MCB 419 PARMACETUTICAL MICROBIOLOGY

- Introduction to pharmaceutical microbiology

- Scope of pharmacology
- Sources of drugs & the importance of microorganisms
- Action of antimicrobial agents on microorganisms
- Antibiotic production.
- Microbiological assay of antimicrobial agents

INTRODUCTION TO PHARMACEUTICAL MICROBIOLOGY

Pharmaceutical microbiology is the branch of microbiology that focuses on all aspects of

pharmacy or pharmaceutical sciences especially as it relates to the discovery, manufacture and

quality control of pharmaceuticals and other biological agents such as antimicrobial agent, water

for injection and vaccines.

It is an applied branch of microbiology that focuses on the study of microorganisms that are

directly or indirectly involved in the manufacture of pharmaceutical products. Pharmaceutical

microbiologists ensure that starting raw materials for the manufacture of pharmaceuticals

including water are sterile enough and free from any form of contaminating microorganisms.

They carry out series of tests on starting materials for the manufacture of pharmaceuticals as well

as test the finished products to ensure their safety and efficacy. Pharmaceutical microbiologists

has contributed significantly in quality healthcare delivery across the world especially in the area

of producing, testing, and delivering novel biologicals and medications for the effective

management and treatment of infectious and non-infectious diseases globally.

Pharmaceutical companies around the world are investing heavily in research and development

(R&D); and they are also in high demand for pharmaceutical microbiologists due to the
relevance of this branch of microbiology in the manufacture of safe, effective and good-quality

pharmaceuticals.

Pharmaceutical microbiology also deals with the controlling of microorganisms that cause

spoilage of pharmaceutical products, and this area of microbiology is also keenly interested in

harnessing the metabolic activities of microorganisms to develop novel and potent medicines and

other pharmaceuticals for the healthcare and other related sector. The production of novel drugs

from herbal plants and other natural products are also the subject of pharmaceutical

microbiology.

Scope of Pharmacology
Pharmacology essentially is a science that deals with the study of drug production and its action.

Drug on its own is any substance used in medicine for the treatment of ailments.

Pharmaceutical microbiology essentially deals with the study of drug production and its actions
against disease causing agents.

In general, drugs can be produced from plants, animals, microorganisms or synthesized in the
laboratory. Production of drugs from plants and animals entails the isolation of the active
principles from the tissues or organs of the plants or animals.

Drugs from microorganisms include culturing of microorganisms on synthetic media &

subsequent isolation of the microbial agent from a culture medium or from lysed cells. These
chemical substances from microorganisms (antibiotics) can be purified and used as drugs.
Generally, the mechanism of action of drugs depends on the type of drugs and type of ailment. In
infectious diseases caused my microorganism, drugs used for the cure of such diseases act by
SELECTIVE TOXICITY, this is achieved by either killing the organisms or preventing its
growth without damaging the host cells.
The site of action could be cell wall, cell membrane, interference with protein synthesis or DNA
synthesis. Some drugs simply act as septic agent thereby preventing microbial colonization and
allowing natural healing to take place. E.g wounds

Others may simply act by correcting metabolic disorders e.g insulin used for the treatment of
diabetes which regulates glucose metabolism in man. The production sale and uses of drugs are
governed by laws formulated by different nations or internationally.

These laws are then enforced by different agencies in different countries e.g NAFDAC in
Nigeria. Some drug are internationally not accepted to be used under certain conditions e.g
steroids.

In pharmaceutical industries, microbiological standards are ensure to maintain acceptable

standards, such standards includes:

- The drugs must be free of contaminating organisms (pathogenic)

- In syrups, preservatives are normally incorporated to prevent microbial contamination
and colonization
- Antibiotics must be prepared in sufficient concentration to ensure maximum action
against the microorganisms to which they are directed.

SOURCES OF DRUGS AND IMPORTANCE OF MICROORGANISMS

Generally, drugs can be synthetic or obtained from living organisms. Synthetic drugs are those
obtained as a product of chemical reactions in the lab while those of living organisms could be
from plants, animals or microorganisms.

SOURCES OF DRUGS FROM PLANT

The use of herb for the various treatment of various sources of ailment is also old as man
himself. Such plants are commonly referred to as medicinal plants. It can be defined as any plant
which one or more of its organs contain subs that can be used in any form for therapeutic
purposes or which are precursors for the synthesis of useful drugs. These subs i.e the bio active
agents in plants a widely distributed in plant parts and usually localized in specific portions of
the plant such as the leaves, stem, root, seed or the back of the stem. The bio active agents which
have been found in plant that are therapeutic value includes; tannins, alkaloids and saponins.

Although the mechanisms of actions of these agents are not well understood. Some have been
found to be capable of precipitating proteins from solutions. Though in traditional medicine,
these plants parts are used in combination without refining. Today efforts are being made for the
purification of these active components.

Basically in this procedure, the plant parts are mixed either in dry or fresh form after which
organ subs such as alcohol or inorganic solvent such as H20 is used for the extraction of the
active component. The extracted material is then evaporated and re- suspended in appropriate
liquid medium, this can be further purified by column chromatography technique, thin layer
chromatography e.t.c.

