphate
A second energy reaction involves synthesis of ATP by a che-
miosmotic mechanism similar to that shown in figures 10.18 and Glucose
10.19. Channels in the thylakoids of the granum actively pump H+ Fructose intermediates
into the inner chamber, producing a charge gradient. ATP synthase Figure 10.26 The Calvin cycle. The main events of the
located in this same thylakoid uses the energy from H+ transport to reactions in photosynthesis that do not require light. It is during this
phosphorylate ADP to ATP. Because it occurs in light, this process cycle that carbon is fixed into organic form using the energy (ATP and
is termed photophosphorylation. Both NADPH and ATP are NADPH) released by the light reactions. The end product, glucose,
released into the stroma of the chloroplast, where they drive the can be stored as complex carbohydrates, or it can be used in various
reactions of the Calvin cycle. amphibolic pathways to produce other carbohydrate intermediates or
amino acids.
Light-Independent Reactions
The subsequent photosynthetic reactions that do not require light Other photosynthesizers such as green and purple bacteria possess
occur in the chloroplast in or the cytoplasm of cyanobacteria. bacteriochlorophyll, which is more versatile in capturing light. They
These reactions use energy produced by the light phase to synthe- have only a cyclic photosystem I, which routes the electrons from
size glucose by means of the Calvin cycle (figure 10.26). the photocenter to the electron carriers and back to the photosystem
The cycle begins at the point where CO2 is combined with again. This pathway generates a relatively small amount of ATP,
a doubly phosphorylated 5- carbon acceptor molecule called and it may not produce NADPH. As photolithotrophs, these bacte-
ribulose-1,5-bisphosphate (RuBP). This process, called carbon ria use H2, H2S, or elemental sulfur rather than H2O as a source of
fixation, generates a 6-carbon intermediate compound that imme- electrons and reducing power. As a consequence, they are anoxy-
diately splits into two 3-carbon molecules of 3-phosphoglyceric genic (non-oxygen-producing), and many are strict anaerobes.
acid (PGA). The subsequent steps use the ATP and NADPH While most of the mechanisms just described involve chlo-
generated by the photosystems to form high-energy intermedi- rophyll or bacteriochlorophyll as the light-absorbing pigment,
ates. First, ATP adds a second phosphate to 3-PGA and produces archaea use a pigment called bacteriorhodopsin. You may recog-
1,3-bisphosphoglyceric acid (BPG). Then, during the same step, nize the root rhodopsin, which is a pigment present in vertebrate
NADPH contributes its hydrogen to BPG, and one high-energy eyes (in the rods and cones). This type of photosynthesis does not
phosphate is removed. These events give rise to glyceraldehyde- involve electron transport but instead uses a light-driven proton
3-phosphate (PGAL). This molecule and its isomer dihydroxyac- pump. Through chemiosmosis, it generates ATP.
etone phosphate (DHAP) are key molecules in hexose synthesis
leading to fructose and glucose. You may notice that this pathway 10.5 Learning Outcomes—Assess Your Progress
is very similar to glycolysis, except that it runs in reverse (see
figure 10.15). Bringing the cycle back to regenerate RuBP requires 19. Summarize the overall process of photosynthesis in a
PGAL and several steps not depicted in figure 10.26. single sentence.
20. Discuss the relationship between light-dependent and
Other Mechanisms of Photosynthesis light-independent reactions.
The oxygenic, or oxygen-releasing, photosynthesis that occurs in 21. Explain the role of the Calvin cycle in the process of
plants, algae, and cyanobacteria is the dominant type on the earth. photosynthesis.
These activities are designed for you to use on your own with a study group—either a face-to-face group or a virtual one,
consisting of 3–5 members. Studying together can be very helpful, but there are effective and ineffective ways to do it. For example,
getting together without a clear structure is often not a good use of your time. Use your time efficiently by using one or more of the
exercises below.
FACE-TO-FACE GROUPS
Use one or more of the activities below.
Peer Instruction: Assign numbers to your group members to use all semester long. Now look at these five concepts from this
chapter. Each group member prepares a 5-minute lesson on the topic corresponding to their number. Don’t worry if you have fewer
than 5 members; just use however many you have! During your group study time, each member presents their lesson, and the group
spends another 5–10 minutes discussing that lesson.
1. Redox reaction
2. Electron carriers
3. Glycolysis
4. Krebs cycle
5. Fermentation
Concept Maps: Each member of the group should use this list of terms from this chapter to generate their own concept map. This
can be hand-drawn or created using software (see Appendix C for guidelines). During group study time, compare each other’s
concept maps and help each other make sure they are correct. Of course, there are many different “correct” maps. Examining
each member’s map will help you talk through the varied concepts and how they are related.
Concept Terms:
metabolism catabolism products enzymes
anabolism substrates pH
Table Topics: Each group member should identify a concept or topic from this week’s class assignments with which they are
having trouble and share it during group study time. The other group members can then help to clarify confusing issues or share
how they figured it out. Aim for a maximum of 15 minutes per topic. If the topic remains unclear to the group, bring it up during
class or use the instructor’s office hours or e-mail to ask for help. Taking the time to struggle with a difficult concept first makes
your questions much more specific and more likely to yield helpful answers.
(continued)
VIRTUAL GROUPS
Not everyone has the time or opportunity to meet with group members outside of class time. You or your instructor can
create a virtual group using e-mail or the course software.
Weekly Discussion Board: This forum can be used as a way for groups to discuss topics, via e-mail, or other learning management
systems or online platforms, before they are covered in class. As each member of the group answers the current week’s question, they
should send their responses to every other member of their group. It’s best to agree on a deadline based on how your class schedule
works (Saturday for the next week’s topics, for example). Then, after the topic is discussed in class, each member should send a
response that all group members will see with a follow-up post on the same topic. If you cover more than one chapter in a week,
someone can be designated to choose which chapter Discussion Board question you will use. Or simply decide up front that you will
always use the first-chapter-of-the week’s question, to keep the schedule simple.
Discussion Question
Use the concept of electron carriers to explain how the electron transport chain works. Then explain why ATP synthase is so
important to the life of a cell.
Chapter Summary
MICROBIAL METABOLISM
THE METABOLISM OF MICROBES
10.1
• Metabolism is the sum of cellular chemical and physical activities. It consists of anabolism,
energy-requiring reactions that convert small molecules into larger molecules, and catabolism,
in which large molecules are broken down and energy is produced.
• Metabolism is made possible by organic catalysts called enzymes.
• Enzymes are not consumed in the reactions they catalyze. Each enzyme acts specifically on its
matching molecule or substrate.
• Enzymes are active only within narrow operating ranges of temperature, osmotic pressure, and pH.
• Enzymes can be regulated through activators or repressors binding to them, or they can be
regulated during the genetic process that gives rise to enzymes.
This 21 Question Grid takes the topics from this chapter and arranges them with respect to the American Society for Microbiology’s
Undergraduate Curriculum guidelines—all six of the important “Concepts” as well as the important “Competency” of scientific literacy. Three
questions are supplied, which cover chapter content referring to the Concept or Competency in increasing levels of Bloom’s taxonomy for learning.
ASM Concept/ A. Bloom’s Level 1, 2—Remember B. Bloom’s Level 3, 4—Apply C. Bloom’s Level 5, 6—Evaluate
Competency and Understand (Choose one.) and Analyze and Create
Evolution 1.
The electron transport system 2.
Speculate on why glycolysis, 3. Provide evidence in support
in bacteria is located on the which is the beginning point of or refuting the following
and in of many catabolic pathways, statement: The evolution
eukaryotic cells on the does not require oxygen. of aerobic respiration was
. driven by the success of
a. plastid, chloroplast photosynthetic microbes.
b. cytoplasmic membrane,
mitochondrion
c. cell wall, mitochondrion
d. mitochondrion, cytoplasmic
membrane
c. proteins.
d. substrates.
ASM Concept/ A. Bloom’s Level 1, 2—Remember B. Bloom’s Level 3, 4—Apply C. Bloom’s Level 5, 6—Evaluate
Competency and Understand (Choose one.) and Analyze and Create
Microbial Systems 13. Enzymes that can be shut 14. Which of the three major 15. Write a paragraph explaining
down or activated based on catabolic strategies do you why metabolic pathways
the presence of chemicals in think the last common ancestor that are amphibolic are
their environment are called possessed? Defend your advantageous for cells.
a. constitutive. answer.
b. repressed.
c. holoenzymes.
d. regulated.
Impact of 16. The type of microbial 17. Defend this statement: 18. Construct an argument for the
Microorganisms metabolic pathway that is most Microbes (and their metabolic possibility that scientists will
often exploited to make acids strategies) are absolutely continue to discover novel
and alcohols industrially is essential to plant life on earth. metabolic strategies that are
a. aerobic respiration. not yet known.
b. anaerobic respiration.
c. fermentation.
d. none of the above.
Scientific Thinking 19. Which of the following is true? 20. Find three enzymatic 21. Polymerase chain reaction (PCR)
a. The suffix “-ase” indicates an capabilities that are commonly is a lab technology that requires
enzyme. used to identify microbial high temperatures to make
b. Often enzymes are named for species in a clinical lab. copies of DNA. The copies are
the substrates they act upon. made using an enzyme called
a polymerase. PCR would not
c. Enzymes are larger than their
have been possible before
substrates.
thermophilic archaea were
d. All of the above are true. discovered. Explain why.
Visual Connections
These questions use visual images or previous content to connect content within and between chapters.
These terms and concepts are most critical for your understanding of this chapter—and may be the most difficult. Have you mastered them?
Concepts Terms
Electron flow Metabolism
Redox reactions Catabolism
Electron carriers Anabolism
Glycolysis ATP
Krebs cycle Enzyme
Electron transfer system Substrate
Aerobic and anaerobic respiration Fermentation
Amphibolism Pyruvic acid
Photosynthesis
280
Much of the time in our daily existence, we take for granted
Disinfection destroys most microbial life, reducing contamination
tap water that is drinkable, food that is not spoiled, shelves full on inanimate surfaces.
of products to eradicate “germs,” and drugs to treat infections. (also
Antisepsis called degermation) is the same as disinfection
Controlling our exposure to potentially harmful microbes is a except a living surface is involved.
monumental concern in our lives, and it has a long and eventful
history. Salting, smoking, pickling, and drying foods as well as
Decontamination (also called sanitization) is the mechanical
exposing food, clothing, and bedding to sunlight were prevalent removal of most microbes from an animate or inanimate surface.
practices among early civilizations. The Greeks and Romans The flowchart in figure 11.1 summarizes the major applications
burned clothing and corpses during epidemics, and they stored and aims in microbial control.
water in copper and silver containers. During the great plague
pandemic of the Middle Ages, it was common to bury corpses Relative Resistance of Microbial Forms
in mass graves, burn the clothing of plague victims, and ignite The primary targets of microbial control are microorganisms
aromatic woods in the houses of the sick in the belief that fumes capable of causing infection or spoilage. These are constantly
would combat the disease. Each of these early methods, although present in the external environment and on the human body. This
crude, laid the foundations for microbial control methods that are population is usually not simple or uniform. It often contains
still in use today. mixtures of microbes with big differences in their resistance and
harmfulness. Some of the microbes that can be dangerous if not
controlled include bacterial vegetative cells and endospores, fun-
General Considerations in Microbial Control gal hyphae and spores, yeasts, protozoan trophozoites and cysts,
The methods of microbial control used outside of the body are worms, viruses, and prions. Figure 11.2 compares the general
designed to result in four possible outcomes: sterilization, disin- resistance these forms have to physical and chemical methods of
fection, antisepsis, or decontamination. control.
Actual comparative figures on the requirements for destroying Methods of Microbial Control
various groups of microorganisms are shown in table 11.1. Bacte-
The terminology for describing and defining measures that con-
rial endospores have traditionally been considered the most resist-
trol microbes can get a little jumbled. The official designations
ant microbial entities, being as much as 18 times harder to destroy
are those shown in figure 11.1, but in practice, people use the
than their counterpart vegetative cells. Because of their resistance
terms loosely. For example, someone may ask you to sterilize a
to microbial control, destroying them is the goal of sterilization
patient’s skin, even though this is not technically correct. It does
because any process that kills endospores will invariably kill all
not fit the technical definition of the term. It’s best to start with
less-resistant microbial forms. Other methods of control (disinfec-
a good groundwork, so let’s look at concepts, definitions, and
tion, antisepsis) act primarily upon microbes that are less hardy
usages in antimicrobial control.
than endospores.
Protozoan cysts
Protozoan trophozoites
Nonenveloped viruses
Enveloped
En
En viruses
Less resistant
SARS-CoV-2 is in this group!
–Kelly & Heidi
Table 11.1 Comparative Resistance of Bacterial Endospores microorganisms but do not destroy the more resistant endospore and
and Vegetative Cells to Control Agents cyst stages. Keep in mind that the destruction of endospores is not
always necessary because most of the infectious diseases of humans
Required
and animals are caused by non-endospore-forming microbes.
Required to Destroy Endospores
to Destroy Vegetative Are ___ More Disinfection refers to the use of a physical process or a
Method Endospores Forms Resistant* chemical agent (a disinfectant) to destroy vegetative patho-
gens but not bacterial endospores. Disinfectants are normally
Heat (moist) 120°C 80°C 1.5×
used only on inanimate objects because, in the concentrations
Radiation 4,000 Grays 1,000 Grays 4× required to be effective, they can be toxic to human and other
(X-ray) dosage animal tissue. Disinfection processes also remove the harmful
Sterilizing gas 1,200 mg/L 700 mg/L 1.7× products of microorganisms (toxins) from materials. Examples
(ethylene oxide) of disinfection include applying a solution of 5% bleach to an
examining table, boiling food utensils used by a sick person, and
Sporicidal 3h 10 min 18×
immersing thermometers in an iodine solution between uses.
liquid (2%
glutaraldehyde)
Sepsis is defined as the growth of microorganisms in the
blood and other tissues. The term asepsis refers to any practice
*The greater resistance of endospores versus vegetative cells given as an average figure. that prevents the entry of infectious agents into sterile tissues and
thus prevents infection. Aseptic techniques commonly practiced in
Terminology health care range from sterile methods to antisepsis. In antisepsis,
chemical agents called antiseptics are applied directly to exposed
Sterilization is a process that destroys or removes all viable micro- body surfaces (skin and mucous membranes), wounds, and surgi-
organisms, including viruses. Any material that has been subjected cal incisions to destroy or inhibit vegetative pathogens. Examples
to this process is said to be sterile. These terms should be used of antisepsis (also called degermation) include preparing the skin
only in the strictest sense for methods that have been proved to before surgical incisions with iodine compounds, swabbing an open
sterilize. An object cannot be slightly sterile or almost sterile—it root canal with hydrogen peroxide, using alcohol wipes on the skin,
is either sterile or not sterile. Control methods that sterilize are and performing the surgical hand scrub. To recap, degermation is
generally reserved for inanimate objects because sterilizing the a synonym for antisepsis, and both are included in the term aseptic
human body or its parts would call for such harsh treatment that it techniques. Sterile methods are also grouped under the term aseptic
would be dangerous and impractical. technique. It sounds complicated, but if you are going to be work-
The ability to sterilize products—surgical instruments, ing in a clinical setting, different people will use different terms.
