Day 2

Prof. Eman El-Seidi

Antimicrobial
Chemotherapy
Definitions
Antimicrobial chemotherapeutic agents: are
chemically synthesized substances that are used to treat
infectious diseases by killing or inhibiting the growth (or
multiplication) of microorganisms.
Antibiotics: are low-molecular weight antimicrobial
substances that are produced as secondary metabolites by
certain groups of microorganisms, especially Streptomyces,
Bacillus and a few moulds (Penicillium & Cephalosporium).
Some antibiotics are currently made synthetically
(synthetic antibiotics).
Chemical modification of certain antibiotics,
to achieve the desired properties, has been a prominent
method of new drug development (semisynthetic
antibiotics).
Definitions 2
Bacteriostatic agent: is an antimicrobial agent that is capable
of inhibiting bacterial multiplication. Multiplication resumes upon
removal of the agent.

Bactericidal agent: is an antimicrobial agent that is capable of

killing bacteria. Multiplication can not be resumed.

Selective toxicity: is the ability of an antimicrobial agent to

harm a pathogen without harming the host.

Spectrum of activity: the range of microorganisms that are

affected by a certain antibiotic is expressed as its spectrum of action.
Antibiotics which kill or inhibit the growth of a wide range of Gram-
positive and Gram-negative bacteria are said to be broad spectrum. If
effective mainly against either Gram-positive or Gram-negative
bacteria, they are narrow spectrum. If effective against a single
organism or disease, they are referred to as limited spectrum.
Mechanisms of Action of
Antimicrobial Drugs
 Mechanisms of Action of
Antimicrobial Drugs
A- Inhibition of bacterial cell wall synthesis
Examples:
β-lactam antibiotics: e.g. penicillins &
cephalosporins
Glycopeptides: e.g. vancomycin &
teicoplanin
Cycloserine & bacitracin

These antibiotics are bactericidal with minimal

tissue toxicity (safe).
The β-lactam drugs inhibit the last steps of
peptidoglycan synthesis by binding to certain
cell receptors known as penicillin-binding
proteins (PBPs).

On the other hand, glycopeptides and

cycloserine inhibit early steps in the
biosynthesis of peptidoglycan, inside the
cytoplasmic membrane.

Therefore, the mechanism of resistance to β-

lactam antibiotics is different from that for
other groups. Subsequently, vancomycin could
be used successfully in infections caused by β-
lactam resistant staphylococci.
B- Interference with the cell membrane
function

Examples:
Antibacterial agents: e.g. polymyxin & colistin.
Antifungal agents: e.g. amphotericin B, nystatin
and imidazoles.

These agents are microbicidal. They are highly toxic

as they have narrow margin of selective toxicity.
C- Inhibition of bacterial protein synthesis
Bacteria have 70S ribosomes (with 30S & 50S
subunits) whereas mammalian cells have 80S
ribosomes (40S & 60S subunits):
Agents acting on the 30S ribosomal subunit:
e.g. tetracycline & aminoglycosides
(gentamicin, amikacin, streptomycin).

Agents acting on the 50S ribosomal subunit:

e.g. macrolides (erythromycin, clarithromycin,
azithromycin), lincomycins (clindamycin),
streptogramins (quinupristin/dalfopristin),
oxazolidinones (linezolid), chloramphenicol
and fusidic acid.
D- Inhibition of bacterial nucleic acid synthesis
Inhibition of RNA synthesis through the strong
binding to DNA-dependent RNA polymerase: e.g.
rifampin.

Inhibition of DNA synthesis through blocking DNA

gyrase: e.g. quinolones & novobiocin.

Inhibition of dihydrofolic acid reductase leading to

inhibition of folic acid synthesis, which is important
for purine synthesis → inhibition of nucleic acid
formation. Examples: trimethoprim &
pyrimethamine.

Inhibition of folic acid synthesis by competitive

antagonism: e.g. sulfonamides are structural
analogues of PABA, competing with it for the
enzyme involved in folic acid synthesis.
Summation

A. Inhibition of bacterial cell wall synthesis.

B. Interference with the cell membrane

function.

C. Inhibition of bacterial protein synthesis.

C. Inhibition of bacterial nucleic acid

synthesis.
 Guidelines for Proper Choice of
an Antimicrobial Agent for
Therapy
1. Know the patient condition: If the infected patient is
seriously ill, start empiric therapy without delay but after
collecting specimens for culture. In closed lesions, where
there is no available sample, empiric therapy is also
indicated.
Depending on the type of infection, there will be a
short list of bacteria most likely to be causing that
infection.
Depending on the type of bacteria, there will be an
antibiotic most likely to successfully treat that
infection.
"Best guess" treatment is not always successful or
appropriate as many bacteria have unpredictable
susceptibilities to antimicrobial agents.
2. Know the nature of infection whether bacterial,
viral, fungal, or parasitic: the primary treatment of an
abscess is immediate drainage, not antibiotics.
Another common mistake is to give an antibacterial
agent for a viral infection.

