MEDICAL MICROBIOLOGY

Course for medical students

Associate Professor L. Yocheva, Ph.D.

Department of Biology, Medical Genetics and

Microbiology
Medical Faculty
SU “St. Kliment Ohridski”

Email: [email protected]
 Lecture 8
Antimicrobial Chemotherapy
(Part two)
1. Major classes of antibiotics. Mechanisms of
action and major representative
˗ Characteristics of the antibiotics inhibiting
the protein synthesis
˗ Characteristics of the antibiotics inhibiting
the nucleic acid synthesis
2. Resistance of bacteria to antimicrobial
agents (drug resistance). Mechanisms of
resistance. Antibiotic policy.
 Key words

• Aminoglycosides
• Tetracyclines
• Macrolides
• Oxazolidinones
• Lincosamides
• Streptogramins
• Quinolones
• Sulfonamides
• Trimethoprim
• Rifamycins
• Metronidazole
 Major Antibacterial Agents

• Antibiotics can be classified according to

their mechanism of action as:

1. Inhibitors of cell wall synthesis

2. Inhibitors of cell membrane function

3. Inhibitors of protein synthesis

4. Inhibitors of nucleic acid synthesis

INHIBITORS OF THE
PROTEIN SYNTHESIS
 Main Features
• Target – ribosomes
• In order to reach the ribosomes they have
to pass trough:
– the outer membrane in Gram-negative
or cell wall in Gram-positive bacteria
– the cytoplasmic membrane of both
• They inhibit the synthesis of proteins
affecting the function of 30S or 50S
subunits
• They are bactericidal or bacteriostatic
 Agents Inhibiting Protein Synthesis

• Аminoglycosides
• Tetracyclines
• Glycylcyclines
• Оxazolidinones
• Chloramphenicol
• Macrolides
• Clindamycin
• Streptogramines
Mechanism of Action of the Different Agents
 Aminoglycosides

• Most used are:

– Tobramycin
– Gentamicin
– Amikacin (broadest spectrum)
• Others:
– Neomycin
– Kanamycin
– Streptomycin (treatment of M. tuberculosis)
Chemical Structure of Aminoglycosides

• Aminocyclitol ring
• Aminosugars connected to the ring by
glycoside bonds
 Producers of Aminoglycosides

Streptomyces griseus – producer of

streptomycin (SEM)

Producers of aminoglycosides are

mainly representatives of the order Мicromonospora purpurea –
Actinomycetales producer of gentamicin
 Mechanism of Action

• After passing through the outer and the inner

membrane antibiotics bind irreversibly to the
30S ribosomal subunit

• This leads to:

– production of aberrant proteins
– interruption of protein synthesis

• Bactericidal effect
 Mechanism of Action

• Transport across the membrane is oxygen-

dependent (using the transmembrane
potential of the respiratory chains)

• The result is a resistance of all anaerobic

bacteria to aminoglycosides. (Anaerobic
bacteria do not possess the enzymes of the
respiratory chains)
 Mechanism of Action

• Antibiotics of this group can not be absorbed

from the gastro-intestinal tract

• They also can not cross the blood-brain

barrier and do not pass into the CSF

• In order to ensure a desired blood level a

intravenous application is used
 Spectrum of Activity
• Active mostly against Gram-negative bacteria
• Narrow spectrum of activity and bactericidal effect
• Streptococci and enterococci (Gram-positive) are
resistant - aminoglycosides can not penetrate the
cell wall
• In these cases two groups of antibiotics are used as
combination:
1. Inhibitors of the cell wall synthesis (β-lactams or
glycopeptides)
2. Aminoglycosides
• Examples of some synergistic combinations are
ampicillin and gentamycin or vancomycin and
gentamycin
 Side Effects and Risk Factors in
Treatment with Aminoglycosides

• Ototoxic and nephrotoxic effects

• Toxicity - a major problem in clinical use

• Age, over dosage, impaired renal function,

usage for more than 5 days, etc. are risk
factors in treatment
 Resistance to Aminoglycosides
• Different ways of the development of the
resistance exist
• The most common mechanism of resistance is
production of several groups of enzymes that
can modify the molecule of aminoglycosides
• This is accomplished by the action of
enzymes:
˗ phosphotransferases
˗ adenyltransferases
˗ acetyltransferases
 Tetracyclines

