General microbiology

MLAB 213

antimicrobial agents (part 2)

Medical laboratory sciences

 Mechanisms of resistance to antibiotics
Antibiotic resistant microorganisms may:

- lack the structure targeted by the antibiotic (ex: Mycoplasmas lack cell walls,
hence resistant to penicillins)

- be impermeable to the antibiotic (ex: most Gram-ve’s are impermeable to

Penicillin G)

- modify the antibiotic into an inactive form (ex: staphylococci contain B-

lactamases that break penicillin)

- modify the target of the drug

- develop a resistant biochemical pathway (Ex: many pathogens develop

resistance to sulfonamides by modifying their metabolism in a way that
enable them to take up folic acid from the environment)

- pump the drug out of the cell (tetracyclines, chloramphenicol)

Mechanisms of Resistance:

1. Alteration in Membrane Permeability

2. Alteration in Antibiotic Binding Site
3. Enzyme Production
4. Bypass of Metabolic Process
5. Efflux Pump
Mechanisms by which bacteria become resistant to antimicrobial agents
 Alteration in Membrane Permeability

• In Gram -ve bacteria, the first

line of resistance against an
antibiotic is the outer
membrane.

• The outer membranes of Gram

-ve bacteria contain proteins
called “Porins” that regulate
the passage of various
substances in & out of the
bacterial cells.
 Alteration in Membrane Permeability May Result From:

• Narrow Porins –
Reducing antibiotic entry
eg.E.coli with Ceph. & B-
Lactams.

• Decrease number of
porins – eg. Quinolones
& Carbapenem resistant
Pseudomonas &
Acinetobacter sp.
 Alteration in Antibiotic Binding Site

• Chromosomal mutations alter the target sites for

antibiotics so they can no longer bind to those binding sites
& exert their effect, thereby, conferring resistance on the
bacteria.

Examples:
• Alteration to Penicillin Binding Proteins (PBPs), account for
the resistance of the Beta-Lactam antibiotics specifically for
Gram +ve bacteria.
Staphylococcus aureus
Enteroccus (Van operon)
• VanS is a membrane-associated sensor (of vancomycin) that controls the level of phosphorylation of VanR.
• VanR is a transcriptional activator of the operon encoding VanH, VanA and VanX.
• VanH is a dehydrogenase that reduces pyruvate to D-Lac
• VanA is a ligase that catalyses the formation of an ester bond between D-Ala and D-Lac
• VanX is a dipeptidase that hydrolyses the normal peptidoglycan component D-Ala-D-Ala
• VanY is a D,D-carboxypeptidase that hydrolyses the terminal D-Ala residue of late peptidoglycan precursors
 Enzyme Production

• Production of Beta-Lactamase enzymes is the most common

form of resistance to beta-lactam antibiotics.

• Beta-lactamases are capable of hydrolyzing the beta-lactam ring

responsible for the antimicrobial action of beta-lactam
antibiotics.

• Beta-Lactamase resistance is an important & growing problem.

Beta lactamases (example 1)
 Extended spectrum beta lactamase

• Extended-spectrum beta-lactamases (ESBLs) are mutant enzymes with a

broader range of activity than their parent molecules

• They:
– Hydrolyze 3rd and 4th gen cephalosporins and aztreonam
– Should report it as "resistant" to all penicillins, cephalosporins, and
aztreonam

– Remain susceptible to beta-lactamase inhibitors

Aminoglycosidases (example 2)

• The phosphotransferases (APHs)

• The adenyltransferases (ANTs)
• The acetyltransferases (AACs)
 Bypass of Metabolic Process

• Antibiotics that target a specific metabolic event become

ineffective if the bacterium can carry out the reaction via a
different pathway.