syringes, and some packaged foods, just to name a few—is essen-
tial to human well-being. Although most sterilization is performed
with a physical agent such as heat, a few chemicals can be classified The Agents versus the Processes
as sterilizing agents because of their ability to destroy endospores. The terms sterilization, disinfection, etc., refer to processes. There
In many situations, sterilization is neither practical nor necessary, are other terms that describe the agents used in the process. Two
and only certain groups of microbes need to be controlled. Some anti- of these are the terms bactericidal and bacteristatic. The root
microbial agents eliminate only the susceptible vegetative states of -cide, meaning “to kill,” can be combined with other terms to
define an antimicrobial agent aimed at destroying a certain group
of microorganisms. For example, a bactericide is a chemical that
A Note About Prions destroys bacteria (except for those in the endospore stage). It may
or may not be effective on other microbial groups. A fungicide
Prions are in a class of their own when it comes to “sterilization” is a chemical that can kill fungal spores, hyphae, and yeasts. A
procedures. This chapter defines sterile as the absence of all virucide is any chemical known to inactivate viruses, especially
viable microbial life—but none of the procedures described in on living tissue. A sporicide is an agent capable of destroying
this chapter are necessarily sufficient to destroy prions. Prions bacterial endospores. A sporicidal agent can also be considered a
are extraordinarily resistant to heat and chemicals. If instruments sterilant because it can destroy the most resistant of all microbes.
or other objects become contaminated with these unique agents, Germicide and microbicide are additional terms for chemical
either they must be discarded as biohazards or, if this is not agents that kill microorganisms.
possible, enhanced sterilization procedures must be applied in The Greek words stasis and static mean “to stand still.”
accordance with CDC guidelines. The guidelines themselves are They can be used in combination with various prefixes to
constantly evolving as new information becomes available. In describe a condition in which microbes are prevented from
the meantime, this chapter discusses sterilization using bacterial multiplying but are not killed outright. Although killing or
endospores as the toughest form of microbial life. When tissues, permanently inactivating microorganisms is the usual goal of
fluids, or instruments are suspected of containing prions, con- microbial control, microbistasis does have meaningful applica-
sultation with infection control experts and/or the CDC is recom- tions. Bacteristatic agents prevent the growth of bacteria on
mended when determining effective sterilization conditions. tissues or on objects in the environment, and fungistatic chemi-
cals inhibit fungal growth. The general terms for the killing
or inhibition of microbes are microbicidal and microbistatic, or crutches. They require only low-level disinfection unless they
respectively. Chemicals used to control microorganisms in the become contaminated with blood or body fluids.
body (antiseptics and drugs) often are chosen for their microbi- A remarkable variety of substances can require sterilization.
static effects because the ones that are microbicidal can be They range from durable solids such as rubber to sensitive liquids
highly toxic to human cells. such as serum. An unusual case of having to sterilize an entire
office building, without destroying the records and equipment
in it, occurred in 2001 when the Hart Senate Office Building in
Decontamination (Sanitization)
Washington, D.C., was deliberately contaminated with Bacillus
Sanitization is any cleansing technique that mechanically removes anthracis endospores. (It was eventually accomplished using steri-
microorganisms as well as other debris to reduce contamination to lizing gas.) Hundreds of situations requiring sterilization confront
safe levels. A sanitizer is a compound such as soap or detergent the network of persons involved in public health care, whether
used to perform this task. technician, nurse, doctor, or manufacturer, and no method works
Cooking utensils, dishes, bottles, cans, and clothing that have well in every case.
been washed and dried may not be completely free of microbes, but Considerations such as cost, effectiveness, and method of dis-
they are considered safe for normal use (sanitary). Air sanitization posal are all important. For example, disposable plastic items such
with ultraviolet (UV) lamps reduces airborne microbes in hospital as catheters and syringes that are used in invasive medical proce-
rooms, veterinary clinics, and laboratory installations. Note that dures have the potential for infecting tissues. These must be steri-
some sanitizing processes (such as dishwashing machines) may lized during manufacture by a nonheating method (gas or radiation)
be rigorous enough to sterilize objects, but this is not true of all because heat can damage plastics. After these items have been
sanitization methods. Also note that sanitization is often preferable used, it is often necessary to destroy or decontaminate them before
to sterilization. In a restaurant, for example, you could be given a they are discarded because of the potential risk to the handler.
sterile fork with someone else’s old food on it and a sterile glass Steam sterilization, which is quick and sure, is a sensible choice
with lipstick on the rim, but it is preferable to have a sanitized glass at this point because it does not matter if the plastic is destroyed.
with no remnants of the previous guest. On top of this, steriliza-
tion procedures add greatly to the cost of doing business. In other
words, usefulness of a technique depends on the context. What Is Microbial Death?
Death is defined as the permanent termination of an organism’s
vital processes. Signs of life in complex organisms such as ani-
Practical Concerns in Microbial Control
mals are often self-evident, and death is made clear by loss of
Numerous considerations should go into selecting a method of nervous function, respiration, or heartbeat. In contrast, death in
microbial control: microscopic organisms that are composed of just one or a few
1. Does the item in question require sterilization, or is disinfec- cells is often hard to detect because they reveal no conspicuous
tion adequate? In other words, must endospores be destroyed, vital signs to begin with. Lethal agents (such as radiation and
or is it necessary to destroy only vegetative pathogens? chemicals) do not necessarily alter the overt appearance of micro-
2. Is the item to be reused or permanently discarded? If it will bial cells. Even the loss of movement in a motile microbe does not
be discarded, then the quickest and least expensive method, indicate death. This fact has made it necessary to develop special
which is often destructive, can be used. ways to define and delineate microbial death.
3. If it will be reused, can the item withstand heat, pressure, The destructive effects of chemical or physical agents occur
radiation, or chemicals? at the level of a single cell. As the cell is continuously exposed
4. Is the control method suitable for a given application? (For to an agent such as intense heat or toxic chemicals, various cell
example, UV radiation can possibly kill endospores, but it structures become dysfunctional. The entire cell can sustain
will not penetrate solid materials.) Or, in the case of a chemi- irreversible damage in the process. At present, the most practical
cal, will it leave an undesirable residue? way to detect this damage is to determine if a microbial cell can
5. Will the agent penetrate to the necessary extent? still reproduce when exposed to a s uitable environment. If the
6. Is the method cost- and labor-efficient, and is it safe? microbe has sustained metabolic or structural damage to such
an extent that it can no longer reproduce, even under ideal envi-
One useful framework for determining how devices that ronmental conditions, then it is no longer viable. The permanent
come in contact with patients should be handled is whether they loss of reproductive capability, even under optimum growth
are considered critical, semicritical, or noncritical. Critical conditions, has become the accepted microbiological definition
medical devices are those that are expected to come in contact of death.
with sterile tissues. Examples include a syringe needle or an
artificial hip. These must be sterilized before use. Semicritical
devices are those that come in contact with mucosal membranes. Factors That Affect Death Rate
An endoscopy tube is an example. These must receive at least The cells of a culture can show significant variation in suscepti-
high-level disinfection and, preferably, should be sterilized. bility to a given microbicidal agent. Death of the whole popula-
Noncritical items are those that do not touch the patient or are tion is not instantaneous but begins when a certain threshold of
only expected to touch intact skin, such as blood pressure cuffs microbicidal agent (some combination of time and concentration)
is met. Death continues in a logarithmic manner as the time (fig- of microbe but a mixture of bacteria, fungi, endospores,
ure 11.3a) or concentration of the agent is increased. Because and viruses, presenting a broad spectrum of microbial
many microbicidal agents target the cell’s metabolic processes, resistance.
active cells (younger, rapidly dividing) tend to die more quickly 3. The type of microbial growth. Planktonic bacterial populations
than those that are less metabolically active (older, inactive). Read are those that grow freely within fluid environments and do not
that again—it might seem counterintuitive at first, but it is an attach to surfaces. In general, they are more susceptible to con-
important property to keep in mind when trying to control micro- trol agents as compared to the microbes within well-developed
bial populations inside or outside of the body. Eventually, a point biofilms adhering to surfaces such as medical devices or human
is reached at which survival of any cells is highly unlikely. This tissues.
point is equivalent to sterilization. 4. The temperature and pH of the environment.
The effectiveness of a particular agent is governed by several 5. The concentration (dosage, intensity) of the agent. For exam-
factors besides time. These additional factors influence the action ple, UV radiation is most effective at 260 nm, and most disin-
of antimicrobial agents: fectants are more active at higher concentrations.
6. The mode of action of the agent (figure 11.3d). How does it
1. The number of microorganisms (figure 11.3b). A higher load
kill or inhibit the microorganism?
of contaminants requires more time to destroy.
7. The presence of solvents, interfering organic matter, and
2. The nature of the microorganisms in the population
inhibitors. Saliva, blood, and feces can inhibit the actions of
(f igure 11.3c). In most actual circumstances of disinfection
disinfectants and even of heat.
and sterilization, the target population is not a single species
108
108 107 High load
107 106
106 105
Low
105 104 load
104 103
103 102
102 101
101 Sterilization
2 4 6 8 10 12 14 16 18 20 10 14
Time (Minutes) Time for Sterilization
Agent added
Endospores
108 Vegetative cells 108 Cells still capable of
growth (if microbistatic
107 107
Number of Viable Cells
Time Time
Figure 11.3 Factors that influence the rate at which microbes are killed by antimicrobial agents. (a) Length of time the agent is
in contact with the microbes. During exposure to a chemical or physical agent, all cells of a microbial population, even a pure culture, do not die
simultaneously. Over time, the number of viable organisms remaining in the population decreases logarithmically, giving a straight-line relationship
on a graph. (b) Effect of the initial microbial load (how many microbes are present). (c) Relative resistance of endospores versus vegetative forms.
(d) The difference between microbistatic and microbicidal agents.
Complete Denaturation
(b)
Substrate
Substrate
Pasteurization: Disinfection of Beverages Fresh beverages Milk, wine, beer, other beverages
such as milk, fruit juices, beer, and wine are easily contaminated
during collection and processing. Because microbes have the
potential for spoiling these foods or causing illness, heat is
frequently used to reduce the microbial load or destroy pathogens.
Pasteurization is a technique in which heat is applied to liquids to
kill potential agents of infection and spoilage while retaining the
liquid’s flavor and food value.
Ordinary pasteurization techniques require special heat
exchangers that expose the liquid to 71.6°C for 15 seconds (flash
method) or to 63°C to 66°C for 30 minutes (batch method). The
first method is preferable because it is less likely to change flavor
John A. Rizzo/Photodisc/Getty Images
and nutrient content, and it is more effective against certain
resistant pathogens such as Coxiella and Mycobacterium.
James King-Holmes/Science Source Although these treatments inactivate most viruses and destroy
the vegetative stages of 97% to 99% of bacteria and fungi,
they do not kill endospores or particularly heat-resistant microbes (mostly nonpathogenic
lactobacilli, micrococci, and yeasts). Milk is not sterile after regular pasteurization. In fact, it
can contain 20,000 microbes per milliliter or more, which explains why even an unopened carton
of milk will eventually spoil. (Newer techniques can also produce “shelf-stable” milk that has a
storage life of 3 months. This milk is processed with ultrahigh temperature [UHT]—134°C—for
1 to 2 seconds.)
Steam Under Pressure: Autoclaving At sea level, normal atmospheric pressure is 15 pounds per Heat-resistant materials such as glassware,
square inch (psi), or 1 atmosphere. At this pressure, water will boil (change from a liquid to a gas) at cloth, metal instruments, liquids, paper, some
100°C, and the resultant steam will remain at exactly that temperature, which is too low to reliably kill media, and some heat-resistant plastics. If
all microbes. In order to raise the temperature of steam, the pressure at which it is generated must be items are heat-sensitive (plastic Petri dishes)
increased. As the pressure is increased, the temperature at which water boils and the temperature of the but will be discarded, the autoclave is still a
steam produced both rise. For example, at a pressure of 20 psi (5 psi above normal), the temperature of good choice. It is ineffective for sterilizing
steam is 109°C. As the pressure is increased to 10 psi above normal, the steam’s temperature rises to substances that repel moisture (oils, waxes), or
115°C, and at 15 psi above normal (a total of 2 atmospheres), it will be 121°C. It is not the pressure by for those that are harmed by it (powders).
itself that kills the microbes but the increased temperature it produces. Sterilizing technique.
Such pressure-temperature combinations can be achieved only with a special device that can subject
pure steam to pressures greater than 1 atmosphere. Health and commercial industries use an autoclave for
this purpose, (figure 11.6) and a comparable home appliance is the pressure cooker. The most efficient
pressure-temperature combination for achieving sterilization is 15 psi, which yields 121°C. It is important
to avoid overpacking or haphazardly loading the chamber, which prevents steam from circulating freely
around the contents and impedes the full contact that is necessary. The length of the process is adjusted
according to the bulkiness of the items in the load (thick bundles of material or large flasks of liquid) and
how full the chamber is. The range of holding times varies from 10 minutes for light loads to 40 minutes
for heavy or bulky ones; the average time is 20 minutes.
microgen/Getty Images
The hot-air oven provides another means of dry-heat Glassware, instruments, powders, and oils
sterilization. The so-called dry oven is usually electric that steam does not penetrate well. Not
(occasionally gas) and has coils that radiate heat within suitable for plastics, cotton, and paper, which
an enclosed compartment. Heated, circulated air may burn at the high temperatures, or for
transfers its heat to the materials in the oven. liquids, which will evaporate.
Sterilization requires exposure to 150°C to 180°C for Sterilizing technique.
2 to 4 hours, which ensures thorough heating of the
objects and destruction of endospores.
Method
DNA
(breakages)
Radiation Barrier Cell
source
(a)
Radiation Barrier
source
(b)
Tom Pantages
DNA
(abnormal bonds) unpalatable, or even unsafe by its having been subjected to ion-
izing radiation. But irradiated food has been extensively studied,
(c) and each of these concerns has been addressed.
Figure 11.8 Cellular effects of irradiation. (a) Ionizing Irradiation may lead to a small decrease in the amount of thia-
radiation can penetrate a solid barrier, bombard a cell, enter it, and mine (vitamin B1) in food, but this change is small enough to be
dislodge electrons from molecules. Breakage of DNA creates massive inconsequential. The irradiation process does produce short-lived
mutations, and damage to proteins prevents them from repairing it. free radical oxidants, which disappear almost immediately (this
(b) A solid barrier cannot be penetrated by nonionizing radiation. same type of chemical intermediate is produced through cooking
(c) Nonionizing radiation enters a cell, strikes molecules, and excites as well). Certain foods do not irradiate well and are not good can-
them. The effect on DNA is mutation by formation of abnormal bonds. didates for this type of antimicrobial control. The whites of eggs
become milky and liquid, grapefruits get mushy, and alfalfa seeds
do not germinate properly. Finally, it is important to remember
Applications of Ionizing Radiation that food is not made radioactive by the irradiation process. Many
studies, in both animals and humans, have concluded that there are
Foods have been subject to irradiation in limited circumstances for no negative effects from eating irradiated food.
more than 50 years. From flour to pork and ground beef to fruits
and vegetables, radiation is used to kill not only bacterial patho-
gens but also insects and worms and even to inhibit the sprouting Nonionizing Radiation: Ultraviolet Rays
of white potatoes. As soon as radiation is mentioned, however, Ultraviolet (UV) radiation ranges in wavelength from approxi-
consumer concern arises that food may be made less nutritious, mately 100 nm to 400 nm. It is most lethal from 240 nm to 280 nm
Osmotic Pressure
In chapter 9, you learned about the effects of osmotic pressure on
cells. This fact has long been exploited as a means of preserving
Filter Pores
food. Adding large amounts of salt or sugar to foods creates a hyper-
tonic environment for bacteria in the foods, causing plasmolysis and
making it impossible for the bacteria to multiply. People knew that
these techniques worked long before the discovery of bacteria. This
is why meats are “cured,” or treated with high salt concentrations,
so they can be kept for long periods without refrigeration. High
sugar concentrations in foods like jellies have the same effect.
safety), many solid or gaseous antimicrobial chemicals are chemical is added to 200 parts of water by volume. Solutions such as
dissolved in water, alcohol, or a mixture of the two to produce a chlorine that are effective in very diluted concentrations are expressed
liquid solution. Solutions containing pure water as the solvent are in parts per million (ppm). In percentage solutions, the solute is added
termed aqueous, whereas those dissolved in pure alcohol or water- to water to achieve a certain percentage in the solution. Alcohol, for
alcohol mixtures are termed tinctures. instance, is used in percentages ranging from 50% to 95%.
