Medical Microbiology Dr. Saleh M.Y.

Phase II MBBS Medical Students

1

Lecture 7-
Medical Microbiology
MBBS-Phase II- ims- MSU
Date: Monday; 18/10/2010

Antibiotics and antimicrobial chemotherapy

Chemotherapy is the treatment of infectious diseases by administration of drugs (antibiotics) which
are lethal or inhibitory to the causative organisms.

History
Rapid development in antimicrobial chemotherapy began in 1935 with the discovery of
(1)
sulphonamides by Domagk. In 1928, Alexander Fleming noted that the product of a mould
Penicillium notatum can inhibit the growth of staphylococci and other organisms. The active part of
this product was successfully purified by Florey et al., in 1940, and named penicillin. This was
followed by the isolation of several antibiotics from the filtrate of Streptomyces e.g. streptomycin,
tetracyclines, chloramphenicol, etc.

An antibiotic is an antimicrobial substance produced by a living microorganism and is active in high

dilutions. In recent years, many of these antibiotics have been chemically synthesized. The term is used
for any antimicrobial chemotherapeutic agent whether naturally produced or synthetic.

Types of action of antimicrobial chemotherapeutics

From their behavior toward bacterial populations antibacterial agents are divided into two classes:

1- Bactericidal drugs: these have a rapid lethal action against the pathogenic agents e.g.
penicillins, cephalosporins, and aminoglycosides.
2- Bacteriostatic drugs: these merely inhibit the division of the pathogenic agents i.e. growth of
organisms e.g. sulphonamides, tetracyclines and chloramphenicol.

(1)
Originally noticed by a French medical student, Ernest Duchesne, in 1896 and rediscovered by Alexander Fleming, a
Scottish bacteriologist in London, discovered penicillin by mistake when he was trying to study staphylococci bacteria in
1928. He was running experiments with the bacteria in his laboratory at London's St. Mary's Hospital, and set a laboratory
dish containing the bacteria near an open window. Upon returning to the experiment, he found that some mold blown in
through the open window onto the dish, contaminating the bacteria. Instead of throwing away his spoiled experiment,
Fleming looked closely at it under his microscope. Surprisingly, he saw not only the mold growing on the staphylococci
bacteria, but a clear zone around the mold. The Penicillium mold, the precursor to penicillin, was dissolving the deadly
staphylococci bacteria.
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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Range of Action of Antimicrobial Chemotherapeutics:

Antibiotics fall into three main categories:
1. Active mainly against gram-positive organisms e.g. penicillin, erythromycin and lincomysin.
2. Active mainly against gram-negative organisms e.g. polymyxin and nalidixic acid.
3. Active against both gram-positive and gram-negative organisms (broad-spectrum activity)
e.g. tetracyclines, chloramphenicol, and ampicillin.

Broad Spectrum Antibiotics

Broad-spectrum antibiotics are antibiotics which are designed to work against a broad spectrum of
bacteria, rather than narrow-spectrum antibiotics, which are only effective against a smaller range of
bacteria. These medications are classically used in cases in which a doctor is not sure about the identity
of a disease-causing organism and wants to provide a patient with medication which will rapidly attack
the infection, rather than waiting for culture results and prescribing a narrow-spectrum antibiotic which
is more targeted in effect.

Some examples of broad-spectrum antibiotics include;

1- penicillin,
2- cephalosporin,
3- tetracycline,
4- ciprofloxacin,
5- levofloxacin.

These drugs work on both gram-negative and gram-positive organisms. When a patient appears to have
a bacterial infection, a broad-spectrum antibiotic is the most likely to provide effective treatment
without knowing which organism is behind the infection. For example, when a patient comes to a
doctor with bronchitis, the doctor may prescribe a general antibiotic medication to treat the infection
without taking a culture.