3. Identify the site of infection (e.g. respiratory,

cerebrospinal, urinary … etc): It is important to select
an antibiotic that is able to penetrate to the site of
infection and achieve effective concentration, e.g.
certain drugs are able to pass the blood-brain barrier
(e.g., chloramphenicol), others are highly
concentrated in urine (e.g., amoxycillin &
nitrofurantoin).

4. Obtain microbiological specimens for culture &

sensitivity: The organism should be isolated,
identified and tested for susceptibility. Based on this,
give the appropriate dose of the antibiotic to which
that organism is likely to respond.
5. Choose the spectrum of antibiotic activity:
Choose as narrow an antibiotic spectrum as you can.
When you get the results of culture and susceptibility,
revise your treatment to 'narrow-down' the spectrum
as far as possible. The use of broad spectrum
antibiotics is likely to faster induce resistance to
antibiotics and may be complicated by superinfection.

6. Know the potential of the drug to produce

toxicity: In the critically ill, some drugs known to
be of low toxicity will exert high toxicity if they
accumulate in the blood due to liver or kidney
dysfunction.
7. Use antibiotics that are only safe for the
pregnant & lactating women and for infants &
children.

8. Define the duration of antibiotic therapy:

which should be based on good evidence that the
patient has responded well so that cessation of
antibiotic therapy is indicated. Undue prolonged use
may result in drug toxicity and antibiotic resistance.

9. Know that the in vitro response is not

always same as in vivo: There are some agents
that appear to be effective in vitro, but will not work
in vivo. Always look at sensitivity result in the light
of your knowledge of the microbe and the patient,
especially the site of infection.
10. Choose bactericidal antibiotics if possible:
Bactericidal drugs are generally preferred
over bacteriostatic drugs.

11. Justify a single drug or a combination: The

ideal rule in antimicrobial therapy is
monotherapy which means choosing one drug
effective against a particular organism.
However, there are conditions which
necessitate the use of more than one antibiotic
in order to achieve a successful clinical
response.
 Indications of Empiric therapy
(best guess)

1. In seriously ill patients: started

without delay but after specimen
collection for culture.

2. In closed lesions, where is no available

sample (e.g., brain abscess).
 Combined therapy
(The rule is monotherapy)
1. Seriously ill patients / serious infections:
e.g. Immunocompromised patients / bacterial meningitis.

2. Febrile neutropenia
3. TB:
• to delay emergence of drug-resistant mutants.
• to reduce toxicity.

4. To achieve bactericidal action through

synergism
5. Mixed infections
 Effects of Combined therapy

1. Synergism (1 + 1 = > 2).

2. Antagonism (1 + 1 = < 1).

3. Indifference (1 + 1 = 1).

4. Addition (1 + 1 = 2).
In Vitro Microbial Susceptibilities
to Antimicrobial Agents
and its Relation to In Vivo Activity
Microorganisms vary in their susceptibility to
different chemotherapeutic agents and
susceptibilities can change over time.

The activity of an antimicrobial agent against

an organism is dependent on its concentration.

the effectiveness of a chemotherapeutic

agent can be obtained from determining the
minimal inhibitory concentration (MIC). The
MIC is defined as the lowest concentration of a
drug that prevents growth of a test organism.
 The breakpoint of an antimicrobial
agent is the concentration that can be
achieved in the serum with optimal
dose:

Relation between MIC & breakpoint

- MICs ≤ breakpoint = susceptible

- MICs > breakpoint = resistant

 The routine in vitro susceptibility
testing can be done by one of the
following methods:

1. Disc diffusion methods.

2. Dilution methods such as tube

broth dilution.

3. Gradient diffusion (E test)

methods.
Disc Diffusion method
E-test method
COMLICATIONS OF CHEMOTHERAPY
1. Toxicity: dose-dependent or independent, e.g.:
Tetracycline → staining of teeth in infants.
Streptomycin → 8th cranial nerve damage →
vestibular dysfunction.
Aminoglycosides → may cause nephrotoxicity.
Chloramphenicol → can cause bone marrow
depression.

2. Allergy (hypersensitivity): usually not dose-

dependent, e.g.:
Penicillins → urticaria, anaphylactic shock or
serum sickness.
Local application of sulphonamides → contact
dermatitis.
3. Emergence of resistant strains: The abuse
of antibiotics (low dosage, interrupted course,
no real indication, and improper choice) →
emergence of resistant mutants. These
mutants will overgrow and replace the
originally susceptible bacteria.
4. Superinfection: due to outgrowth of resistant
members of normal flora when the sensitive
ones are eradicated during antibiotic therapy
e.g.:
Pseudomembranous colitis caused by
outgrowth of Clostridium difficile.
Oral thrush caused by overgrowth of the
yeast Candida.
RESISTANCE TO ANTIMICROBIAL
AGENTS

Antibiotic resistance is a global problem

faced today in the treatment of infectious
diseases.

