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Introduction

At a meeting organized by WHO in Geneva in 1977,1 concern was expressed about the worldwide increase in antibiotic resistance associated with the growing, and frequently indiscriminate, use of antibiotics in both man and animals. In recent years, drug-resistant bacteria have given rise to several serious outbreaks of infection, with many deaths. This has led to a need for national and international surveillance programmes to monitor antibiotic resistance in bacteria by susceptibility testing using reliable methods that generate comparable data. The availability of microbiological and epidemiological information would help clinicians in selecting the most appropriate antimicrobial agent for the treatment of a microbial infection.

If predictions are to be valid, the susceptibility test must be performed by an accurate and reproducible method, the results of which should be directly applicable to the clinical situation. The ultimate criterion of the reliability of any susceptibility testing method is its correlation with the response of the patient to antimicrobial therapy.

The WHO meeting considered that the modified disc technique of Kirby-Bauer, for which requirements had been established by WHO in I976,2 could be recommended for clinical and surveillance purposes in view of its technical simplicity and reproducibility. The method is particularly suitable for use with bacteria belonging to the family Enterobacteriaceae, but it can also be recommended as a general purpose method for all rapidly growing pathogens, except strict anaerobes. It was therefore recommended that the details of this test be made available for laboratory workers.3

1 WHO Technical Report Series, No. 624, 1978 (Surveillance for the prevention and control of health hazards due to antibiotic-resistant enterobacteria: report of a WHO Meeting).

2 WHO Technical Report Series, No. 610, 1977 (Twenty-eighth report of the WHO Expert Committee on Biological Standardization), Annex 5.

3 A comparable method, based on the same principles and quality control requirements as the Kirby-Bauer method, is the NEO-SENSITABS method, produced by ROSCO Diagnostica, Taastrup, Denmark. This method uses 9-mm colour-coded, antibiotic tablets, instead of paper discs. The tablet form results in an extraordinary stability with a shelf-life of four years, even at room temperature. This increased stability is very important for laboratories in tropical countries.

General principles of antimicrobial susceptibility testing

Antimicrobial susceptibility tests measure the ability of an antibiotic or other antimicrobial agent to inhibit bacterial growth in vitro. This ability may be estimated by either the dilution method or the diffusion method.

The dilution test

For quantitative estimates of antibiotic activity, dilutions of the antibiotic may be incorporated into broth or agar medium, which is then inoculated with the test organism. The lowest concentration that prevents growth after overnight incubation is known as the minimum inhibitory concentration (MIC) of the agent. This MIC value is then compared with known concentrations of the drug obtainable in the serum and in other body fluids to assess the likely clinical response.

The diffusion test

Paper discs, impregnated with the antibiotic, are placed on agar medium uniformly seeded with the test organism. A concentration gradient of the antibiotic forms by diffusion from the disc and the growth of the test organism is inhibited at a distance from the disc that is related, among other factors, to the susceptibility of the organism.

There is an approximately linear relation between log MIC, as measured by a dilution test, and the inhibition zone diameter in the diffusion test. A regression line expressing this relation can be obtained by testing a large number of strains by the two methods in parallel (see Fig. 7 and 8).

What factors affect disk diffusion method?

Fig. 7. Graphic representation of the relationship between log2 MIC and the inhibition zone diameter obtained by the diffusion test using discs containing a single concentration of antibiotic

What factors affect disk diffusion method?

Fig. 8. Interpretation of zone sizes as susceptible, intermediate, and resistant by their relationship to the MIC

Clinical definition of terms “resistant” and “susceptible”: the three-category system

The result of the susceptibility test, as reported to the clinician, is the classification of the microorganism in one of two or more categories of susceptibility. The simplest system comprises only two categories: susceptible and resistant. This classification, although offering many advantages for statistical and epidemiological purposes, is too inflexible for the clinician to use. Therefore, a three-category classification is often adopted. The Kirby-Bauer method and its modifications recognize three categories of susceptibility and it is important that both the clinician and the laboratory worker understand the exact definitions and the clinical significance of these categories.

· Susceptible. An organism is called “susceptible” to a drug when the infection caused by it is likely to respond to treatment with this drug, at the recommended dosage.

· Intermediate susceptibility covers two situations. It is applicable to strains that are “moderately susceptible” to an antibiotic that can be used for treatment at a higher dosage because of its low toxicity or because the antibiotic is concentrated in the focus of infection (e.g., urine).

The classification also applies to strains that show “intermediate susceptibility” to a more toxic antibiotic that cannot be used at a higher dosage. In this situation, the intermediate category serves as a buffer zone between susceptible and resistant.

As most clinicians are not familiar with the subtle, although clinically important, distinction between intermediate and moderate susceptibility, many laboratories use the designation “intermediate” for reporting purposes.

· Resistant. This term implies that the organism is expected not to respond to a given drug, irrespective of the dosage and of the location of the infection.

For testing the response of staphylococci to benzylpenicillin, only the categories “susceptible” and “resistant” (corresponding to the production of b-lactamase) are recognized.

The ultimate decision to use a particular antibiotic, and the dosage to be given, will depend not only on the results of the susceptibility tests, but also on their interpretation by the physician. Other factors, such as pathogenic significance of the microorganism, side-effects and pharmacokinetic properties of the drug, its diffusion in different body sites, and the immune status of the host, will also have to be considered.

Indications for routine susceptibility tests

A susceptibility test may be performed in the clinical laboratory for two main purposes:

· to guide the clinician in selecting the best antimicrobial agent for an individual patient;

· to accumulate epidemiological information on the resistance of microorganisms of public health importance within the community.

Susceptibility tests as a guide for treatment

Susceptibility tests should never be performed on contaminants or commensals belonging to the normal flora, or on other organisms that have no causal relationship to the infectious process. For example, the presence of Escherichia coli in the urine in less than significant numbers is not to be regarded as causing infection, and it would be useless and even misleading to perform an antibiogram.

Susceptibility tests should be carried out only on pure cultures of organisms considered to be causing the infectious process. The organisms should also be identified, as not every microorganism isolated from a patient with an infection requires an antibiogram.

Routine susceptibility tests are not indicated in the following situations:

· When the causative organism belongs to a species with predictable susceptibility to specific drugs. This is the case for Streptococcus pyogenes and Neisseria meningitidis, which are still generally susceptible to penicillin. (However, there have recently been a few reports of sporadic occurrences of penicillin-resistant meningococci.) It is also the case for faecal streptococci (enterococci), which, with few exceptions, are susceptible to ampicillin. If resistance of these microorganisms is suspected on clinical grounds, representative strains should be submitted to a competent reference laboratory.

· If the causative organism is slow-growing or fastidious and requires enriched media, e.g., Haemophilus influenzae and Neisseria gonorrhoeae, disc-diffusion susceptibility tests may give unreliable results.

The emergence of b-lactamase-producing variants of these species has led to the introduction of special tests, such as the in vitro test for b-lactamase production. It will be the responsibility of the central and regional laboratories to monitor the susceptibility of pneumococci, gonococci, and Haemophilus. If problems arise with resistant strains, the peripheral laboratories should be alerted and instructions should be given on appropriate testing methods and on alternative treatment schemes.

· In uncomplicated intestinal infections caused by salmonellae (other than S. typhi or S. paratyphi), susceptibility tests are not routinely needed. Antibiotic treatment of such infections is not justified, even with drugs showing in vitro activity. There is now ample evidence that antimicrobial treatment of common salmonella gastroenteritis (and indeed of most types of diarrhoeal disease of unknown etiology) is of no clinical benefit to the patient. Paradoxically, antibiotics prolong the excretion and dissemination of salmonellae and may lead to the selection of resistant variants.

