Antibiotic Resistance – What is it?
•Antibiotics – drugs that fight infections caused by bacteria
•Antibiotic resistance – when bacteria acquired an ability to resist the effectiveness of the drug to which it is normally susceptible.
•How it happens?
•Bacteria causes interruption or disturbance of one or more of the steps of antimicrobial action result in partial or complete loss of antibiotic effectiveness
•Bacteria survive antibiotic control and continue to multiply into resistant strains
Antibiotic Resistance
•Resistant bacteria usually have a gene that makes the antibiotic ineffective
–Surviving bacteria will replicate
•Bacteria have plasmids that allow genes to move between different types of bacteria
–Bacteria that was previously susceptible to antibiotic now have the resistant gene
•Dead bacterial cells give off DNA that can be incorporated into living bacteria allowing it to become resistant

Origin of Drug Resistance

A. Genetic

i. Chromosomal

ii. Extra chromosomal

B. Non-genetic basis

Genetic:

i. Chromosomal

- resistance may be acquired by spontaneous mutation in the gene that code for either the target of the drug or the transport system in the membrane

Example: Mutation in the PBP – making bacteria resistance to ß- lactam drugs

ii. Extra chromosomal/Plasmid mediated resistance:

•Bacteria have some plasmids( R factors) that carry gene for a variety of enzymes that can degrade antibiotics and modify membrane transport system.
•This plasmids allow resistance genes to move between different types of bacteria
–Bacteria that was previously susceptible to antibiotic now have the resistant gene
•Gene transfer occur by following mechanisms-
•Transduction-transfer of cell DNA by bacteriophage.
•Conjugation – transfer of genetic material from one bacteria to others by sex pili
•Transformation - Dead bacterial cells give off DNA that can be incorporated into living bacteria allowing it to become resistant
•Plasmid mediated resistance is very important from a clinical point of view for three reasons-

i. plasmid frequently mediate resistance to multiple drugs.

ii. plasmid have a high rate of transfer from one cell to

another

iii. it occurs in many different species

B. Non-genetic basis of drug resistance

There are several non-genetic reasons for the failure of drugs to inhibit the growth of bacteria

1. Bacteria can be walled off within an abscess

cavity –so the drug cannot penetrate effectively

2. Bacteria can be in a resting state

3. Loss their cell wall survive as protoplast

4. Failure of the drug to reach the appropriate

site in the body

5. Failure of the patient to take the drug

( noncompliance)

Mechanisms of Drug Resistance

1. Bacteria produce enzyme that inactivate drug

–ß-lactamases inactivate penicillin & cephalosporin

2. Decreased permeability to drug or increased elimination of drug from cell

–acquired or mutation

3. Change in drug receptors/ altered antimicrobial targets

–mutation or acquisition

4. Change in metabolic patterns

–mutation of original enzyme

5. Any combination of mechanisms 1 through 5

Contributing Factors to Resistance
•Overuse in humans
ØUse multiple antibiotic when one would be sufficient
ØPrescribe unnecessarily long courses of antibiotic therapy
ØUse antibiotic in self limited infection
ØOver use antibiotics for prophylaxis before and after surgery
•Animal and agricultural use
ØUsed in animal feed to prevention/treatment of infection and growth promotion
ØEvidence of resistant strains in livestock
Factors contributing to the emergence and dissemination of antimicrobial resistance among bacteria:
•1. Emergence of ‘new’ genes( e.g. MRSA, VRE)
•2. Spread of ‘old’ genes to new host( penicillin resistant N. gonorrhoeae)
•3. Mutation of ‘old’ genes resulting in more potent resistance gene
•4. Emergence of intrinsically resistant opportunistic bacteria ( e.g. Stenotrophomonas maltophilia)
Prevention of Antibiotic Resistance
1. Prevent transmission

2. Use antimicrobial wisely –

•Only use an antibiotic when they are likely to be beneficial
•Treat infection not contamination or colonization
•Do not save any of your antibiotic prescription
•Stop the treatment when infection is cured or unlikely
•