Most compounds require adequate contact time to allow the
Selecting a Microbicidal Chemical chemical to penetrate and to act on the microbes present. The com-
The choice and appropriate use of antimicrobial chemical agents position of the material being treated must also be considered, as
are of constant concern in medicine and dentistry. Here are some it may greatly impact the effectiveness of these germicidal agents.
guidelines to be considered, and optimized to the extent possible: Smooth, solid objects are more reliably disinfected than are those
with pores or pockets that can trap soil. An item contaminated
1. rapid action even in low concentrations, with common biological matter such as serum, blood, saliva, pus,
2. solubility in water or alcohol and long-term stability, fecal material, or urine presents a problem in disinfection. Large
3. broad-spectrum microbicidal action without toxicity to amounts of organic material can hinder the penetration of a disin-
human and animal tissues, fectant and, in some cases, can form bonds that possibly reduce its
4. penetration of inanimate surfaces to sustain a cumulative or activity. Cleaning is different from disinfecting. Cleaning (physi-
persistent action, cally removing organic matter) is always a necessary first step
5. resistance to becoming inactivated by organic matter, before antimicrobial chemicals can be used.
6. noncorrosive or nonstaining properties, Also, common sense is an important factor in successful
7. sanitizing and deodorizing properties, and microbial control. For example, many hospitals use detergent wet
8. affordability and ready availability. wipes to clean surfaces. Studies have shown that when the wipes
No chemical can completely fulfill all of those requirements, but are used on a contaminated surface and then used on a second sur-
glutaraldehyde and hydrogen peroxide approach this ideal. face, the second surface becomes contaminated with whatever was
Germicides are evaluated in terms of their effectiveness in on the contaminated surface. So new wipes are required for each
destroying microbes in medical and dental settings. Echoing the surface. In May 2020, the World Health Organization issued guid-
language we used earlier in the chapter, referring to medical devices ance on the cleaning and disinfection of environmental surfaces
as critical, semicritical, or noncritical, three levels of chemical decon- in the context of COVID-19. The main chemicals recommended
tamination procedures exist. These are high, intermediate, and low. were chlorine (a halogen) and ethyl alcohol.
High-level germicides kill endospores and, if properly used, are ster-
ilants. These germicides are used on critical items such as catheters, Germicidal Categories According
heart-lung equipment, and implants. These devices are not heat- to Chemical Group
sterilizable and are intended to enter the body tissues during medical Several general groups of chemical compounds are widely used for
procedures. Intermediate-level germicides kill fungal (but not bacte- antimicrobial purposes in medicine and commerce. Prominent agents
rial) spores, resistant pathogens such as the tubercle bacillus, and include halogens, heavy metals, alcohols, phenolic compounds,
viruses. They are used to disinfect semi-critical items (respiratory oxidizers, aldehydes, detergents, and gases. These groups are summa-
equipment, thermometers). Low levels of disinfection eliminate only rized in table 11.6. Note that each agent is described with respect to its
vegetative bacteria, vegetative fungal cells, and some viruses. They specific forms, modes of action, indications for use, and limitations.
are used to clean noncritical materials such as electrodes, straps, and The following section points out some pertinent facts about
pieces of furniture that touch the skin surfaces but not the mucous each of these groups.
membranes.
The Halogen Antimicrobial Chemicals
Factors Affecting the Microbicidal Activity The halogens are fluorine, bromine, chlorine, and iodine, a group
of Chemicals of nonmetallic elements. These elements are highly effective com-
Factors that affect the usefulness of a germicide include the nature ponents of disinfectants and antiseptics because they are microbi-
of the microorganisms being treated, the nature of the material cidal and not just microbistatic, and they are sporicidal with longer
being treated, the degree of contamination, the time of expo- exposure. For these reasons, halogens are the active ingredients in
sure, and the strength and chemical action of the germicide. The nearly one-third of all antimicrobial chemicals currently marketed.
strength of a germicide also plays a role in its ability to act upon Iodophors are complexes of iodine and alcohol. This formula-
a microbial population. The modes of action of most germicides tion allows the slow release of free iodine and increases its degree of
are to attack the cellular targets discussed earlier: proteins, nucleic penetration. These compounds have largely replaced free iodine solu-
acids, the cell wall, and the cell membrane. tions in medical antisepsis because they are less prone to staining or
A chemical’s strength or concentration is expressed in various irritating tissues. Common iodophor products marketed as Betadine,
ways, depending on the method of preparation. In dilutions, a small Povidone (PVP), and Isodine contain 2% to 10% of available iodine.
volume of the liquid chemical (solute) is diluted in a larger volume of In 2017, the Food and Drug Administration banned many
solvent to achieve a certain ratio. For example, a common phenolic iodophors from consumer products because of unsatisfactory
disinfectant such as Lysol is usually diluted 1:200; that is, 1 part of evidence of efficacy and issues with safety.
Aldehydes Kill endospores Organic substances Glutaraldehyde can irreversibly Glutaraldehyde kills rapidly and Glutaraldehyde is somewhat
and all bearing a —CHO disrupt the activity of is broad-spectrum; used to unstable, especially
other functional group enzymes and other proteins sterilize respiratory equipment, with increased pH and
microbes on the terminal within the cell scopes, kidney dialysis temperature
carbon machines, dental instruments
Ortho-phthalaldehyde Ortho-phthalaldehyde is safer than Ortho-phthalaldehyde is
Can sterilize. glutaraldehyde and just as much more expensive than
effective glutaraldehyde
Gaseous Ethylene Ethylene oxide Ethylene oxide reacts Ethylene oxide is used to disinfect Ethylene oxide is
sterilants/ oxide kills is a colorless vigorously with functional plastic materials and delicate explosive—it must
disinfectants endospores; substance that groups of DNA and instruments; can also be used be combined with a high
other exists as a gas at proteins, blocking both to sterilize syringes, surgical percentage of carbon
gases less room temperature DNA replication and supplies, and medical devices dioxide or fluorocarbon
effective enzymatic actions that are prepackaged It can damage lungs, eyes,
Chlorine dioxide is a strong and mucous membranes if
alkylating agent contacted directly
Can sterilize. Ethylene oxide is rated as
a carcinogen by the
government
Acids and alkalis Some bacteria, Organic acids in food pH alteration Food manufacture, deodorants, Large changes in pH can be
viruses, production deodorizers corrosive
fungi
297
Phenol and Its Derivatives Applications of Alcohols Ethyl alcohol, also called ethanol
or grain alcohol, is known for being germicidal, nonirritating,
Phenol (carbolic acid) is a poisonous compound derived from the
and inexpensive. Solutions of 70% to 95% are routinely used as
distillation of coal tar. First adopted by Joseph Lister in 1867 as
skin degerming agents because the surfactant action removes
a surgical germicide, phenol was the major antimicrobial chemi-
skin oil, soil, and some microbes sheltered in deeper skin layers.
cal until other phenolics with fewer toxic and irritating effects
One limitation to its effectiveness is the rate at which it evapo-
were developed. Substances chemically related to phenol are
rates. Ethyl alcohol is occasionally used to disinfect electrodes,
often referred to as phenolics. Solutions of phenol are now used
face masks, and thermometers, which are first cleaned and then
only in certain limited cases, but phenol remains one standard
soaked in alcohol for 15 to 20 minutes. Most alcohol-based hand
against which other phenolic disinfectants are rated. The phenol
sanitizers and foams contain 60% to 70% ethyl alcohol and are
coefficient quantitatively compares a chemical’s antimicrobial
more resistant to evaporation, but to be microbicidal they must
properties to those of phenol. Hundreds of these chemicals are
still be exposed to skin for 30 seconds. Insight 11.1 addresses
now available.
how often health care workers use alcohol hand rubs and raises
Perhaps the most widely known phenolic is triclosan, chemi-
some questions about what effect they have. Isopropyl alcohol,
cally known as dichlorophenoxyphenol. Until its ban in consumer
sold as rubbing alcohol, is even more microbicidal and less
products in 2017, it was added to dozens of products, from soaps
expensive than ethanol, but these benefits must be weighed
to kitty litter. It acts as both disinfectant and antiseptic and is
against its toxicity. It must be used with caution in disinfection
broad-spectrum in its effects. It is still licensed for use in health
or skin cleansing because inhalation of its vapors can adversely
care settings, but the consumer ban came about because research
affect the nervous system.
established that widespread use of triclosan can lead to the devel-
opment of resistance—both to triclosan itself and to unrelated
antibiotics—in microbes. Oxidizing Agents
Hydrogen peroxide (H2O2) is a colorless, caustic liquid that
Chlorhexidine decomposes into water and oxygen gas in the presence of light,
The compound chlorhexidine (Hibiclens, Hibitane, Peridex) is a metals, or catalase. The germicidal effects of hydrogen peroxide
complex organic base containing chlorine and two phenolic rings. are due to the direct and indirect actions of oxygen. Oxygen forms
Its mode of action targets both cell membranes (lowering surface hydroxyl free radicals (.OH), which, like the superoxide radical
tension until selective permeability is lost) and protein structure (see chapter 9), are highly toxic and reactive to cells. Although
(causing denaturation). At moderate to high concentrations, it is most microbial cells produce catalase to inactivate the metabolic
bactericidal for both gram-positive and gram-negative bacteria but hydrogen peroxide, it cannot neutralize the amount of hydrogen
inactive against endospores. Its effects on viruses and fungi vary. It peroxide entering the cell during disinfection and antisepsis.
possesses distinct advantages over many other antiseptics because Hydrogen peroxide is bactericidal, virucidal, fungicidal, and, in
of its mildness, low toxicity, and rapid action. It is not absorbed higher concentrations, sporicidal.
into deeper tissues to any extent. It is sold in over-the-counter
Applications of Hydrogen Peroxide As an antiseptic,
mouthwashes as well.
3% hydrogen peroxide serves a variety of needs, including skin
and wound cleansing, bedsore care, and mouthwashing. It is espe-
Alcohols as Antimicrobial Agents cially useful in treating infections by anaerobic bacteria because of
Alcohols are colorless hydrocarbons with one or more —OH the lethal effects of oxygen on them. Hydrogen peroxide is also a
functional groups. Of several alcohols available, only ethyl and versatile disinfectant for contact lenses, surgical implants, plastic
isopropyl are suitable for microbial control. Methyl alcohol is not equipment, utensils, bedding, and room interiors.
particularly microbicidal, and more complex alcohols are either Vaporized hydrogen peroxide can also be used as a sterilant
poorly soluble in water or too expensive for routine use. Alcohols in enclosed areas. Hydrogen peroxide plasma sterilizers exist for
are used alone in aqueous solutions or as solvents for tinctures those applications involving small industrial or medical items. For
(iodine, for example). larger, enclosed spaces, such as isolators and pass-through rooms,
How many times have you heard that one of the best ways to your environment and they stay only transiently. Contaminants are
prevent infections in hospitals is for health care workers to usually easily washed off, while colonizers are embedded deeper
wash their hands? OK, but now that we know about how impor- in your skin oils and the top layers of epithelial cells.
tant our resident microbiota is, we should ask what frequent One question raised, but not answered, by this study is whether
hand washing does to that. A recent study followed 34 health altering the normal microbiome of the hand by frequent hand
care workers in a surgical intensive care unit. Twenty-four of them hygiene practices might make it (a) easier or (b) more difficult for
were registered nurses, six were respiratory therapists, and four pathogens to contaminate and/or colonize caregivers’ hands.
were nurse technologists.
During a typical 12-hour shift,
∙∙ 53% of them reported washing their hands with soap and
water between 6 and 20 times,
∙∙ 41% used alcohol rubs more than 20 times, and
∙∙ 62% donned more than 40 pairs of gloves.
The researchers then tested for common healthcare-
associated pathogens and found that approximately 45% of the
health care workers’ dominant hands were positive for Staphylo-
coccus aureus. They detected methicillin-resistant Staphylococcus
aureus (MRSA) on 3.9% of the dominant hands. About 4% of
hands were positive for the fungus Candida albicans.
These rates look very similar to the rates found in the general O. Dimier/PhotoAlto
population—those of us who are not washing our hands 20 times
a day. This might be an illustration of the difference between
colonization—when microbes become part of your “normal” Source: Rosenthal, et al. “Healthcare Workers’ Hand Microbiome May Mediate Carriage
microbiota—and contamination—when you pick up microbes from of Hospital Pathogens,” Pathogens. 2014 Mar: 3(1): 1–13.
Applications of Heavy Metals A silver compound with sev- viruses. Chief among its disadvantages are an inability to reliably
eral applications is silver nitrate (AgNO3) solution. In the late 19th destroy endospores and, on a more practical note, its tendency to
century, it was introduced for preventing gonococcal infections in stain proteins, including those in human skin.
the eyes of newborn infants who had been exposed to an infected
birth canal. This preparation is not used as often now because many Gaseous Sterilants and Disinfectants
pathogens are resistant to it. It has been replaced by antibiotics in
Processing inanimate substances with chemical vapors, gases, and
most instances. Solutions of silver nitrate (1% to 2%) can also be
aerosols provides a versatile alternative to heat or liquid chemicals.
used as topical germicides on mouth ulcers and occasionally root
Currently, those vapors and aerosols having the broadest applications
canals. Silver sulfadiazine ointment, when added to dressings, effec-
are ethylene oxide (ETO), propylene oxide, and chlorine dioxide.
tively prevents infection in second- and third-degree burn patients,
Ethylene oxide is a colorless substance that exists as a gas
and pure silver is now incorporated into catheters to prevent urinary
at room temperature. It is very explosive in air, a feature that can
tract infections in the hospital. Colloidal silver preparations are mild
be eliminated by combining it with a high percentage of carbon
germicidal ointments or rinses for the mouth, nose, eyes, and vagina.
dioxide or fluorocarbon. Like the aldehydes, ETO is a very strong
Silver ions are increasingly incorporated into many hard surfaces,
alkylating agent, and it reacts vigorously with functional groups
such as plastics and steel, as a way to control microbial growth on
of DNA and proteins. Through these actions, it blocks both DNA
items such as toilet seats, stethoscopes, and even refrigerator doors.
replication and enzymatic actions. Ethylene oxide is one of a very
Companies have even found ways to impregnate textiles with silver
few gases generally accepted for chemical sterilization because,
and quaternary ammonium compounds to produce antimicrobial
when employed according to strict procedures, it is a sporicide. A
fabrics that stay stain- and odor-free over long periods of use.
specially designed ETO sterilizer called a chemiclave, a variation on
the autoclave, is equipped with a chamber; gas ports; and it controls
Aldehydes as Germicides for temperature, pressure, and humidity. Ethylene oxide is rather
Organic substances bearing a —CHO functional group (a strong penetrating but relatively slow-acting, requiring from 90 minutes to
reducing group) on the terminal carbon are called aldehydes. Sev- 3 hours. Some items absorb ETO residues and must be aerated with
eral common substances such as sugars and some fats are techni- sterile air for several hours after exposure to ensure dissipation of as
cally aldehydes. The two aldehydes used most often in microbial much residual gas as possible. For all of its effectiveness, ETO has
control are glutaraldehyde and formaldehyde. some unfortunate features. Its explosiveness makes it dangerous to
Glutaraldehyde is a yellow liquid with a mild odor. Its mecha- handle. Also, it can damage the lungs, eyes, and mucous membranes
nism of activity involves cross-linking protein molecules on the cell if contacted directly. It is rated as a carcinogen by the government.