Susceptibility tests as an epidemiological tool

Routine susceptibility tests on major pathogens (S. typhi, shigellae) are useful as part of a comprehensive programme of surveillance of enteric infections. They are essential for informing the physician of the emergence of resistant strains (chloramphenicol-resistant S. typhi, co-trimoxazole-resistant and ampicillin-resistant shigellae) and of the need to modify standard treatment schemes. Although susceptibility testing of non-typhoid salmonellae serotypes causing intestinal infection is not relevant for treating the patient, the appearance of multiresistant strains is a warning to the physician of the overuse and misuse of antimicrobial drugs.

Continued surveillance of the results of routine susceptibility tests is an excellent source of information on the prevalence of resistant staphylococci and Gram-negative bacilli that may be responsible for cross-infections in the hospital. Periodic reporting of the susceptibility pattern of the prevalent strains is an invaluable aid to forming a sound policy on antibiotic usage in the hospital by restriction and/or rotation of life-saving drugs, such as the aminoglycosides and cephalosporins.

Choice of drugs for routine susceptibility tests in the clinical laboratory

The choice of drugs used in a routine antibiogram is governed by considerations of the antibacterial spectrum of the drugs, their pharmacokinetic properties, toxicity, efficacy, and availability, as well as their cost to both the patient and the community. Among the many antibacterial agents that could be used to treat a patient infected with a given organism, only a limited number of carefully selected drugs should be included in the susceptibility test.

Table 12 indicates the drugs to be tested in various situations. The drugs in the table are divided into two sets. Set 1 includes the drugs that are available in most hospitals and for which routine testing should be carried out for every strain. Tests for drugs in set 2 are to be performed only at the special request of the physician, or when the causative organism is resistant to the first-choice drugs, or when other reasons (allergy to a drug, or its unavailability) make further testing justified. Many antibiotics with good clinical activity have been omitted from the table, but it must be emphasized that they are rarely needed in the management of the infected patient. In very rare cases, one or more additional drugs should be included when there is a special reason known to the physician, or when new and better drugs become available. Periodic revision of this table is therefore desirable, and this should be done after appropriate discussions with clinical staff. Many problems arise in practice, because clinicians are not always aware that only one representative of each group of antimicrobials is included in routine tests. The result obtained for this particular drug may then be extrapolated to all, or most, of the other members of the group. Difficulties arise in some countries when the physician is familiar only with the commercial brand name of the drug and not with its generic nonproprietary name. A serious effort should be made to inform medical personnel about the international nonproprietary names of pharmaceutical substances, and to encourage their use.1

1 International Nonproprietary Names for Pharmaceutical Substances, Cumulative List No. 7. Geneva, World Health Organization, 1988.

Table 12. Basic sets of drugs for routine susceptibility testsa


Staphylococcus

Enterobacteriaceae

Pseudomonas aeruginosa



Intestinal

Urinary

Blood and tissues


Set 1
First choice

benzylpenicillinoxacillinerythromycintetracycline

chloramphenicol

ampicillinchloramphenicolco-trimoxazolenalidixic acid

tetracycline

sulfonamidetrimethoprimco-trimoxazoleampicillinnitrofurantoinnalidixic acid

tetracycline

ampicillinchloramphenicolco-trimoxazoletetracyclinecefalotin

gentamicin

piperacillingentamicin

tobramycin

Set 2
Additional drugs

gentamicinamikacinco-trimoxazole

clindamycin

norfloxacin

norfloxacinchloramphenicol

gentamicin

cefuroximeceftriaxoneciprofloxacinpiperacillin

amikacin

amikacin

a Notes on the individual antibacterial agents are given in the text.

1. The benzylpenicillin disc is used to test susceptibility to all b-lactamase-sensitive penicillins (such as oral phenoxymethylpenicillin and pheneticillin). Isolates of staphylococci that fall into the resistant category produce b-lactamase and should be treated with a b-lactamase-resistant penicillin or with another antibiotic, such as erythromycin.

2. Oxacillin. The oxacillin disc is representative or the whole group of b-lactamase-resistant penicillins (including meticillin, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin). Moreover, there is good clinical evidence that cross-resistance exists between the meticillin and the cephalosporin groups. Therefore, it is useless and misleading to include cefalotin in the antibiogram for staphylococci. Resistance to meticillin and related drugs is often of the heterogeneous type, i.e., the majority of cells may be fully susceptible and produce a wide inhibition zone, while the resistant part of the population appears in the form of minute discrete colonies growing within the inhibition zone. This type of resistance is more apparent when the temperature of the incubator is set at 35 °C1 or when the incubation time is prolonged.

A serious disadvantage of meticillin, as a representative disc for the b-lactamase-resistant penicillins, is its great lability even under conventional storage conditions. The oxacillin disc is much more resistant to deterioration and is therefore preferred for the standardized diffusion test. The cloxacillin and dicloxacillin discs are not used as they may not indicate the presence of a heteroresistant strain.

1 SAHM, D. F. et al. Current concepts and approaches to antimicrobial agent susceptibility testing. In: Cumitech 25, Washington, DC, American Society for Microbiology, 1988.

3. The results for the tetracycline disc may be applied to chlortetracycline, oxytetracycline, and other members of this group. However, most tetracycline-resistant staphylococci remain normally sensitive to minocycline. A disc of minocycline may thus be useful to test multiresistant strains of staphylococci.

4. The result with the chloramphenicol disc may be extrapolated to thiamphenicol, a related drug with a comparable antibacterial spectrum, but without known risk of aplastic anaemia.

5. Only one representative sulfonamide (sulfafurazole) is required in the test.

6. The co-trimoxazole disc contains a combination of trimethoprim and a sulfonamide (sulfamethoxazole). Although the use of combinations of drugs in discs has been condemned in previous WHO reports,2 co-trimoxazole is an exception because the two components of this synergistic combination have comparable pharmacokinetic properties and generally act “as a single drug”.

2 WHO Technical Report Series, No. 796, 1990 (The use of essential drugs: fourth report of the WHO Expert Committee).

7. Ampicillin is the prototype of a group of broad-spectrum penicillins with activity against many Gram-negative bacteria. As it is susceptible to b-lactamase, it should not be used for testing staphylococci. Generally, the susceptibility to ampicillin is also valid for other members of this group: amoxycillin, pivampicillin, talampicillin, etc. (though amoxycillin is twice as active against salmonellae and only half as active against shigellae and H. influenzae).

8. Cefalotin. Only cefalotin needs to be tested routinely, as its spectrum is representative of all other first-generation cephalosporins (cefalexin, cefradine, cefaloridine, cefazolin, cefapirin). Where second- and third-generation cephalosporins and related compounds (cefamycins) with an expanded spectrum are available, a separate disc for some of these new drugs may be justified in selected cases (cefoxitin, cefamandole, cefuroxime, cefotaxime, ceftriaxone). Although some cephalosporins can be used to treat severe staphylococcal infections, the susceptibility of the infecting strain can be derived from the result with oxacillin as already mentioned under 2 above.

9. Erythromycin is used to test the susceptibility to some other members of the macrolide group (oleandomycin, spiramycin).

10. Aminoglycosides. This group of chemically related drugs includes streptomycin, gentamicin, kanamycin, netilmicin and tobramycin. Their antimicrobial spectra are not always closely enough related to permit assumption of cross-resistance, but against susceptible pathogens these agents have been shown to be equally effective. Numerous studies have compared the nephrotoxicity and ototoxicity of gentamicin, netilmicin and tobramycin, but there is no conclusive evidence that any one of the drugs is less toxic than the others. It is strongly recommended that each laboratory select a single agent for primary susceptibility testing. The other agents should be held in reserve for treatment of patients with infections caused by resistant organisms.

11. Nitrofurantoin is limited to use only in the treatment of urinary tract infections, and should not be tested against microorganisms recovered from material other than urine.