SOURCES OF DRUGS FROM ANIMAL

The tissues and organs of animals could also serve as sources of drugs. Animals mainly used are
cattle, sheep & goats. The drugs could be induced in these animals or naturally present in them.
E.g insulin used for treatment of diabetes is obtained from pancreas of cattle. Interferon from the
blood of animal can serve as anti-viral & anti-cancer agents. The vaccines used for immunization
are generally of animal origin. In general, the production of these chemical substance are from
the tissues and organs of animals

MICROORGANISM AS SOURCE OF DRUGS

Today, microorganism constitute one of the largest group of organisms as sources of drugs. The
most available & widely used drugs from microorganisms are ANTIBIOTICS, these are
chemical substance produced by microorganisms to killing or inhibiting the growth of other
organisms when applied in small concentration.

More than 90,000 different types of antibiotics have been detected but only the few are used
because of the toxicity of others to the host cell. Most commonly used antibiotics are produced
by bacterial especially actinomycetes and Bacillus sp. Some are also produced from filamentous
fungi: the production of these drugs involves these general steps;
 - Isolation & selection of the organism.
- Fermentation on suitable medium for the production of the antibiotic.
- Extraction and Purification.
- Crystallization.

ACTION OF ANTI-MICROBIAL AGENTS ON MICROORGANISM

Antimicrobial agents are substance that either inhibit the growth of microorganism or kill

them. These subs that specially kill or inhibit growth of bacterial are referred to as

ANTIBACTERIAL AGENTS while those that affect are called ANTIFUNGAL AGENTS.

Those directed against viruses are ANTIVIRAL AGENTS. Generally, antimicrobial agents can

be classified into two major groups which are (i) Physical Agents (ii) Chemical Agents.

The physical antimicrobial agent include heat, radiation, α, β & Ȣ rays while example of

chemical antimicrobial agent include; antibiotics, organic and inorganic acids, high pH or low

pH values, antiseptic substance, alcohol etc.

Generally, the action of antimicrobial agents may be able to only cause death in

organisms, such mode of action whereby the microorganisms is killed is referred to as CIDAL

e.g Microbiocidal. Another one is static under this condition, addition of the agent however only

inhibit the growth of the microorganism and when such agent are removed from the medium of

growth, organisms survives. Such antimicrobial agents are classified as STATIC. e.g

bacteriostatic actions. Some antimicrobial agents may be highly specific in action e.g some

antibiotics affect Gram positive bacteria & not negative bacteria and vice-versa. However, some

can kill or inhibit the growth of negative & positive such as classified as having BROAD

SPECTRUM activities.
ANTIBIOTICS AS AN ANTIMICROBIAL AGENTS /CHEMICAL ANTIMICROBIAL

Antibiotics are chemical substance produced my microorganisms which either inhibits

the growth of other microorganisms or kill them when applied in small concentration. Some

antibiotics affect only positive bacteria but not negative. Such antibiotics are classified as

NARROW SPECTRUM. However, some antibiotics will kill or inhibit the growth of both

positive & negative bacteria and are classified as BROAD SPECTRUM. As found in other

antimicrobial agents, some antibiotics have static or cidal activities under certain conditions, two

or more antibiotic may be used as directed against microorganisms. The action of such

combination may lead to increase activity higher from the individual. Under this condition, the

consumed effect is referred to as SYNERGETIC on the other hand, the combination of two or

more antibiotics may lead to lower activities when compared with the individual, such action is

referred to as an ANTAGONISTIC. The mechanism of action of antibiotic as chemical

antimicrobial agent varies between one antibiotic and the other.

MODE OF ACTION OF ANTIMICROBIAL AGENTS

Antimicrobial agents exert their inhibitory activities on microorganisms in different ways

by interfering with various activities of the microbial cells and invariably causing their death.
Processes that are interfered with in microbial cells include:

1. Cell wall synthesis

2. Function of cell membrane
3. Protein synthesis
4. Nucleic acid metabolism
5. Intermediary metabolic activities.

Action on microbial cell wall

 As earlier mentioned, the bacterial cell with the exception of few, is surrounded by a rigid

wall that gives the organisms shape and protection against damages from the environment. For

instance, when a bacterial cell assimilates low molecular weight substances from the immediate

surrounding, an increased osmotic pressure within the cell that may be several times more than

that of the surrounding medium may occur. It is therefore the function of the cell wall to protect

the protoplasmic membrane against such a situation. The uniqueness of bacterial cell wall with

materials that are absent in eukaryotic cells explains the high specificity and toxicity of the such

agents that act on this site in bacteria. Antibiotics which act primarily on the bacterial cell wall

include β- lactam antibiotics (e.g penicillins and cephalosporins ), bactrim, cycloserin

fosfomycin and vancomycin.

The actual destruction of the cell wall is probably an effect of mucopeptidases which

function by lysing the inner part of the wall in a growing bacteria cell while at the same time,

allowing the increasing cytoplasm to expand. The outer part of the cell wall enlarges by synthesis

of the necessary materials as the cell grows. Thus, penicillins and all β-lactams are selective

inhibitors of bacterial cell synthesis by acting as structural analogues of acetylmuramic acid.