If an infection persists or it appears unusual in nature, cultures will be done. In a culture, a sample from
the patient is collected and cultured on suitable media in the laboratory to find out which organism is
responsible for the infection. Furthermore, a culture can also be used to test antibiotics in case an
organism is antibiotic-resistant. In this case, the culture is used to find the drug which will be most
effective so that the patient does not have to try several unsuccessful broad-spectrum antibiotics before
finding one which works.
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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One problem with broad-spectrum antibiotics which began to grow in the late 20th century was the
emergence of antibiotic resistance in bacteria. Almost as soon as humans started developing antibiotics,
bacteria started swapping genes which they could use to survive antibiotic therapy. In some cases,
organisms developed resistance to multiple broad-spectrum antibiotics, making treatment of infections
involving these organisms very challenging (complicated). More advanced classes of antibiotics were
developed in response, but bacteria also adapted to address these. A broad-spectrum antibiotic is only
useful as long as it kills most bacteria and organisms which can quickly adapt to resist antibiotics
present a significant challenge.

Mechanism of Action of Antimicrobial Chemotherapeutics

An ideal antimicrobial agent should have selective toxicity i.e. it can kill or inhibit the growth of a
microorganism in concentrations that are not harmful to the cells of the host. Disinfectants e.g. phenol
and antiseptics e.g. alcohol and iodine, destroy bacteria but they are highly toxic to tissue cells and are
unsuitable for use as chemotherapeutic agents.

Thus, the mechanism of action of a chemotherapeutic must depend on the inhibition of a metabolic
channel or a structure that is present in the microbe but not in the host cell. Several mechanisms are
known:

1- Inhibition of cell wall synthesis: Due to its unique structure and function, the bacterial cell
wall is an ideal point of attack by selective toxic agents. Some antibiotics e.g. penicillin,
cephalosporins and vancomycin, interfere with cell wall synthesis and cause bacteriolysis.
2- Inhibition of cytoplasmic membrane function: Some antibiotics cause disruption of the
cytoplasmic membrane and leakage of cellular proteins and nucleotides leading to cell death.
Polymyxins, amphotericin B, and nystatin are examples.
3- Inhibition of protein synthesis: Many antimicrobial chemotherapeutics block protein synthesis
by acting on the 30s or 50s subunits of the bacterial ribosome. Examples are chloramphenicol,
tetracycline, erythromycin and the aminoglycosides e.g. tobramycin, gentamycin and
streptomycin.
4- Inhibition of nucleic acid synthesis: These can act on any of the steps of DNA or RNA
replication e.g. quinolones, trimethoprim, rifampicin, nalidixic acid, novobiocin and
metronidazole.
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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5- Competitive inhibition: in which the chemotherapeutic agent competes with an essential

metabolite for the same enzyme e.g. p-aminobenzoic acid (PABA) is an essential metabolite
for many organisms. They use it as a precursor in folic acid synthesis which is essential for
nucleic acid synthesis. Sulphonamides are structural analogues to PABA so they enter into the
reaction in place of PABA and compete for the active center of the enzyme thus inhibiting folic
acid synthesis.

Mechanisms of Resistance to Antimicrobial Agents

In the treatment of infectious diseases, one of the serious problems commonly faced with, is the
development of bacterial resistance to the antibiotic used. The mechanisms by which the organism
develops resistance may be one of the following:

1- The organism produces enzymes that destroy the drug e.g. production of:
a. -lactamase - that destroys penicillin by penicillin resistant staphylococci
b. Acetyltransferase produced by gram negative bacilli destroys chloramphenicol.
2- The organism changes its permeability to the drug, by modification of protein in the outer cell
membranes, thus impairing its active transport into the cell e.g. resistance to polymyxins.
3- The organism develops an altered receptor site for the drug e.g. resistance to aminoglycosides
is associated with alteration of a specific protein in the 30s subunit of the bacterial ribosome
that serves as a binding site in susceptible organisms.
4- The organism develops an altered metabolic pathway that bypasses the reaction inhibited by the
drug e.g. sulphonamide-resistant bacteria acquire the ability to use preformed folic acid with
no need for extracellular PABA.