The modified Kirby-Bauer method

The disc diffusion method, originally described in 1966,1 is well standardized and has been widely evaluated. Official agencies have recommended it, with minor modifications, as a reference method which could be used as a routine technique in the clinical laboratory.

1 BAUER, A. W. et al. Antibiotic susceptibility testing by a standardized single disc method. American journal of clinical pathology, 44: 493-496 (1966).

Reagents

Mueller-Hinton agar

1. Mueller-Hinton agar should be prepared from a dehydrated base according to the manufacturer’s recommendations. The medium should be such that control zone sizes within the published limits are produced (see Table 13). It is important not to overheat the medium.

2. Cool the medium to 45-50 °C and pour into the plates. Allow to set on a level surface, to a depth of approximately 4 mm. A 9-cm plate requires approximately 25 ml of medium.

3. When the agar has solidified, dry the plates for immediate use for 10 - 30 minutes at 35 °C, by placing them in the upright position in the incubator with the lids tilted.

4. Any unused plates may be stored in a plastic bag, which should be sealed and placed in the refrigerator. Plates stored in this way will keep for 2 weeks.

To ensure that the zone diameters are sufficiently reliable for testing susceptibility to sulfonamides and co-trimoxazole, the Mueller-Hinton agar must have low concentrations of the inhibitors thymidine and thymine. Each new lot of Mueller-Hinton agar should therefore be tested with a control strain of Enterococcus faecalis (ATCC 29212 or 33186) and a disc of co-trimoxazole. A satisfactory lot of medium will give a distinct inhibition zone of 20 mm or more that is essentially free of hazy growth or fine colonies.

Antibiotic discs

Any commercially available discs with the proper diameter and potency can be used. Stocks of antibiotic discs should preferably be kept at - 20°C; the freezer compartment of a home refrigerator is convenient. A small working supply of discs can be kept in the refrigerator for up to 1 month. On removal from the refrigerator, the containers should be left at room temperature for about 1 hour to allow the temperature to equilibrate. This procedure reduces the amount of condensation that occurs when warm air reaches the cold container. If a disc-dispensing apparatus is used, it should have a tight-fitting cover and be stored in the refrigerator. It should also be allowed to warm to room temperature before being opened.

Table 13. Zone diameter limits for control strainsa



Diameter of zone of inhibition (mm)

Antibiotic

Disc potency

S. aureus
(ATCC 25923)

E. coli
(ATCC 25922)

P. aeruginosa
(ATCC 27853)

amikacin

30 mg

20-26

19-26

18-26

ampicillin

10 mg

27-35

16-22

-

benzylpenicillin

10 IU

26-37

-

-

cefalotin

30 mg

29-37

17-22

-

ceftriaxone

30 mg

22-28

29-35

17-23

cefuroxime

30 mg

27-35

20-26

-

chloramphenicol

30 mg

19-26

21-27

-

ciprofloxacin

100 mg

22-30

30-40

25-33

clindamycin

2 mg

24-30

-

-

co-trimoxazole

25 mg

24-32

24-32

-

erythromycin

15 mg

22-30

8-14

-

gentamicin

10 mg

19-27

19-26

16-21

nalidixic acid

30 mg

-

22-28

-

nitrofurantoin

300 mg

18-22

20-25

-

norfloxacin

10 mg

17-28

28-35

22-29

oxacillin

1 mg

18-24

-

-

piperacillin

100 mg

-

24-30

25-33

sulfonamideb

300 mg

24-34

18-26

-

tetracycline

30 mg

19-28

18-25

-

tobramycin

10 mg

19-29

18-26

19-25

trimethoprim

5 mg

19-26

21-28

-

a National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disc susceptibility tests. Tentative standard. 4th ed. Villanova, PA, USA, NCCLS, 1988.

b Sulfafurazole.

Turbidity standard

Prepare the turbidity standard by pouring 0.6 ml of a 1% (10 g/litre) solution of barium chloride dihydrate into a 100-ml graduated cylinder, and filling to 100 ml with 1% (10 ml/litre) sulfuric acid. The turbidity standard solution should be placed in a tube identical to the one used for the broth sample. It can be stored in the dark at room temperature for 6 months, provided it is sealed to prevent evaporation.

Swabs

A supply of cotton wool swabs on wooden applicator sticks should be prepared. They can be sterilized in tins, culture tubes, or on paper, either in the autoclave or by dry heat.

Procedure

To prepare the inoculum from the primary culture plate, touch with a loop the tops of each of 3 - 5 colonies, of similar appearance, of the organism to be tested.

What factors affect disk diffusion method?

Figure

Transfer this growth to a tube of saline.

What factors affect disk diffusion method?

Figure

When the inoculum has to be made from a pure culture, a loopful of the confluent growth is similarly suspended in saline.

Compare the tube with the turbidity standard and adjust the density of the test suspension to that of the standard by adding more bacteria or more sterile saline.

What factors affect disk diffusion method?

Figure

Proper adjustment of the turbidity or the inoculum is essential to ensure that the resulting lawn of growth is confluent or almost confluent.

Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess inoculum by pressing and rotating the swab firmly against the side of the tube above the level of the liquid.

What factors affect disk diffusion method?

Figure

Streak the swab all over the surface of the medium three times, rotating the plate through an angle of 60 ° after each application. Finally, pass the swab round the edge of the agar surface. Leave the inoculum to dry for a few minutes at room temperature with the lid closed.

What factors affect disk diffusion method?

Figure

The antibiotic discs may be placed on the inoculated plates using a pair of sterile forceps. It is convenient to use a template (see Fig. 10) to place the discs uniformly.

What factors affect disk diffusion method?

Figure

A sterile needle tip may also be used to place the antibiotic discs on the plate.

What factors affect disk diffusion method?

Figure

Alternatively, an antibiotic disc dispenser can be used to apply the discs to the inoculated plate.

What factors affect disk diffusion method?

Figure

A maximum of seven discs can be placed on a 9-10 cm plate. Six discs may be spaced evenly, approximately 15 mm from the edge of the plate, and 1 disc placed in the centre of the plate. Each disc should be gently pressed down to ensure even contact with the medium.

The plates should be placed in an incubator at 35 °C within 30 minutes of preparation. Temperatures above 35 °C invalidate results for oxacillin/meticillin.

Do not incubate in an atmosphere of carbon dioxide.

After overnight incubation, the diameter of each zone (including the diameter of the disc) should be measured and recorded in mm. The results should then be interpreted according to the critical diameters shown in Table 14.

The measurements can be made with a ruler on the under-surface of the plate without opening the lid.

What factors affect disk diffusion method?

Figure

If the medium is opaque, the zone can be measured by means of a pair of calipers.

What factors affect disk diffusion method?

Figure

A template (see Fig. 9) may be used to assess the final result of the susceptibility tests.

What factors affect disk diffusion method?

Figure

The endpoint of inhibition is judged by the naked eye at the edge where the growth starts, but there are three exceptions:

· With sulfonamides and co-trimoxazole, slight growth occurs within the inhibition zone; such growth should be ignored.

· When b-lactamase-producing staphylococci are tested against penicillin, zones of inhibition are produced with a heaped-up, clearly defined edge; these are readily recognizable when compared with the sensitive control, and regardless of size of zone of inhibition, they should be reported as resistant.

· Certain Proteus species may swarm into the area of inhibition around some antibiotics, but the zone of inhibition is usually clearly outlined and the thin layer of swarming growth should be ignored.

Interpretation of the zone sizes

· Using a template. When the zone sizes are compared with the template (see Fig. 9) the result - susceptible, resistant, or intermediate - can be read at once: “susceptible”, when the zone edge is outside the black circle; “resistant”, when there is no zone, or when it lies within the white circle; and “intermediate”, when the edge of the zone of inhibition lies on the black circle.