surface. In this process, amino acids are alkylated, meaning that Chlorine dioxide is another gas that has of late been used as a
a hydrogen atom on an amino acid is replaced by the glutaralde- sterilant. Despite the name, chlorine dioxide works in a completely
hyde molecule itself. It can also irreversibly disrupt the activity of different way from the chlorine compounds discussed earlier in
enzymes within the cell. Glutaraldehyde is rapid and broad-spectrum the chapter. It is a strong alkylating agent, which disrupts proteins
and is one of the few chemicals officially accepted as a sterilant and and is effective against vegetative bacteria, fungi, viruses, and
high-level disinfectant. It destroys endospores in 3 hours and fungi endospores. Although chlorine dioxide is used for the treatment of
and vegetative bacteria (even Mycobacterium and Pseudomonas) in drinking water, wastewater, food-processing equipment, and medi-
a few minutes. Viruses, including the most resistant forms, appear cal waste, its most well-known use was in the decontamination of
to be inactivated after relatively short exposure times. Glutaralde- the Senate offices after the anthrax attack of 2001.
hyde retains its potency even in the presence of organic matter, is
noncorrosive, does not damage plastics, and is less toxic or irritating Acids and Alkalis
than formaldehyde. Its principal disadvantage is that it is somewhat
Conditions of very low or high pH can destroy or inhibit microbial
unstable, especially with increased pH and temperature.
cells, but they are of limited use due to their corrosive and hazard-
Formaldehyde is a sharp, irritating gas that readily dissolves
ous nature. Aqueous solutions of ammonium hydroxide are a com-
in water to form an aqueous solution called formalin. Pure forma-
mon component of detergents, cleansers, and deodorizers. Organic
lin is a 37% solution of formaldehyde gas dissolved in water. The
acids are widely used in food preservation because they prevent
chemical is microbicidal through its attachment to nucleic acids
endospore germination, inhibit bacterial and fungal growth, and
and functional groups of amino acids. Formalin is an intermediate-
are generally regarded as safe to eat. For example, acetic acid (in the
to high-level disinfectant, although it acts more slowly than glu-
form of vinegar) is a pickling agent that inhibits bacterial growth;
taraldehyde. Formaldehyde’s extreme toxicity (it is classified as a
propionic acid is commonly incorporated into breads and cakes to
carcinogen) and irritating effects on the skin and mucous mem-
inhibit molds; lactic acid is added to sauerkraut and olives to pre-
branes greatly limit its clinical usefulness.
vent growth of anaerobic bacteria; and benzoic and sorbic acids are
A third aldehyde, ortho-phthalaldehyde (OPA), is another high-
added to beverages, syrups, and margarine to inhibit yeasts.
level disinfectant. OPA is a pale blue liquid with a barely detectable
odor and can be most directly compared to glutaraldehyde. It has a
mechanism of action similar to glutaraldehyde, is stable, is nonir- Essential Oils
ritating to the eyes and nasal passages, and, for most uses, is much In recent years, essential oils have become more prominent in
faster acting than glutaraldehyde. It is effective against vegetative western medicine. They have, of course, been used for centuries
bacteria, including Mycobacterium and Pseudomonas; fungi; and in traditional medicine. Components of essential oils (which all
come from plants) have been studied extensively, especially in 16. List advantages and disadvantages to the use of phenolic
countries where simple antibiotics were unavailable. But now that compounds as control agents.
industrialized countries are experiencing a loss in effectiveness 17. Explain the mode of action of alcohols and their limitations
of antibiotics, these plant extracts are being closely considered. as effective antimicrobials.
Some are indeed effective at stopping microbial growth, especially 18. Pinpoint the most appropriate applications of oxidizing
those that are phenolics and derivatives of phenolics. The FDA is agents.
hustling to keep up with approving those that work, and also pro-
19. Define the term surfactant, and explain this antimicrobial’s
tecting consumers from those that don’t. There will certainly be a
mode of action.
future for essential oils that are truly effective.
For a look at the antimicrobial chemicals found in some com- 20. Identify examples of some heavy metal control agents and
mon household products, see table 11.7. their most common applications.
21. Discuss the advantages and disadvantages of aldehyde
agents in microbial control.
11.3 Learning Outcomes—Assess Your Progress
22. Identify applications for ethylene oxide sterilization.
14. Name the desirable characteristics of chemical control agents.
15. Discuss several different halogen agents and their uses in
microbial control.
These activities are designed for you to use on your own with a study group—either a face-to-face group or a virtual one,
consisting of 3–5 members. Studying together can be very helpful, but there are effective and ineffective ways to do it. For example,
getting together without a clear structure is often not a good use of your time. Use your time efficiently by using one or more of the
exercises below.
FACE-TO-FACE GROUPS
Use one or more of the activities below.
Peer Instruction: Assign numbers to your group members to use all semester long. Now look at these five concepts from this
chapter. Each group member prepares a 5-minute lesson on the topic corresponding to their number. Don’t worry if you have fewer
than 5 members; just use however many you have! During your group study time, each member presents their lesson, and the group
spends another 5–10 minutes discussing that lesson.
1. -cidal vs. -static agents
2. Microbial death
3. What does “sterile” mean?
4. Which physical methods can achieve sterilization?
5. Which chemical methods can achieve sterilization?
Concept Maps: Each member of the group should use this list of terms from this chapter to generate their own concept map. This
can be hand-drawn or created using software (see Appendix C for guidelines). During group study time, compare each other’s
concept maps and help each other make sure they are correct. Of course, there are many different “correct” maps. Examining
each member’s map will help you talk through the varied concepts and how they are related.
Concept Terms:
halogens alcohol phenolics silver
endospores prions autoclaving
Table Topics: Each group member should identify a concept or topic from this week’s class assignments with which they are
having trouble and share it during group study time. The other group members can then help to clarify confusing issues or share
how they figured it out. Aim for a maximum of 15 minutes per topic. If the topic remains unclear to the group, bring it up during
class or use the instructor’s office hours or e-mail to ask for help. Taking the time to struggle with a difficult concept first makes
your questions much more specific and more likely to yield helpful answers.
VIRTUAL GROUPS
Not everyone has the time or opportunity to meet with group members outside of class time. You or your instructor can
create a virtual group using e-mail or the course software.
Weekly Discussion Board: This forum can be used as a way for groups to discuss topics, via e-mail, or other learning management
systems or online platforms before they are covered in class. As each member of the group answers the current week’s question, they
should send their responses to every other member of their group. It’s best to agree on a deadline based on how your class schedule
works (Saturday for the next week’s topics, for example). Then, after the topic is discussed in class, each member should send a
response that all group members will see with a follow-up post on the same topic. If you cover more than one chapter in a week,
someone can be designated to choose which chapter Discussion Board question you will use. Or simply decide up front that you will
always use the first-chapter-of-the week’s question, to keep the schedule simple.
Discussion Question
Triclosan and some other antimicrobial compounds have been banned from consumer products. Explain why not every chemical
that can be used to kill microbes should be used.
Chapter Summary
PHYSICAL AND CHEMICAL CONTROL OF
MICROBES
CONTROLLING MICROORGANISMS
11.1
• Controlling microbes in the environment and on the surface of the body calls for the use of
physical and chemical agents to eliminate them or reduce their numbers.
• The final outcomes of microbial controls are named sterilization, antisepsis, disinfection,
and decontamination.
• Some antimicrobial agents are -cidal (kills), and others are -static (inhibits growth).
• Microbial death is defined as the permanent loss of reproductive capacity in
microorganisms.
• The four major cell targets for antimicrobial agents are the cell wall, cell membrane,
biosynthesis pathways for DNA or RNA, and protein (enzyme function).
This 21 Question Grid takes the topics from this chapter and arranges them with respect to the American Society for Microbiology’s
Undergraduate Curriculum guidelines—all six of the important “Concepts” as well as the important “Competency” of scientific literacy. Three
questions are supplied, which cover chapter content referring to the Concept or Competency in increasing levels of Bloom’s taxonomy for learning.
ASM Concept/ A. Bloom’s Level 1, 2—Remember B. Bloom’s Level 3, 4—Apply and C. Bloom’s Level 5, 6—Evaluate
Competency and Understand (Choose one) Analyze and Create
Evolution 1. D
NA repair mechanisms can 2. E
xplain why all cells in 3. T
riclosan and other
help alleviate the effects of a population do not die antimicrobial compounds
a. UV radiation. instantaneously when exposed have been banned from most
b. alcohol disinfection. to an antimicrobial agent, consumer products. Explain
even when they are of a single why we should not be using
c. autoclaving.
species. every chemical available to us
d. dry heat. to combat microbes.
c. endospores
d. cysts
Metabolic Pathways 7.
Cytoplasmic enzymes are most 8. M
ost antimicrobials that 9. T
here is a bacterium that has
likely to be disrupted by target protein function are a metabolic pathway that
a. UV light. nonselective as to the microbes enables it to survive in the
b. low temperatures. they affect. Explain why that presence of acid. What kind of
might be so. pathway or product would you
c. high temperatures.
expect in this case?
d. detergents.
Microbial Systems 13. Disinfection procedures must 14. Provide a rationale for how 15. What additional considerations
take into account a particular microbe could would you have to address if
a. the mixture of microbes be considered a serious the device you were to trying
being targeted. contaminant in one situation to disinfect—such as a water
b. whether the microbes in and completely harmless in line in a dental office—was
biofilms are dispersed. another. known to contain a microbial
biofilm?
c. the amount of organic matter
associated with the microbes.
d. all of the above.
ASM Concept/ A. Bloom’s Level 1, 2—Remember B. Bloom’s Level 3, 4—Apply and C. Bloom’s Level 5, 6—Evaluate
Competency and Understand (Choose one) Analyze and Create
Scientific Thinking 19. The most versatile method 20. Precisely what is microbial 21. Devise an experiment that
for sterilizing heat-sensitive death? will differentiate between
liquids is bactericidal versus
a. UV radiation. bacteristatic effects.
b. exposure˙ to ozone.
c. peracetic acid.
d. filtration.
Visual Connections
This question uses a visual image to connect content within and between chapters.
These terms and concepts are most critical for your understanding of this chapter—and may be the most difficult. Have you mastered them?
Concepts Terms
-cidal vs. -static agents Sterilization
Microbial death Disinfection
Factors influencing microbial death Antisepsis/degermation
Physical vs. chemical control Decontamination/sanitation
Desirable qualities in germicidal chemicals Thermal death time
Which physical methods can sterilize Thermal death point
Which chemical methods can sterilize
This article reported the results of a study on the gut microbes of mice that were collected from residential settings
in NYC. A team of researchers from Columbia University genetically analyzed the microbes found in these mice, and they
discovered that almost 40% of the mice harbored pathogenic bacteria. Bacteria that can cause gastrointestinal disease, such
as Salmonella, E. coli, C. difficile, and Shigella, were some examples. A deeper look showed that some of the bacteria isolated
from these mice matched the exact strains that had caused recent human disease outbreaks. Many of the mice also carried
antibiotic-resistant forms of these bacteria.
Dr. Ian Lipkin, the leading researcher on the project, has also conducted other studies on the microbiome of mice, and,
based on his findings, he suggested that mice should certainly be considered as potential sources when disease outbreaks
occur in people.
■■ What is the intended message of the article?
■■ What is your critical reading of the summary of the article? Remember that in this context, “critical reading” does not
necessarily mean What criticism do you have? but asks you to apply your knowledge to interpret whether the article is
factual and whether the facts support the intended message.
■■ How would you interpret the news item for your nonmicrobiologist friends?
■■ What is your overall grade for the news item—taking into account its accuracy and the accuracy of its intended effect?
Media Under The Microscope Wrap-Up appears at the end of the chapter.
307
12.1 Principles of Antimicrobial Therapy toxic to the infectious agent (or cripple its ability to multiply) while
being nontoxic to the host and must remain active in the body as
A hundred years ago in the United States, one out of three chil- long as needed yet be safely and easily broken down and excreted.
dren was expected to die of an infectious disease before the age Also, microbes in biofilms often require different drugs than when
of 5. Early death or severe, lifelong debilitation from scarlet they are not in biofilms. In short, the perfect drug does not exist;
fever, diphtheria, tuberculosis, meningitis, and many other bac- but by balancing drug characteristics against one another, we can
terial diseases was a fearsome yet undeniable fact of life to most often achieve a satisfactory compromise (table 12.1).
of the world’s population. The introduction of modern drugs to
control infections in the early to mid-1900s was a medical revo-
lution that has added significantly to the life span and health of
humans. It is no wonder that for many years, antibiotics in par-
Table 12.1 Characteristics of the Ideal Antimicrobial Drug
ticular were regarded as miracle drugs. Although antimicrobial
∙ Toxic to the microbe but nontoxic to the host
drugs have greatly reduced the incidence of certain infections,
∙ Microbicidal rather than microbistatic
they have definitely not eradicated infectious disease and prob-
∙ Soluble in body fluids
ably never will. In fact, you will read later that we are danger-
ously close to a post-antibiotic era, wherein the drugs we have ∙ Remains potent long enough to act and is not broken down or excreted
prematurely
are no longer effective.
∙ Slow or nonexistent development of antimicrobial resistance
The goal of antimicrobial treatment is simple: Administer a
drug to an infected person that destroys the infective agent without ∙ Complements or assists the activities of the host’s defenses
harming the host’s cells. In reality, this goal is difficult to achieve ∙ Remains active in tissues and body fluids
because many (often contradictory) factors must be taken into ∙ Readily delivered to the site of infection
account. The ideal drug should be easy to administer yet be able ∙ Does not disrupt the host’s health by causing allergies or predisposing
to reach the infectious agent anywhere in the body. It should be the host to other infections
Starting Treatment
Before antimicrobial therapy using any type of drug can begin, it
Testing to See Which Drug Will Work
is best that at least three factors be known: Determining which antimicrobial agent or agents are most effec-
tive against the microbes that are actually in the patient is essential
1. the identity of the microorganism causing the infection, in those groups of bacteria commonly showing resistance, such
2. the degree of the microorganism’s susceptibility (also called as Staphylococcus species, Neisseria gonorrhoeae, Streptococ-
sensitivity) to various drugs, and cus pneumoniae, Enterococcus faecalis, and the aerobic gram-
3. the overall medical condition of the patient. negative enteric bacilli. However, not all infectious agents require
Sometimes antibiotic treatment must be started before the antimicrobial sensitivity testing. Some bacteria are reliably sus-
identity of the microbe is known, but that is not ideal. ceptible to certain antibiotics, such as group A streptococci and
penicillin. In these cases, susceptibility testing is not necessary.