Resistance to antibiotics is more prevalent

in hospitals, especially ICUs due to the
higher antibiotic use.

Resistance to antimicrobial agents may be

either intrinsic or acquired.
Intrinsic (inherent or natural) resistance

Definition: Natural resistance to an antibiotic

without the acquisition of resistance factors.
It is consistent and can be expected once the
organism is known e.g. Enterococcus is
cephalosporin resistant.
Examples:
1. Streptomycetes has a gene that protects it from the
antibiotics it produces.
2. Gram-negative cell membrane has pores too small
to allow large antibiotic molecules (e.g. nafcillin) to
penetrate.
3. An organism lacks the target or receptor for the
antibiotic e.g. resistance of mycoplasmas to
penicillins & resistance of Enterococcus species to
cephalosporins.
Acquired resistance

It results from altered bacterial physiology and

structure due to changes in the bacterial
genome.

It is inconsistent and unpredictable. The

unpredictable nature of this resistance is the
1ry reason why laboratory methods to detect
resistance are necessary.
 Genetic Mechanisms of Acquired
Resistance

1. Mutation & selection (vertical

evolution): Exposure of an organism to an
antibiotic exerts a selective pressure on the
organism and leads to mutation. The more
frequent the exposure to the antibiotic the
greater the potential resistance.

2. Exchange of genes between strains and

species (horizontal evolution). Resistance
genes can be encoded on plasmids, phages,
transposons and integrons.
Mechanisms of Acquired Resistance
1. Reduction of the intracellular concentration
of the antibiotic by:

A. Decrease in influx of antibiotic through:

 e.g. imipenem resistance in Pseudomonas.
 e.g. aminoglycosides resistance in
Staphylococcus aureus.
B. Efflux pumps:

The antibiotic is pumped out across the

cytoplasmic membrane faster than it can
diffuse in, so the concentration of
antibiotic remains too low to be effective.

This mechanism plays a role in the

resistance to many antibiotics including
macrolides, tetracycline, quinolones and β-
lactams by organisms such as Escherichia
coli, Shigella and S. aureus.
2. Inactivation of the antibiotic, e.g.:

Production of β-lactamases leads to

hydrolysis of the β-lactam ring, thus
inactivating penicillins and cephalosporins.

Production of acetyl transferase results in

chloramphenicol resistance.

Production of aminoglycosides-
modifying enzymes results in
aminoglycoside resistance.
3. Target modification:

Modification of the penicillin-binding

proteins (PBPs): e.g.,
Resistance to penicillin in pneumococci.
Resistance to all β-lactam antibiotics in MRSA.

Alteration of the 50S ribosomal subunit

reduces the affinity of macrolides,
linezolid and streptogramins for the
ribosome.

Alteration of the 30S ribosomal subunit

reduces the affinity of aminoglycosides
for the ribosome.
4. Target elimination by developing new
metabolic pathways that bypass the
original target: e.g. resistance to
trimethoprim (which acts by inhibition
of dihydrofolic acid reductase needed
for folic acid synthesis).
 Antimicrobial
Chemoprophylaxis
Chemoprophylaxis is the administration of an
effective antimicrobial agent to prevent rather
than to treat infection with a certain microbe,
thus preventing development of a disease.

Prevention rather than

treatment
Examples:
Long acting penicillin (or erythromycin): is
given to rheumatic patients to prevent
reinfection with S. pyogenes.

Rifampicin: is given to close contacts of

meningococcal meningitis for 2 days to
prevent meningitis.

Penicillin or erythromycin: is given to

individuals with abnormal heart valves prior to
dental procedures to prevent endocarditis.

Preoperative in some surgical operations.

STERILIZATION
&
DISINFECTION
Definition and principles of
terms
 Sterilization
Validated process used to
render a product free of all
forms of viable microorganisms
including all bacterial spores.

Sterilization is essential for

 Culture media,
 Critical items intended to
enter the vascular system
and sterile tissues such as
vascular catheters and
surgical instruments
 Disinfection
 It is a process that eliminates most, if not all,
pathogenic microorganisms except
spores.

 Disinfection is required for devices or

equipment that do not penetrate tissues but
used in contact with the skin or mucous
membranes.

 Examples:
 Stethoscope diaphragm swabbed with
70% alcohol
 Immersion of endoscopes in 2% ortho-
phthalaldehyde (OPA) or 2% alkaline
glutaraldehyde (pH 7.5-8.5) for 12
minutes
 Cleaning (or pre-cleaning)
• Removal of foreign material (organic or inorganic
contaminants) from medical devices as part of
decontamination process.

• Usually done with water & soap, detergents or enzymatic

products.

• Cleaning must always precede disinfection or sterilization.

• Surgical instruments should be pre-soaked or rinsed to

prevent drying of blood and to soften or remove blood
from the instruments.