Although the exact mode of action β-lactams still remain unclear, the agents are supposed to

interact with some specific target in the membrane such as penicillin-binding proteins normal

cell growth. Transpeptidase enzymes which are involved in cross-linking of peptidoglycan

molecules are blocked by the β-lactam antibiotics, hence the rigid cell wall structure is not

formed. This leads to higher susceptibility to osmotic shock and eventual cell lysis

Action of antimicrobial agents on the cell membrane

The cell or cytoplasmic membrane of bacteria which encloses the cytoplasm plays a significant

role in the cell by controlling the movement of materials between the internal and external

environment of the cell. It also ensures that metabolites and nutrients are concentrated within the

cell and serves as a site for respiratory and some biosynthetic activities.

There are some antibiotics which interfere with one or more of these functions, thus

leading to serious disruption of the viability of the organism. In some microorganisms, notably

bacteria and fungi, cell membrane is the target of action by agents such as amphotericin B,

nystatin and the polymyxins. The polyenes (amphotericin and nystatin) selectively inhibit

organisms whose cell membrane contain sterol and so interfere with the osmotic barrier of the

cells. These agents, therefore, are active only against fungi, yeasts and some other eukaryotic

cells and not against prokaryotic cells since their membrane lacks sterol.
THE PRODUCTION PROCESS OF SOME ANTIBIOTICS

PENICILIN: This is an antibiotics whose mode of action is inhibition of

bacteria cell wall synthesis. There are different types of penicillin which include:

 Penicillin G
 Penicillin X
 Penicillin K e.t.c and
 Procaine Penicillin

The production processes of the above are similar. The steps involve in
penicillin production include:

(a) Strain selection

(b) Fermentation
© Extraction
(d) Purification
(e) Crystallization
A STRAIN SELECTION: Although Penicillin has long been shown to
be produced by a wide range of organism such as Penicillium species,
Aspagillus species, Cephalosporium species and Streptomyces. Today
penicillin is produced mainly from strain of Penicillium notatum or P.
chrysogenum. In the production process a high yielding stain is selected
from the several strain of this organism.

ANTIBIOTIC PRODUCTION AND ISOLATION

Antibiotics are chemical substance produced by micro-organism which are

capable of killing low concentration. Most often they are product of
secondary metabolism, today more than 9,000 antibiotics substances are known
and several 100 are discovered daily.

However, only small proportion of known antibiotics used clinically, because the
rest are too toxic. At though many antibiotic can be chemical produced but cos of
chemical complexity and cost involve majority of antibiotics are produce
commercially by microbial fermentation.

Most commercially useful antibiotic are produced by bacteria of the actinomycetes

and endospore forming groups and by some filamentous fungi e.g

Bacitracin-Bacillus licheniformis

Erthromycin- Streptomyces erythrens

Kanamycin- Streptomyces kanamyceticus

Tetracycline- Streptomyce rimosus

Penicillin-Penicillium chrysogenum

The economic significance of antibiotic shown by the fact that over 100,000 tons
of anti-biotic are produced annually and amounting to several trillions of naira

ISOLATION OF ANTIBIOTICS
The majority of micro-organism that provides antibiotic today are known to be
isolated from the soil. The isolation can mainly be done by screening. The 2 major
form of screening approaches are:

Primary screening (10)

Secondary screening (20)

Primary screening: the method involved in Primary screening screening facilitate

the preliminary screening of anti-biotic producer on an easily visualized zone in
confluent growth of test organism

This method include:

1. The crowed plate method

2. The direct soil inoculation method

3. The cross-streak method

4. The agar plug method

5. The replica planting method

A. THE CROWED PLATE METHOD: this method in used for the isolation of
soil micro-organism that in capable of providing anti-biotic against another soil
micro-microbes organism. It involves plating a heavy soil suspension (1:10 or
1:100) on agar to allow for confluent growth. Anti-biotic producing organism will
form a clear zone round its colonies, this can then be purified for further studies

DISADVANTAGES

1. Slow growing microbe’s e.g actinomycetes maybe over grown

 2. Susceptibility of soil m

,.,icro-organism may not be clinically related to some important organism.

B. DIRECT SOIL INOCULATION METHOD: thin in the method used when

anti-biotic against known organism in required. In thin method pour plate of the test
organism in first prepared then the soil crumbs or dilution placed on the plate.
Antibiotic producing organism will grow and from one to clearance round itself. If
the test organism is susceptible it can then be picked and purified

C. CROSS STREAK METHOD: the method used to test the ability of purified
isolate against test mycoses from soil) to produce anti-biotic against test organism in
this reach. The pre isolate in streaked across the upper 3rd end of the plate containing
medium that support growth of the isolate and that test organism after streaking, the
plate is incubated for seven days during which growth and the production of
antibiotics which growth and the production of antibiotics that diffuse away from
the streak would have occurred. The test organism are then streaked at right angle to
the original isolates. After incubation at appropriate temperature, the extents of
inhibition of the various test is observed.

D AGAR PLUCT METHOD: This method in very useful which when the
test organism are firstly prepared using sterile corn borer plugs of about 0.5cm in
diameter are prepared for progressive distances from the isolate. The plug are
them placed on the plate with different organism. The diameter of zone of
inhibition are the measure of anti-biotic production

6. REPLICA PLATING METHOD: this method is used for the rapid

screening of large number of organism for AB production. In this tech a sterile
paid in placed on the plate containing discreet colonies of organism to be tested
for anti-biotic properties. The pad in then carefully touched on plate seeded with
the test organism clearance zone are then observed on tested organism and the
isolate producing the zone.

