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Chapter: Microbiology and Immunology: Antimicrobial Agents Therapy and Resistance

Antibiotic Sensitivity Testing

Antibiotic sensitivity testing is carried out to determine the appropriate antibiotic agent to be used for a particular bacte-rial strain isolated from clinical specimens. Antibiotic sensitiv-ity testing can be carried out by two broad methods, as follows: a) Disc diffusion tests b) Dilution tests

Antibiotic Sensitivity Testing

Antibiotic sensitivity testing is carried out to determine the appropriate antibiotic agent to be used for a particular bacte-rial strain isolated from clinical specimens. Antibiotic sensitiv-ity testing can be carried out by two broad methods, as follows:

a)        Disc diffusion tests

b)       Dilution tests

Disc Diffusion Tests

Disc diffusion tests are the most commonly used methods in a laboratory to determine susceptibility of bacteria isolates to antibiotics. In this method, as the name suggests, discs impregnated with known concentrations of antibiotics are placed on agar plate that has been inoculated with a culture of the bacterium to be tested. The plate is incubated at 37°C for 18–24 hours. After diffusion, the concentration of antibi-otic usually remains higher near the site of antibiotic disc, but decreases with distance. Susceptibility to the particular antibi-otic is determined by measuring the zone of inhibition of bacte-rial growth around the disc (Fig. 9-3).

Selection of media

The medium that supports both test and control strains is selected for carrying out antibiotic susceptibility testing of the bacteria. For example, Mueller–Hinton agar is used for testing Gram-negative bacilli and Staphylococcus spp., blood agar for Streptococcus spp. and Enterococcus spp. species, chocolate agarfor Haemophilus influenzae, and Wellcotest medium for sulfon-amides and cotrimoxazole.

The medium is prepared by pouring onto the flat horizon-tal surface of Petri dishes of 100 mm to a depth of 4 mm. The pH of the medium is maintained at 7.2–7.4. More alkaline pH increases the activity of tetracyclines, novobiocin, and fusidic acid, whereas an acidic pH reduced the activity of aminogly-cosides and macrolides, such as erythromycin. The plates after preparation may be stored at 4°C for up to 1 week.

Preparation of the inoculum

For testing antibiotic sensitivity, the bacteria are first isolated in pure culture on a solid medium. At least three to four morpho-logically similar colonies of the bacteria to be tested are touched and inoculated into appropriate broth and incubated at 37°C for 4–6 hours. The density of bacterial suspension in the broth is adjusted to 1.5 108 cfu/mL by comparing its turbidity with that of 0.5 McFarland opacity standard tube. The broth is inoculated on the medium by streaking with sterile swabs. A sterile cotton swab is dipped into the broth and excess broth is removed by rota-tion of the swab against the sides of the tube above the fluid level.

Antibiotic discs

Only the clinically relevant antibiotics are tested in antibiotic susceptibility tests. Antibiotic discs (6-mm filter paper discs) can be prepared from pure antimicrobial agents in laborato-ries or can be obtained commercially. The discs are applied with sterile forceps, a sharp needle, or a dispenser onto the surface of the medium, streaked with test strains, and the reading is reported after incubating the plate for 18–24 hours at 37°C aerobically.

Types of disc diffusion tests

Disc diffusion tests are of the following types:

a)        Kirby–Bauer disc diffusion method

b)       Stokes disc diffusion method

c)        Primary disc diffusion test

Kirby–Bauer disc diffusion method: Kirby–Bauer discdiffusion method is the most common method used routinely for determination of antibiotic sensitivity of bacteria isolated from clinical specimens. In this method, both the test strains and the control strains are tested in separate plates.

The test is performed by inoculating the test organism in a suitable broth solution, followed by incubation at 37°C for 2–4 hours. Then 0.1 mL of the broth is inoculated on the surface of the agar medium by streaking with a sterile swab. In this method, either nutrient agar or Mueller–Hinton agar in Petri dishes is used. The inoculated medium is incubated overnight at 37°C. The susceptibility of drug is determined from the zones of inhibition of bacterial growth surrounding the anti-biotic discs. The diameters of the zone of inhibition are calcu-lated with vernier calipers or a thin transparent millimeter scale to the nearest millimeter. The point of abrupt diminution of the zone is considered as the zone edge. A maximum of six anti-biotic discs are tested in a Petri dish of 85 mm size (Fig. 9-4).

Interpretation of the zone size is done as per the interpreta-tion chart. Depending on the zone size, bacteria can be consid-ered sensitive, intermediate or resistant to antibiotics.

Stokes method: This is a disc diffusion method, which makesuse of inbuilt controls against many variables. In this method, the Petri dish containing the Mueller–Hinton agar is divided horizontally into three parts. The test strain is inoculated in the central area and the control strains on the upper and lower third of the plate. In modified Stokes method, control strain is inoculated in the central part but test strains are inoculated on the upper and lower third of the plate. The plates are incubated at 37°C and observed for zones of bacterial inhibition around the discs (Fig. 9-5). A maximum of six antibiotic discs can be applied on a 100 mm Petri dish.

    Reporting of the result is carried out by comparing the zones of inhibition of test and control bacteria. The zone sizes are measured from the edge of the disc to edge of the zone. It is interpreted as follows:

·           Sensitive (S): The zone of test bacterium is equal to or morethan that of control strain. The differences between the zone sizes of control and test strains should not be more than 3 mm if the zone size of the test bacterium is smaller than that of control.

