ANTIMICROBIAL SUSCEPTIBILITY TESTING

Susceptibility of organism is important factors in treatments of any bacterial infection especially the
resistant cases (patients not responding to drugs) so it is important to establish how susceptible or
resistant of organism to drugs.
Two methods of testing susceptibility
 Tube dilution methods
 Diffusion methods

I. Tube dilution
The drug is incorporated with media mainly in form of broth and this drugs media is inoculated in test
organism. Dilution is made in serial dilution as you can know what concentration of drug can kill the
bacteria then incubated and record growth which is indicate as cloudiness.
The lowest concentration of antibiotic that show no growth is known as minimal inhibitory
concentration (MIC) however the lowest concentration where growth does not occur after re-culture of
MIC is known as minimal bacterialcidal concentration (MBC).

II. Diffusion method

Further sub divided into;
Agar diffusion methods
Here the surface of agar is inoculated with the test organism using cotton pad to ensure uniform growth of
bacteria, antibiotics is placed inform of a dish or punch holes on agar plate and put a drop of antibiotics in
the hole so that microorganism is exposure to continuous gradient of antibiotics and incubated. When
organism reach area of antibiotic will be either be inhibitor from growing and will grow if it is resistant to
drugs.

Kirby Bauer method

Allow rapidly determination of efficacy of drug by measuring the diameter of zone of
inhibition that result from diffusion of drug from media surrounding the dish. Filter paper
dish of uniform size that are impregnated with specific concentration of different
antibiotic are then placed on surface of agar plate that have be inoculated with test of
choice organism. Agar use is Mueller Hinton plate is the incubated at correct temperature
and examine for growth or growth inhibition. This is normally indicated by clear zone
around the dish that was drugs; the susceptibility of organism is determined by the size of
clear zone.

Clear zone size determine by:

 Ability of drug to diffuse into the media
 Numbers of organism inoculated
 Growth rate of organism
 Degree of susceptibility of organism to antibiotic

Synergistic effect of drug combination

This is the use of two or more microbial agents to treat infections; the rationale of this is expectation that
combination may have the following effect.
I. May lower incidence of bacteria resistant
II. It may reduce host toxicity to antimicrobial agent because when using two or more drug
you decrease dosage.
III. It enhances agents bactericidal activity , this is known as synergism

The most common methods use to demonstrated synergistic is the Kirby Bauer method.
Diag. 1
If drugs are not working in combination each of them will show zone of inhibition
Diag. 2

Drugs work together

Diag. 3

Antimicrobial agent used to treat infection diseases

Important properties of antimicrobial agents to patient are Selective toxicity, the agents act to inhibits or
kill bacteria pathogen but has no or little effect on animal taking the drug. These mean biochemical
processes in bacteria are different from those in the body of the animal.
Antimicrobial may have two effects on bacterial
 May kill bacterial hence bactericidal
 May inhibit the growth hence bacteristatics
The range of bacteria that have be affected by certain antibiotics is express as spectrum action those
antibiotics effectively against wide range of bacteria i.e. Gram positive &Gram negative are called Broad
spectrum, if antibiotics inhibit one group of bacteria either Gram+ve or Gram -ve is called narrow
spectrum of action while if it is effectively only against one organism is to have Limited spectrum of
action.

ANTIBACTERIAL AGENTS

Definitions:
Antimicrobial agents: substance with inhibitory or destroy or kill microorganism properties against
microorganism.
Antibiotic: substance produced by microorganism that inhibits the growth of (bacteriostatic) or kills
(bactericidal).

Modes of action of antibiotics

i. INHIBITORS OF CELL-WALL SYNTHESIS
Most bacteria, unlike mammalian cells, have a cell wall. The main groups of antimicrobial agents that
act selectively on the bacteria cell wall are the β-lactam and glycopeptides.
Β-lactam antibiotics
Structure: this is a large group of compounds, all with a β-lactam ring
Action: These antibiotic interfere with the cross-linking of cell-wall polymer peptidoglycan by inhibiting
carboxypeptidase and transpeptidase (penicillin-binding protein˂ PBPs˃) reaction, which form a link
between the building block of peptidoglycan, N-acetylmuramic acid and N-acetylglucosamine this
weakens the cell wall and results in rupture (lysis) of the microorganism. Examples penicillin,
cephalosporins and carbapenems.

Antibacterial resistance
  Alteration in target site e.g. meticillin-resistant staphylococcus aures can produce a penicillin-
binding protein penicillin-binding protein with low affinity for β-lactams and therefore continue
to function in the presence of the antibiotic.
 Cell membrane alteration, reducing uptake or increasing loss from the cell by drug efflux pumps
in Gram-negative bacteria

Cephalosporins: Are a large group of antimicrobial agents based on cephalosporin C with a structure
similar to the penicillins and produce by cephalosporins fungi.