• Eg. Trimethoprim-sulfamethoxazole inhibits folic acid synthesis,

but resistance has developed in gram-ve organisms such as
E.coli, N.gonorrhoeae & N.meningitidis.
 Efflux pump
• Antibiotic enters the bacterial cell & is pumped out inactive

Efflux pump genes and proteins are present in both

antibiotic-susceptible and antibiotic-resistant bacteria.

Pumps may be specific for one substrate or may

transport a range of structurally dissimilar compounds
(including antibiotics of multiple classes); such pumps can
be associated with multiple drug (antibiotic) resistance
(MDR)
Example of pseudomonas aeruginosa resistance to beta lactams and
fluoroquinolones
 Slowing the emergence and spread of antimicrobial resistance

1. Responsibilities of Physicians: must work to identify microbe and

prescribe suitable antimicrobials, must educate patients

2. Responsibilities of Patients: need to carefully follow instructions

3. Educate Public: must understand appropriateness and limitations

of antibiotics ; antibiotics not effective against viruses

4. Global Impacts: organism that is resistant can quickly travel to

another country in some countries antibiotics available on non-
prescription basis
 UNDESIRABLE EFFECTS OF
ANTIMICROBIAL AGENTS

• Selection of resistant organisms

• The patient may become allergic to the agent

• Many antimicrobial agents are toxic to humans

• With prolonged use, broad spectrum antibiotics may

destroy the normal flora of the mouth, intestine, or
vagina.
 Did antibiotic resistance emerge following the use of
antibiotics by man ??

- it seems that R plasmids existed in natural microbial populations

before antibiotics were discovered

however, inappropriate extensive use of antimicrobial drugs is the

leading cause of rapid development of drug-resistance in pathogens.
This pressure selects for R plasmid propagation

- Due to this inappropriate and indiscriminate use of antibiotics

almost all pathogenic microorganisms have developed resistance to
some chemotherapeutic agents

Few pathogens even developed resistance to all drugs

 How to avoid emergence of drug resistance

- use antibiotics for treatment of susceptible diseases

- use in large dozes and sufficient time to avoid emergence

of mutants

- use of a combination of drugs

- search for new more effective drugs

 How to detect drug resistance

Antimicrobial Susceptibility testing:

1. Minimum Inhibitory Concentration (MIC)

2. Minimum Bactericidal Concentration (MBC)

3. Disk Diffusion Method

4. Molecular diagnosis
 AST Methods Interpretation

• agar disk diffusion method provides

qualitative interpretive category results of
susceptible, intermediate, and resistant

• microdilution and agar gradient diffusion

methods provide a quantitative result, a
minimum inhibitory concentration
 Disk-diffusion Method (Kirby-Bauer Method):

 The disk-diffusion method (Kirby-Bauer) is more suitable for routine testing in

a clinical laboratory where a large number of isolates are tested for
susceptibility to numerous antibiotics.

 An agar plate is uniformly inoculated with the test organism

 A paper disk impregnated with a fixed concentration of an antibiotic is placed

on the agar surface.

 Growth of the organism and diffusion of the antibiotic commence

simultaneously resulting in a circular zone of inhibition in which the amount
of antibiotic exceeds inhibitory concentrations.

 The diameter of the inhibition zone is a function of the amount of drug in the
disk and susceptibility of the microorganism.
Selection of a Colony to Test

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 MacFarland 0.5 and
Adjusted Test Organism

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 Use of Disk Dispensers
• Advantages
– practical, rapid
– increase reproducibility

• Risks:
– contamination
– reduces personal
judgment skills

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Disk Susceptibility Testing Problems

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Disk Susceptibility Testing Problems
 Measuring Conditions

Calipers Ruler

read with good light, and from the back of the plate
zone size reading is drug specific
magnification may help
millimeters matter

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 This test must be rigorously standardized since zone size is also dependent on:

 inoculums’ size
 medium composition
 temperature of incubation
 excess moisture
 thickness of the agar

 Zone diameter can be correlated with susceptibility as measured by the

dilution method.