3.Diagnose & treat effectively

•Appropriate drug should be use( correct regimen, timing, dosage, route & duration)

4.Prevent infection

•Vaccinate
•Get the catheters out

Antibiotic Susceptibility test
•Main purposes:

As a guide for treatment

- to select both effective antimicrobial drug & dose

As an epidemiological tool

–The emergence of resistant strains of major pathogens (e. g. Shigellae, Salmonella typhi)
–Continued surveillance of the susceptibility pattern of the prevalent strains (e. g. Staphylococci, Gram-negative bacilli)


Laboratory antimicrobial susceptibility testing can be performed using:

1. Drug dilution method

2. Disc diffusion method


1. Drug dilution method

–making dilutions (2-fold) of antibiotic in broth
–followed by inoculation of test bacteria, incubation, overnight
–

2. Disc diffusion method (Kirby-Bauer method)

–Put a filter disc, containing measured quantity of drugs, and placed on a plate of sensitivity testing solid medium that has been seeded with test bacteria
•Broth dilution/ Agar dilution methods
•Permit quantitative results:
–Indicating amount of a given drug necessary to inhibit (bacteriostatic activity) or kill (bactericidal activity) the microorganisms tested
–This provides physician to choice of both the drug and dose
•Drug dilution method results commonly reported as MIC & MBC
•Minimum Inhibition Concentration (MIC)
–The lowest concentration of antimicrobial agent that can inhibits bacterial growth/ multiplication
–
•Minimum Bactericidal Concentration (MBC) or Minimum Lethal Concentration (MLC)
–The lowest concentration of antimicrobial agent that required to kill bacteria.

•Procedure
–Making dilutions (2-fold) of antibiotic in broth
•Mueller-Hinton, Tryptic Soy Broth
–Inoculation of bacterial inoculum, incubation, overnight
•Controls: no inoculum, no antibiotic
–Turbidity visualization à MIC
–Subculturing of non-turbid tubes, overnight
Growth (bacterial count) à MBC
•Disadvantages :
–Only one antibiotic & one organism can be tested each time
–Time-consuming & laborious
•Disc diffusion method : The Kirby-Bauer test
–Antibiotic-impregnated filter disc*
–Susceptibility test against more than one antibiotics by measuring size of “inhibition zone ”
–1949: Bondi and colleaguesà paper disks
–1966: Kirby, Bauer, Sherris, and Tuck à filter paper disks
•Demonstrated that the qualitative results of filter disk diffusion assay correlated well with quantitative results from MIC tests
•Procedure

(Modified Kirby-Bauer method: National Committee for Clinical Laboratory Standards. NCCLS.)

–Prepare applx. 108 CFU/ml bacterial inoculum in a saline or tryptic soy broth tube (TSB) or Mueller-Hinton broth (5 ml)
•Pick 3-5 isolated colonies from plate
•Adjust the turbidity to the same as the McFarland No. 0.5 standard.*
–Streak the swab on the surface of the Mueller-Hinton agar (3 times in 3 quadrants)
Leave 5-10 min to dry the surface of agar
–Place the appropriate drug-impregnated disc on the surface of the inoculated agar plate
–Invert the plates and incubate them at 35 oC, o/n (18-24 h)
–Measure the diameters of inhibition zone in mm
•Interpretation of results
–By comparing with the diameters with “standard tables”

–Susceptible
–Intermediate susceptible
•Low toxic antibiotics: Moderate susceptible
•High toxic antibiotics: buffer zone btw resistant and susceptible
–Resistant
•Salient features of quality control
–Use antibiotic discs of 6 mm diameter
–Use correct content of antimicrobial agent per disc
–Store supply of antimicrobial discs at -20 oC
–Use Mueller-Hinton medium for antibiotic sensitivity determination
–Use appropriate control cultures
- Keep the antibiotic discs at room temperature for one hour before use
- Incubate the sensitivity plates for 16-18 hours before reporting
- Incubate the sensitivity plates at 35oC
- Space (2mm) the antibiotic discs properly to avoid overlapping of inhibition zone
- Measure zone sizes precisely
- Interpret zone sizes by referring to standard charts