Origin of Resistance to Antimicrobial Agents

These mechanisms may be of non genetic or genetic origin:

A- Non genetic Drug Resistance:

i. Metabolic inactivity: Most antimicrobial agents act effectively only on replicating cells.
Non multiplying organisms are phenotypically resistant to drugs. Tubercle bacilli survive
for several years in tissues and their resistance to antituberculous drugs is due in part to their
metabolic inactivity (dormancy).
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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ii. Loss of target structure: L-forms of bacteria are penicillin resistant, having lost their cell
wall which is the structural target site of the drug.

B- Genetic Drug Resistance

i- Plasmid mediated resistance

Resistance (R) factors are a class of plasmids that mediate resistance to one or more antimicrobial
agent. Plasmids frequently carry genes that code for the production of enzymes that inactivate or
destroy antimicrobial agents e.g. p-Iactamase which destroys the p-Iactam ring in penicillin and
cephalosporins. Plasmids may result in epidemic resistance among bacteria by moving from one to the
other by conjugation, transduction, or transformation.

ii- Transposon-mediated resistance

Many transposons(2) carry genes that code for drug resistance. As they move between plasmids and
chromosomes they can transfer this property to bacteria. The process is called transposition.

iii- Chromosomal drug resistance

This develops as a result of spontaneous mutation in a gene that controls susceptibility to an
antimicrobial agent. The most common result of chromosomal mutation is alteration of the receptors
for a drug. For example, streptomycin resistance can result from a mutation in the chromosomal gene
that controls the receptor for streptomycin located in the 30s bacterial ribosome.

(2)
Transposons are sequences of DNA that can move or transpose themselves to new positions within the genome of a
single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create
phenotypically significant mutations and alter the cell's genome size. Barbara McClintock's discovery of these jumping
genes early in her career earned her a Nobel prize in 1983.
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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Complications of Antibacterial Chemotherapy

1- Development of drug resistance
This is one of the most serious complications of chemotherapy. The emergence of resistant mutants is
encouraged by:
a- Inadequate dosage,
b- Prolonged treatment,
c- The presence of a closed focus of infection and
d- The abuse of antibiotics without in vitro susceptibility testing

The problem is more serious when resistant strains develop in the community, e.g. in hospitals it is
common to find that about 90% of strains of Staph. aureus are resistant to penicillin.

2- Drug toxicity
Many of the antibacterial drugs have toxic side effects. This can be due to;
a- Over-dosage,
b- prolonged use or narrow margin of selective toxicity e.g. streptomycin affects the 8th
cranial nerve leading to deafness, chloramphenicol may cause depression of the bone
marrow, the aminoglycosides (e.g. garamycin, netilmycin, tobramycin) are nephrotoxic.
Tetracyclines inhibit growth and development of bones and teeth in the developing fetus
and infants.

3- Super-infection

a- Superinfection may occur by pre-existing resistant strains present in the environment e.g.
penicillin resistant Staph. aureus in hospital infections.
b- Another type of superinfection is due to suppression of normal flora by the antibiotic used
and their replacement with drug-resistant. organisms which cause disease e.g.:
i. Overgrowth of Candida in the vagina causing vaginitis or in the mouth causing
oral thrush.
ii. Prolonged oral chemotherapy leading to suppression of intestinal flora and
overgrowth of staphylococci causing staphylococcal enterocolitis or C. difficile
which causes pseudomembranous colitis.
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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iii. Overgrowth of gram-negative organisms naturally drug-resistant e.g.

pseudomonas, proteus or enterobacter, may account for respiratory tract
superinfection.

4- Hypersensitivity
The drug may act as a hapten, binds to tissue proteins, and stimulates an exaggerated immune response
leading to tissue damage i.e. hypersensitivity. Any type of hypersensitivity reaction can occur with
several antibiotics. The most serious is anaphylactic shock, this may occur with penicillin or
cephalosporins. Milder manifestations may be urticaria, purpural eruptions, skin rash, diarrhoea,
vomiting and' jaundice.