· Using a ruler. When the zone sizes are measured in mm, the results should be interpreted according to the critical diameters given in Table 14.

Direct versus indirect susceptibility tests

In the standardized method outlined above, the inoculum is prepared from colonies on a primary culture plate or from a pure culture. This is called an “indirect sensitivity test”. In certain cases, where a rapid answer is important, the standardized inoculum may be replaced by the pathological specimen itself, e.g., urine, a positive blood culture, or a swab of pus. For specimens of urine, a microscopical examination of the sediment should first be made in order to see if there is evidence of infection, i.e., the presence of pus cells and/or organisms. The urine may then be used as the inoculum in the standard test. Likewise, susceptibility tests may be performed on incubated blood cultures showing evidence of bacterial growth, or a swab of pus may be used as a direct inoculum, when a Gram-stained smear shows the presence of large numbers of a single type of organism. This is called a “direct susceptibility test”; its advantage over the indirect test is that a result is obtained 24 hours earlier. The main disadvantage is that the inoculum cannot be properly controlled. When the susceptibility plate shows too light or too heavy growth, or when the culture is a mixture, the results should be interpreted with caution, and the test repeated on pure cultures.

Inoculum density

If the inoculum is too light, the inhibition zones will be larger although the sensitivity of the organism is unchanged. Relatively resistant strains may then be reported as susceptible. Conversely, if the inoculum is too heavy, the zone size will be reduced and susceptible strains may be reported as resistant. Usually optimal results are obtained with an inoculum size that produces near confluent growth.

What factors affect disk diffusion method?

Fig. 9. Zone diameters for the determination of susceptibility with the standard disc diffusion method

Table 14. Interpretative chart of zone sizesa


Diameter of zone of inhibition (mm)

Antibiotic or chemotherapeutic agent

Disc potency

Resistant

Intermediate/
moderately susceptible

Susceptible

amikacin

30 mg

£ 14

15-16

³ 17

ampicillin when testing:





- Enterobacteriaceae

10 mg

£ 13

14-16

³ 17

- Enterococcus faecalis

10 mg

£ 16

-

³ 17

benzylpenicillin when testing staphylococci

10 IU

£ 28

-

³ 29

ceftriaxone

30 mg

£ 13

14-20

³ 21

cefuroxime sodium

30 mg

£ 14

15-17

³ 18

cefalotin

30 mg

£ 14

15-17

³ 18

chloramphenicol

30 mg

£ 12

13-17

³ 18

clindamycin

2 mg

£ 14

15-20

³ 21

co-trimoxazole

25 mg

£ 10

11-15

³ 16

erythromycin

15 mg

£ 13

14-22

³ 23

gentamicin

10 mg

£ 12

13-14

³ 15

nalidixic acid

30 mg

£ 13

14-18

³ 19

nitrofurantoin

300 mg

£ 14

15-16

³ 17

oxacillin when testing:





- staphylococci

1 mg

£ 10

11-12

³ 13

- pneumococci

1 mg

£ 19

-

³ 20

piperacillin when testing:





- Enterobacteriaceae

100 mg

£ 17

18-20

³ 21

- Pseudomonas

100 mg

£ 14

15-17

³ 18

sulfonamides

300 mg

£ 12

13-16

³ 17

tetracycline

30 mg

£ 14

15-18

³ 19

tobramycin

10 mg

£ 12

13-14

³ 15

trimethoprim

5 mg

£ 10

11-15

³ 16

a National Committee for Clinical Laboratory Standards. Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

Timing of disc application

If the plates, after being seeded with the test strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take place before the discs are applied. This causes a reduction in the zone diameter and may result in a susceptible strain being reported as resistant.

Temperature of incubation

Susceptibility tests are normally incubated at 35 °C for optimal growth. If the temperature is lowered, the time required for effective growth is extended and larger zones result. When a heterogeneous resistant strain of Staphylococcus aureus is being tested against meticillin (oxacillin), the resistant portion of the population can be detected at 35 °C. At higher temperatures the entire culture appears to be susceptible. At 35 °C or lower temperatures, resistant colonies develop within the zone of inhibition. These resistant colonies can be seen more easily if the plate is left for several hours at room temperature before the result is read. Such colonies should always be identified to check whether they are contaminants.

What factors affect disk diffusion method?

Fig. 10. Template for uniform placement of susceptibility discs on plates of 90 mm diameter

Incubation time

Most techniques adopt an incubation period of between 16 and 18 hours. In emergencies, however, a provisional report may be made after 6 hours. This is not recommended as a routine and the result should always be confirmed after the conventional incubation time.

Size of plate, depth of agar medium, and spacing of the antibiotic discs

Susceptibility tests are usually carried out with 9 - 10 cm plates and no more than 6 or 7 antibiotic discs on each plate. If larger numbers of antibiotics have to be tested, two plates, or one 14-cm diameter plate, is to be preferred. Excessively large inhibition zones may be formed on very thin media; the converse is true for thick media. Minor changes in the depth of the agar layer have negligible effect. Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or deformation near the edge of the plates (see Fig. 10).

Potency of the antibiotic discs

The diameter of the inhibition zone is related to the amount of drug in the disc. If the potency of the drug is reduced owing to deterioration during storage, the inhibition zone will show a corresponding reduction in size.

Composition of the medium

The medium influences the size of the zone by its effect on the rate of growth of the organism, the rate of diffusion of the antibiotic, and the activity of the agent. It is essential to use the medium appropriate to the particular method.

The many factors influencing the zone diameters that may be obtained for the same test organism clearly demonstrate the need for standardization of disc diffusion methods. Only if the conditions laid down in a particular method are closely followed can valid results be obtained. Alteration of any of the factors affecting the test can result in grossly misleading reports for the clinician.

The precision and accuracy of the method should be monitored by establishing the quality control programme described below. Variations can then be immediately investigated and corrective action taken to eliminate them.

Quality control

The need for quality control in the susceptibility test

The final result of a disc diffusion test is influenced by a large number of variables. Some of the factors, such as the inoculum density and the incubation temperature, are easy to control, but a laboratory rarely knows the exact composition of a commercial medium or the batch-to-batch variations in its quality, and it cannot take for granted the antimicrobial content of the discs. The results of the test must therefore be monitored constantly by a quality control programme, which should be considered part of the procedure itself.

The precision and accuracy of the test are controlled by the parallel use of a set of control strains, with known susceptibility to the antimicrobial agents. These quality control strains are tested using exactly the same procedure as for the test organisms. The zone sizes shown by the control organisms should fall within the range of diameters given in Table 13. When results regularly fall outside this range, they should be regarded as evidence that a technical error has been introduced into the test, or that the reagents are at fault. Each reagent and each step in the test should then be investigated until the cause of the error has been found and eliminated.

Standard procedure for quality control

The quality control programme should use standard reference strains of bacteria that are tested in parallel with the clinical cultures. They should preferably be run every week, or with every fifth batch of tests, and, in addition, every time that a new batch of Mueller-Hinton agar or a new batch of discs is used.

Standard strains for quality control

Staphylococcus aureus (ATCC 25923)
Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC 27853)

These cultures can be obtained from national culture collections. They are commercially available in the form of pellets of desiccated pure cultures.

Cultures for day-to-day use should be grown on slants of nutrient agar (tryptic soy agar is convenient) and stored in the refrigerator. They should be subcultured on to fresh slants every 2 weeks.

Preparing the inoculum

The cultures may be inoculated into any type of broth, and incubated until the broth is turbid. Each broth should be streaked on to an agar plate and incubated overnight. Single colonies should then be picked off and submitted to susceptibility tests as described on “Procedure”.

Placing antimicrobial discs

After the inoculum has been streaked on to the plates, as described on “Procedure”, the appropriate discs should be applied. The discs to be selected for each control strain are listed in Table 13.