FERMENTATION

The production of penicillin during the fermentation stage involves the use of large
culture vessels (fermentation) that can hold up to 400, 00 litres of culture medium.
The inoculum for such a large volume of culture in derived from laboratory culture
maintained either on agar slant or agar plate. It developed through culture volumes
of increasing size up to a final volume of 5-10% of the total culture medium. The
medium for open killing production molasses. Most often molasses in used since it
is a byproduct of sugar production from sugar cane. The nitrogen sources include
soya beans extract, cottons seed source include soya beans. Extract, cottons seed or
corn steep liquor (extract of water from glucose production form high molecular
substance) calcium carbonates or calcium phosphate maybe used as butter system
to maintain the pH blow 6.8 and 7.4 by authomatic titration of either a safe or an
acid usually the carbohydrate sources in added first to the culture medium to
promote active growth of the mycelia. During fermentation the temperature must
be maintained at about 25-300c

Penicillin production in a highly aerobic process and efficient aeration is therefore

very necessary. The entire process of fermentation for penicillin production can be
divided into 3 phases:

1. Trophophase: during this phase there is rapid growth of mycelia cells

without the production of penicillin thin normally takes about 30hours to
achieve

2. Todo phase: At this phase the carbon source in the medium in depleted,
active growth cease but there in large amount of penicillin produced change
 is secreted into the culture medium. This normally last for 5-7 days. At the
end of this fermentation process when the carbon source as produced would
have been excreted into the culture medium

3. Lysis stage: at this phase all the carbon and nitrogen source are completely
depleted the mycelia lyse to release the remaining penicillin and ammonia

EXTRACTION PURIFICATION

At the end of the fermentation, the broth is transferred into setting time and cooled
rapidly to 5-100c accompanying by reduction to pit 6 with mineral acids that broth
is than filter to remove the fungi mycelia and the filter to remove the fungi mycelia
an the filtrate to remove the fungi mycelia and the filtrate adjusted to PH2 using
mineral acid

The antibiotic in the filtered broth then extracted with Amylor Butylacetate, the
aqueous is then separated from the organic solvent containing the antibiotic by
centrifugation (Counter current separate). The organic solvent is then pass through
activated charcoal to remove impurities and bacteria extracted with 2 0c phosphate
buffer PH 7.5. The buffer with the penicillin in then acid (phosphoric acids) and
then extracted again with smaller volume of amyl acetate. The procedure may be
repeated several times in order to concentrate and purify the penicillin. The
penicillin may then be crystallized or converted to stable salt from in one of these
ways

 React with Caco3- ca salt derivative

 React with Na or K buffers to give salts of these ions or
  Maybe precipitated with organic acids such as trimethylamine procaine
penicillin is obtained by dissolving Na or penicillin in procaine hydro-
chloride

Semi-synthetic penicillin: today, penicillin G and U are the only natural penicillin
produced others semi-synthetic ( to take care of broad spectrum activity) produce
pen, G or V and then replace the acylor (R) grp by chemical or alkylase produced
by a number of microorganism then reacted with appropriate carboxylic acid
change then extracted by solvent ctrustion---oral

Production of tetracycline (Streptomyces remosus)

The tetracycline are an important groups of anti-biotic which find wide spread use
in medicine (They are broad spectrum anti-biotic (active against G+ve and G-ve
bacteria). They are produced mainly by strains of Streptomyces remosus , S.
aureofacrem

A production scheme for chlorotetracycline in shown bellows:

Inoculum (spores in agar slant)

Agar plate constituents : 2% meat extract, 0.05% asparagines, 1% glucose , 0.5%, K 2HPO4

Spore as inoculums 1.3% agar

Flask shake

24HRS

Pre fermentor

50% Innoculum, 19-24 hrs, pH 5.2-6.2

Main culture fermentor composition, 2% (NHa) 2 Hpo4, K2HPO4, o.1% CaCo3,
0.25MgSo4.7H20, 0.000331Cuso4.5H20,
0.00511ZnSo4.7H20,
0.0006331Mncl2.4H20

Purification Chanel products to precipitation by Colum chromatography

PRODUCTION OF STREPTOMYCIN

 Streptomycin is a broad spectrum antibiotic (antimycobacterial) belonging to

oligosaccharide antibiotic/aminoglycoside family.
 The industrial production of streptomycin is carried out using submerged fermentation
processes.
 Streptomyces spores are maintained as soil stocks or lyophilized and are used for
inoculating sporulation medium, which is then transferred to germinator where biomass
is increased for inoculating fermenters.
 The fermentation media consists of glucose, starch, dextrin, soy meal, corn steep liquor,
sodium sulphate.
 The streptomycin fermentation requires high aeration and agitation.
 The fermentation is carried out at 28-30ºC with pH maintained at 7.6-8 for good
productivity.
 The fermentation lasts for 5-7 days with of yield of 1-3 g/L of the fermentation broth.