Chemoprophylaxis

Chemoprophylaxis is the use of antimicrobial agents to prevent rather than to treat infectious diseases.
The following are principal conditions for which prophylactic antibiotics are positively indicated:
1- The use of benzathine penicillin G injections every 3-4 weeks to prevent
reinfection with Strept. pyogenes in rheumatic patients.
2- A single large dose of amoxycillin given immediately prior to dental
procedures is recommended for patients with congenital or rheumatic heart
disease to prevent endocarditis.
3- The oral administration of rifampicin 600 mg twice a day for 2 days to
exposed persons during epidemics of meningococcal meningitis.
4- Oral administration of tetracycline to prevent cholera caused by Vibrio
cholerae.
5- Chemoprophylaxis in surgery: little is known about its effectiveness.
However, conditions in which chemoprophylaxis is indicated are:
a- Large bowel surgery.
b- Major orthopedic and cardiac surgery.
c- Amputation of an ischaemic limb.
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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Clinical Use of Antibiotics:

The objective of antibiotic therapy is to cure the patient with minimal complications. At the same time,
it is important to discourage the emergence of drug-resistant organisms. The following principles
should be observed:

1- Antibiotics should not be given for trivial infections.

2- They should be used for prophylaxis only in special circumstances.
3- Treatment should be based on a clear clinical and bacteriological diagnosis. Suitable specimens
should be sent to the laboratory before treatment is begun. However, treatment can be started
after taking the sample; but should be modified later according to results of antibiotic sensitivity
testing in vitro.
4- Antibiotics for systemic treatment should be given in full therapeutic doses for adequate period.
5- Combined therapy with two or more antibiotics is required in some conditions e.g. :

a. Serious resistant infections e.g. infective endocarditis or meningitis.

b. In treatment of tuberculosis 2 or 3 drugs are given in combination to delay emergence
of resistant mutants. Also to decrease toxic effects of the drugs by lowering the dose of
each.
c. Severe mixed infections e.g. peritonitis following perforation of the colon.
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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Antimicrobial Chemotherapeutics

Killing pathogens, but not us!

Definitions

Chemotherapeutics

Antimicrobials

Antibiotics

Antifungals

Strategies

Look for metabolic targets not found in human cells

Bacteria some

Fungi fewer

Protozoa even less

Viral very few

Targets in Bacteria

Cell Wall

Protein synthesis

Cytoplasmic Membrane

General metabolic pathways

Nucleic Acid synthesis

Inhibition of Attachment

Targets of Antimicrobials

Cell Wall

Penicillin

Cephalasporin

Vancomycin
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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Protein synthesis

Streptomycin

Tetracycline

Erythromycin

Cytoplasmic Membrane

Amphotericin B & Fluconazole

Polymyxins

General metabolic pathways

Sulfanilamide

Protease Inhibitors

Nucleic Acid synthesis

Rifampin - RNA

Ciprofloxacin helicase

AZT & Acyclovir

Inhibition of Attachment

Antivirals - pleconaril

Spectrum of Activity

Determining Efficacy of Antibiotics

Kirby Bauer Test

MIC

Broth Dilution

Etest

MBC

Effect of Route of Administration

Side Effects
 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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Kidney Damage

Nerve Damage

Allergies

Immunities

Interactions with Calcium

Disruption of Normal Microbiota

Hairy Tongues

Metronidazole

Developing Resistance

Mutation and Selection

Horizontal Gene Transfer

Mechanisms

Alteration of target

Membrane & Cell wall permeability

Enzymes

Changes in metabolic pathway

Cross Resistance

Countering Drug Resistance

Completing the regimen

Synergism

Using Narrow spectrum drugs

Limiting use

Developing new drugs

Multi-drug treatments

Are bacteria winning the war against antibiotics?

 Medical Microbiology Dr. Saleh M.Y. Phase II MBBS Medical Students
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Antiprotozoans & Antihelminths

Quinolones antimalarial

Mebendazole Pinworms and protozoans

Ivermectin flaccid paralysis

Cultures for some microorganisms that produce antibiotics