• Tetracyclin, doxycycline, minocycline

• In general bacteriostatic antibiotics
• Inhibit protein synthesis by binding to the 30S
ribosomal subunits, thus blocking the binding
of aminoacyl-transfer RNA (tRNA) to the
ribosome (prevent introduction of new amino
acids to the peptide chain)
• Broad spectrum of activity - against Gram-
positive and some Gram-negative bacteria
• Effective in the treatment of infections caused
by Mycoplasma, Chlamydia, and Rickettsia
Tetracyclines
 Side Effects

• Strong affinity for calcium

• Deposition in the teeth and the bony
structures during the fetus stage and the
first 6 years of life
• Yellowish-brown discoloration of the teeth
• Considerations:
- should not be given to children < 8 yrs or
to women in last trimester of pregnancy -
irreversibly discolors permanent teeth
- tetracycline during 1st trimester of
pregnancy can cause birth defects
 Resistance to Tetracyclines

• The most common cause of resistance is

active efflux of the antibiotic out of the cell

• Another mechanisms:
– Decreased penetration of the antibiotic
into the bacterial cell
– Alteration of the ribosomal target site
– Enzymatic modification of the antibiotic
 Glycylcyclines*

• Glycylcyclines - a new class of

antibiotics synthetic analogues of
tetracyclines
• Tigecycline* (1999 г.) – only one agent
available for use in the group
• Broad spectrum against Gram-positive,
Gram-negative and anaerobic bacteria
• For the treatment of infections, caused by
MRSA, MDR S. pneumoniae, enterococci,
Acinetobacter, Bacteroides fragilis
 Tigecycline – Mechanisms of Action

• Tigecycline binds to the 30S ribosomal

subunit, inhibiting protein synthesis in a
fashion similar to that of the
aminoglycosides, macrolides and
linezolid

• It is generally bacteriostatic except for

Streptococcus pneumoniae and
Legionella spp. where it is bactericidal
 Oxazolidinones*
• Linezolid – active against Gram-positive
bacteria (a narrow-spectrum class of
antibiotics)
• Binds to the 50S ribosomal subunit and
blocks initiation of protein synthesis
• Linezolid is active against all staphylococci,
streptococci, and enterococci (including
those strains resistant to penicillins,
vancomycin, and aminoglycosides)
• The use of linezolid is generally reserved for
the difficult to treat multidrug-resistant
bacteria
 Chloramphenicol

• Chloramphenicol
• Binding to the 50S ribosomal subunit
• Broad spectrum, similar to that of tetracyclines
• Bacteriostatic
• Penetrates well into the cerebrospinal fluid
(CSF)
• Side effects:
– aplastic anemia (the most serious side effect)
– bone marrow suppression
• Discontinued use in many countries
• Main application – meningitis or typhoid fever, if
no other option for treatment
 Macrolides
• Erythromycin – basic macrolide antibiotic
• Modification of the macrolide structure leads to
the development of Azithromycin,
Clarithromycin, and Roxithromycin
• Bind to the ribosomal 50S subunit and
block polypeptide elongation
• Generally bacteriostatic, they may be
bactericidal at high doses
• Broad spectrum of activity
˗ Macrolides mainly affect Gram-positive cocci,
some Gram-negative bacteria, Mycoplasma, and
intracellular pathogens such as Chlamydia and
Legionella
Macrolides

• 14-, 15-,16-, 17-, 18-

membered lactone ring
• Erythromycin - basic
antibiotic; producer
Streptomyces erythreus
 Clinical Use of Macrolides

• To treat infections caused by:

˗ Mycoplasma, Legionella, and Chlamydia
species
˗ Gram-positive bacteria (especially if the
patients are allergic to penicillin)
• Most Gram-negative bacteria are resistant to the
macrolides, but Campylobacter and
Helicobacter are susceptible

• Azithromycin and clarithromycin have also

been used to treat infections caused by
mycobacteria
 Side Effects of Microlides

• Usually mild:
– nausea
– vomiting
– diarrhea
 Fidaxomicin*

• A new representative of macrolides

• 18-membered lactone ring
• It inhibits RNA polymerase at a different
site than rifampicin
• Bactericidal effect
• Spectrum - acts on Gram-positive
anaerobes
• It is mainly used to treat
pseudomembranous colitis caused by
Clostridium difficile
 Clindamycin
• Clindamycin (semisynthetic derivative
Lincomycin)

• Like chloramphenicol and the macrolides,

Clindamycin blocks protein elongation by
binding to the 50S ribosome subunit

• High activity against staphylococci and

anaerobic Gram-negative rods

• Generally inactive against aerobic Gram-

negative bacteria
 Clinical Use

• Clindamycin is used primarily to treat

anaerobic infections - dental infections
and infections of the respiratory tract,
skin, and soft tissue, and peritonitis