·           Intermediate (I): The zone size of the test bacterium shouldbe at least 2 mm, and the differences between the zone of test and control strain should be at least 3 mm.

·           Resistant (R): The zone size of the test bacterium is 2 mmor less.

Interpretation of disc diffusion tests:Results of disc diffusiontests, such as Kirby–Bauer and Stokes method, are interpreted as follows:

·           Sensitive (S): Infection treatable by the normal dosage ofthe antibiotic.

·           Intermediate (I): Infection may respond to higher dosage.

·           Resistant (R): Unlikely to respond to usual dosage of theantibiotics.

However, for certain bacteria and antibiotic discs, the following may be kept in mind while interpreting results of disc diffusion tests:

a)         Proteus mirabilis, Proteus vulgaris, and other bacteria producingswarming produce a thin film on agar surface often extend-ing into the zones of inhibition. In such situations, the zones of swarming should be ignored and the outer clear margin should be measured to determine the zones of inhibition.

b)       Many strains of MRSA grow very slowly in the presence of methicillin. They produce growth within the zone of inhi-bition on incubation for more than 48 hours. This prob-lem can be overcome by incubating the bacteria at 30°C or by using 5% salt agar and incubating at 37°C.

c)        Penicillinase producing strains of Staphylococcus often fail to secrete enough enzymes to neutralize penicillin close to the antibiotic disc. In such situation, it may show a zone of inhibition, but with the presence of large colonies at the edge of the zone and without any gradual fading away of the growth of the bacteria toward the disc. In such condi-tion, the zones of inhibition should not be considered, and it should be reported resistant irrespective of the zone size.

d)       Trimethoprim and sulfamethoxazole should be tested separately to know whether the bacterium is sensitive to both or only to one of these. These should never be tested in combination, the way these two drugs are used in com-bination in clinical practice.

Dilution Tests

Dilution tests are performed to determine the minimum inhib-itory concentration (MIC) of an antimicrobial agent. MIC is defined as the lowest concentration of an antimicrobial agent that inhibits the growth of organisms. Estimation of the MIC is useful to:

·           Regulate the therapeutic dose of the antibiotic accurately in the treatment of many life-threatening situations, such as bacterial endocarditis.

·           Test antimicrobial sensitivity patterns of slow-growing bacteria, such as M. tuberculosis.

Following methods are carried out to determine the MIC:

a)        Broth dilution method

b)       Agar dilution method

c)        Epsilometer test (E-test)

Broth dilution method

The broth dilution method is a quantitative method for deter-mining the MIC of an antimicrobial agent that inhibits the growth of organisms in vitro. In this method, the antimicrobial agent is serially diluted in Mueller–Hinton broth by doubling dilution in tubes and then a standard suspension of the broth culture of test organism is added to each of the antibiotic dilu-tions and control tube. This is mixed gently and incubated at 37°C for 16–18 hours. An organism of known susceptibility is included as a control. The MIC is recorded by noting the lowest concentration of the drug at which there is no visible growth as demonstrated by the lack of turbidity in the tube. The main advantage of this method is that this is a simple procedure for testing a small number of isolates. The added advantage is that using the same tube, the minimum bactericidal concentration (MBC) of the bacteria can be determined.

The MBC is determined by subculturing from each tube, showing no growth on a nutrient agar without any antibiotics. Subcultures are made from each tube showing no growth into the nutrient agar plates without any antibiotics. The plates are examined for growth, if any, after incubation overnight at 37°C. The tube containing the lowest concentration of the drug that fails to show any growth on subculture plate is considered as the MBC of the antibiotic for that strain. Broth dilution may be of two types—macrodilution and microdilution. Broth microdilu-tion is done using microtiter plates and is considered the “gold standard.”

Agar dilution method

Agar dilution method is a quantitative method for determining the MIC of antimicrobial agent against the test organism.

    Mueller–Hinton agar is used in this method. Serial dilution of the antibiotic are made in agar and poured onto Petri dishes. Dilutions are made in distilled water and added to the agar that has been melted and cooled to not more than 60°C. One control plate is inoculated without antibiotics. Organism to be tested is inoculated and incubated overnight at 37°C. Plates are examined for presence or absence of growth of the bacte-ria. The concentration at which bacterial growth is completely inhibited is considered as the MIC of the antibiotic.

The organisms are reported sensitive, intermediate, or resis-tant by comparing the test MIC values with that given in CLSI guidelines. The main advantage of the method is that a num-ber of organisms can be tested simultaneously on each plate containing an antibiotic solution.

Epsilometer test (E test)

Epsilometer test (E test), based on the principle of disc diffu-sion, is an automated system for measuring MIC of a bacterial isolate. In this method, an absorbent plastic strip with a con-tinuous gradient of antibiotic is immobilized on one side. MIC interpretative scale corresponding to 15 twofold MIC dilu-tions is used on the other side. The strip is placed on the agar plate inoculated with the test organism with the MIC scale fac-ing toward the opening side of the plate. An elliptical zone of growth inhibition is seen around the strip after incubation at 37°C overnight. The MIC is read from the scale at the intersec-tion of the zone with the strip. The end point is always read at complete inhibition of all growth including hazes and isolated colonies. E test is a very useful test for easy interpretation of the MIC of an antibiotic (Fig. 9-6).

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