• Cleaning is done manually in use areas without

mechanical units (e.g., ultrasonic cleaners or washer-
disinfectors) OR for fragile or difficult-to-clean
instruments)
 Decontamination
• Reduction of pathogenic microorganisms to a
level at which items are safe to handle.

• Decontamination includes sterilization and

disinfection processes.
Disinfectant & Antiseptic
Disinfectant
Usually a chemical agent (but sometimes a
physical agent) that achieves disinfection. It refers
to substances applied to inanimate objects.

Antiseptic
A chemical disinfectant which can be safely
applied to skin and mucous membranes but not
suitable for systemic administration.

Examples
– 70% isopropyl alcohol to prepare skin for
injection,
– Preoperative skin preparation with alcohol-based
iodine compound
Germicide & Sterilant
Germicide:
Agent that destroys microorganisms; may be
named indicating the microorganisms the
germicide kills
Virucide,
Bactericide,
Fungicide,
Sporicide,
Tuberculocide

The term germicide includes both antiseptics and

disinfectants.

Sterilant:
Chemical germicide that achieves sterilization
(e.g., 2% alkaline glutaraldehyde for 10-12
hours)
 Decreasing order of resistance of microorganisms to
disinfection and sterilization

Level
Resistant Prions Prion processing
Bacterial spores Sterilization
Coccidia Disinfection
Mycobacteria High
Non-lipid or Small viruses
Fungi Intermediate
Vegetative bacteria
Susceptible Lipid or medium-sized viruses Low
Sterilization

Usually done in the Central Sterile

Services Department (CSSD), an
integrated place in hospitals and
other healthcare facilities that
performs sterilization on medical devices,
equipment and consumables; for
subsequent use.
 Methods of Sterilization
I. Heat:
1. Steam sterilization (moist heat)
2. Dry heat sterilization

II. Low temperature sterilization methods:

1. Hydrogen peroxide gas plasma
2. Ethylene oxide gas sterilization
3. Peracetic acid sterilization

III. Other sterilization methods:

1. Ionizing radiation
2. Filtration
3. Ozone
4. Formaldehyde steam
5. Infrared radiation
Steam Sterilization

The most safe and most commonly used method of

sterilization.

It is accomplished in an autoclave
uses moist heat

Four parameters of steam sterilization:

1. Steam,
2. Pressure,
3. Temperature,
4. Time
Steam Sterilization
Sterilization temperature and exposure time:

at 121°C for 20-30 minutes

at 132-134°C for 3-6 minutes

N.B., prions can be inactivated by autoclaving

at 134°C for 60 minutes.

Advantages
Non-toxic,
Inexpensive,
Rapidly heats and penetrates fabrics
Monitors of steam sterilizers (autoclaves)

Chemical
Mechanical Biological
indicators or
indicators indicators (BI)
integrators (CI)
Monitors of steam sterilizers (autoclaves)

1. Mechanical Indicators

A printout or graph
that monitors the
time, temperature
& pressure of the
sterilization cycle.
Monitors of steam sterilizers (autoclaves)

2. Chemical Indicators

Chemically impregnated paper strips that must be

used with each sterilization cycle to monitor the
temperature or time & temperature.

Visible colour changes occur at specified

temperature and time.
Monitors of steam sterilizers
(autoclaves)

3. Biological Indicators
• Paper strips impregnated with spores of
Geobacillus stearothermophilus (formerly Bacillus
stearothermophilus).
• They are placed at the coldest point of the
chamber.

• After finishing the cycle of sterilization, they are

incubated in fluid medium at 37°C for 48h.

• Absence of bacterial growth indicates an

efficient sterilization cycle.
 Biological Indicators
Sterilization cannot be proved except by
culturing

Efficient sterilization cycle

Inefficient sterilization cycle
Dry heat sterilization

1. Incineration: is particularly applicable for

dead animal bodies, infectious hospital waste
such as used surgical dressings,
needles….etc.
Dry heat sterilization

2. Red heat:
Inoculating wires, loops and points of forceps
are sterilized by holding them in the flame
until they are red hot.
 Dry heat sterilization

3. Dry Heat Sterilizers or hot air ovens

Dry hot air is the sterilizing agent

The most common time-temperature relationships are

o 170oC for 60 minutes,
o 160oC for 120 minutes,
o 150oC for 150 minutes

Bacillus atrophaeus spores should be used as a

biological indicator.
Dry heat sterilization
3. Dry Heat Sterilizers or hot air ovens (cont.)

Used for materials that might be damaged by moist heat

(e.g., powders, oils, petroleum products, sharp instruments)

Advantages :
Non-toxic,
Relatively inexpensive,
Non-corrosive for metal and sharp instruments

Disadvantages:
Slow rate of heat penetration,
Time-consuming,
High temperatures are not suitable for most materials
Low temperature (LT) sterilization methods
1. Hydrogen peroxide gas plasma
Mode of action
1. The free radicals interact with essential cell
components (e.g., enzymes, nucleic acids) → →
→ → → disrupt the metabolism of
microorganisms,
2. Direct inactivation by hydrogen peroxide

Total time of sterilization cycle is about 50 minutes.