Reading the plates

After 16 to 18 hours’ incubation, the diameters of the inhibition zones should be measured with a ruler and recorded, together with the date of the test, on a special quality control chart. This chart should display data for each disc - strain combination. The chart is labelled in millimetres, with an indication of the range of acceptable zone sizes. An example of such a chart is shown in Fig. 11. When the results consistently fall outside the acceptable limits, action should be taken to improve the quality of the test.

Grossly aberrant results, which cannot be explained by technical errors in the procedure, may indicate contamination or sudden changes in the susceptibility or growth characteristics of the control strain. If this occurs, a fresh stock-strain should be obtained from a reliable source.

What factors affect disk diffusion method?

Fig. 11. Quality control chart for antimicrobial susceptibility testing

Control disc: Ampicillin disc 10 mg. Control strain: E. coli ATCC 25922


Page 2

Inoculum density

If the inoculum is too light, the inhibition zones will be larger although the sensitivity of the organism is unchanged. Relatively resistant strains may then be reported as susceptible. Conversely, if the inoculum is too heavy, the zone size will be reduced and susceptible strains may be reported as resistant. Usually optimal results are obtained with an inoculum size that produces near confluent growth.

What factors affect disk diffusion method?

Fig. 9. Zone diameters for the determination of susceptibility with the standard disc diffusion method

Table 14. Interpretative chart of zone sizesa


Diameter of zone of inhibition (mm)

Antibiotic or chemotherapeutic agent

Disc potency

Resistant

Intermediate/
moderately susceptible

Susceptible

amikacin

30 mg

£ 14

15-16

³ 17

ampicillin when testing:





- Enterobacteriaceae

10 mg

£ 13

14-16

³ 17

- Enterococcus faecalis

10 mg

£ 16

-

³ 17

benzylpenicillin when testing staphylococci

10 IU

£ 28

-

³ 29

ceftriaxone

30 mg

£ 13

14-20

³ 21

cefuroxime sodium

30 mg

£ 14

15-17

³ 18

cefalotin

30 mg

£ 14

15-17

³ 18

chloramphenicol

30 mg

£ 12

13-17

³ 18

clindamycin

2 mg

£ 14

15-20

³ 21

co-trimoxazole

25 mg

£ 10

11-15

³ 16

erythromycin

15 mg

£ 13

14-22

³ 23

gentamicin

10 mg

£ 12

13-14

³ 15

nalidixic acid

30 mg

£ 13

14-18

³ 19

nitrofurantoin

300 mg

£ 14

15-16

³ 17

oxacillin when testing:





- staphylococci

1 mg

£ 10

11-12

³ 13

- pneumococci

1 mg

£ 19

-

³ 20

piperacillin when testing:





- Enterobacteriaceae

100 mg

£ 17

18-20

³ 21

- Pseudomonas

100 mg

£ 14

15-17

³ 18

sulfonamides

300 mg

£ 12

13-16

³ 17

tetracycline

30 mg

£ 14

15-18

³ 19

tobramycin

10 mg

£ 12

13-14

³ 15

trimethoprim

5 mg

£ 10

11-15

³ 16

a National Committee for Clinical Laboratory Standards. Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

Timing of disc application

If the plates, after being seeded with the test strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take place before the discs are applied. This causes a reduction in the zone diameter and may result in a susceptible strain being reported as resistant.

Temperature of incubation

Susceptibility tests are normally incubated at 35 °C for optimal growth. If the temperature is lowered, the time required for effective growth is extended and larger zones result. When a heterogeneous resistant strain of Staphylococcus aureus is being tested against meticillin (oxacillin), the resistant portion of the population can be detected at 35 °C. At higher temperatures the entire culture appears to be susceptible. At 35 °C or lower temperatures, resistant colonies develop within the zone of inhibition. These resistant colonies can be seen more easily if the plate is left for several hours at room temperature before the result is read. Such colonies should always be identified to check whether they are contaminants.

What factors affect disk diffusion method?

Fig. 10. Template for uniform placement of susceptibility discs on plates of 90 mm diameter

Incubation time

Most techniques adopt an incubation period of between 16 and 18 hours. In emergencies, however, a provisional report may be made after 6 hours. This is not recommended as a routine and the result should always be confirmed after the conventional incubation time.

Size of plate, depth of agar medium, and spacing of the antibiotic discs

Susceptibility tests are usually carried out with 9 - 10 cm plates and no more than 6 or 7 antibiotic discs on each plate. If larger numbers of antibiotics have to be tested, two plates, or one 14-cm diameter plate, is to be preferred. Excessively large inhibition zones may be formed on very thin media; the converse is true for thick media. Minor changes in the depth of the agar layer have negligible effect. Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or deformation near the edge of the plates (see Fig. 10).

Potency of the antibiotic discs

The diameter of the inhibition zone is related to the amount of drug in the disc. If the potency of the drug is reduced owing to deterioration during storage, the inhibition zone will show a corresponding reduction in size.

Composition of the medium

The medium influences the size of the zone by its effect on the rate of growth of the organism, the rate of diffusion of the antibiotic, and the activity of the agent. It is essential to use the medium appropriate to the particular method.

The many factors influencing the zone diameters that may be obtained for the same test organism clearly demonstrate the need for standardization of disc diffusion methods. Only if the conditions laid down in a particular method are closely followed can valid results be obtained. Alteration of any of the factors affecting the test can result in grossly misleading reports for the clinician.

The precision and accuracy of the method should be monitored by establishing the quality control programme described below. Variations can then be immediately investigated and corrective action taken to eliminate them.


Page 3

Inoculum density

If the inoculum is too light, the inhibition zones will be larger although the sensitivity of the organism is unchanged. Relatively resistant strains may then be reported as susceptible. Conversely, if the inoculum is too heavy, the zone size will be reduced and susceptible strains may be reported as resistant. Usually optimal results are obtained with an inoculum size that produces near confluent growth.

What factors affect disk diffusion method?

Fig. 9. Zone diameters for the determination of susceptibility with the standard disc diffusion method

Table 14. Interpretative chart of zone sizesa


Diameter of zone of inhibition (mm)

Antibiotic or chemotherapeutic agent

Disc potency

Resistant

Intermediate/
moderately susceptible

Susceptible

amikacin

30 mg

£ 14

15-16

³ 17

ampicillin when testing:





- Enterobacteriaceae

10 mg

£ 13

14-16

³ 17

- Enterococcus faecalis

10 mg

£ 16

-

³ 17

benzylpenicillin when testing staphylococci

10 IU

£ 28

-

³ 29

ceftriaxone

30 mg

£ 13

14-20

³ 21

cefuroxime sodium

30 mg

£ 14

15-17

³ 18

cefalotin

30 mg

£ 14

15-17

³ 18

chloramphenicol

30 mg

£ 12

13-17

³ 18

clindamycin

2 mg

£ 14

15-20

³ 21

co-trimoxazole

25 mg

£ 10

11-15

³ 16

erythromycin

15 mg

£ 13

14-22

³ 23

gentamicin

10 mg

£ 12

13-14

³ 15

nalidixic acid

30 mg

£ 13

14-18

³ 19

nitrofurantoin

300 mg

£ 14

15-16

³ 17

oxacillin when testing:





- staphylococci

1 mg

£ 10

11-12

³ 13

- pneumococci

1 mg

£ 19

-

³ 20

piperacillin when testing:





- Enterobacteriaceae

100 mg

£ 17

18-20

³ 21

- Pseudomonas

100 mg

£ 14

15-17

³ 18

sulfonamides

300 mg

£ 12

13-16

³ 17

tetracycline

30 mg

£ 14

15-18

³ 19

tobramycin

10 mg

£ 12

13-14

³ 15

trimethoprim

5 mg

£ 10

11-15

³ 16

a National Committee for Clinical Laboratory Standards. Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

Timing of disc application

If the plates, after being seeded with the test strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take place before the discs are applied. This causes a reduction in the zone diameter and may result in a susceptible strain being reported as resistant.