Identifying the Infection Selecting the correct antimicrobial agent begins by testing
the in vitro activity of several drugs against the infectious agent
Identification of infectious agents from body specimens should be
by means of standardized methods. In general, these tests involve
attempted as soon as possible. It is especially important that such
exposing a pure culture of the bacterium to several different drugs
specimens be taken before any antimicrobial drug is given—in
and determining whether they inhibit the growth of the microbe.
case the drug eliminates the infectious agent. Direct examina-
The Kirby-Bauer technique is an agar diffusion test that pro-
tion of body fluids, sputum, or stool is a rapid initial method for
vides useful data on antimicrobial susceptibility. In this test, the
detecting and perhaps even identifying bacteria or fungi. A doctor
surface of a plate of special medium is spread with the test bac-
often begins the therapy on the basis of such immediate findings
terium, and small discs containing a premeasured amount of anti-
or even on the basis of an informed best guess. For instance, a
microbial are dispensed onto the bacterial lawn. After incubation,
Gram stain of pus produced from a gonorrhea infection reveals
the zone of inhibition surrounding the discs is measured and com-
the characteristic appearance: bean-shaped gram-negative diplo-
pared with a standard for each drug (table 12.3 and figure 12.2).
cocci. This appearance is unique enough (when combined with
Here are some things to note about the test:
the symptoms) that the physician can assume it is Neisseria gon-
orrhoeae and begin treatment accordingly. In most cases, though, ∙∙ Each disc contains a different antibiotic.
further testing is needed to identify the offending microbe. These ∙∙ Here you see that the vancomycin disc (“VA”) is not inhibi-
methods may involve culturing the microbe or may instead rely on tory at all; so the bacteria are “S” (susceptible).
a variety of nonculture methods. Sometimes identification efforts ∙∙ Each antibiotic has a different zone of inhibition size that
are inconclusive. In these cases, epidemiological understanding determines whether a bacterium is resistant, susceptible, or of
may be required to predict the most likely agent in a given infec- intermediate susceptibility. That means that a bacterium that
tion. For example, Streptococcus pneumoniae accounts for the exhibits a 30 mm zone of inhibition to Drug A may still be
majority of cases of meningitis in children, followed by Neisseria considered resistant to it, and when it exhibits a 22 mm zone
meningitidis. of inhibition to Drug B it could be considered susceptible to it.
Antibiotic-
impregnated disk ENR
5
0cm 1 5 2 3 4
∙∙ The zone is measured from edge to edge of no growth, straight antimicrobials (figure 12.4). In many clinical laboratories, these anti-
across the antibiotic disc. microbial testing procedures are performed in automated machines
that can test dozens of drugs simultaneously.
This profile of antimicrobial sensitivity is called an antibiogram.
An alternative diffusion system that provides additional informa-
The MIC and Therapeutic Index
tion on drug effectiveness is the Etest® (figure 12.3).
More sensitive and quantitative results can be obtained with The results of antimicrobial sensitivity tests guide the physician’s
tube dilution tests. First, the antimicrobial is diluted serially in tubes choice of a suitable drug. If therapy has already begun, it is used
of broth; then each tube is inoculated with a small, uniform sample to determine if the tests bear out the use of that drug. Once therapy
of pure culture, incubated, and examined for growth (turbidity). The has begun, it is important to observe the patient’s clinical response
smallest concentration—which is the highest dilution—of drug that because the in vitro activity of the drug is not always correlated with
visibly inhibits growth is called the minimum inhibitory concentra- its in vivo effect. In a controlled situation—such as in a hospital—
tion (MIC). The MIC is useful in determining the smallest effective when antimicrobial treatment fails, the failure is due to
dosage of a drug and in providing a comparative index against other 1. the inability of the drug to diffuse into that body compartment
(the brain, joints, skin);
2. resistant microbes in the infection that did not make it into the
sample collected for testing; or
3. an infection caused by more than one pathogen (mixed), some
of which are resistant to the drug.
In outpatient situations, you have to also consider the possibility
that the patient did not take the antimicrobials correctly.
Disease Connection
Antibiotic failure can be caused by the inability of an antibiotic to
diffuse into the target tissue. This may be due to poor circulation.
For example, older patients, especially older patients with diabetes,
sometimes show little to no signs of wound healing after complet-
ing a prescribed course of oral antibiotics to treat leg or foot ulcers.
Why? When peripheral vascular disease (a condition that affects the
blood vessels beyond the heart and brain) is present, the antibiotic
may not be delivered to the lower extremities due to their poor cir-
culation. These patients often require potent intravenous antibiotics
or topical antibiotics to treat infection, rather than oral antibiotics.
Figure 12.3 Alternative to the Kirby-Bauer procedure.
Another diffusion test is the Etest®, which uses a strip to produce the
zone of inhibition. The advantage of the Etest® is that the strip contains
a gradient of drug (azithromycin in this photo example) calibrated in
Other factors influence the choice of an antimicrobial drug
micrograms. This way, the MIC can be measured by observing the mark besides microbial sensitivity to it. The nature and spectrum of the
on the strip that corresponds to the edge of the zone of inhibition. drug, its potential adverse effects, and the condition of the patient
Dr. Richard Facklam/CDC can be critical. When more than one antimicrobial drug would be
Figure 12.4 Tube dilution test for determining the minimum inhibitory concentration (MIC). (a) The antibiotic is diluted serially through tubes
of liquid nutrient from right to left. Then all tubes are inoculated with an identical amount of a test bacterium and then incubated. The first tube on the left is a
control that lacks the drug and shows maximum growth. The first tube in the series that shows no growth (no turbidity) contains the concentration of antibiotic
that is the MIC. (b) Microbroth dilution in a multiwell plate. Here, amphotericin B, flucytosine, and several azole drugs are tested on a pathogenic yeast. Pink
indicates growth, and blue indicates no growth. Numbers indicate the dilution of the MIC, and the X in each row shows the first well without growth.
(b) Dr. David Ellis
effective for treating an infection, other factors are considered. special precautions. For example, age can diminish gastrointesti-
It is always best to choose the one that has the fewest effects on nal absorption and organ function, and most antimicrobial drugs
microbes other than the one being targeted. This decreases the cross the placenta and could affect fetal development.
potential for a variety of adverse reactions. Patients must be asked about other drugs they are taking because
Because drug toxicity is of concern, it is best to choose the incompatibilities can result in increased toxicity or failure of one or
one with high selective toxicity for the infectious agent and low more of the drugs. For example, the combination of aminoglycosides
human toxicity. The therapeutic index (TI) is defined as the ratio and cephalosporins increases toxic effects on the kidney; antacids
of the dose of the drug that is toxic to humans to its effective dose. reduce the absorption of isoniazid; and the interaction of rifampin
The closer these two figures are (the smaller the ratio), the greater with oral contraceptives can abolish the contraceptive’s effect.
the potential is for toxic drug reactions. For example, a drug that Some drug combinations (penicillin with certain aminoglycosides,
has a therapeutic index of or amphotericin B with flucytosine) act synergistically, so reduced
doses of each can be used in combined therapy. Often, patients are
10 μg/ml: toxic dose also taking over-the-counter medicines and supplements, many of
TI = 1.1
9 μg/ml (MIC) which can affect the way antimicrobials work.
Adding to the complexity of choosing an antimicrobial is our
is a riskier choice than one with a therapeutic index of new knowledge of the human microbiome, especially in the gut.
Oral antimicrobials are chemically modified by the microbes in the
10 μg/ml
TI = 10 gut, in different ways depending on which microbes are there. This
1 μg/ml
accounts for substantial variability in how different humans respond
to the drugs, and is an active area of research.
When a series of drugs being considered for therapy have similar
MICs, the drug with the highest therapeutic index usually has the
widest margin of safety. Another term, which refers to concentra- Affecting Microbes, Affecting Humans
tions of the drug in the blood, is the therapeutic window. This is The goal of antimicrobial drugs is either to disrupt the cell
the range of blood level of the drug in which it is producing the processes or structures of pathogens or to inhibit their replica-
desired effect without toxicity. tion. Drugs can interfere with the function of enzymes required
The physician must also take a careful history of the patient to synthesize or assemble macromolecules, or they can destroy
to discover any preexisting medical conditions that will influ- structures already formed in the cell. Above all, drugs should be
ence the activity of the drug or the response of the patient. A selectively toxic, which means they should kill or inhibit micro-
history of allergy to a certain class of drugs precludes the use of bial cells without simultaneously damaging host tissues. This
that drug and any drugs related to it. Underlying liver or kidney concept of selective toxicity is central to antibiotic treatment, and
disease will influence the choice of drug therapy because these the best drugs in current use are those that block the actions or
organs play such an important part in metabolizing or excret- synthesis of molecules in microorganisms but not in vertebrate
ing the drug. Infants, the elderly, and pregnant women require cells. (We will see later that the drugs of the future may in fact
DNA DNA/RNA
inhibit microbial growth by attacking some aspect of the host cell cells), DNA, RNA, proteins, and cell (or cytoplasmic) membrane.
they utilize.) Examples of drugs with excellent selective toxicity Consequently, current antimicrobial drugs are divided into catego-
are those that block the synthesis of the cell wall in bacteria (peni- ries based on which of these metabolic targets they affect. These
cillins). They have low toxicity and few direct effects on human categories are outlined in figure 12.5 and include
cells because human cells lack the chemical peptidoglycan and
1. inhibition of cell wall synthesis;
for that reason are unaffected by this action of the antibiotic. The
2. inhibition of nucleic acid (RNA and DNA) structure and function;
most toxic to human cells are drugs that act upon a structure that
3. inhibition of protein synthesis, particularly involving ribosomes;
both the infective agent and the host cell have, such as the cell
4. interference with cell membrane structure or function; and
membrane (an example is amphotericin B, used to treat fungal
5. inhibition of folic acid synthesis.
infections). As the characteristics of the infectious agent become
more and more similar to those of the host cell, selective toxicity
becomes more difficult to achieve and undesirable side effects are The Effects of Antimicrobials on Humans
more likely to occur. We examine that subject in more detail in a
Although selective toxicity is the ideal constantly being sought,
later section.
antimicrobial therapy, by its very nature, involves contact with
foreign chemicals that can harm human tissues. In fact, estimates
Mechanisms of Drug Action on Microbes indicate that at least 5% of all persons taking an antimicrobial
If the goal of antimicrobial usage is to disrupt the structure or func- drug experience some type of relatively serious adverse reaction
tion of an organism to the point where it can no longer survive, then to it. The major side effects of drugs fall into one of three catego-
the first step is to identify the structural and metabolic needs of the ries: direct damage to tissues through toxicity, allergic reactions,
living microbe. Once those have been determined, methods of and disruption in the balance of normal microbial biota. The
removing, disrupting, or interfering with these requirements can damage caused by antimicrobial drugs can be short-term and
be figured out. The metabolism of an actively dividing cell is reversible or permanent, and it ranges in severity from cosmetic
marked by the production of new cell wall components (in most to lethal (table 12.4).
Table 12.4 Major Adverse Toxic Reactions therapy is diarrhea, which can progress to severe intestinal irrita-
to Common Antimicrobials tion or colitis. Although some drugs directly irritate the intestinal
Primary Damage or lining, the usual gastrointestinal complaints are caused by disrup-
Antimicrobial Drug Abnormality Produced tion of the intestinal microbiota.
Antibacterials
Human Allergic Responses to Drugs
Penicillin G Skin abnormalities One of the most frequent drug reactions is allergy. This reaction
Cephalosporins Inhibition of platelet function occurs because the drug acts as an antigen (a foreign material
Decreased circulation of white capable of stimulating the immune system) and stimulates an
blood cells allergic response. This response can be provoked by the intact drug
Nephritis molecule or by substances that develop from the body’s metabolic
Sulfonamides Formation of crystals in kidney; alteration of the drug. In the case of penicillin, for instance, it is
blockage of urine flow not the penicillin molecule itself that causes the allergic response
Hemolysis but a product, benzylpenicilloyl. Allergic reactions have been
Reduction in number of red reported for every major type of antimicrobial drug, but the peni-
blood cells cillins account for the greatest number of antimicrobial allergies,
Polymyxins Kidney damage followed by the sulfonamides.
Weakened muscular responses People who are allergic to a drug become sensitized to it dur-
ing the first contact, usually without symptoms. Once the immune
Quinolones (ciprofloxacin, Headache, dizziness, tremors,
norfloxacin) GI distress
system is sensitized, a second exposure to the drug can lead to
a reaction such as a skin rash (hives); respiratory inflammation;
Antifungals and, rarely, anaphylaxis, an acute, overwhelming allergic response
Amphotericin B Disruption of kidney function that develops rapidly and can be fatal. (This topic is discussed in
greater detail in chapter 17.) Note: Allergy is a negative effect
Flucytosine Decreased number of white
instigated by the human’s inappropriate response to the drug. Tox-
blood cells
icity is a direct effect of the drug on human tissue.
Antiprotozoan Drugs
Metronidazole Nausea, vomiting Suppression and Alteration of the Human
Antihelminthics
Microbiota by Antimicrobials
With the explosion of knowledge about the microbiome, we have
Pyrantel Irritation
Headache, dizziness to take into consideration the effects of antibiotic usage on the
human microbiome. Antibiotics may be causing a wide array of
Antivirals unintended effects by changing the nature of our microbiome. See
Amantadine Nervousness, light-headedness Insight 12.1 for an example of this.
Nausea Even before we had a clear picture of the absolute importance
AZT Immunosuppression, anemia
of the microbiome, it was obvious that antibiotic usage could have
disruptive effects on our health. If a broad-spectrum antimicrobial
is introduced into a host to treat infection, it will destroy microbes
regardless of their roles as normal biota, affecting not only the
Toxicity to Human Organs targeted infectious agent but also many others in sites far removed
Drugs most often adversely affect the following organs: the liver from the original infection. When this therapy destroys beneficial
(hepatotoxic), kidneys (nephrotoxic), gastrointestinal tract, cardio- resident species, other microbes that were once in small numbers
vascular system and blood-forming tissue (hemotoxic), nervous can begin to overgrow and cause disease. This complication is
system (neurotoxic), respiratory tract, skin, bones, and teeth. Some called a superinfection.
potential toxic effects of drugs on the body, along with the drugs Some common examples demonstrate how a disturbance in
that may cause them, are detailed in table 12.4. microbial biota leads to replacement biota and superinfection. A
The skin is a frequent target of drug-induced side effects. The broad-spectrum cephalosporin used to treat a urinary tract infection
skin response can be a symptom of drug allergy or a direct toxic by Escherichia coli will cure the infection, but it will also destroy
effect. Some drugs interact with sunlight to cause photoderma- the lactobacilli in the vagina that normally maintain a protective
titis, a skin inflammation. Tetracyclines are contraindicated (not acidic environment there. The drug has no effect, however, on Can-
advisable) for children from birth to 8 years of age because they dida albicans, a yeast that also resides in healthy vaginas. Released
bind to the enamel of the teeth, creating a permanent gray-brown from the inhibitory environment provided by lactobacilli, the yeasts
discoloration. Pregnant women should avoid tetracyclines because multiply and cause symptoms. Candida can cause similar superin-
they can cause liver damage. They also cross the placenta and can fections of the oropharynx (thrush) and the large intestine.
be deposited in the developing fetal bones and teeth. However, Oral therapy with tetracyclines, clindamycin, and broad-
the most common complaint associated with oral antimicrobial spectrum penicillins and cephalosporins is associated with a serious
There are some who say that the rise of obesity in this country and Lean Weight (g) Body Fat (%)
around the world coincides in time with the discovery and rise in
the use of antibiotics. Before the 1950s, Americans were all slim + no antibiotic 15.3 20.0
(control)
and dashing, right? They were if they survived childhood diseases,
that is. + Penicillin 14.8 24.0
This is clearly just observational speculation. And here is
+ Vancomycin 15.0 21.8
another observation that we do not understand: For decades,
farmers have been feeding their livestock antibiotics to increase + Penicillin and 14.8 23.6
their growth. Of course, that practice has come under scrutiny with Vancomycin
the realization that antibiotic overuse contributes to the develop- + Chlortetracycline 15.2 21.5
ment of resistance.