• It is also used to treat bone and joint

S. aureus infections
 Streptogramins
• Streptogramin A and Streptogramin B are used
in combination
• Act synergistically to inhibit protein synthesis
• Combination of both is
Quinopristin/Dalfopristin (trade name
Synercid)
• Gram-positive spectrum of activity
• Active against staphylococci,
streptococci, Enterococcus faecium
• Use is restricted for vancomycin-resistant
E. faecium
 INHIBITORS OF
NUCLEIC ACID SYNTHESIS
 Antibiotics Inhibiting the Synthesis
of Nucleic Acids

• Quinolones
• Rifampin
• Metronidazole
• Antimetabolites
 Quinolones

• Chemotherapeutics
• Nalidixic acid is the first quinolone to be
developed (narrow spectrum of activity)
• Manipulations of the basic molecule of nalidix
acid (fluorination), have led to the synthesis
of fluoroquinolones that have enhanced
antibacterial efficacy
• Drugs inhibit bacterial DNA topoisomerase
type II (gyrase) or topoisomerase type IV
 Fluoroquinolones

• Broad spectrum of activity

• Bactericidal activity
• Therapeutic applications (respiratory, urogenital
tract infections, gastrointestinal infections, etc.)
• Their use in children and pregnant is not
recommended
• Fluoroquinolones are not absolutely
contraindicated for these groups and for certain
severe infections where other antibiotics are not
an option, their use can be justified
Structure of Fluoroquinolones
 Fluoroquinolones Positives

• Can be taken orally with good absorption

• Deep penetration into tissues
• Can enter into cells of the host
• Some possess a very broad spectrum of
activity
• Well tolerated from the macroorganism
Classification of Quinolones
 Spectrum of Activity of Fluoroquinolones

• They are active against several clinically

important aerobic Gram-negative bacteria
(like those belonging to family
Еnterobacteriaceae (E. coli and others),
Pseudomonas aeruginosa, or H. influenzae
• They are also active against Gram-positive
cocci like S. pneumoniae, S. aureus and β-
haemolytic streptococci
• C. pneumoniae, Mycoplasma pneumoniae,
Legionella pneumophila are also susceptible
• Anaerobic cover is limited (moxifloxacin only)
 Rifampin

• Rifampin (trade name Tubocin)

• Blocks bacterial DNA-dependent RNA
polymerase and inhibits the synthesis of mRNA
• Broad spectrum of activity, bactericidal effect
• Activity against staphylococci, streptococci, M.
tuberculosis
• For the treatment of tuberculosis
• Chromosomal mutations can lead to resistance
• Recommended use is combination with one or
more other antibiotics in order to prevent rapid
development of antimicrobial resistance
 Antimetabolites - Sulfonamides

• The first synthetic antibacterial agents for

general use in human medicine
• In recent years - less use:
– due to the availability of new and more active
antibiotics
– due to increased resistance
• Prevent the synthesis of folic acid by
competing with p-aminobenzoic acid
• Bacteriostatic effect
• Effective against a broad range of Gram-
positive, Gram-negative bacteria and
some protozoa
 Antimetabolites –
Trimethoprim and Biseptol
• Trimethoprim is an antimetabolite that interferes
with folic acid metabolism (inhibition of synthesis of
the acid)
• Used in combination with sulfamethoxazole
(Co-trimoxazole, Biseptol)
• The combination is synergistic and inhibit
synthesis of folic acid
• Bactericidal effect
• Against Gram-positive and Gram-negative bacteria
(broad spectrum)
• Drug of choice for the treatment of acute and
chronic urinary or pulmonary infections
 Mechanism of Action of
Sulfonamides and Trimethoprim
 Metronidazole

• Metronidazole is a chemotherapeutic (azole

derivative)
• Produces a toxic effect on DNA (disrupts
the DNA molecule)
• Antiprotozoal drug
• Active against anaerobes
• Can be used in combination with another
antibiotics in cases of mixed infections
RESISTANCE OF BACTERIA TO
ANTIMICROBIAL AGENTS
 Bacterial Resistance
• Bacterial resistance is the capacity of bacteria to
withstand the effects of antibiotics that are intended
to kill or suppress them
• Only chance for survival of bacteria is the
development of a resistance to antibiotics
• Bacteria have broad spectrum of possibilities to do
this
• Bacterial resistance is present in all parts of the
world with new resistance mechanisms constantly
emerging and spreading globally
• Today highly antimicrobial-resistant microbes exist:
– in the health care units
– in the community
Bacteria Have to Evolve
 Selective Pressure of Antibiotics
Before selection
• Antibiotics select resistant
bacteria by eliminating
susceptible bacteria
• Selected resistant bacteria
After selection
multiply and transfer their
resistant genes to the next
generation
Final population
• This is known as selective
pressure
• Antibiotic resistance is a direct Level of resistance
consequence of antibiotic use
 Two Types of Bacterial Resistance