Medical materials and devices that cannot tolerate
high temperatures and humidity, such as some
plastics, electrical devices, and corrosion-susceptible
metal alloys, can be sterilized by this method.
Biological indicator: Geobacillus
stearothermophilus spores
Low temperature sterilization methods
2.Ethylene oxide (EO) gas sterilization
Exposure time is long and varies from 3 - 6 hours
Expensive & toxic
Need for 24 hours post-conditioning (aeration or
desorption time)
Uses: instruments that cannot be subjected to
steam
Biological indicator: Bacillus atrophaeus
(formerly B. subtilis) spores
Since 1980s, EO has been considered unfavorable
because of the risks posed to employees and the
environment.
 Low temperature sterilization methods

3. Peracetic acid sterilization

It is used to sterilize medical, surgical,

and dental instruments (e.g.,
endoscopes, arthroscopes).

Peracetic acid:
1. denatures proteins,
2. disrupts cell wall,
3. oxidizes proteins and enzymes of
microbes
Ionizing radiation

Two types of ionizing radiation used for

sterilization:
Cobalt 60 gamma rays
Electron accelerators (β-rays)

Ionizing radiation has a high penetrating power

Used for sterilization of pre-packed heat-
sensitive items such as bone grafts, surgical
sutures, disposable plastic syringes, gloves,
catheters and intravenous (IV) infusion sets.

Monitoring of radiation sterilization:

Bacillus pumilus spores
Filtration

A process used to remove bacteria from

thermolabile pharmaceutical fluids (antibiotic
solutions, hormones, vitamins) that cannot be
purified by any other means.

Fluids passed through bacterial membrane filters

with pore size as small as 0.22 µm.

The endopigment producing Serratia marcescens

may be used to test the efficiency of bacterial
membrane filters.
 Filtration (cont.)

Filters can also be used to remove

microorganisms from air supplied to critical
areas such as operating rooms, drug factories
and laboratory biosafety cabinets.

Such filters are known as high efficiency

particulate air (HEPA) filters which can
provide sterile air at the filter face.

• Spores of the fungus Aspergillus may be used

to test the efficiency of HEPA filters.
 Ozone

Ozone (O3) consists of O2 with a

loosely bonded third oxygen atom that
makes ozone a powerful oxidant that
destroys microorganisms.
Formaldehyde Steam
(Not FDA approved for use in healthcare facilities)

Low-temperature sterilization method that involves

use of formalin, which is vaporized into a
formaldehyde gas.

Used to sterilize heat-sensitive medical equipment

such as the mechanical ventilator and incubators
for neonates.

Formaldehyde is a mutagen and a potential human

carcinogen, therefore must be regulated and fully
contained to guarantee the permissible exposure
limit of healthcare workers for formaldehyde.
Disinfection
Categories (Levels) of Disinfectants
High Level Disinfectants
Germicides that kill all microbial pathogens
except large numbers of bacterial spores.

Examples:
OPA for endoscopes
Hydrogen peroxide for contact lenses
Chlorine for blood spills
Intermediate Level Disinfectants

Germicide that kills all microbial

pathogens (including M. tuberculosis)
except bacterial spores.

Examples:
Isopropyl alcohol 70%

Iodophors (e.g., povidone iodine)

Low Level Disinfectants

Germicide that kills:

Most vegetative bacteria (except tubercle

bacilli)

Lipid-enveloped & medium-sized viruses

e.g., human immunodeficiency virus &

hepatitis B virus

Example: Quaternary ammonium compounds for

disinfection of floors and food preparation areas.
MAIN METHODS OF DISINFECTION

1. Chemical

5. 2. Boiling water
Ultraviolet
Disinfection
Methods

4. Thermal 3. Pasteurization
 Main Methods of Disinfection – 1

1. Chemical disinfectants (examples)

2. Boiling water:
• Boiling (100°C) for 20 minutes achieves high
disinfection → useful in emergencies if sterilizer is
not available.
3. Pasteurization of milk
• Heating at 63°C for 30 min. or at 72°C for 20 sec.,
followed by rapid cooling (<10oC) destroys important
pathogens e.g. M. tuberculosis, Brucella, Salmonella
and Coxiella burnetti.
 Main Methods of Disinfection – 2

4. Thermal disinfection (by hot water) in special washing

machines
• e.g. for linen in hospital laundry, dishes and devices which
cannot withstand higher temperature.