Recently researchers in New York decided to put some of 16 26
these speculations to the experimental test. They took five groups *
*
of mice and fed them all the same amount and kind of laboratory 15 24
Ct
Ct
P
V
V
P
V
V
l
C
Al
C
Al
P+
P+
in the table to the right, and also in the graphs underneath it. The Source: Cho, Ilseung et al. “Antibiotics in Early Life Alter the Murine Colonic
“Lean weight” graph shows us that there is no significant difference Microbiome and Adiposity.” Nature 488.7413 (2012): 621–626.
between any of the antibiotic groups (light green bars) and the control
group (dark green bar). If there is a significant difference, there is
an asterisk placed over the light green bar. In the “Body fat” graph
on the right, you see that each of the antibiotic bars has an asterisk, The researchers looked at other consequences of antibiotic
meaning there is a significant difference between them and the usage, including the metabolism of short-chain fatty acids, which
control group. This was true even though their overall body weights have a role in carbohydrate metabolism. Those results showed
were comparable to one another. When researchers analyzed the gut differences for the antibiotic groups as well.
microbiome of the mice, they found that (1) the numbers of microbes These are early studies, and the number of things that influ-
in the mouse guts were comparable among groups, but (2) the types ence the gut microbiome and body weight are too many to count.
of microbes were different in the control group compared to the anti- This by no means should turn us away from antibiotics. They save
biotic groups. Specifically, the ratios of Firmicutes to Bacteroidetes lives. Physicians are now prescribing them sparingly, and we learn
(see chapter 1) were much higher in the antibiotic groups. again, “moderation in all things.”
and potentially fatal condition known as antibiotic-associated colitis 3. Summarize two methods for testing antimicrobial
(pseudomembranous colitis). This condition is due to the overgrowth susceptibility.
in the bowel of Clostridioides difficile (C. diff), an endospore- 4. Define therapeutic index, and identify whether a high or a
forming bacterium that is resistant to many antibiotics. It invades low index is preferable in a drug.
the intestinal lining and releases toxins that induce diarrhea, fever, 5. Explain the concept of selective toxicity.
and abdominal pain. It is very difficult to eradicate, which is why
6. Describe the five major targets of current antimicrobial
experimental treatments such as fecal transplants are sometimes con-
agents, and list major drugs associated with each.
sidered. Figure 12.6 gives an overview of how the microbiome can
be affected by antibiotic usage throughout an individual’s lifetime. 7. Identify which categories of drugs are most selectively
toxic, and explain why.
8. Distinguish between drug toxicity and allergic reactions to
12.1 Learning Outcomes—Assess Your Progress drugs.
1. State the main goal of antimicrobial treatment. 9. Define the term superinfection, and summarize how it
2. Identify sources of the most commonly used antimicrobial develops in a patient.
drugs.
At or 0 95
surrounding
birth • Increased risk of infections, asthma,
allergies, type 1 diabetes
• May increase risk of childhood obesity
Anytime 0 95
post-puberty
• Increased risk of Clostridioides difficile
0 95
Anytime
throughout • Loss of microbial diversity and enrichment
life for resistance genes in microbiome
• With repeated use, increased risk of
type 2 diabetes
Figure 12.6 Depiction of the effects of antibiotics on the microbiome throughout life.
12.2 Survey of Major Antimicrobial There are some antibiotics we will consider that aren’t techni-
cally in one of these classes, or that constitute a (small) class of
Drug Groups their own. One of the most useful ways of categorizing antimicro-
Earlier we said that antibiotics belonged to classes. Each class bials is to designate them as either broad-spectrum or narrow-
contains multiple individual drugs that are assigned to that spectrum. Broad-spectrum drugs are effective against more than
class based on the similarity of their chemical structure. In this one group of bacteria, while narrow-spectrum drugs generally
section we will consider antibacterial drugs in the following target a specific group. Table 12.5 demonstrates that tetracyclines
classes: are broad-spectrum, while polymyxin and even some members of
the penicillin group are narrow-spectrum agents.
β-lactams Glycopeptides The designation of antibiotic classes has meaning to the
Sulfa drugs Oxazolidinones chemists and pharmacologists who developed them. Clinicians
Aminoglycosides Ansamycins also categorize antibiotics based on what their target in the bacte-
Tetracyclines Quinolones rial cell is. One important thing to keep in mind is that antibiotics
Macrolides Streptogramins that are targeted to anything in the cytoplasm of the cell have
Pleuromutilins Lipopeptides a very difficult task of getting through at least one membrane.
Polymyxin
Carbapenems
Tetracyclines
Sulfonamides
Cephalosporins
Penicillins
*Note that some members of a bacterial group may not be affected by the antibiotics indicated, due to acquired or natural resistance.
In other words, exceptions do exist.
Because gram-negative bacteria have an extra membrane with Nobel Prize in Medicine for the discovery and production of
highly negatively charged LPS on the outside, it is more difficult penicillin. The natural product of the fungus can be used either
to find or design drugs that affect gram-negatives. in unmodified form or to make semisynthetic derivatives. All
Now we will look at each target in the bacterial cell and dis- penicillins consist of three parts: a thiazolidine ring, a β-lactam
cuss the antibiotics currently used for that target. ring, and a variable side chain that determines its microbicidal
activity (f igure 12.7).
Antibacterial Drugs Targeting the Cell Wall Subgroups and Uses of Penicillins Penicillins G and V are
the most important natural forms. Penicillin is considered the drug
The Penicillin Group of β-Lactams of choice for infections by sensitive, gram-positive cocci (some
The β-lactam (bey′-tuh-lak′-tam) class of antibiotics is a large, streptococci) and some gram-negative bacteria (such as the syphi-
diverse group of compounds, most of which end in the suffix lis spirochete). Certain semisynthetic penicillins such as ampicil-
-cillin. They are named for the chemical ring they all possess, lin and amoxicillin have broader spectra and can be used to treat
called a β-lactam ring. Penicillin is the earliest representative of infections by gram-negative enteric rods.
this class. Penicillin was discovered in 1928 when scientist Alex- Many bacteria produce enzymes that are capable of destroy-
ander Fleming noticed a contaminant on one of many agar dishes ing the β-lactam ring of penicillin. The enzymes are referred to
of Staphylococcus he had been working with. He noticed that the as penicillinases or β-lactamases, and they make the bacteria
Staphylococcus colonies were not growing in the vicinity of the that possess them resistant to many penicillins. Researchers have
white fuzzy contaminant. Since the world had been searching counted more than 500 different β-lactamases in bacteria. This
for antibacterial compounds for some time, he wondered what points to how versatile bacteria can be in resisting our attacks on
the chemical from the contaminant could be, and if it could reli- them. In response, scientists have created penicillinase-resistant
ably inhibit the growth of bacteria. The contaminant turned out penicillins such as methicillin, nafcillin, and cloxacillin, some of
to be the fungus Penicillium, and the antibacterial compound which are useful in treating infections caused by penicillinase-
that was isolated was named penicillin. Interestingly, Fleming producing bacteria.
was unable to actually isolate and produce the compound in any All of the -cillin drugs are relatively mild and nontoxic because
useful quantities. This was finally accomplished by two other of their specific mode of action on cell walls (which humans
scientists in Oxford in 1942. The three of them shared the 1945 lack). The primary problems in therapy include allergy, which is
Common structure than penicillins. Although some cephalosporins are given orally,
(Aminopenicillanic acid) many are poorly absorbed from the intestine and must be admin-
R Group istered parenterally (par-ehn′-tur-ah-lee) by injection into a
Thiazolidine muscle or a vein.
β-lactam
Cephalosporins are spoken of in generations, referring to the
earliest form that was isolated and utilized, and subsequent chemi-
S CH3
CO N
cal changes that were then licensed for use. First-generation ceph-
CH3 alosporins such as cephalothin and cefazolin are most effective
N against gram-positive cocci and a few gram-negative bacteria. A
Nafcillin O COOH third-generation drug, ceftriaxone (Rocephin), is a semisynthetic
broad-spectrum drug for treating a wide variety of respiratory,
S CH3
CH CO N skin, urinary, and nervous system infections. The fifth-generation
CH3 drug ceftobiprole exhibits activity against methicillin-resistant
COONa N Staphylococcus aureus (MRSA) and against penicillin-resistant
O COOH
S gram-positive and gram-negative bacteria.
Ticarcillin
The Carbapenem Group of β-Lactams
Cl
S CH3
Newer antibiotics such as doripenem and imipenem belong to a
CO N new class of cell wall antibiotics called carbapenems. They are
CH3 powerful but potentially dangerous and reserved for use in hospi-
N
N O COOH tals when other drugs are not working. Recently, the appearance
O CH3 of a gene in gram-negative bacteria coding for an enzyme called
Cloxacillin NDM-1 has caused great concern because it confers resistance
to carbapenems and is highly transmissible from bacterium to
S CH3 bacterium.
CH CO N
CH3 Because of similarities in chemical structure among peni-
COONa N cillins, cephalosporins, and carbapenems, allergies to penicil-
O COOH
Carbenicillin lin often are accompanied by allergies to cephalosporins and
carbapenems.
Figure 12.7 Chemical structure of penicillins. All penicillins
contain a thiazolidine ring (yellow) and a β-lactam ring (red), but each
differs in the nature of the side chain (R group). The R group is also Other Drugs Targeting the Cell Wall
responsible for differences in biological activity. Bacitracin is a narrow-spectrum antibiotic produced by a strain
of the bacterium Bacillus subtilis. Since it was first isolated, its
greatest claim to fame has been as a major ingredient in a com-
altogether different from toxicity, and resistant strains of pathogens.
mon drugstore antibiotic ointment (Neosporin) for combating
Clavulanic acid is a chemical that inhibits β-lactamase enzymes,
superficial skin infections by streptococci and staphylococci. For
thereby increasing the effectiveness of β-lactam antibiotics in the
this purpose, it is usually combined with neomycin (an aminogly-
presence of penicillinase-producing bacteria. For this reason, cla-
coside) and polymyxin.
vulanic acid is often added to semisynthetic penicillins to augment
Isoniazid (INH) is a synthetic drug that is bactericidal to
their effectiveness. For example, a combination of amoxicillin and
Mycobacterium tuberculosis, but only against growing cells.
clavulanate is marketed under the trade name Augmentin. Zosyn
It is generally used in combination with two or three addi-
is a similar combination of a β-lactamase inhibitor and piperacillin
tional drugs in active tuberculosis cases. Vancomycin is a
that is used for a wide variety of systemic infections.
narrow-spectrum antibiotic in the glycopeptide class. It is most
effective in treating staphylococcal infections in cases of peni-
The Cephalosporin Group of β-Lactams cillin and methicillin resistance or in patients with an allergy to
The cephalosporins are a group of antibiotics that were originally penicillins.
isolated in the late 1940s from the mold Acremonium, which was
previously known as Cephalosporium acremonium. Cephalospor-
ins are similar to penicillins. They have a β-lactam structure that Antibacterial Drugs Targeting Protein
can be synthetically altered and have a similar mode of action. The Synthesis (Ribosomes)
generic names of these compounds have the root cef, ceph, or kef The Aminoglycoside Drugs
in their names.
Antibiotics composed of one or more amino sugars and a 6-carbon
Subgroups and Uses of Cephalosporins The cephalo- ring are referred to as aminoglycosides (figure 12.8). These com-
sporins are versatile. They are relatively broad-spectrum, resist- plex compounds are the products of various species of actinomy-
ant to most penicillinases, and cause fewer allergic reactions cetes in the genera Streptomyces and Micromonospora.
50 H3C
S
aa N
Prevent
aa
initiation and H3C CH3
Oxazolidinones block ribosome
assembly. HO OH
H3C OH CH3
H3C O
O
mRNA N(CH3)2
HO
H3C O O
30S O CH3
OCH3
CH3
50S O HO
CH3
aa mRNA is
aa CH3
misread;
protein is
incorrect. Figure 12.10 A typical macrolide antibiotic. Azithromycin: Its
central feature is a large lactone ring to which two hexose sugars are
Aminoglycosides mRNA attached.
30S
50S
aa aa Formation
of peptide
Pleuromutilins bonds is
blocked.
mRNA
30S
50S
tRNA is
Tetracyclines blocked;
Glycylcyclines no protein is
synthesized.
30S Figure 12.11 Sulfa drug containing silver, commonly used
mRNA topically for burn wounds.
Smith Collection/Gado/Archive Photos/Getty Images
50S
Another product of the genus Streptomyces is rifamycin,
aa aa Ribosome which is altered chemically into rifampin. It is somewhat limited
is prevented
Erythromycin from
in spectrum because the molecule cannot pass through the cell
translocating. envelope of many gram-negative bacteria. It is mainly used to treat
infections by several gram-positive rods and cocci and a few gram-
negative bacteria. Rifampin figures most prominently in treating
mRNA
30S mycobacterial infections, especially tuberculosis and leprosy,
but it is usually given in combination with other drugs to prevent
Figure 12.9 Sites of inhibition on the bacterial development of resistance. It is a member of the ansamycin class
ribosome and major antibiotics that act on these sites.
of antibiotics.
All have the general effect of blocking protein synthesis. Blockage
actions are indicated by a red X.
Antibacterial Drugs Targeting Cell Membranes
against gram-positive organisms, including some that are resistant to Every cell has a membrane. As you know, gram-negative bacteria
other drugs. The most well-known example is levofloxacin. Fourth- have two bilayer membranes. Figure 12.12 illustrates a gram-
generation quinolones are effective against anaerobic organisms; an negative envelope and drugs targeted to it. Bacillus polymyxa is
example is moxifloxacin. Side effects that limit the use of quinolones the source of the p
olymyxins, narrow-spectrum peptide antibiot-
can include seizures and other brain disturbances. ics with a unique fatty acid component that contributes to their
CNRI/Science Source
Streptogramins Quinupristin and A combined antibiotic; effective against Staphylococcus and Enterococcus
dalfopristin (Synercid) species that cause endocarditis and surgical infections, including resistant strains
Oxazolidinones Linezolid Synthetic drug; inhibits the initiation of protein synthesis; used to treat antibiotic-
resistant organisms such as MRSA and VRE
Pleuromutilins Altabax Used for skin infections currently
Table 12.6 Specific Antibacterial Drugs and Their Metabolic Targets (continued)
Drug Class/Mechanism of Action Subgroups Uses/Characteristics
Drugs That Target Folic Acid Synthesis
Sulfa drugs Sulfamethoxazole, Used to treat shigellosis, acute Sulfa drug
Interfere with folate metabolism by trimethoprim urinary tract infections, certain (inhibitor)
blocking enzymes required for the protozoan infections
synthesis of tetrahydrofolate, which is
needed by the cells for folic acid synthesis
and eventual production of DNA, RNA,
and amino acids Enzyme
Treatment for Other Protozoan Infections antiprotozoan activities are quinacrine (a quinine-based drug),
sulfonamides, and tetracyclines.