• Antibiotic resistance can be

classified into two main categories:
– intrinsic resistance
– acquired resistance
 Intrinsic Resistance
• Non responsiveness to an antibiotic is due to:
– absence of target
– impermeability to the drug
– lack of enzymes to convert the pro-drug to the
active form
• Present in all strains of a bacterial species
• Stable and well known
• Examples of intrinsic resistance:
– Streptococci or anaerobes are resistant to
aminoglycosides
– Enterococci are resistant to cephalosporines
 Acquired Resistance

• Found in a certain part of the strains of a

species
• Vary geographically and with time
• It is transmitted within a species, but also
between different species
• Unpredictable
• May be lost if there is no selective
antibiotic pressure
• Check the sensitivity with in vitro tests is
required
 Genetic Origin of Drug Resistance

• Most drug resistant microbes emerge as a

result of genetic change and subsequent
selection process

• Mechanisms of genetic resistance are:

– chromosomal resistance
– extrachromosomal resistance (through gene
transfer)
 Chromosomal Resistance

• Develops as a result of spontaneous mutations

in a bacterial chromosome
• More commonly found are low
frequency mutations of 10-12 to 10-7
• However, chromosomal mutants resistant to
Rifampin occurs with high frequency (10-7 to
10-5)
• As a result treatment with rifampin as the sole
drug often fails.
 Extrachromosomal Resistance
• Bacteria often contain extrachromosomal genetic
elements that can be transferred by transduction
(bacteriophages) or by conjugation (plasmids)
• Some plasmids carry genes for antibiotic resistance
to one and often to several antimicrobial drugs
– Some bacteria have more than one plasmid
– Some times transfer is possible between different
species
• Transposons can also carry genes for antibiotic
resistance and can transfer them
• Transformation is another possibility of bacteria to
accept external genes of resistance
Genetic Mechanisms of Antibiotic
Resistance
 Development of Extrachromosomal
Resistance of S. aureus

• The first step in the development of resistance

of S. aureus is to penicillin, followed by
resistance to methicillin (MRSA)
– Infections caused with MRSA have been associated
with high morbidity and mortality rates
– MRSA are the strains of S. aureus that are resistant to
all antibiotics from the antistaphylococcal penicillins
– Also, MRSA offers cross-resistance to all currently
licensed β-lactam antibiotics
• The treatment is possible with vancomycin
(glycopeptide antibiotic)
Vancomycin-resistant S. aureus (VRSA) as an
Example of Next Step in Resistance Development

• Emergence of strains MRSA, resistant to

Vancomycin in recent years is highly problematic
• It has been suggested that patients at risk for
VRSA are co-infected or co-colonized with
vancomycin-resistant enterococci (VRE) and
MRSA
• This enables transfer of vanA gene (located on
plasmid) from VRE to MRSA leading to VRSA
strain
Transfer of Gene for Vancomycin Resistance between
Different Species

• Blue and red wavy lines - chromosomal

DNA of Enterococcus and S. aureus,
respectively
• Blue circle - enterococcal plasmid with a
broad host range of transfer; red circle -
resident staphylococcal plasmid
• Tn1546 is a transposon with gene,
encoding resistance to vancomycin
• GRE = glycopeptide-resistant
enterococci (resistant to vincomycin)
 VRSA Treatment

• Infections caused by VRSA strains lead to

the vancomycin treatment failure

• Newer antimicrobial agents (Linezolid,

Daptomycin, Tigecycline, etc.) for
drug-resistant Gram-positive
pathogens are treatment options
 VRSA Emergence

• Now VRSA strains exist in very low level

(single cases), but their presence is
alarming
• In 2002, the first clinical isolate of
vancomycin-resistant S. aureus (VRSA) from
a patient in Michigan was reported
• By the end of June 2013, VRSA isolates
have been reported for the first time in
Europe and in Latin America
 Main Biochemical Mechanisms of
Antibiotic Resistance