5. Ultraviolet radiation (UV): can be artificially produced by

mercury lamps
• UV have weak penetration power → used only for air &
surface disinfection (e.g., laboratory safety cabinets).
NORMAL FLORA OF THE
HUMAN BODY
Definition

Microorganisms
that inhabit the
skin and mucous
membranes of
healthy normal
people.
 Types of Normal Flora
Resident flora Transient flora
Resident Flora
Relatively fixed types of organisms regularly
found in a given site at a given age.
If disturbed, it promptly re-establishes itself.

Role of Resident Flora

In the GIT, they synthesize vitamin K & help in the absorption
of nutrients.

They prevent colonization by pathogens through “bacterial

interference”:
- Competition for receptors on host cells & for nutrients.

- Inhibition/killing of pathogens by toxic products or

bacteriocin production.
Transient Flora
Non pathogenic or potentially
pathogenic organisms found for some
time and derived from the environment.

They do not establish themselves

permanently on the surface

They do not produce disease except

when the resident flora is disturbed.
 Normal Flora may Produce
Disease Under Certain Conditions

1. Changes of their normal habitat

Viridans streptococci (normal flora of the
URT)  infective endocarditis in
predisposed heart.
E. coli (normal flora of the intestine) 
most common cause of UTI.

2. Disturbance of the resident flora

by broad spectrum antibiotics, may produce
super-infection.
Normal Flora of the Skin

Staphylococcus epidermidis
Diphtheroids & propionobacterium
Viridans streptococci
Enterococci (in the perineum)
Fungi & yeasts (in skin folds)
Acid-fast NTM (e.g. Mycobacterium
smegmatis in external genitalia)
Normal Flora of Mouth, Throat & Pharynx
Gram positive cocci: Staph. epidermidis,
viridans streptococci and enterococci
Gram negative cocci: commensal Neisseria &
Branhamella
Diphtheroids
Spirochaetes: e.g. Trep. macrodentium and
Trep. microdentium

Yeasts (Candida species)

Anaerobic bacteria, specially with bad oral

hygiene  responsible for foul smelling, e.g.
Lactobacilli, Prevotella melaninogenicus,
Actinomyces and Fusobacteria
Normal Flora of the Intestine 1

Normally, the stomach is sterile due to

its acidity.

The duodenum & upper part of small

intestine are also sterile.

The lower part of small intestine & the

colon contain a large amount of
bacterial flora varying according to age
and diet.
 Normal Flora of the Intestine 2
A- Anaerobic bacteria: ~99% of the intestinal flora,
e.g.:
Bacteroides fragilis
Clostridium especially Cl. perfringens
Peptostreptococci
Lactobacilli
B- Aerobic & facultative anaerobic bacteria: only
form 1-4% of the flora, these include:
E. coli & some other members of
Enterobacteriaceae
Pseudomonas
Enterococci (E. faecalis)
C- Yeasts: e.g. Candida species
Normal Flora of the Vagina 1

In the normal adultvagina,

Lactobacillus doderleins usually
predominates and is responsible
for the vaginal acidity, which
resists infections.
Normal flora of the vagina 2

If lactobacilli diminish in number, e.g. after

menopause or antibiotic treatment, mixed flora
predominates (resulting in a condition known as
“Bacterial vaginosis”:
Staph. epidermidis
Group B, β-haemolytic streptococci
Peptostreptococci
Gardnerella vaginalis
Ureaplasma urealyticum
Yeasts (Candida albicans)
Carriers
Apparently healthy individuals
harbouring a pathogenic organism,
without having clinical manifestations &
can transmit this organism to others.

According to the duration of the

carriage state,
(a) transient carriers e.g. during the
incubation period & early
convalescence.
(b) chronic carriers e.g. HBV carriers.
Carriers
Carriers are more dangerous than
cases as a source of infection
because:

They are not known to public.

They are not easily detected.
They are not restricted to bed.
They carry the organism in the inter-
epidemic periods.
Carriers
Carriers play an important role in:

1. Enteric fever (gall bladder)

2. Cholera (intestine)
3. Epidemic cerebrospinal meningitis
(nasopharynx)
4. Diphtheria (throat)
5. Hepatitis B, C virus infection (blood)
6. Staph. aureus infections (skin and nose)
 Laboratory Diagnosis
of Infection

We ask the lab for a diagnosis, expecting a yes or

no, but often end up with just a may be…
Diagnosis of Bacterial Infection

Patient Non-microbiological
Clinical investigations
diagnosis
Radiology

Haematology
Biochemistry

Sample
Take the specimen correctly
Label & package the
specimen up correctly
Appropriate transport &
storage of specimen
Specimens
Specimens must be collected & delivered
promptly in a suitable transport system for
processing in the lab.

Prior discussion with the microbiologist helps

the clinician to know when and how to collect
appropriate samples.

