ANTIMICROBIAL SUSCEPTIBILITY TESTING

The purpose of antimicrobial susceptibility testing is to guide the clinician in the selection of an antimicrobial agent to which the clinical condition being treated will respond. There are three principal methods of antimicrobial susceptibility testing in common use: disc diffusion, broth dilution agar dilution.

The agar disc diffusion technique is probably the most commonly used method in veterinary diagnostic laboratories, particularly in smaller laboratories. The broth microdilution method give data on minimum inhibitory concentrations (MIC).

Disc Diffusion Method Disc diffusion is the simplest method to perform. It entails the placing of discs impregnated with antimicrobial agents onto an agar plate seeded with the bacterium to be tested. . The antimicrobial agents diffuse into the agar creating a zone saturated with the agent, in which an organism susceptible to that agent will not grow.

The method should only be used for the rapidly growing pathogens. In order for the results to be clinically reliable the technique must be carried out in a standardized manner.

Factors affecting the size of the zone of inhibition One of the reasons for the strict standardization of the test procedures is that many factors can influence the size of the zone of inhibition including: • The bacterial concentration of the inoculum: this is of great importance and is usually addressed by ensuring the turbidity of the inoculum is adjusted to a 0.5 McFarland opacity standard. The aim is to have a dense lawn of bacterial growth with the individual colonies just touching each other (Figs 6.1 and 6.2). • The test medium: Mueller–Hinton or modifications of this medium (Iso-sensitest agar, Oxoid) is usually chosen for routine susceptibility tests. It gives good batch-to-batch reproducibility; is low in

• For bacteria, such as streptococci, that are unable to grow on Mueller–Hinton agar, blood agar with 5–10% defibrinated sheep blood can be used. However, the zone size, particularly for nafcillin, novobiocin and meticillin, will be 2–3 mm smaller than the normal control limits (Figs 6.3 and 6.4). • The depth of the agar and the pH of the medium must be standardized as these may also have an effect on the zone size (see Quality Control Methods).

Quality Control Procedures. • Incubation conditions: these have been standardized for routine susceptibility tests to aerobic incubatio at 35°C for 16–18 hours and 24 hours for the staphylococci. The plates must not be incubated under an increased concentration of carbon dioxide

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Routine test procedure for the disc diffusion method Standard method • At least four to five well-isolated colonies of the same morphological type are selected from a non-selective agar plate. Just the tops of the colonies are touched and the growth transferred to a tube containing 4–5 mL of soybean-casein digest broth or an equivalent (tryptone soya broth, Oxoid). • The inoculated broth is incubated at 35–37°C until a slight visible turbidity appears, this is usually within two to eight hours.

Alternative method • The colonies are selected as before and a suspension is made in saline or broth without the preincubation in broth. This is suggested as the best method for testing staphylococci, especially with suspected meticillin-resistant strains. • The turbidity of both the pre-incubated broth and the suspension of bacteria (alternative method) is adjusted by comparison with a 0.5 McFarland turbidity standard. The standard and the test suspension are placed in similar 4–6 mL thin-glass tubes or vials. The turbidity of the test suspension is adjusted with broth or saline and compared with the turbidity standard, against a white background with contrasting black lines until the turbidity of the test suspension equates to that of the turbidity standard. Alternatively, turbidity can be measured using an instrument such as a Densimat (bioMérieux).

McFarland 0.5 turbidity standard Solution A (0.048 M BaC12) 1.175 g BaC12.2H20 Make up to 100 mL with distilled water. Solution B (0.36 N H2SO4) 1.0 mL H2SO4 (Analar grade, sp.gr. 1.84) Make up to 100 mL with distilled water. Stock Solution 0.5 mL Solution A (0.048 M BaC12) 99.5 mL Solution B (0.36 N H2SO4) Shake vigorously and dispense into 4–6 mL sealed tubes or screw-capped vials. Store in the dark at room temperature and replace three months after preparation. Always agitate the turbidity standard before use.

Test procedure: A sterile, non-toxic swab on an applicator stick is dipped into the standardized suspension of bacteria and excess fluid is expressed by pressing and rotating the swab firmly against the inside of the tube above the fluid level. • The swab is streaked in three directions over the entire surface of the agar with the objective of obtaining a uniform inoculation. A final sweep with the swab can be made against the agar around the rim of the Petri dish. • The test agar must be Mueller–Hinton agar or a satisfactory equivalent such as Iso-sensitest agar (Oxoid). The exception is the use of 5–10% sheep blood agar for streptococci or Trueperella (Arcanobacterium) pyogenes that are unable to grow on Mueller–Hinton agar.

The surface of the agar should be moist but no droplets of moisture should be visible on the surface of the agar. • The inoculated plates are allowed to stand for three to five minutes, but no longer than 15 minutes, for any excess moisture from the inoculum to be absorbed by the agar before applying the antimicrobial discs.

• The discs are placed onto the agar surface using sterile forceps or an antibiotic disc dispenser (Fig. 6.5). Each disc is gently pressed with the point of sterile forceps to ensure complete contact with the agar surface. The discs should be placed no closer together than 24 mm (centre-to-centre). This is equivalent to six discs per standard 90 mm Petri dish. • The plates are inverted and placed in a 35°C incubator, within 15 minutes of applying the discs and incubated aerobically for 16–18 hours or 24 hours for staphylococci. • After incubation the diameters of the zones of inhibition are measured to the nearest mm using a ruler or calipers. The diameters are read from the back of the plate when the test is on the comparatively clear Mueller–Hinton medium but over the surface of the agar with streptococci grown

on blood agar. The diameter of the zones should be read across the centre of the discs. Automated devices for reading and interpretation of zone sizes are available (e.g,. Mastascan, Mast Group Ltd.). An interpretation of the size of the zones of inhibition is made with reference to available interpretative tables such as those produced by CLSI. Table 6.1 is modified from CLSI (2008) document M31-A3. The bacterium is reported as susceptible, intermediately

Agar dilution tests These tests are similar to the broth dilution method except that the antibiotic dilutions are incorporated into an agar medium in a series of Petri dishes. These are spot inoculated with a number of test bacteria, between 20–36 bacterial isolates can be accommodated on a 90 mm Petri dish. A series of control bacteria, of known sensitivity, should be included on each plate.

E test Several commercial systems are available for determining MIC values, not all of which involve preparation of a dilution series of the antimicrobial agent. The E test (AB Biodisk) is illustrated (Fig. 6.6). This test consists of a thin, inert plastic test carrier with a predefined exponential gradient of antibiotic. After incubation, the MIC value is read from the scale at the point of intersection between the zone edge and the test carrier. .