 Further correlations using zone diameter allow the designation of an organism

as "susceptible", "intermediate", or "resistant" to concentrations of an
antibiotic which can be attained in the blood or other body fluids of patients
requiring chemotherapy.
 Reference Strains

 E. coli ATCC 25922

 S. aureus ATCC 25923
 P. aeruginosa ATCC 27853

QC organisms must be obtained from reputable source

Use specific QC organisms to test different groups of “drug-

bug” combinations

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• Minimum inhibitory concentration (MIC) is
the lowest concentration of an antimicrobial
that will inhibit the visible growth of a
microorganism after overnight incubation.

• The minimal bacterial concentration (MBC) is

the concentration required to kill a particular
bacterial isolate in vitro and results in a 99.9%
reduction in the initial bacterial density
 1.

Minimum Inhibitory Concentration (MIC) tube test

Principle:

 The tube dilution test is the standard method for determining

levels of resistance to an antibiotic.

 Serial dilutions of the antibiotic are made in a liquid medium

which is inoculated with a standardized number of organisms
and incubated for a prescribed time.

 The lowest concentration of antibiotic preventing appearance of

turbidity is considered to be the minimal inhibitory
concentration (MIC).
 Antimicrobial Gradient Testing
E-test®

Read plates
after
recommended
Incubation
Read MIC
where elipse
intersects
scale
Etest – antimicrobial gradient method

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AST Methods
 Zone of inhibition and
concentration are
proportional
NO growth
growth
growth

4 32
8

The smaller the zone size the higher the MIC

should be to inhibit the growth
 References
 Clinical and Laboratory Standards Institute
 French Society of Microbiology
 British Society for Antimicrobial Chemotherapy

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Example from an excerpt from Reference:
Selection of Drug to Test

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Combination of Drug
Synergistic combination: clavulanic acid- Antagonistic combination:
amoxicillin in the middle surrounded by erythromycin-spiramycin.
cefotaxime, ceftazidime, aztreonam. Staphylococcus aureus with inducible
resistance to the macrolides.
Antifungal agents :

• By interfering with sterol synthesis. When the biosynthesis of ergosterol in a fungal

membrane is interrupted, the membrane becomes excessively permeable, killing the cell ----
ex: Polyenes (amphotericin B), Azoles, (e.g. clotrimazole and miconazole)

• By inhibiting the biosynthesis of glucans, resulting in an incomplete cell wall and cell lysis.----
ex: echinocandins

• By inhibiting nucleic acid synthesis: (ex: flucytosine, an analog of the pyrimidine cytosine).
The fungal cell is able to to convert flucytocine into 5-fluorouracil, which is incorporated into
RNA and eventually disrupts protein synthesis

• By blocking mitosis (e.g., griseofulvin). mode of action is primarily to block microtubule

assembly, which interferes with mitosis and thereby inhibits fungal reproduction

Antiprotozoal drugs are usually quite toxic to the host and work by

• Interfering with DNA and RNA synthesis (e.g., chloroquine, pentamidine, and
quinacrine)

• Interfering with protozoal metabolism (e.g., metronidazole; brand name Flagyl). it acts not
only against parasitic protozoa but also against obligately anaerobic bacteria
 Antihelminthes drugs

Niclosamide: inhibits ATP production under aerobic conditions

Praziquantel is about equally effective for the treatment of tapeworms

it kills worms by altering the permeability of their plasma membranes.
It causes the helminths to undergo muscular spasms and also makes them
susceptible to attack by the immune system
its action exposes surface antigens, which antibodies can then reach.

Mebendazole and albendazole are broad-spectrum antihelminthics

The mode of action of both drugs is to inhibit the formation of microtubules in
the cytoplasm, which interferes with the absorption of nutrients by the
parasite
 Anti-viral agents
• Nucleoside and Nucleotide Analogs (acyclovir)
• Enzyme inhibitors
• interferons: inhibits further spread of the infection