• Impermeability of the bacterial cell to the

drug
• Increased efflux
• Alteration of target or circumvention of the
target
• Enzyme inactivation (the most common
mechanism)
Main Biochemical Mechanisms of
Antibiotic Resistance
 Reduced Access to the Target
(Impermeability)
• Impermeability through the outer membranes of Gram-
negative bacteria
• Loss of outer membrane proteins (porins) or altered
membrane transport systems

• Example: P. aeruginosa –
resistance to Imipenem
• Innate resistance or more often
acquired through mutations
 Efflux Systems
• Found in many types of bacteria
• Block the action of quinolones, tetracyclines and
others
• The drugs may not reach optimal intracellular
concentration which is due to active efflux of the
drug
• Efflux mechanism: expulsion of
the molecule by active transport
 Absence or Modification of the Target or
Target with Low Affinity

• No target - Mycoplasma (resistance to

beta-lactams)
• Modification of Penicillin-Binding
Proteins (PBPs):
– MRSA (resistance to group methicillin, oxacillin,
etc.)
– S. pneumoniae (resistance to penicillin)
• Low-affinity PBPs - Enterococcus
(resistance to cephalosporins)
Modification of the Antimicrobial Target
Resulting in Reduced Binding
 Enzymatic Inactivation of the Antibiotic
• The most common mechanism
• Bacteria produce two types of enzymes:
– Hydrolyzing enzymes (against
beta-lactam antibiotics)
– Modifying enzymes
(against
aminoglycosides)
 Enzymes β-Lactamases

• More than 200 different β-lactamases

have been described

• They are hydrolases that destroy the beta-

lactam ring of antibiotics and they loss
their antibacterial activity

• Some enzymes are specific for penicillins

(i.e., penicillinases), or for cephalosporins
(i.e., cephalosporinases), or for
carbapenems (i.e., carbapenemases)
Enzymes Extended-Spectrum β-Lactamases
• Some β-lactamases have activity against both all
penicillins and cephalosporins as a result of simple
point mutations

• These β-lactamases are referred to as extended-

spectrum β-lactamases (ESBLs)

• ESBLs are most often associated with E. coli and

Klebsiella but other pathogens can produce them

• ESBLs are problematic, because most are encoded

on plasmids that can be transferred from organism to
organism very fast
 Multiple Resistance and Cross-
Resistance

• Resistance to only one antibiotic is

possible
• Multiple drug resistance (MDR) –
resistance to more than one antibiotic
• Cross-resistance - resistance to one drug
means resistance to another
• On the basis of:
– similarity in the structure
– catching similar places
 Types of Resistance According to
Distribution in the Society
• Resistance in bacteria that cause
community-acquired infections
• Resistance in bacteria causing infections in
hospitals (hospital acquired or nosocomial
infections)
• Resistance in “Asymptomatic colonization”
– Patients may harbor the resistant bacteria in
their intestinal tracts as normal human flora
– It does not make them sick, but they are infectious
for others
– Eradication of this normal bacteria will harm the
patient
 Transfer of Resistant Strains From
Animals to Humans

• Antibiotics are also used in veterinary

medicine and animal agriculture
• Antibiotic use in animals has led to the
emergence of resistant bacteria
• Sometimes these resistant bacteria can be
transferred from animals to humans by direct
contact or by handling and/or consuming
contaminated food
 Antibiotics in Animal Feed as Growth
Factor

• Use of antibiotics as growth promoters in

animal feeds has been permitted in the
member states of the European Union during
the last 50 years

• However, concerns about development of

antimicrobial resistance and about transference
of antibiotic resistance genes from animal to
human microbiota led to the withdrawal of the
approval for antibiotics as growth promoters in
the European Union since January 1, 2006
 Antibiotic Resistance - a Public Health
Problem of Increasing Magnitude

• Increase in resistance (especially multidrug

resistance) of bacteria

• Decline in the production of new antibiotics

• The situation of “Bad bugs, no drugs” is

problematic today
 Monitoring of Antibiotic Resistance and
Consumption

• Antibiotic resistance needs monitoring

(surveillance):
– of antimicrobial resistance
– antibiotic consumption
• The prevalence of resistance may vary
geographically and with time for selected
species
• Monitoring programs exist at local, national
and international level
 Antibiotic Policies

1.Restrictive policies:
– Use antibiotic only when necessary.
Inappropriate and excessive use of
antibiotics is a major factor contributing to
antibiotic resistance
– Restriction of self-medication. This is an
important public health problem throughout
the world, since it is a fairly common practice
– Reducing the spread of antimicrobial
resistant microorganisms in hospitals,
institutions, communities and agriculture
 Antibiotic Policies (cont.)