A widely used amoebicide, metronidazole (Flagyl), is effective in
treating mild and severe intestinal infections and hepatic disease
caused by Entamoeba histolytica. Given orally, it also has applica- Antihelminthic Drug Therapy
tions for infections by Giardia lamblia and Trichomonas vaginalis Flukes, tapeworms, and roundworms are much larger parasites
(described in chapters 23 and 24, respectively). Other drugs with than other microorganisms and, being animals, have greater
similarities to human physiology. Also, the usual strategy of using Several antiviral agents mimic the structure of nucleotides and
drugs to block their reproduction is usually not successful in compete for sites on replicating DNA. When these “fake” nucleo-
eradicating the adult worms. The most effective drugs immobilize, tides are incorporated into a growing DNA strand, all synthesis
disintegrate, or inhibit the metabolism of all stages of the life cycle stops. Valacyclovir (Valtrex) and its relatives are synthetic purine
but especially nondividing helminths. compounds that block DNA synthesis in a small group of viruses,
Albendazole is a broad-spectrum antiparasitic drug used in particularly the herpesviruses. Some derivatives of this drug are
several roundworm intestinal infestations. The drug works in the valganciclovir, famciclovir, and penciclovir.
intestine to inhibit the function of the microtubules of worms, eggs, HIV is classified as a retrovirus, meaning it carries its genetic
and larvae. This means the parasites can no longer utilize glucose, information in the form of RNA rather than DNA (HIV and AIDS
which leads to their demise. Another compound, pyrantel, paralyzes are discussed in chapter 19). Upon infection, the RNA genome is
the muscles of intestinal roundworms. Consequently, the worms are used as a template by the enzyme reverse transcriptase (RT) to
unable to maintain their grip on the intestinal wall and are expelled produce a DNA copy of the virus’s genetic information. Because
along with the feces by the normal peristaltic action of the bowel. this particular reaction is not seen outside of the retroviruses, it
Two other antihelminthic drugs are praziquantel, a treatment for offers two ideal targets for chemotherapy. The first is interfering
various tapeworm and fluke infections, and ivermectin, a veterinary with the synthesis of the new DNA strand, which is accomplished
drug now used for strongyloidiasis and onchocerciasis in humans. using nucleoside reverse transcriptase inhibitors (nucleotide
(It has been used “off-label”—which means for infections for which analogs), while the second involves interfering with the action of
it has not been approved—in low-income countries in an attempt to the enzyme responsible for the synthesis, which is accomplished
treat COVID-19 patients, but as of late 2021 its real efficacy has not using nonnucleoside reverse transcriptase inhibitors.
been borne out in well-controlled studies.) Helminthic diseases are Azidothymidine (AZT or zidovudine), the first drug approved
described in chapter 23 because these organisms spend at least some for HIV/AIDS in 1987, is a thymine analog that exerts its effect by
part of their life cycles in the digestive tract. incorporating itself into the growing DNA chain of HIV and ter-
minating synthesis. It is still in use, but several newer drugs based
on the same principle are in use today. Nonnucleoside reverse
Antiviral Agents transcriptase inhibitors (such as nevirapine) accomplish the same
Treating viral infections presents unique problems. With a virus, goal (preventing reverse transcription of the HIV genome) by
we are dealing with an infectious agent that relies upon the host binding to the reverse transcriptase enzyme itself, inhibiting its
cell for the vast majority of its metabolic functions. In currently ability to synthesize DNA.
used drugs, disrupting viral metabolism requires that we disrupt Assembly and release of mature viral particles are also tar-
the metabolism of the host cell. Put another way, selective toxic- geted in HIV through the use of protease inhibitors. These drugs
ity with regard to viral infection is difficult to achieve because a (indinavir, saquinavir), usually used in combination with nucleo-
single metabolic system is responsible for the well-being of both tide analogs and reverse transcriptase inhibitors, have been shown
virus and host. Although viral diseases such as measles, mumps, to reduce the HIV load to undetectable levels by preventing the
and hepatitis are routinely prevented by the use of effective vac- maturation step of virus particles in the cell. Refer to table 12.8 for
cinations, relatively few viral infections have effective treatments. a summary of HIV drug mechanisms and see chapter 21 for further
The currently used antiviral drugs were developed to target coverage of this topic.
specific points in the infectious cycle of viruses. Three major One antiviral drug that has been approved for the treatment
modes of action are of severe COVID-19, remdesivir, is metabolized into an analog
of adenosine. When the virus is creating new nucleic acid strands,
1. barring penetration of the virus into the host cell;
it uses the analog instead of actual adenosine, and replication is
2. blocking the replication, transcription, and translation of viral
stopped.
molecules; and
A logical alternative to artificial drugs has been a human-
3. preventing the maturation of viral particles.
based substance, interferon (IFN). Interferon is a glycoprotein
Table 12.8 presents an overview of the most widely used produced primarily by fibroblasts and leukocytes in response to
antiviral drugs. The following examples provide some additional various immune stimuli. It has numerous biological activities,
detail about the principles in table 12.8. Although antiviral drugs including antiviral and anticancer properties. Studies have shown
protect uninfected cells by keeping viruses from being synthesized that it is a versatile part of animal host defenses, having a major
and released, most are unable to destroy extracellular viruses or role in natural immunities.
those in a latent state.
Fuzeon (generic name enfuvirtide), an anti-HIV drug, keeps 12.2 Learning Outcomes—Assess Your Progress
the virus from attaching to its cellular receptor and thereby pre-
vents the initial fusion of HIV to the host cell. Relenza and Tamiflu 10. Distinguish between broad-spectrum and narrow-spectrum
medications can be effective treatments for influenza A and B as antimicrobials, and explain the significance of the distinction.
well as useful prophylactics. Because one action of these drugs is 11. Trace the development of β-lactam antimicrobials, and
to inhibit the fusion and uncoating of the virus, they must be given identify which microbes they are effective against.
rather early in an infection. Also, viruses can quickly become 12. Describe the action of β-lactamases, and explain their
resistant to antivirals. importance in drug resistance.
Drug molecule
Nucleus
Nucl
N ucl
c
Amantadine and its relatives, Block entry of influenza virus by
zanamivir (Relenza), interfering with fusion of virus with
oseltamivir (Tamiflu) cell membrane (also release); stop the Influenza 2
action of influenza neuraminidase, 3
virus
required for entry of virus into cell
(also assembly) 2 3
Drug
ug molecules
m
No infection
N i f ti
RT
Drug
Nevirapine, efavirenz, Nonnucleotide analog reverse molecule 6
delavirdine transcriptase inhibitors; attach to
HIV RT binding site, stopping its
action 6
No reverse
transcription
13. List examples of other β-lactam antibiotics. do occur. The end result varies from slight changes in micro-
14. Describe common cell wall antibiotics that are not in the bial sensitivity—which can be overcome by larger doses of the
β-lactam class of drugs. drug—to complete loss of sensitivity.
15. Identify the targets of several antibiotics that inhibit protein Then we have the occurrence of resistance originating
synthesis. through horizontal transfer from plasmids called r esistance (R)
factors that are transferred through conjugation, transformation,
16. Identify the cellular target of quinolones, and provide two
or transduction. Studies have shown that plasmids encoded with
examples of these drugs.
drug resistance are naturally present in microorganisms before
17. Name two drugs that target the cellular membrane. they have been exposed to the drug. Such traits are “lying in wait”
18. Describe the unique problems in treating biofilm infections. for an opportunity to be expressed and to confer adaptability on the
19. Name the four main categories of antifungal agents, and species. Many bacteria also maintain transposable drug resistance
provide one example of each. sequences (transposons) that are duplicated and inserted from one
20. List two antiprotozoal drugs and three antihelminthic drugs plasmid to another or from a plasmid to the chromosome. Chromo-
used today. somal genes and plasmids containing codes for drug resistance are
21. Describe three major modes of action of antiviral drugs. faithfully replicated and inherited by all subsequent progeny. This
sharing of resistance genes accounts for the rapid proliferation of
drug-resistant species. As you have read in earlier chapters, gene
transfers are extremely frequent in nature, with genes coming from
12.3 Antimicrobial Resistance totally unrelated bacteria, viruses, and other organisms living in
the body’s normal biota and the environment.
Interactions Between Microbes and Drugs:
Fungi have these two options for becoming antibiotic-resistant,
The Acquisition of Drug Resistance as well as a third option discovered in 2014. In at least some species
One unfortunate development in the use of antimicrobials is the of fungi, a small regulatory RNA known as interfering RNA—or
growth of microbial drug resistance, in which microorganisms RNAi—has been found to bind to a genetic sequence temporarily.
begin to tolerate an amount of drug that would ordinarily be When it is bound, the gene is silenced and the target of the antibi-
inhibitory. The property of drug resistance can be intrinsic or otic is not manufactured by the fungus. This renders it temporarily
acquired. Resistance is called intrinsic when it is a fixed trait of resistant to that drug. This provides the fungus more flexibility,
a microbe. For example, all microbes are intrinsically resistant to allowing it to express the gene later when the antibiotic is no longer
antibiotics they themselves produce. Of much greater importance present. This reversible mechanism is called an epigenetic event,
is the acquisition of resistance to a drug by a microbe that was and the gene silencing is called an epimutation.
previously sensitive to the drug. In our context, the term antibi-
otic resistance will refer to this last type of acquired resistance.
Specific Mechanisms of Drug Resistance
The actual changes that take place inside the cell as a result of
How Does Antimicrobial Resistance Arise? these gene changes mostly fall into five categories. Think of the
Contrary to popular belief, antibiotic resistance is not a recent two pathways to acquring resistance as the “how” and these five
phenomenon. It is tempting to think that resistance arises only mechanisms as the “what” (illustrated in figure 12.13):
after the antibiotic has been in use for a very long time. But the
1. New enzymes are synthesized. These inactivate the drug (only
first bacteria that became resistant to penicillin were found in the
occurs through the acquisition of new genes).
1940s, before the antibiotic had even been released to the public.
2. Permeability or uptake of drug into bacterium is decreased
If there is an antibiotic around, there will be a bacterium that can
(usually occurs via mutation).
resist it. And when millions of doses of that antibiotic have been
3. Drug is immediately eliminated using a transmembrane pump
administered, the resistance can become widespread. The scope
(usually occurs via acquisition of new genes).
of the problem in terms of using the antibiotics as treatments for
4. Binding sites for drug are decreased in number or affinity (can
humans became apparent in the 1980s and 1990s, when scientists
occur via mutation or acquisition of new genes).
and physicians observed treatment failures on a large scale. Now
5. An affected metabolic pathway is shut down or an a lternative
it is such a large problem that an economist recently predicted that
pathway is used (occurs due to mutation of gene(s) for origi-
worldwide deaths from antibiotic-resistant microbes will surpass
nal enzyme or enzymes).
deaths from cancer by 2050.
Bacteria become newly resistant to a drug after one of the Some bacteria can become resistant indirectly by slowing down
following two events occurs: (1) spontaneous mutations in criti- metabolism and lapsing into dormancy. That is because most
cal chromosomal genes or (2) acquisition of entire new genes or antibiotics work best on fast-growing populations. Many spe-
sets of genes via horizontal transfer from another species. The cies of bacteria produce persisters under these circumstances,
chance that such a mutation will be advantageous is small, and bacteria that are so slow-growing that they are not affected by
the chance that it will confer resistance to a specific drug is lower the antibiotic. When the antibiotic goes away, the persisters can
still. Nevertheless, given the huge numbers of microorganisms in begin growing again. Persister formation is particularly common
any population and the constant rate of mutation, such mutations in biofilm infections. The most stark example of this is in patients
Mechanism
Permeability or uptake of
the drug into the bacterium Cell surface Cell surface
Drug of microbe Normal of microbe
is decreased (occurs via Differently shaped
receptor
mutation). receptor
Drug is immediately
eliminated (occurs through Cell surface Cell surface
Drug of microbe of microbe
the acquisition of new New active
genes). drug pump
C1 D1
with cystic fibrosis. This condition causes buildup of mucus in the cephalosporins, rendering the drugs inactive. Two β-lactamases—
lungs, which limits access of the immune response to the Pseu- penicillinase and cephalosporinase—disrupt the structure of certain
domonas aeruginosa bacteria that colonize these patients. The penicillin or cephalosporin molecules, so their activity is lost. So
bacteria are indeed in biofilms and antibiotics are not effective in many strains of Staphylococcus aureus produce penicillinase that
eliminating the infections. This is true even though the bacteria, regular penicillin is rarely a possible therapeutic choice. Differ-
when examined, are still found to be susceptible to the antibiotics. ent forms of β-lactamases have spread among other pathogenic
The persistence of the infection is due to the biofilms producing bacteria as well.
significant numbers of persisters, which are dormant, and unaf-
2. Decreased Drug Permeability The resistance of some
fected by the drugs.
bacteria can be due to a mechanism that prevents the drug from
1. Drug Inactivation Mechanisms Some microbes inactivate entering the cell and acting on its target. Antibiotics often have to
drugs by producing enzymes that permanently alter drug struc- bind either to an external protein on the surface of the bacterium or
ture. One prominent example, bacterial enzymes called β-lactamases, to an internal structure, like a ribosomal protein. If a mutation has
hydrolyze the β-lactam ring (a critical structure) of penicillins and changed the amino acid sequence of the binding site, the antibiotic
How Antibiotic
Resistance Happens
1 2 Antibiotics kill most of the 3 But the drug-resistant 4 Some bacteria give
Lots of bacteria.
bacteria causing the illness (the bacteria are now allowed their drug-resistance to other
A few are drug resistant.
ones sensitive to the drug). Usually to grow and take over. bacteria, causing more problems.
this is enough to stop the disease
progression.
will no longer be able to attach, and the bacterium will become (figure 12.14), sensitive individuals are inhibited or destroyed,
impervious to the drug. and resistant forms survive and proliferate. During subsequent
population growth, offspring of these resistant microbes will
3. Drug Is Eliminated Many bacteria possess multidrug-
inherit this drug resistance. In time, the replacement population
resistant (MDR) pumps that actively transport drugs and other
will have a majority of the drug-resistant forms and can eventually
chemicals out of cells. These pumps are proteins encoded by
become completely resistant. In ecological terms, the environmen-
plasmids or chromosomes. They are stationed in the cell mem-
tal factor (in this case, the drug) has put selection pressure on the
brane and expel molecules by a proton-motive force similar to
population, allowing the more “fit” microbe (the drug-resistant
ATP synthesis (see figure 12.13). They confer drug resistance on
one) to survive, and the population has evolved to a condition of
many gram-positive pathogens (Staphylococcus, Streptococcus)
drug resistance.
and gram-negative pathogens (Pseudomonas, E. coli). Because
they lack selectivity, one type of pump can expel a broad array
of antimicrobial drugs, detergents, and other toxic substances. The Human Role in Antimicrobial Resistance
A recent study found that 75% of antimicrobial prescriptions are for
4. Change of Drug Receptors Bacteria can become resistant
throat, sinus, lung, and upper respiratory infections. A fairly high
to aminoglycosides when point mutations in ribosomal proteins
percentage of these are viral in origin and will have little or no ben-
arise (see figure 12.13). Erythromycin and clindamycin resistance
efit from antibacterial drugs. In the past, many physicians tended to
is associated with an alteration on the 50S ribosomal binding site.
use a “shotgun” antimicrobial therapy for minor infections, which
Penicillin resistance in Streptococcus pneumoniae and methicillin
involves administering a broad-spectrum drug instead of a more
resistance in Staphylococcus aureus are related to an alteration in
specific, narrow-spectrum one. This practice led to superinfections
the binding proteins in the cell wall. Enterococci have acquired
and other adverse reactions. Importantly, it also caused the devel-
resistance to vancomycin through a similar alteration of cell wall
opment of resistance in “bystander” microbes (normal biota) that
proteins. Fungi can become resistant by decreasing their synthesis
were exposed to the drug. This helped to spread antibiotic resistance
of ergosterol, the principal receptor for certain antifungal drugs.
to pathogens. Also, tons of excess antimicrobial drugs produced in
5. Changes in Metabolic Patterns The action of drugs this country are exported to developing countries, where controls
directed at metabolic products can be avoided by a microbe if it are not as strict. It is common for people in these countries to self-
develops an alternative metabolic pathway or enzyme. For example, medicate without understanding the correct medical indication.
sulfonamide and trimethoprim resistance develop when microbes Drugs used in this way are largely ineffective, but worse yet, they
deviate from the usual patterns of folic acid synthesis. Fungi can are known to be responsible for the emergence of drug-resistant
acquire resistance to flucytosine by completely shutting off certain bacteria that subsequently cause epidemics.
metabolic activities.