The streptomycin fermentation proceeds through three phases:

 In first phase, the organism produces proteases which digest the soybean meal and
release ammonia and carbohydrates.
 These are utilized for increasing the biomass. Glucose is slowly utilized and net
production of streptomycin is low during this phase.
 The pH of the medium increases from 6.7 or 6.8 to 7.5 or higher.
 This phase lasts for 24h.
 2. The next phase is the idiophase or the stationary phase during which maximum
streptomycin (secondary metabolite) is produced.
 It ranges from 24h to 6-7 days.
 Rapid utilization of ammonia and glucose occurs with no mycelial growth and pH during
this phase remains fairly constant at 7.6 to 9.0.
 3. In the last phase (death phase) the sugars have been completely depleted in the
medium and streptomycin production ceases completely.
 The ammonia released due to the cell lysis raises medium pH. Fermentation broth is
generally harvested before the last phase begins
Recovery of Streptomycin

 On completion of fermentation, the mycelium is separated from the broth by filtration.

 The remaining liquid is then percolated through cation exchange resin columns
where streptomycin gets adsorbed and is finally eluted by washing with buffer as
streptomycin sulphate.
 Further impurities are removed by treating it with sodium hypochlorite, EDTA and
activated carbon.
 The purified streptomycin sulphate solution is concentrated under vacuum and dried
aseptically.

Phases of Streptomycin production:

Phase 1: Rapid growth producing mycelial biomass. Little production of Streptomycin is

obtained.

Phase 2: additional producing of mycelium. Streptomycin accumulates in the medium

Phase 3: Process has completed. Finally the mycelium is separated by filtration and antibiotics
reserved.

Note : Proteolytic activity of the microbes releases NH3 to the medium from the soybean meal
causing a rise in Ph.

Also, the glucose and NH3 releases are consumed during the third phase.

The pH remains fairly constant between 7.6 and 9.0.

2. Secondary screening

After antibiotics has been identified from the 1 0 Screening, certain test are
identified in the 20 screening essentially to establish the safety of such
antibiotics. The test include:-

(a) Toxicity test

(b) Test for stability

© Hemolytic test

(d) Heat and acid stability test

(e) Teratogenicity test

A TOXICITY TEST
This is normally carried out to ascertain the toxicity level of the new
antibiotics. It is carried out by injecting known concentration of the
antibiotics into experimental rats the rats are then observed for death or any
other abnormal characteristics.

B. TEST FOR STABILITY

This test is important for antibiotics that are administered orally. It involves
subjecting the antibiotics to extracts from digestive systems and other organs
of the body after which then antibiotics is assay for activity.

C. TERATOGENITY TEST: This test is carried out to determine the

effect f the antibiotic on developing fetus. The test is carried out by
injecting pregnant rats with known concentration of the antibiotics the rats
are then examine for abortion or abnormal in rats that are born.

D. HEAMOLYTIC TEST:- This test is carried out to determine the

ability of the antibiotics to cause lysis of the red blood cell. It is carried
out by introducing known quantify of the antibiotics into blood agar, the
agar is then observed for zone of clearance around the point of introduction
of the antibiotics.

E. HEAT AND ACID STABILITY TEST

This test is carried out to determine the stability of the antibiotics when
exposed to high temperature and extreme of acidity. In temperature stability
test the antibiotics is exposed to temperature higher than the normal body
temperature at the end of exposure the antibiotics is tested for a activities.
In acidity pH value ranging from 1- 12 at the end of exposure, the
antibiotics is tested for activity.
Sensitivity and Resistance of microorganisms to antibiotic:

Antibiotics are chemical agents produced by micro-organism in oral concentration

inhibit or kill the growth of other m/organism. The mechanism of action of anti-
biotic varies from the anti-biotic to another

Generally, an anti-biotic could be narrow spectrum or bread spectrum. An activity

when an organism respond to the inhibition of growth of that organism, or death of
the organism it is said that the organism the susceptible to the anti-biotic
susceptibility of an organism to A.B can be affected by various factors such as:

 The age of the micro-organism

 The nutritional status of the organism
 Temperature
 PH
 Water activity
 The age of an organism may greatly influence the

Susceptibility of such micro-organism to A.B the in because anti-biotic are more

effective against actively growing cells than an old cell, this be because metabolic
status in the aged cell maybe at its dearest minimum. The nutritional status of
organism may effect susceptibility because in an unfavorable nutrients the rate of
growth of organism is greatly retarded (c) increase in temperature tends to increase
susceptibility of organism to A.B, this is because increase in temperature may
increase metabolic rate in the cell and also increased rate of transport of A.B into
the cell (d) water activity in simply the biotic of H20 available to cell for metabolic
activities. When the H20 activity be very low, metabolic activity in reduced
F. PH influenced growth of micro-organism growing in an unfavorable PH
leads to reduce of growth hence metabolic activity in the cell in also reduced
under such condition susceptibility of the micro-organism be also reduced

Test for anti-microbial susceptibility

Difference type of micro-organism have difference degree of susceptibility to

difference anti-microbial agent different test can be used to indicate changes Anti-
microbial agents in likely to inhibit the growth of micro-organism. The 2 most
widely used method for detecting the susceptibility of a micro-organism to Anti-
magnesium are:

 DISC DIFFUSION METHOD

 BROTH DILUTION TUBE METHOD

DISC DIFFUSION METHOD: (Kirbylanter method) Dis Method is widely used,

because it is simple in expensive. The only disadvantage it has is that it cannot be
used to determine whether the action of the A-M agents is static or cell in the
techniques the appropriate agents medium (Muller Hinton agar), it allows diffusion
of agent very easily) in prepared in a petri dish, and then uniformly inoculated over
a surface with aba studied amount of the broth culture of the organism which had
been incubated for 24hours. Then a filter paper which had been incubated for
24hours. Then a filter paper disc impregnated with antimicrobial agnet of known
concentration placed on the solidified agar medium. During incubation, the Anti-
microorganism agents has effect on the micro-organism, a zone of clearance (zone
of inhibition forms around the disc). The diameter of zone of inhibition is an
indicative of sensitivity of the organism to the anti-microbial agnet. This diameter
is then compared with a STD. table and concentration. The result in reported as the
 organism being sensitive intermediate or resistance (can also be used to detect
susceptibility of several AB to a particular organism