Antimicrobial spectrum of activity: Variable but generally include S. aureus, streptococci, many Gram-
negative species, including Neisseriae, Haemophilus and coliforms (E.coli).

Toxicity/side effects: Hypersensitivity with rashes, usually maculopapular (a rash with both flat and raised parts).
Resistance: Similar to the penicillins

Bacitracin: Produce by Bacillus species, it inhibit the cell wall synthesis by inhibit Teichoic acid
synthesis. It has high toxicity hence limited to its used and because it has poor absorption by gut hence
given to sterilize gut before surgery so as to get rid of organism before surgery which may cause
infection.

ii. ANTIMICROBIAL AGENTS ACTING ON CELL MEMBRANES

Only a few agents target the microbial membrane, its function is to disorganize the structure and function
leading to dead of microorganism. However because of similarity of phospholipids of bacterial and
animal cell membrane their action is not specific.

Polymyxin: Active on gram –ve and limited to topical usage only normally bind to membrane
phospholipids and interferes with function of membrane. It’s given to patients with urinary infection
cause by pseudomonas specifically if they are resistant to drugs which have be used earlier.
iii. INHIBITORS OF PROTEIN SYNTHESIS
This group includes the aminoglycosides, macrolides, chloramphenicol and tetracycline, which target
the difference between human and bacterial ribosomes, the latter having 30 s and 50 s subunits. Always
attack protein synthesis at ribosomal level and they work mainly on 70 s ribosomal in bacterial rather than
80 s ribosomes found in animal.

CHLORAPHENICOL: Broad spectrum drugs but bacteriastatic

Antimicrobial spectrum of activity: Effective against a wide range of organism, including Gram
negative and Gram positive bacteria, chlamydiae, mycoplasmae and rickettsiae.
Toxicity/side effect – has a dose related, but reversible, depressant effect on bone marrow; rarely,
irreversible, potentially fatal, marrow aplasia (disappearance of the cells in the bone marrow that are responsible for blood
production). Toxicity in neonates causes grey baby syndrome.
Resistance – Occurs as a result of inactivation by an inducible acetylase enzyme; reduced permeability.

TETRACYCLINES: Bacteria are able to transport into their body and concentrating it at concentration
that is 50 times higher than in blood hence bacteria is kill by it concentration.
Antimicrobial spectrum of activity: Broad spectrum of activity against many Gram positive and Gram
negative bacteria; chlamydiae, mycoplasmae and rickettsiae.
Toxicity/side effect – Gastrointestinal intolerance; skin rashes
Resistance – Efflux from cell; less commonly by protection of target from the tetracycline.

iv. Inhibition of nucleic acid synthesis

Several classes of antimicrobial agents act on microbial nucleic acid (are against synthesis of DNA &
RNA of bacteria by binding to nucleic acid thus prevent their action and bacteria will dead), including the
quinolones, sulphonamides, diaminopyrimidines, rifampcin and nitroimidazoles.

SULPHONAMIDES e.g. sulphadiazine, sulphadimidine

Mechanism of action: Act on folic acid synthesis as competitive antagonists of ρ-aminobenzoic acid,
inhibiting purine and thymidine synthesis
Antimicrobial spectrum of activity: Broad spectrum of activity, including streptococci, neisseria, H.
influenza
Toxicity: Hypersensitivity reaction, causing renal damage, rash.
Resistance: Many enterobacteriaceae are now resistant, as a result of production of an altered
dihydropteroate synthetase with decreased affinity for sulphonamide.

Rifamycins
Mechanism of action: Binds to RNA polymerase and blocks synthesis of mRNA
Antimicrobial spectrum of activity: Active against many staphylococci, streptococci, H. influenza,
neisseriae and legionella, however mycobacteria, Enterobacteriaceae and P. aeruginosa are resistant.
Toxicity/side effect – Adverse reaction include skin rashes and transient liver function abnormalities; a
rare cause of hepatic failure.
Resistance – Resistant mutants emerge when used as a single agent; resistance is the result of the change
in a single amino acid of the RNA polymerase target site.

Resistance to antimicrobial agents

Mechanisms

There are six main mechanisms by which bacteria become resistant to antimicrobial agents
a) Alteration of the target site to reduce or eliminate binding of the drug to the target.
b) Destruction/inactivation of the target
c) Blockage of transport of the agent into the cell
d) Metabolic bypass, providing the cell with a replacement for the metabolic step inhibited by the
drug.
e) Increased loss of drug from the cell by increased production of efflux pump.
f) Protection of the target site by a bacterial protein.