Good communication not only helps in deciding

which tests to request but also influence how to
interpret the results & thereafter proper
management of patients.
 General Guidelines 1
1. Collect specimens from the actual infection site, during
the acute phase of infection.
2. Under strict aseptic conditions.
3. Collect sufficient specimen.
4. Before starting antibiotic therapy, whenever possible.
5. Use sterile collection devices & containers.
6. Number of specimens for the best chance of recovery of
the causative microorganisms:
e.g.: - at least 2 sets of blood cultures in 24 hrs.
- 3 successive morning sputum samples for TB.
- Paired sera for serological investigations.
 General Guidelines 2
7. Proper skin disinfection is extremely important
before doing a blood culture.

8. Pus is always superior to a swab.

9. Label the specimens appropriately.

10. All clinical specimens should be considered as

potential biohazards and should be handled with
care using universal precautions.
 Types of Specimens

1. Solid (e.g., tissue, biopsy and

formed stool).

2. Liquid (e.g.: blood, CSF, urine,

sputum and aspirate).

3. On swabs
 General Guidelines 3
Collection of good quality samples
depends on the time, type & quantity
of the collected specimen.

Minimal contamination with normal

flora is always desirable & especially
important if quantitative culture is to
be done.
General Guidelines 4

Careful skin antisepsis

before specimen collection
(e.g., blood culture, lumbar
puncture).

Try to bypass anatomic

areas containing normal
flora (e.g., protected brush
bronchoscopy for VAP).
General Guidelines 5

Thought convenient & most commonly

used for specimen collection, sterile swab
absorbs a small volume of specimen
material & provides the poorest conditions
for microbial survival.

Positive findings from specimens collected

directly from normally sterile tissues &
body fluids are almost always diagnostic.
Collecting the correct specimen

Endocervical swabs for gonococci

Pernasal swabs for pertussis

Sputum, not saliva for TB

Blood culture bottles, not clotted blood

Pus, not swabs

Collecting the specimen correctly
Take a mid-stream urine
avoid contamination with perineal flora
CSF
Avoid contamination
Avoid bloody tap
Throat swab
Make the patient gag!
Blood cultures
Avoid contamination with skin organisms
 Organisms may die out in
specimens for various reasons

• Bacterial multiplication may cause an

adverse change in pH.

• Growth of one species may inhibit another.

• Some organisms are very sensitive to

oxygen (strict anaerobes) & some to drying
(e.g., Neisseria gonorrheae).
 Specimen Transport 1
Every effort should be made to ensure that the
organism sought is viable when it reaches the lab:

• All specimens must be transported preferably within

2 hours.

• Refrigeration at 4-10oC can help to preserve cells

and reduce the multiplication of commensals.

• Specimens for the isolation of Haemophilus, S.

pneumoniae or Neisseria species must never be
refrigerated because cold kills these pathogens.

• Specimens should not be frozen before culture.

Never refrigerate CSF, genital, eye or internal ear
specimens.
 Specimen Transport 2
• Specimens should never be allowed to dry out.

• Specimens for bacterial culture should not be stored

for more than 24 hours.

• Optimal transport of specimens depends primarily

on the volume of material obtained.

• Rapid transport of samples to the lab is always

desirable. If this is not possible, media should be
inoculated directly from the sampling site or placed in
a suitable transport medium.
Specimen Transport 3
Should be ASAP (preferably within 30 minutes).

Some organisms are susceptible to environmental

conditions (e.g.:
Presence of oxygen (anaerobes).
Changes in temperature (N. meningitidis).
Changes in pH (Shigella).

Use transport media for suspected delay.

Transport media are usually buffered fluids or

semi-solid media that contain minimal nutrients
(to minimize bacterial growth during transport).
 Specimen Transport 4

Problems in delay or inappropriate storage:

- delay in diagnosis & treatment
pathogens die
contaminants overgrow
Blood cultures directly into incubator
not refrigerated!
CSF straight to lab
Don't put an entire surgical specimen into
formalin! But send a portion to microbiology lab in a
sterile container in saline (e.g., gastric biopsy
specimens for H. pylori).
Specimen Rejection 1

Errors in specimen collection &

transport are the most common
reasons for failure to ascertain
aetiologic diagnosis of infections.

Consequently, the lab must adhere to a

strict policy of specimen acceptance &
rejection.
Criteria for Specimen Rejection 2
It does not have proper patient
identification.
It is received in an inappropriate
condition (Dry swab or leaking container or
specimens that are obviously
contaminated).
Discrepancy between identity on
specimen & request form.
Specimens unsuitable for request
(anaerobic request from aerobic
transport), i.e., An unrewarding
investigation is requested.
Prolonged transport.
Criteria for Specimen Rejection 3
A single small volume specimen submitted
for multiple requests (e.g. aerobes,
anaerobes, fungus & TB).
Multiple specimens on the same day for the
same test request.
Sputum specimen consisting of saliva only
(sputum is rejected if > 10 SEC/LPF are
seen).
Urine held at room temperature for >2 hrs.
Urinary catheter tip for culture.
Any specimen received in formalin.
 Handling Specimens

When received in the lab, priority is given to

most critical specimens (blood, CSF, sterile
body fluids, tissues).