Temperature of incubation

Susceptibility tests are normally incubated at 35 °C for optimal growth. If the temperature is lowered, the time required for effective growth is extended and larger zones result. When a heterogeneous resistant strain of Staphylococcus aureus is being tested against meticillin (oxacillin), the resistant portion of the population can be detected at 35 °C. At higher temperatures the entire culture appears to be susceptible. At 35 °C or lower temperatures, resistant colonies develop within the zone of inhibition. These resistant colonies can be seen more easily if the plate is left for several hours at room temperature before the result is read. Such colonies should always be identified to check whether they are contaminants.

What factors affect disk diffusion method?

Fig. 10. Template for uniform placement of susceptibility discs on plates of 90 mm diameter

Incubation time

Most techniques adopt an incubation period of between 16 and 18 hours. In emergencies, however, a provisional report may be made after 6 hours. This is not recommended as a routine and the result should always be confirmed after the conventional incubation time.

Size of plate, depth of agar medium, and spacing of the antibiotic discs

Susceptibility tests are usually carried out with 9 - 10 cm plates and no more than 6 or 7 antibiotic discs on each plate. If larger numbers of antibiotics have to be tested, two plates, or one 14-cm diameter plate, is to be preferred. Excessively large inhibition zones may be formed on very thin media; the converse is true for thick media. Minor changes in the depth of the agar layer have negligible effect. Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or deformation near the edge of the plates (see Fig. 10).

Potency of the antibiotic discs

The diameter of the inhibition zone is related to the amount of drug in the disc. If the potency of the drug is reduced owing to deterioration during storage, the inhibition zone will show a corresponding reduction in size.

Composition of the medium

The medium influences the size of the zone by its effect on the rate of growth of the organism, the rate of diffusion of the antibiotic, and the activity of the agent. It is essential to use the medium appropriate to the particular method.

The many factors influencing the zone diameters that may be obtained for the same test organism clearly demonstrate the need for standardization of disc diffusion methods. Only if the conditions laid down in a particular method are closely followed can valid results be obtained. Alteration of any of the factors affecting the test can result in grossly misleading reports for the clinician.

The precision and accuracy of the method should be monitored by establishing the quality control programme described below. Variations can then be immediately investigated and corrective action taken to eliminate them.


Page 4

Inoculum density

If the inoculum is too light, the inhibition zones will be larger although the sensitivity of the organism is unchanged. Relatively resistant strains may then be reported as susceptible. Conversely, if the inoculum is too heavy, the zone size will be reduced and susceptible strains may be reported as resistant. Usually optimal results are obtained with an inoculum size that produces near confluent growth.

What factors affect disk diffusion method?

Fig. 9. Zone diameters for the determination of susceptibility with the standard disc diffusion method

Table 14. Interpretative chart of zone sizesa


Diameter of zone of inhibition (mm)

Antibiotic or chemotherapeutic agent

Disc potency

Resistant

Intermediate/
moderately susceptible

Susceptible

amikacin

30 mg

£ 14

15-16

³ 17

ampicillin when testing:





- Enterobacteriaceae

10 mg

£ 13

14-16

³ 17

- Enterococcus faecalis

10 mg

£ 16

-

³ 17

benzylpenicillin when testing staphylococci

10 IU

£ 28

-

³ 29

ceftriaxone

30 mg

£ 13

14-20

³ 21

cefuroxime sodium

30 mg

£ 14

15-17

³ 18

cefalotin

30 mg

£ 14

15-17

³ 18

chloramphenicol

30 mg

£ 12

13-17

³ 18

clindamycin

2 mg

£ 14

15-20

³ 21

co-trimoxazole

25 mg

£ 10

11-15

³ 16

erythromycin

15 mg

£ 13

14-22

³ 23

gentamicin

10 mg

£ 12

13-14

³ 15

nalidixic acid

30 mg

£ 13

14-18

³ 19

nitrofurantoin

300 mg

£ 14

15-16

³ 17

oxacillin when testing:





- staphylococci

1 mg

£ 10

11-12

³ 13

- pneumococci

1 mg

£ 19

-

³ 20

piperacillin when testing:





- Enterobacteriaceae

100 mg

£ 17

18-20

³ 21

- Pseudomonas

100 mg

£ 14

15-17

³ 18

sulfonamides

300 mg

£ 12

13-16

³ 17

tetracycline

30 mg

£ 14

15-18

³ 19

tobramycin

10 mg

£ 12

13-14

³ 15

trimethoprim

5 mg

£ 10

11-15

³ 16

a National Committee for Clinical Laboratory Standards. Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

Timing of disc application

If the plates, after being seeded with the test strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take place before the discs are applied. This causes a reduction in the zone diameter and may result in a susceptible strain being reported as resistant.

Temperature of incubation

Susceptibility tests are normally incubated at 35 °C for optimal growth. If the temperature is lowered, the time required for effective growth is extended and larger zones result. When a heterogeneous resistant strain of Staphylococcus aureus is being tested against meticillin (oxacillin), the resistant portion of the population can be detected at 35 °C. At higher temperatures the entire culture appears to be susceptible. At 35 °C or lower temperatures, resistant colonies develop within the zone of inhibition. These resistant colonies can be seen more easily if the plate is left for several hours at room temperature before the result is read. Such colonies should always be identified to check whether they are contaminants.

What factors affect disk diffusion method?

Fig. 10. Template for uniform placement of susceptibility discs on plates of 90 mm diameter

Incubation time

Most techniques adopt an incubation period of between 16 and 18 hours. In emergencies, however, a provisional report may be made after 6 hours. This is not recommended as a routine and the result should always be confirmed after the conventional incubation time.

Size of plate, depth of agar medium, and spacing of the antibiotic discs

Susceptibility tests are usually carried out with 9 - 10 cm plates and no more than 6 or 7 antibiotic discs on each plate. If larger numbers of antibiotics have to be tested, two plates, or one 14-cm diameter plate, is to be preferred. Excessively large inhibition zones may be formed on very thin media; the converse is true for thick media. Minor changes in the depth of the agar layer have negligible effect. Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or deformation near the edge of the plates (see Fig. 10).

Potency of the antibiotic discs

The diameter of the inhibition zone is related to the amount of drug in the disc. If the potency of the drug is reduced owing to deterioration during storage, the inhibition zone will show a corresponding reduction in size.

Composition of the medium

The medium influences the size of the zone by its effect on the rate of growth of the organism, the rate of diffusion of the antibiotic, and the activity of the agent. It is essential to use the medium appropriate to the particular method.

The many factors influencing the zone diameters that may be obtained for the same test organism clearly demonstrate the need for standardization of disc diffusion methods. Only if the conditions laid down in a particular method are closely followed can valid results be obtained. Alteration of any of the factors affecting the test can result in grossly misleading reports for the clinician.

The precision and accuracy of the method should be monitored by establishing the quality control programme described below. Variations can then be immediately investigated and corrective action taken to eliminate them.


Page 5

Introduction

At a meeting organized by WHO in Geneva in 1977,1 concern was expressed about the worldwide increase in antibiotic resistance associated with the growing, and frequently indiscriminate, use of antibiotics in both man and animals. In recent years, drug-resistant bacteria have given rise to several serious outbreaks of infection, with many deaths. This has led to a need for national and international surveillance programmes to monitor antibiotic resistance in bacteria by susceptibility testing using reliable methods that generate comparable data. The availability of microbiological and epidemiological information would help clinicians in selecting the most appropriate antimicrobial agent for the treatment of a microbial infection.