ANTIBIOGRAM OF SENSITIVITY TEST

The pattern of sensitivities of a given strain towards a range of antibiotics is called

an antibiogram. When this method is used. It is possible to compare sensitivity or
otherwise of a bacteria Spp. to difference antibiotics. The zone of inhibition when
compared can be classified as follows:

1. Resistant, sensitive, intermediate, enzyme inactivation selective action,

synergism, Antagonism

ANTIBIOTIC ZONE OF INHIBITION

Disc potency Resistance Intermediate Susceptible

Sensitive
Ampicillin 10Ng <11mm Between 12-13 >14

Chloramphenicol 30Ng <12mm Between 13-17 >18

Erythromycin 5Ng <13mm Between 14-17 >18
Penicillin 18units <20mm Between 21-28 >28
polymycin 300units <8mm Between 9-11 >12

1. AGAR DIFFUSION METHOD (see ppt)

This method involves seeding an agar with organism and challenging the organism
with difference concentration of the anti-microbial agents

First the plate with appropriate agar is inoculated with the organism a cork borer of
appropriate size in used to punch holes in the agar medium. The holes re filled with
appropriate concentration of the antimicrobial agents. The gents in allowed to drain
into the gar before incubation for 24.48hours zone of inhibition in then observed
for antimicrobial activity.

2. BROTH DILUTION METHOD ( see ppt)

This test in used not only to determine the susceptibility of micro-organism to anti-
microbial agents but also used to determine minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC). In thin experiment
difference concentration of anti-biotic is prepared can be either in peptone H20 or
nutrient broth. Each of the tube in then inoculated with standardized broth culture
at appropriate temperature after 24hours, the tube are then observe for growth
usually. The lowest concentration that prevent visible growth is the minimum
inhibitory concentration. (MIC). All the tubes that did not show agar medium that
does not contain the anti-microbial agents the plate are then incubated for 24hours.
The agar medium in the minimum bactericidal concentration (MBC)

AGAR PLUG METHOD

The agar plug method in often us for testing anti-fungi activities of an anti-
microbial agent in this technique a known concentration of the anti-microbial
agents is incorporated into the agar medium and allowed to solidify. A corn borer
is then used to remove a plug form the tip of the colony of activity growing fungus,
the plug them put on the solid agar medium and incubated for 24-48hours. A
control is also set up along with experimental by seeding the agar medium without
any anti-microbial agents with the plug of the fungus. The diameter of growth of
the experiment are compared for sensitivity. The degree of the diameter of growth
is a measure of sensitivity

DRUG RESISTANCE

Drug resistance simply implies the ability of micro-organism to be insensitive

action of a particular drug or an anti-microbial agents. Although resistance can be
developed to ritually all chemotherapeutic agents. The most widely study group to
which micro-organism are resistance to are anti-biotic. This is known to occur
within an organism and also under experimental condition. It is now well
established that in appropriate and extensive use of anti-biotic in leading to the
rapid antibiotics resistance in disease causing micro-organism (Reveal mode of
action of antibiotics)

MECHANISM OF RESISTANCE

The resistance of micro-organism to antibiotics can be brought about by various

mechanism such resistance in micro-organism such resistance in microorganism
can be due to inherent property to the organism or it can be acquired .
antibiotics resistance that is acquired is mostly mediated by the presence of
plasmids in the cell of the organisms which develop such resistance .The
major mechanisms of antibiotics resistance include:-

1 Lack of structure in the organism chain antibiotics inhibit.

2 The organism may be impermeable to the antibiotics

3. The organism may be able to alter the antibiotics to an inactive form.

4 The organism may modify the target of the antibiotics

5 There may be alteration in the metabolic pathway in which the antibiotics

blocks.

6 The organism may be able to pump out an antibiotics entering the cell.

A.B resistance may be acquired through plasmids that carry the antibiotics
resistance gene or as a result of mutation at chromosomal level.

1. LACK OF TARGET STRUCTURE: - An organism may lack a target

structure which antibiotics inhibit e.g some bacteria such as mycoplasmas
lack the typical bac- cell wall hence such organism are resistance to A.B that
inhibit cell wall synthesis, as their mode of action e.g penicillin, cephalosporin,
Bacitracin e.t.c

2. IMPERMEABILITY OF ORGANISM: some bacteria may have cell wall

composition that does not permit the entering of some antibiotics perhaps due to
the nature and structure of antibiotics e.g the inability of penicillin G to have effect
on G-ve-organism in thought to be due to the entering of penicillin e.g tissue P.
aeruginosa is belief to have resistance plasmids that code for enzyme which
prevent the uptake of antibiotics