After microscopic examination of each

specimen (to ensure appropriateness &
adequate volume for needed tests), some
specimens undergo initial processing before
culture.
Some Specimen processing

Homogenization (grinding) of tissues.

Concentration by centrifugation of large

volumes of sterile fluids (ascitic or pleural
fluid).

Decontamination to remove commensal

flora which may interfere with recovery of
fastidious pathogenic organisms (e.g.
mycobacteria, Legionella).
Specimens & Infection Control

Please be considerate to lab staff!!

Label hazardous specimens

Don't send specimens to the lab

without proper packing
Leaking or blood-stained specimens
are not acceptable!!!
 Factors limiting usefulness of
bacteriological investigations
Wrong sample
e.g. saliva instead of sputum
Delay in transport / inappropriate
storage
e.g. CSF
Overgrowth by contaminants
e.g. blood cultures
Insufficient sample / sampling error
e.g. in mycobacterial disease
Patient has received antibiotics
Diagnosis of Bacterial Infection
1.microscopy unstained (for motility) or
stained (e.g. Gram stain)

Stain Decolorize Counterstain

2.culture identification by biochemical or

serological tests on pure growth
from single colony

on plates or in broth

& sensitivities by disc diffusion

methods,
breakpoints or
MICs 3. Serodiagnosis 4. DNA technologies
 Microscopy
Unstained preparations

“Wet preparation”: for motility

Dark-ground illumination for syphilis

 Microscopy
Stained preparations

Gram-stain

Acid-fast stain
Ziehl-Neelsen

Fluorescence
Direct, e.g. auramine
Immunofluorescence
 Culture of Bacteria

Solid media
Agar plates
For Identification
For Enumeration
Slopes
For safe long-term storage of culture,
e.g. Lowenstein-Jensen medium for TB

Liquid media (broth)

For enrichment or sensitivity
Advantages of Solid Media

Isolation of single colonies

get bacterium in pure culture

Identify bacteria by colonial

morphology

Quantification by colony-
forming units (CFU)
Molecular
Methods
Nucleic Acid Probes
 Probes are:

1- Specific,
2- single stranded,
3- short sequence,
4- complementary,
5- labeled.
prepared synthetically by DNA-
synthesizing machines for the
detection of target (e.g. microbial)
DNA by hybridization technique.
 Inducible with
DNA Probes Labeled probe
Spot solution of
Denatured DNA
Onto membrane DNA strands
Bound to Wash away
membrane Unhybridized
surface probe
Applications of DNA Probes
Detection of pathogenic organisms,
by nucleic acid probe technology is
likely to be most useful when:
» Difficult or costly to culture.

» Slow or non-growing.

» Present in low concentration in

clinical samples.

» Hazardous to propagate in the

laboratory.
 PCR

Definition:
It’s an enzymatic technique for the
amplification of specific DNA
sequences in vitro (amplification
technique for a particular region of target
DNA selectively in vitro).
Primers are:

1- Specific,
2- single stranded,
3- short sequence,
4- complementary to the flanks of
desired region of target DNA.
5- NOT labeled.
PCR is:

Sensitive (1 copy → million copies).

Specific (due to the use of specific primers).

Rapid, efficient, reproducible.

PCR Master Mix
Buffered cocktail containing:

Extracted DNA
dNTPs
Taq DNA polymerase (acts on DNA target)
Taq buffer
Primers
Distilled water

± Mineral oil
PCR Steps

Denaturation: at 94-95oC

Annealing: at 30-65oC (for

maximum specificity use 62oC-
72oC).

Extension: at 65-75oC (72oC is

optimal for Taq DNA polymerase).
 PCR Steps

1. Denaturation

2. Primer annealing

3. Primer extension
Detection of the Amplified
PCR Product

Gel electrophoresis

Hybridization with specific labelled

probes.

Nucleic acid sequencing.

 Agarose Gel
Electrophoresis
Hybridization with
specific labelled
probes
Automated DNA Sequencer
DNA Sequences
 Disadvantages of Molecular
Diagnostics

More expensive than manual testing

Complicated equipment & expensive reagents
Needs good quality control
Needs talented personnel
 Reverse Transcriptase PCR
(RT-PCR)
RT-PCR is used for the amplification
of RNA target (e.g. HCV).

It requires 2 steps before starting

PCR:
Formation of ssDNA complementary to
the target RNA by the enzyme reverse
transcriptase.

Formation of dsDNA by the enzyme

DNA polymerase.
 RT-PCR

ssRNA ssDNA dsDNA

Reverse DNA
transcriptase
Polymerase

Continue
PCR
Technique
 Real-Time PCR

It’s a sensitive and a specific

quantitative test which ensures
rapid detection.

Faster analysis: approximately two

hours.
LightCycler® Instrument
THANK YOU