Mechanism of Antimicrobial

Resistance

Dr. Nisha Goyal

Lecturer
Department of Microbiology
ANTIMICROBIAL
RESISTANCE
 Refers to development of resistance to
an antimicrobial agent by a
microorganism

 Can be of two types-

 Intrinsic
 Acquired
Intrinsic Resistance
 Refers to innate ability of bacterium to
resist a class of antimicrobial agents due to
its inherent structural or functional
characteristics

 E.g. vancomycin resistance among gram-

negative bacteria and metronidazole
resistance among aerobic bacteria
Acquired Resistance
 Emergence of resistance in bacteria that are
ordinarily susceptible to antimicrobial agents

 By acquiring the genes coding for resistance

 Most prevalent form of antimicrobial

resistance shown by bacteria
Mutational and Transferable
Drug Resistance
Bacteria acquire new genes mainly by two
broad methods:
Mutational Resistance

Transferrable Drug Resistance

Mutational Resistance
 Resistance develops due to mutation of resident genes.

 Usually, a low level resistance

 Develops to one drug at a time; can be overcome by

combination of different classes of drugs

 Typically seen in Mycobacterium tuberculosis (reason for

multidrug therapy using 4-5 different classes of drugs in
tuberculosis treatment)
 Transferrable Drug
Resistance
 Plasmid coded, Horizontal spread;
usually transferred by conjugation or
rarely by transduction, or
transformation

 Resistance coded plasmid (R

plasmid) can carry multiple genes,
each coding for resistance to one class
of antibiotic

 High degree of resistance to multiple

drugs, which cannot be overcome by
using combination of drugs
 Susceptibility test, main purposes:
 As a guide for treatment
 Sensitivity of a given m.o. to known conc. of drugs
 Its concentration in body fluids or tissues

 As an epidemiological tool
 The emergence of resistant strains of major pathogens
 Continued surveillance of the susceptibility pattern of
the prevalent strains
Antimicrobial Susceptibility Testing

 Dilution method
 vary amount of antimicrobial substances incorporated
into liquid or solid media
 followed by inoculation of test bacteria

 Diffusion method
 Put a filter disc, or a porous cup/a bottomless cylinder
containing measured quantity of drugs on the a solid
medium that has been seeded with test bacteria
 Conventional testing methods

 Disc-diffusion method:

 Principle – impregnated disc absorbs moisture from the

agar and antibiotic diffuses into the agar medium

 As distance from disc increases, antibiotic concentration

decreases

 Visible growth of bacteria occurs on the surface of agar

where the concentration of antibiotic falls below the inhibitory
level for the test strain.

 Concentration of diffused antibiotic at the interface of

growing and inhibited bacteria approximates to the MIC
obtained in dilution tests.
Disc Diffusion Method
Antibiotic discs –
 commercially prepared discs 6mm in diameter are used

 Can be prepared from Whatman filter paper no.1, sterilized in hot air
oven

 Antibiotics delivered with 20 gauge wire loop, diameter 2mm. This

delivers 0.005ml antibiotic to each disc

 Discs and disc dispensers should be stored in sealed containers with a

dessicant

 Bulk stock kept at -20oC

 Working stock kept in sealed containers with dessicant , stored at less

than 8 oC
 Turbidity standard for inoculum
preparation –

 To standardize the inoculum density

 BaSO4 turbidity standard, equivalent
to 0.5 McFarland is used
 Prepared by 0.5ml 0.048 mol/L
BaCl2 + 99.5ml 0.18 mol/L H2SO4
 Density verified by using
spectrophotometer
 Absorbance at 625 nm should be
0.008 – 0.10 for 0.5 McFarland
standard
 Densities verified monthly
 Disc Diffusion Method
 Procedure (Modified Kirby-Bauer method: National Committ
ee 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
 Disc Diffusion Method
 Procedure (cont.)
Bacterial growth
 Place the appropriate drug-imp
regnated disc on the surface of
the inoculated agar plate
 Invert the plates and incubate t
hem at 35 oC, o/n (18-24 h)
 Measure the diameters of
inhibition zone in mm
Disk Diffusion
Test
 Prepare inoculum
suspension

Select colonies
 Mix well

Standardize inoculum
suspension
 Swab plate

Remove sample
 Incubate overnight

Add disks
Measure Zones
 Disk Diffusion Test

 Qualitative results

 Susceptible

 Intermediate – may respond if infection is at body

site where drug concentrates (e.g. urine) or if
higher than normal dose can be safely given

 Resistant
Two types of disc diffusion methods:

1) Kirby –Bauer (recommended by the CLSI and

WHO)

1) Same plate comparative disc diffusion test

(Stokes) ( used in British laboratories)
 Kirby-Bauer method
 Sterile cotton swab dipped in the adjusted suspension

 MH agar plate inoculated by streaking the swab over the entire agar
surface

 Predetermined battery of antimicrobial discs are dispensed

 Discs distributed evenly so that they are no closer than 24mm from
center to center

 12 discs should be placed on one 150mm plate or 5 discs on 100 mm

plate

 Once in contact with the agar surface the disc should not be relocated.
Plates incubated in ambient air
 Stokes method

o Tests of organism of unknown sensitivity and control

organism of known sensitivity are set up at the same
time and in identical conditions on the same plate

o The control inoculum are spread in two bands on either

side of the plate, and the test organism is inoculated
onto the central area of the plate

o An uninoculated gap 2-3mm wide should separate the

test and control areas

o Interpretation done by comparing the size of inhibition

zone
 Factors Affecting Size of Zone of Inhibition

 Inoculum density Larger zones with light inoculum and vice versa

 Timing of disc application If after application of disc, the plate is kept for l
onger time at room temperature, small zones may form

 Temperature of incubation Larger zones are seen with temperatures < 3

5 oC

 Incubation time Ideal 16-18 hours; less time does not give reliable results

 Potency of antibiotic discs Deterioration in contents leads to reduced siz

e

 Composition of medium Affects rate of growth, diffusion of antibiotics and

activity of antibiotics
 Factors Affecting Size of Zone of Inhibition

 Acidic pH of medium Tetracycline, novobiocin, methicillin zones are lar

ger

 Alkaline pH of medium Aminoglycosides, erythromycin zones are larger

 Reading of zones Subjective errors in determining the clear edge

 Size of the plate Smaller plates accommodate less number of discs

 Depth of the agar medium (4 mm) Thin media yield excessively large i
nhibition zones and vice versa

 Proper spacing of the discs (2.5 cm) Avoids overlapping of zones

Modify methods for fastidious bacteria

Media recommended
for test of fastidious
bacteria
THANK
YOU