3. INACTIVATION OF ANTIBIOTICS: In most cases of inactive antibiotics

is due to the presence and resistance plasmid in an organism change codes for new
enzyme in that organism that are capable of inactivation the antibiotics by
modifying its structure this modification can either be by phosphorylates (because
by phosphate enzymes) or methylation (e.g by methylases enzymes) or by
acetylation e.g by acetylases enzymes) his modifying forms of anti-biotic then lose
their activities e.g of antibiotic that can be modified by this mechanization include:
(1) The Aminoglycoside antibiotics e.g streptomycin. In the case of penicillin
the lactamase enzyme spit the B-lactan ring of the antibiotics thereby destroying
the molecules. Chloramphenicol resistance is medicated by resistance plasmids
that produce an enzymes that activities the molecule. Example of organism this
type of resistance can be develop include Staph aureus, enteric bacteria ,Neisseria
gonorrhea

Assignment

Draw the structure of the following A-B in the space below

(1) Streptomycin-
(2) Penicillin-
(3) Chloramphenicol
4. ALTERATION OF TARGET SITE: Resistance in microorganism due to
alteration of target site of antibiotics is usually due to modification of these target
site by mutants in the chromosomal gene e.g ribosome. This mechanism of
resistance is common under laboratory experimental condition, hence they are
often isolated from culture that were predominantly antibiotics sensitive but rarely
from isolated patients’ e.g streptomycin against enteric bacteria such as E. coli may
exhibit resistance by this mechanism.

5. DEVELOPMENT OF RESISTANCE TO BIOCHEICAL PATHWAY:

several A.B exert their effect on terminal with metabolic pathway in org e.g folic
acid metabolism maybe interfered with by sulphonamides which act as substance
analogue which the organism cannot utilize but block enzyme action. In organism
such as Staphylococcus aureus and some enteric bacteria alternative path way be
developed due to changes in chromosomal gene by mutation.

6. EFFCUX (to dump out). This is a mechanism of resistance in micro-

organism that may be to pump out an antibiotics entering the cell. Usually this is
due to the presence of resistance factor in an organism change is capable of
coding for enzymes that actively pump out the antibiotics e.g in some enteric
bacteria, resistance to tetracycline may be due to this mechanism

HOW CAN ONE REDUCE ANTIBIOTIC RESISTANCE?

 Do sensitivity test
 Use combine therapy
 Reduce the extensive use of antibiotics
 Limit inappropriate use of antibiotics Form the above, the incidence of
microbial resistance to antibiotics maybe achieved by:
 Carrying out antibiotics sensitivity test on isolate from patients before the
administrations of the antibiotics
 Use of combine therapy i.e using two unrelated chemotherapeutic agents
 A-B should be appropriately used in their correct doses of avoiding the
excessive use of antibiotics

MICROBIOLOGICAL ASSAY OF ANTIMICOBIAL AGENTS

An assay simply refers to technology which can be used to determine the presence
of a chemical constituent in a biological system. The detects of such constituent
maybe aimed at determine the quantity or the quality of such chemical constituent
in the biological system. There are 3 broad techniques that can be used to assay a
biological system these are:
(a) Physical assay

(b) Chemical assay

(c) Microbiological assay

A. The physical method of assaying simply make use of the physical properties
of chemical constituents such physical properties include the color, taste, density
e.t.c in the physical analysis no changes in the chemical properties or physical
properties of the constituents

B. In the chemical analysis/assay technology the nature of the chemical

constituent maybe altered as a result of a reaction between the chemical
constituents of the biological system and another compound change result into
changes that can be measured

C. The microbiological assays make use of micro-organism is detect the

presence or absence of a chemical constituent in a biological system.
Microbiological assay is a highly specialized techniques change can be used to
test for the pressure or absence of a single chemical constituent in a mixture of a
based is on ability of the organic to utilize the chemical constituents either for its
growth or to inhibit its growth. In the situation whereby the chemical substance is
used for growth, the microorganism is not capable of synthesizing the material but
must be supplied in the growth medium. Any biological system that support the
growth medium of the organism indicate the presence of that nutrient. For an A-M
agents that inhibit the growth of micro-organism its presence in a biological system
can be detected by challenging such system with a susceptible microorganism
hence, its presence is indicated by zone of inhibits quantitatively, it is possible
to determine the amount and such a-m agent on the biological system. In this types
of experiment different concentration of the purified antimicrobial agents is
challenged with standardized culture of the susceptible micro-organism. After
incubation the zone of inhibition on the plates are measured, this measurements are
then used to plot standard curves.

After the standard curve has been prepared, unknown quantity of the A-M agents
in the biological system can be determined by changing such system with the
susceptible organism. The zone of inhibition obtained is measured and use to
determine the quantity of the a-M agents from the standard curve.