2. Rotational strategy or removing the

selective pressure:
– Periodic changes of antibiotics used in
treatment might also help to avoid the
emergence of resistant strains by altering
the selective pressures
– Mutation which has induced resistance to an
antibiotic can be affected by another mutation
which removes the resistance
 Conclusion

• Antibiotic resistance is a direct

consequence of antibiotic use

• Both continue to escalate despite many

calls for moderation of antibiotic use in the
hospitals and in the community
 High Priority Antibiotic Resistant
Infections

• Multi-drug resistant Gram-negative bacterial

infections (Klebsiella, E. coli, Enterobacter,
Pseudomonas, Acinetobacter, etc.)
• MRSA infections
• Enterococcal infections
• Tuberculosis
• Multi-drug resistant Pneumococcal
infections
• Gonorrhea
 New Antibiotics for Resistant Bacteria

• Daptomycin
• Linezolid
• Tigecycline
• Quinopristin/Dalfopristin
• Ceftobiprole - fifth-generation cephalosporin
active against a broad range of Gram-positive and
Gram-negative pathogens
 Antimicrobial Susceptibility Testing
• Antimicrobial susceptibility testing is used to
assess the activity of an antimicrobial agent on
a bacteria
• It is performed on bacterial strain, isolated
from individual patients, using different in
vitro methods
• The two main objectives to examinate
antibacterial resistance are:
– individual (to guide the selection and modification of
antimicrobial therapy)
– epidemiological (surveillance of antimicrobial
resistance and its evolution)
 Examination of Antibiotic Resistance –
in vitro Methods

• Serial dilution method for determination

the minimal inhibitory concentration
(MIC) of antibiotics
• Kirby-Bauer disk diffusion method
• E-test for determination of susceptibility and
MIC (gradient diffusion method)
• Detection of genes encoding resistance by
molecular biological methods
 Antimicrobials: Classification Criteria of
Bacterial Strains
• Susceptible (S) - the infection caused by that
strain is highly likely to respond to treatment
with an agent (agent is suitable for treatment)
• Intermediate (I) - the infection is likely to
respond to higher dosing regimens (agent is
not suitable for treatment)
• Resistant (R) - the infection caused by that
strain is unlikely to respond to treatment with
any regimen of the antimicrobial agent (agent
is not suitable for treatment)
 Minimal Inhibitory Concentration (MIC)

• The basic measurement of the susceptibility of

a microorganism to an antimicrobial
• The MIC is defined as the lowest concentration
of a range of antibiotic dilutions that inhibits
visible growth of bacteria within a defined period
of time
• Each microbial species will have an unique MIC
distribution, i.e. not all members of a species will
have exactly the same wild-type MIC
• The strain is said to be Susceptible (S),
Intermediate (I) or Resistant (R)
Minimal Inhibitory Concentration Test

128 64 32 16 8 4 2 1 0,5 Control

µg/ml
 Minimal Inhibitory Concentration Test
Description

• Originally performed in test tubes (macrobroth

dilution)
• Two-fold dilutions of the antimicrobial are made
in a nutrient broth and then each well is
inoculated with a standardized number of
microorganisms
• As defined by standardized methodology, the
plates are incubated at a defined temperature
for a defined period of time
 Minimal Inhibitory Concentration Test
Description

• The lowest concentration of antimicrobial

with no visible growth is the minimum
inhibitory concentration (MIC)
• Modern technology has allowed
miniaturization and automation of broth
dilution methodology which has reduced the
time to results
• In general, for routine bacterial susceptibility
testing, results are available in several hours
to a day
 The Disk Diffusion Method

• The disk diffusion method involves placing

antimicrobial impregnated disks on an
agar surface that has been inoculated with
a standardized suspension of
microorganism

• After a defined period of incubation, the zone of

no growth around each disk is measured and
interpreted based on published interpretive
criteria, which have been developed by
comparison with MIC methods
Kirby-Bauer Disk Diffusion Method
 E-test (Gradient Diffusion Method)

• Antimicrobial gradient diffusion is another

form in which a concentration gradient is
established in an agar medium onto which a
standardized suspension of microorganism
is inoculate

• This method has the capability to generate a

MIC value across an extensive range of
dilutions for a wide range of organism/
antimicrobial combinations
 E-test

Bacterial growth measured according to an antibiotic

concentration gradient, 18 hrs.
E-test

S. pneumoniae
Penicillin G
MIC = 3 µg/ml