If predictions are to be valid, the susceptibility test must be performed by an accurate and reproducible method, the results of which should be directly applicable to the clinical situation. The ultimate criterion of the reliability of any susceptibility testing method is its correlation with the response of the patient to antimicrobial therapy.

The WHO meeting considered that the modified disc technique of Kirby-Bauer, for which requirements had been established by WHO in I976,2 could be recommended for clinical and surveillance purposes in view of its technical simplicity and reproducibility. The method is particularly suitable for use with bacteria belonging to the family Enterobacteriaceae, but it can also be recommended as a general purpose method for all rapidly growing pathogens, except strict anaerobes. It was therefore recommended that the details of this test be made available for laboratory workers.3

1 WHO Technical Report Series, No. 624, 1978 (Surveillance for the prevention and control of health hazards due to antibiotic-resistant enterobacteria: report of a WHO Meeting).

2 WHO Technical Report Series, No. 610, 1977 (Twenty-eighth report of the WHO Expert Committee on Biological Standardization), Annex 5.

3 A comparable method, based on the same principles and quality control requirements as the Kirby-Bauer method, is the NEO-SENSITABS method, produced by ROSCO Diagnostica, Taastrup, Denmark. This method uses 9-mm colour-coded, antibiotic tablets, instead of paper discs. The tablet form results in an extraordinary stability with a shelf-life of four years, even at room temperature. This increased stability is very important for laboratories in tropical countries.

General principles of antimicrobial susceptibility testing

Antimicrobial susceptibility tests measure the ability of an antibiotic or other antimicrobial agent to inhibit bacterial growth in vitro. This ability may be estimated by either the dilution method or the diffusion method.

The dilution test

For quantitative estimates of antibiotic activity, dilutions of the antibiotic may be incorporated into broth or agar medium, which is then inoculated with the test organism. The lowest concentration that prevents growth after overnight incubation is known as the minimum inhibitory concentration (MIC) of the agent. This MIC value is then compared with known concentrations of the drug obtainable in the serum and in other body fluids to assess the likely clinical response.

The diffusion test

Paper discs, impregnated with the antibiotic, are placed on agar medium uniformly seeded with the test organism. A concentration gradient of the antibiotic forms by diffusion from the disc and the growth of the test organism is inhibited at a distance from the disc that is related, among other factors, to the susceptibility of the organism.

There is an approximately linear relation between log MIC, as measured by a dilution test, and the inhibition zone diameter in the diffusion test. A regression line expressing this relation can be obtained by testing a large number of strains by the two methods in parallel (see Fig. 7 and 8).

What factors affect disk diffusion method?

Fig. 7. Graphic representation of the relationship between log2 MIC and the inhibition zone diameter obtained by the diffusion test using discs containing a single concentration of antibiotic

What factors affect disk diffusion method?

Fig. 8. Interpretation of zone sizes as susceptible, intermediate, and resistant by their relationship to the MIC

Clinical definition of terms “resistant” and “susceptible”: the three-category system

The result of the susceptibility test, as reported to the clinician, is the classification of the microorganism in one of two or more categories of susceptibility. The simplest system comprises only two categories: susceptible and resistant. This classification, although offering many advantages for statistical and epidemiological purposes, is too inflexible for the clinician to use. Therefore, a three-category classification is often adopted. The Kirby-Bauer method and its modifications recognize three categories of susceptibility and it is important that both the clinician and the laboratory worker understand the exact definitions and the clinical significance of these categories.

· Susceptible. An organism is called “susceptible” to a drug when the infection caused by it is likely to respond to treatment with this drug, at the recommended dosage.

· Intermediate susceptibility covers two situations. It is applicable to strains that are “moderately susceptible” to an antibiotic that can be used for treatment at a higher dosage because of its low toxicity or because the antibiotic is concentrated in the focus of infection (e.g., urine).

The classification also applies to strains that show “intermediate susceptibility” to a more toxic antibiotic that cannot be used at a higher dosage. In this situation, the intermediate category serves as a buffer zone between susceptible and resistant.

As most clinicians are not familiar with the subtle, although clinically important, distinction between intermediate and moderate susceptibility, many laboratories use the designation “intermediate” for reporting purposes.

· Resistant. This term implies that the organism is expected not to respond to a given drug, irrespective of the dosage and of the location of the infection.

For testing the response of staphylococci to benzylpenicillin, only the categories “susceptible” and “resistant” (corresponding to the production of b-lactamase) are recognized.

The ultimate decision to use a particular antibiotic, and the dosage to be given, will depend not only on the results of the susceptibility tests, but also on their interpretation by the physician. Other factors, such as pathogenic significance of the microorganism, side-effects and pharmacokinetic properties of the drug, its diffusion in different body sites, and the immune status of the host, will also have to be considered.

Indications for routine susceptibility tests

A susceptibility test may be performed in the clinical laboratory for two main purposes:

· to guide the clinician in selecting the best antimicrobial agent for an individual patient;

· to accumulate epidemiological information on the resistance of microorganisms of public health importance within the community.

Susceptibility tests as a guide for treatment

Susceptibility tests should never be performed on contaminants or commensals belonging to the normal flora, or on other organisms that have no causal relationship to the infectious process. For example, the presence of Escherichia coli in the urine in less than significant numbers is not to be regarded as causing infection, and it would be useless and even misleading to perform an antibiogram.

Susceptibility tests should be carried out only on pure cultures of organisms considered to be causing the infectious process. The organisms should also be identified, as not every microorganism isolated from a patient with an infection requires an antibiogram.

Routine susceptibility tests are not indicated in the following situations:

· When the causative organism belongs to a species with predictable susceptibility to specific drugs. This is the case for Streptococcus pyogenes and Neisseria meningitidis, which are still generally susceptible to penicillin. (However, there have recently been a few reports of sporadic occurrences of penicillin-resistant meningococci.) It is also the case for faecal streptococci (enterococci), which, with few exceptions, are susceptible to ampicillin. If resistance of these microorganisms is suspected on clinical grounds, representative strains should be submitted to a competent reference laboratory.

· If the causative organism is slow-growing or fastidious and requires enriched media, e.g., Haemophilus influenzae and Neisseria gonorrhoeae, disc-diffusion susceptibility tests may give unreliable results.

The emergence of b-lactamase-producing variants of these species has led to the introduction of special tests, such as the in vitro test for b-lactamase production. It will be the responsibility of the central and regional laboratories to monitor the susceptibility of pneumococci, gonococci, and Haemophilus. If problems arise with resistant strains, the peripheral laboratories should be alerted and instructions should be given on appropriate testing methods and on alternative treatment schemes.

· In uncomplicated intestinal infections caused by salmonellae (other than S. typhi or S. paratyphi), susceptibility tests are not routinely needed. Antibiotic treatment of such infections is not justified, even with drugs showing in vitro activity. There is now ample evidence that antimicrobial treatment of common salmonella gastroenteritis (and indeed of most types of diarrhoeal disease of unknown etiology) is of no clinical benefit to the patient. Paradoxically, antibiotics prolong the excretion and dissemination of salmonellae and may lead to the selection of resistant variants.

Susceptibility tests as an epidemiological tool

Routine susceptibility tests on major pathogens (S. typhi, shigellae) are useful as part of a comprehensive programme of surveillance of enteric infections. They are essential for informing the physician of the emergence of resistant strains (chloramphenicol-resistant S. typhi, co-trimoxazole-resistant and ampicillin-resistant shigellae) and of the need to modify standard treatment schemes. Although susceptibility testing of non-typhoid salmonellae serotypes causing intestinal infection is not relevant for treating the patient, the appearance of multiresistant strains is a warning to the physician of the overuse and misuse of antimicrobial drugs.