The Hospital Factor
Natural Selection and Drug Resistance The hospital environment continually exposes pathogens to a
Any large population of microbes is likely to contain a few individ- variety of drugs. Hospitals also house susceptible patients with
ual cells that are already drug resistant because of prior mutations weakened defenses and a workforce that may not strictly adhere to
or transfer of plasmids. As long as the drug is not present in the universal precautions. These factors have led to penicillin resistance
habitat, the numbers of these resistant forms are likely to remain in nearly 100% of all Staphylococcus aureus strains within just
low. But if the population is subsequently exposed to this drug 30 years.
Drugs in Animal Feeds Table 12.9 Most Serious Antibiotic Resistance Threats
Nearly 70% of all antibiotics in the United States are given to live- Gram-Positive or
stock, both because they have been found to result in larger animals Most Serious Antibiotic Gram-Negative
and as a general prevention of bacterial disease. The WHO issued Resistance Threats (or Other)
a call to stop using medically important antibiotics in agriculture, Urgent Threats
and some countries have done well with this. Enteric bacteria Carbapenem-resistant Acinetobacter G−
such as Salmonella, Escherichia coli, and enterococci that live as
Candida auris Fungus
normal intestinal biota of these animals readily share resistance
plasmids and are constantly selected and amplified by exposure to Clostridioides difficile G+ endospore-former
drugs. These pathogens subsequently “jump” to humans and cause Carbapenem-resistant Enterobacterales G−
drug-resistant infections, often at epidemic proportions. A bill in Drug-resistant Neisseria gonorrhoeae G−
Congress called the Preservation of Antibiotics for Medical Treat-
Serious Threats
ment Act has been introduced multiple times by Representative
Drug-resistant Campylobacter G−
Louise Slaughter of New York, the only microbiologist in Congress.
It was most recently reintroduced in 2017. As of the printing of this Drug-resistant Candida (other than C. auris) Fungus
textbook, it was still not approved by Congress. ESBL*-producing Enterobacterales G−
Vancomycin-resistant Enterococci (VRE) G−
An Urgent Problem Multidrug-resistant Pseudomonas aeruginosa G−
Textbooks generally avoid using superlatives and exclamation Drug-resistant nontyphoidal Salmonella G−
points. But the danger of antibiotic resistance can hardly be over- Drug-resistant Salmonella serotype Typhi G−
stated. The Centers for Disease Control and Prevention (CDC)
Drug-resistant Shigella G−
issued a “Threat Report” about this issue for the first time in 2013,
and it continues to monitor the situation, which it labels “poten- Methicillin-resistant Staphylococcus aureus G+
tially catastrophic.” (MRSA)
Even though the antibiotic era began less than 80 years ago, Drug-resistant Streptococcus pneumoniae G+
we became so confident it would be permanent that we may have Drug-resistant tuberculosis —
forgotten what it was like before antibiotics were available. Even
Concerning Threats
routine surgeries were life-threatening without prophylactic anti-
Erythromycin-resistant Group A Streptococcus G+
biotics. Certain types of pneumonia had a 50% fatality rate. Strep
throat could turn deadly overnight. Infected wounds often required Clindamycin-resistant Group B Streptococcus G+
amputations or led to death. Yet the effectiveness of our currently *Extended-spectrum β-lactamase
available antibiotics is declining, in some cases very rapidly. There Source: CDC, 2021.
is a real possibility that we will enter a postantibiotic era, in which
some infections will be untreatable. researchers are now convinced that they will need to go back to
The CDC has categorized resistant bacteria into three groups discovering natural antibiotics in bacteria because they can pen-
(Table 12.9). This classification is used to inform the nation’s etrate the membrane. It is interesting that they are now looking
plan for combating antibiotic resistance. Note that this table is not for bacteria that live in animals, not the soil, for these new min-
meant to contain protozoa, helminths, or viruses. ing adventures. Granted, the animals are very small. Nematodes
harbor bacteria that produce antibiotics that antagonize competing
New Approaches to Antimicrobial Therapy bacteria but don’t harm eukaryotic cells.
The past decade has also brought about a revolution in how
The good news is that there is now an uptick in research into novel
we think about antibiotics. There are several new approaches,
antimicrobial strategies. When antibiotic resistance first became
shown here:
widely problematic, the specter of “no new drugs” was very real
because drug makers had drastically slowed their antibiotic devel- ∙∙ The use of nanomaterials, such as graphene oxide and par-
opment. They can perhaps be excused because there was a time ticulate gold particles, causes damage to bacterial membranes
from the 1950s to the 1980s when the medical world assumed that without antibiotics. Other nanoparticles can generate toxic
eventually all infections could be wiped out with antibiotics. That reactive oxygen and nitrogen species inside the bacterial cell.
proved to be a premature assessment, as we have seen. Nanomaterials can also serve as catalysts to convert bacterial
Until recently, most attempts to find new antibiotics were components into toxic chemicals. Alternatively, nanomaterials
based on the old paradigm that the usual cell targets will simply can be used to deliver natural antibiotics more effectively to
need to have new drugs designed to inhibit them. Designing drugs their targets. These studies are still confined to the laboratory.
turned out to be less useful than expected, however, because very ∙∙ Antisense RNAs are a form of small RNAs that can be
few of them could penetrate the gram-negative envelope, and the designed to enter a microbe, bind to its DNA or RNA, and
vast majority of bacteria that are becoming untreatable are gram- shut down the expression of critical genes, leading to cell
negative. (Take another glimpse at table 12.9 to see that.) Many death in a very targeted way.
These activities are designed for you to use on your own with a study group—either a face-to-face group or a virtual one,
consisting of 3–5 members. Studying together can be very helpful, but there are effective and ineffective ways to do it. For example,
getting together without a clear structure is often not a good use of your time. Use your time efficiently by using one or more of the
exercises below.
FACE-TO-FACE GROUPS
Use one or more of the activities below.
Peer Instruction: Assign numbers to your group members to use all semester long. Now look at these five concepts from this
chapter. Each group member prepares a 5-minute lesson on the topic corresponding to their number. Do not worry if you have fewer
than 5 members; just use however many you have! During your group study time, each member presents their lesson, and the group
spends another 5–10 minutes discussing that lesson.
1. Narrow-spectrum vs. broad-spectrum
2. Ways microbes acquire antimicrobial resistance (not mechanisms, see below)
3. Mechanisms of antimicrobial resistance (not ways microbes acquire them, see above)
4. Selective toxicity
5. Minimum inhibitory concentration
Concept Maps: Each member of the group should use this list of terms from this chapter to generate their own concept map. This
can be hand-drawn or created using software (see Appendix C for guidelines). During group study time, compare each other’s
concept maps and help each other make sure they are correct. Of course, there are many different “correct” maps. Examining
each member’s map will help you talk through the varied concepts and how they are related.
Concept Terms:
selective toxicity MIC broad-spectrum therapeutic index
disc diffusion sensitivity resistance therapeutic window
(continued)
Table Topics: Each group member should identify a concept or topic from this week’s class assignments with which they are
having trouble and share it during group study time. The other group members can then help to clarify confusing issues or share
how they figured it out. Aim for a maximum of 15 minutes per topic. If the topic remains unclear to the group, bring it up during
class or use the instructor’s office hours or e-mail to ask for help. Taking the time to struggle with a difficult concept first makes
your questions much more specific and more likely to yield helpful answers.
VIRTUAL GROUPS
Not everyone has the time or opportunity to meet with group members outside of class time. You or your instructor can
create a virtual group using e-mail or the course software.
Weekly Discussion Board: This forum can be used as a way for groups to discuss topics, via e-mail, or other learning management
systems or online platforms, before they are covered in class. As each member of the group answers the current week’s question, they
should send their responses to every other member of their group. It’s best to agree on a deadline based on how your class schedule
works (Saturday for the next week’s topics, for example). Then, after the topic is discussed in class, each member should send a
response that all group members will see with a follow-up post on the same topic. If you cover more than one chapter in a week,
someone can be designated to choose which chapter Discussion Board question you will use. Or simply decide up front that you will
always use the first-chapter-of-the week’s question, to keep the schedule simple.
Discussion Question
A friend was recently diagnosed with strep throat. One week after his treatment, his symptoms returned. Your friend tells you,
“I must have become immune to the drug the doctor gave me.” Discuss the validity of your friend’s statement, providing support
for or refuting their claim.
Chapter Summary
ANTIMICROBIAL
TREATMENT
PRINCIPLES OF ANTIMICROBIAL THERAPY
12.1
• Antimicrobial drugs are produced either synthetically or from natural sources. They inhibit
or destroy microbial growth in an infected host.
• Antimicrobial drugs can be either broad-spectrum or narrow-spectrum. The former are
drugs that act against both gram-positive and gram-negative bacteria, and the latter act
against only one of these, and possibly just one or two bacterial species.
• Bacteria and fungi are the primary sources of most currently used antibiotics. Chemical
changes can improve on these.
• Important terms related to antimicrobial treatment are the minimal inhibitory concentration
(MIC), therapeutic index, and therapeutic window.
• Antimicrobials are best when they are selectively toxic, meaning they are toxic to the
microbe but not to the host.
• The word antibiotic is generally reserved for drugs that target bacteria.
• There are five main cellular targets for antibiotics in cellular microbes: cell wall synthesis,
nucleic acid structure and function, protein synthesis (ribosomes), cell membranes, and
folic acid synthesis.
• There are three broad categories of detrimental effects that antimicrobials can have on
humans/mammals: allergic reactions, toxicity, and the alteration of the microbiome.
ANTIMICROBIAL RESISTANCE
12.3
• Microorganisms are all naturally resistant to various drugs, but acquired antimicrobial
resistance is an inevitable result of using antimicrobials.
• Bacteria naturally experience a large number of mutations every time they replicate. Some
of these instantly make a bacterium resistant to a drug it was formerly sensitive to. In the
presence of that antibiotic, the mutant will grow and displace the sensitive bacteria.
• Bacteria also share, via horizontal gene transfer, entire plasmids that give rise to multiprotein
pumps that can expel antibiotics before they have a chance to act.
• The bacteria that are most critical in terms of antibiotic resistance are mostly gram-negatives,
and new drugs are in development in an attempt to overcome the major barrier that the outer
membrane presents.
• Novel strategies for antibiotics include the use of nanomaterials, both on their own and as
a delivery tool for antibiotics; antisense RNAs to shut down production of various proteins;
CRISPR technology; and bacteriophage therapy (a new “old” strategy).
This 21 Question Grid takes the topics from this chapter and arranges them with respect to the American Society for Microbiology’s
Undergraduate Curriculum guidelines—all six of the important “Concepts” as well as the important “Competency” of scientific literacy. Three
questions are supplied, which cover chapter content referring to the Concept or Competency in increasing levels of Bloom’s taxonomy for learning.
ASM Concept/ A. Bloom’s Level 1, 2—Remember B. Bloom’s Level 3, 4—Apply C. Bloom’s Level 5, 6—Evaluate
Competency and Understand (Choose one) and Analyze and Create
Evolution 1. Microbial resistance to drugs is 2. In the “Media Under The 3. You are invited to participate
acquired through Microscope Wrap-Up,” we in a debate about the concept
a. conjugation. criticize the fact that the author of evolution. Construct an
b. transformation. of the article mentions that the opening statement supporting
mice became resistant to the evolution, using antibiotic
c. transduction.
antibiotic. What is wrong with resistance in microorganisms
d. all of these. that statement? as an example.
Cell Structure and 4. Drugs that prevent the formation 5. Why does the β-lactam class of 6. This chapter discussed five
Function of the bacterial cell wall are antibiotics have milder toxicity major cellular targets for
a. quinolones. than other antibiotics? antibiotic action. Can you
b. penicillins. imagine a different one? Which
one and why?
c. tetracyclines.
d. aminoglycosides.
(continued)
ASM Concept/ A. Bloom’s Level 1, 2—Remember B. Bloom’s Level 3, 4—Apply C. Bloom’s Level 5, 6—Evaluate
Competency and Understand (Choose one) and Analyze and Create
Metabolic Pathways 7. A compound synthesized by 8. Conduct research to find out 9. What kind of organisms do
bacteria or fungi that destroys why drugs blocking folic acid you suppose first possessed
or inhibits the growth of other synthesis are highly selective β-lactamases, and for what
microbes is a/an (not harmful to humans). reason?
a. synthetic drug.
b. antibiotic.
c. interferon.
d. competitive inhibitor.
Information Flow 10. R factors are _______ that 11. You take a sample from a 12. D
evelop an argument about
and Genetics contain a code for ________. growth-free portion of the why the CRISPR technique
a. genes, replication zone of inhibition in the Kirby- could potentially provide
b. plasmids, drug resistance Bauer test and inoculate it a permanent solution to
onto a plate of nonselective antibiotic resistance, where
c. transposons, interferon
medium. What does it mean other “fixes” have been
d. plasmids, conjugation if growth occurs on the new temporary.
plate?
Microbial Systems 13. Treating malarial infections is 14. Can you think of a situation in 15. Scientists have found evidence
theoretically difficult because which it would be better for that exposing some bacteria to
a. the protozoal parasite is a drug to be microbistatic antibiotics makes them much
eukaryotic and therefore rather than microbicidal? more likely to form biofilms.
similar to human cells. Discuss thoroughly. Construct an argument for why
b. there are several species of this might be.
Plasmodium.
c. no single drug can target
all the life stages of
Plasmodium.
d. all of the above are true.
Impact of 16. P
ick the true statement about 17. Speculate on whether having 18. Imagine a postantibiotic era
Microorganisms antibiotics. large concentrations of in which there are no drug
a. Antibiotics were invented by antibiotics in water sources or treatments for microbial
chemists in the 1830s. farmland would be a positive infections. What alternative
b. Antibiotics are natural or negative phenomenon. strategies will make up our
products of microorganisms. Defend your answer. arsenal against morbidity and
mortality due to microbes?
c. Antibiotics are inherently
dangerous for the
environment.
d. None of the above are true.
Scientific Thinking 19. An antimicrobial drug with a 20. A critically ill patient enters 21. You are the director of the
_______ therapeutic index is your emergency room, Centers for Disease Control
a better choice than one with a exhibiting signs and symptoms and Prevention. What are your
______ therapeutic index. of severe septic shock. In this top three recommendations for
a. low; high case, should you immediately halting the spread of antibiotic-
b. high; low begin treatment with a broad- resistant bacteria?
spectrum drug or a narrow-
spectrum drug? Explain your
answer.
Visual Connections
This question uses visual images to connect content within and between chapters.
1. Figure 12.5. Where could penicillinase affect each of these Common structure
antibiotics? (Aminopenicillanic acid)
R Group
Thiazolidine
β-lactam
S CH3
CO N
CH3
N
Nafcillin O COOH
S CH3
CH CO N
CH3
COONa N
O COOH
S
Ticarcillin
These terms and concepts are most critical for your understanding of this chapter—and may be the most difficult. Have you mastered them?
Concepts Terms
Narrow-spectrum vs. broad-spectrum Selective toxicity
Disc diffusion test Susceptibility
Broth dilution test Resistance
Sites of activity for antimicrobials Minimum inhibitory concentration
Why anti-eukaryotic antimicrobials are toxic Therapeutic index
Ways microbes acquire antimicrobial resistance Probiotics
Mechanisms of antimicrobial resistance Prebiotics
Allergy vs. toxicity Superinfection