ASSAY FOR NUTRITIONAL FACTORS

The presence of a quality of a specific nutrient in a biological system can be

assayed by using microbiological techniques such as nutritional factors cannot be
synthesized in cell of the microorganism but it is essential for the growth of the
micro-organism e.g Lactobacillus spp are unable to synthesis Niacin, the factor
can be perished to the organism in a medium that is complete and satisfaction in
all other respect if Niacin is lacking there will be no growth A minute amount
(0.001 mg/ml) in the culture medium stimulate the growth of the micro-organism
and Niacin in a biological system can be detected by using a medium
supplemented change such system of growth occur it will be an indication of the
pressure and Niacin

DISINFECTANTS/ ANTISEPICS
Disinfectants are chemical antimicrobial agents that kill microorganism when use
at their effective concentration. They are usually toxic to living tissue and hence
are use on in animate object e.g mecurricurichloride chlorine gas, quertinary
ammonium compound, copper sulphate e.t.c for a good quality disinfectant it must
possesses the following properties:
 It should be able to attack and kill and types of mic organism i.e the
pathogenic, non-pathogenic and potentially patho-genic organism
ammonium
  It must be rapid in its action
 It must be able to penetrate the material been disinfected
 It must dissolve easily or mix easily with water to from a stable solution i.e
after the appropriate concentration has been prepared it does not dissociate
to losses its activities
 It should not decompose when expose to heat, light rays or unfavorable
whether condition
 It should not have adverse effect on materials being disinfected
 It should not have an unpleasant odor or discolored the material being
disinfected
 It should easily be obtain at a comparatively low cost and also easily
transported
MODE OF ACTION OF DISINFECTIONS
Disinfections exhibits a wide range of mechanism of action when applied to micro-
organism. Some of this include:
1. Cell membrane injury: the cell membrane of most microbial cell action
lipid continents and also play a major regulatory role in the movement of
molecules in and out of the cell. The cell membrane is the target of action
of many disinfection which they act upon by altering their structural
configuration such an injured membrane may lose. It ability to regulate
the movement of molecules in an out of the cell. The consequent of thin
in cell death.
2. As oxidizing agent: may protein in bacterial cell have-sit groups
(Sulphur hydrate groups) that when oxide will make the protein to losses
its function several types of disinfectant have the ability of oxidizing
such proteins thereby rending them non-functional
 3. Protein denaturation: some disinfection have strong coagulant action
and once they coagulate protein within the cells such protein are
denatured and hence lose their activity.
4. Inactivation of vital enzymes of micro-organism: some disinfectants
exact their effect by reacting with specific enzymes of micro-organism to
make them loose their activity check for more of the mechanism of
disinfectant (Asbyn)
FACTORS AFFECTING ACTION OF DISINFECTANT

Several factors affect the effectiveness of disinfectant when applied against

micro-organisms. Some of these factors include:

1 Concentration of the antimicrobial agents: Most often the

effectiveness of a disinfectant increase with the increase in the
concentration. So when disinfectants are apply their effective
concentration must first be determined.

2 Duration of exposure: The longer the population of cells are expose

to disinfectant the more the organisms are likely to be killed.

3. Temperature: Generally, an increase in temperature at which a

disinfectant is applied often entrance its activity.

4 Nature of material to be disinfected: The effectiveness of a

disinfectant may also be influenced by the nature of the material to be
disinfected e.g if the material is not easily penetrated by a disinfectant
then its activity may be lowered.

5 Age and population of the microbial cells: generally actively growing

cells are susceptible to the disinfectant than cells in the stationary
 phase of growth. Similarly vegetable cells are more susceptible than
spores.

HOW TO DETERMINE EFFICIENCY OF DISINFECTANT

(a) Dilution method

(b) Compare with phenol

(a) Phenol Coffee test: In this, the potency of a disinfectant is compared

with that of phenol. A series of dilution of phenol and the expected
disinfectant are inoculated with test bacteria S. typhi & S. aureus then placed
in a 20 or 37o c water bath. These inoculated disinfectant tube are then
subculture to regular fresh medium at 5min. Interval and the subcultures are
incubated for two or more days. The highest dilution that kill the bacteria
after 10min exposure but not after 5minute are used to calculate the phenol
coefficient. The reciprocal of the appropriate test disinfectant dilution is
divided by that for phenol to obtain the coefficient e.g if the phenol was
1/90 and max effective dilution for disinfectant x was 1/450. The phenol
coefficient of x would be 5. The higher the phenol coefficient value, the
more effective the disinfectant under these test condition. A value greater
than 1 means that the disinfectant is more effective than phenol.

2. DILUTION METHOD: This determined experimentally the rate at

which selected bacteria are destroyed with various chemical agents. Stainless
steel cylinders are contaminated with specific bacteria spp under carefully
controlled conditions. The cylinders are dried, briefly immersed in the rest
disinfectant for 10minutes transferred to culture media an incubate for 2days.
The disinfectant concentration that kills the organism in the sample with a
95% level of confidence under these condition determined the dilution rate.
Sulphonamides -- the deadly mimics
Sulphonamides exert their effect by fooling the bacterial cell into thinking they are molecules of
p-aminobenzoic acid (PABA) because of their similar structures (see below).
PABA is a precursor of folic acid, which is required by cells as a coenzyme in the synthesis of
nucleic acids. The sulphonamide acts as a competitive enzyme inhibitor (see Chapter 6),
preventing the synthesis of folic acid, which in turn affects nucleic acid metabolism and leads to
cell death. Because of their close structural resemblance to the PABA, the sulphonamides are
said to be structural analogues; another, equally descriptive name is antimetabolites.
β-lactam antibiotics have a second mode of action
The β-lactams also act by preventing the natural regulation of enzymes called autolysins. These
enzymes function by breaking down peptidoglycan in a controlled fashion, causing breaks to
allow for the addition of new peptidoglycan as the cell grows, and are normally regulated by
naturally occurring inhibitors. The β-lactams neutralise the activity of these inhibitors, leading to
further breakdown of the cell wall.