Continued surveillance of the results of routine susceptibility tests is an excellent source of information on the prevalence of resistant staphylococci and Gram-negative bacilli that may be responsible for cross-infections in the hospital. Periodic reporting of the susceptibility pattern of the prevalent strains is an invaluable aid to forming a sound policy on antibiotic usage in the hospital by restriction and/or rotation of life-saving drugs, such as the aminoglycosides and cephalosporins.

Choice of drugs for routine susceptibility tests in the clinical laboratory

The choice of drugs used in a routine antibiogram is governed by considerations of the antibacterial spectrum of the drugs, their pharmacokinetic properties, toxicity, efficacy, and availability, as well as their cost to both the patient and the community. Among the many antibacterial agents that could be used to treat a patient infected with a given organism, only a limited number of carefully selected drugs should be included in the susceptibility test.

Table 12 indicates the drugs to be tested in various situations. The drugs in the table are divided into two sets. Set 1 includes the drugs that are available in most hospitals and for which routine testing should be carried out for every strain. Tests for drugs in set 2 are to be performed only at the special request of the physician, or when the causative organism is resistant to the first-choice drugs, or when other reasons (allergy to a drug, or its unavailability) make further testing justified. Many antibiotics with good clinical activity have been omitted from the table, but it must be emphasized that they are rarely needed in the management of the infected patient. In very rare cases, one or more additional drugs should be included when there is a special reason known to the physician, or when new and better drugs become available. Periodic revision of this table is therefore desirable, and this should be done after appropriate discussions with clinical staff. Many problems arise in practice, because clinicians are not always aware that only one representative of each group of antimicrobials is included in routine tests. The result obtained for this particular drug may then be extrapolated to all, or most, of the other members of the group. Difficulties arise in some countries when the physician is familiar only with the commercial brand name of the drug and not with its generic nonproprietary name. A serious effort should be made to inform medical personnel about the international nonproprietary names of pharmaceutical substances, and to encourage their use.1

1 International Nonproprietary Names for Pharmaceutical Substances, Cumulative List No. 7. Geneva, World Health Organization, 1988.

Table 12. Basic sets of drugs for routine susceptibility testsa


Staphylococcus

Enterobacteriaceae

Pseudomonas aeruginosa



Intestinal

Urinary

Blood and tissues


Set 1
First choice

benzylpenicillinoxacillinerythromycintetracycline

chloramphenicol

ampicillinchloramphenicolco-trimoxazolenalidixic acid

tetracycline

sulfonamidetrimethoprimco-trimoxazoleampicillinnitrofurantoinnalidixic acid

tetracycline

ampicillinchloramphenicolco-trimoxazoletetracyclinecefalotin

gentamicin

piperacillingentamicin

tobramycin

Set 2
Additional drugs

gentamicinamikacinco-trimoxazole

clindamycin

norfloxacin

norfloxacinchloramphenicol

gentamicin

cefuroximeceftriaxoneciprofloxacinpiperacillin

amikacin

amikacin

a Notes on the individual antibacterial agents are given in the text.

1. The benzylpenicillin disc is used to test susceptibility to all b-lactamase-sensitive penicillins (such as oral phenoxymethylpenicillin and pheneticillin). Isolates of staphylococci that fall into the resistant category produce b-lactamase and should be treated with a b-lactamase-resistant penicillin or with another antibiotic, such as erythromycin.

2. Oxacillin. The oxacillin disc is representative or the whole group of b-lactamase-resistant penicillins (including meticillin, nafcillin, cloxacillin, dicloxacillin, and flucloxacillin). Moreover, there is good clinical evidence that cross-resistance exists between the meticillin and the cephalosporin groups. Therefore, it is useless and misleading to include cefalotin in the antibiogram for staphylococci. Resistance to meticillin and related drugs is often of the heterogeneous type, i.e., the majority of cells may be fully susceptible and produce a wide inhibition zone, while the resistant part of the population appears in the form of minute discrete colonies growing within the inhibition zone. This type of resistance is more apparent when the temperature of the incubator is set at 35 °C1 or when the incubation time is prolonged.

A serious disadvantage of meticillin, as a representative disc for the b-lactamase-resistant penicillins, is its great lability even under conventional storage conditions. The oxacillin disc is much more resistant to deterioration and is therefore preferred for the standardized diffusion test. The cloxacillin and dicloxacillin discs are not used as they may not indicate the presence of a heteroresistant strain.

1 SAHM, D. F. et al. Current concepts and approaches to antimicrobial agent susceptibility testing. In: Cumitech 25, Washington, DC, American Society for Microbiology, 1988.

3. The results for the tetracycline disc may be applied to chlortetracycline, oxytetracycline, and other members of this group. However, most tetracycline-resistant staphylococci remain normally sensitive to minocycline. A disc of minocycline may thus be useful to test multiresistant strains of staphylococci.

4. The result with the chloramphenicol disc may be extrapolated to thiamphenicol, a related drug with a comparable antibacterial spectrum, but without known risk of aplastic anaemia.

5. Only one representative sulfonamide (sulfafurazole) is required in the test.

6. The co-trimoxazole disc contains a combination of trimethoprim and a sulfonamide (sulfamethoxazole). Although the use of combinations of drugs in discs has been condemned in previous WHO reports,2 co-trimoxazole is an exception because the two components of this synergistic combination have comparable pharmacokinetic properties and generally act “as a single drug”.

2 WHO Technical Report Series, No. 796, 1990 (The use of essential drugs: fourth report of the WHO Expert Committee).

7. Ampicillin is the prototype of a group of broad-spectrum penicillins with activity against many Gram-negative bacteria. As it is susceptible to b-lactamase, it should not be used for testing staphylococci. Generally, the susceptibility to ampicillin is also valid for other members of this group: amoxycillin, pivampicillin, talampicillin, etc. (though amoxycillin is twice as active against salmonellae and only half as active against shigellae and H. influenzae).

8. Cefalotin. Only cefalotin needs to be tested routinely, as its spectrum is representative of all other first-generation cephalosporins (cefalexin, cefradine, cefaloridine, cefazolin, cefapirin). Where second- and third-generation cephalosporins and related compounds (cefamycins) with an expanded spectrum are available, a separate disc for some of these new drugs may be justified in selected cases (cefoxitin, cefamandole, cefuroxime, cefotaxime, ceftriaxone). Although some cephalosporins can be used to treat severe staphylococcal infections, the susceptibility of the infecting strain can be derived from the result with oxacillin as already mentioned under 2 above.

9. Erythromycin is used to test the susceptibility to some other members of the macrolide group (oleandomycin, spiramycin).

10. Aminoglycosides. This group of chemically related drugs includes streptomycin, gentamicin, kanamycin, netilmicin and tobramycin. Their antimicrobial spectra are not always closely enough related to permit assumption of cross-resistance, but against susceptible pathogens these agents have been shown to be equally effective. Numerous studies have compared the nephrotoxicity and ototoxicity of gentamicin, netilmicin and tobramycin, but there is no conclusive evidence that any one of the drugs is less toxic than the others. It is strongly recommended that each laboratory select a single agent for primary susceptibility testing. The other agents should be held in reserve for treatment of patients with infections caused by resistant organisms.

11. Nitrofurantoin is limited to use only in the treatment of urinary tract infections, and should not be tested against microorganisms recovered from material other than urine.

The modified Kirby-Bauer method

The disc diffusion method, originally described in 1966,1 is well standardized and has been widely evaluated. Official agencies have recommended it, with minor modifications, as a reference method which could be used as a routine technique in the clinical laboratory.

1 BAUER, A. W. et al. Antibiotic susceptibility testing by a standardized single disc method. American journal of clinical pathology, 44: 493-496 (1966).

Reagents

Mueller-Hinton agar

1. Mueller-Hinton agar should be prepared from a dehydrated base according to the manufacturer’s recommendations. The medium should be such that control zone sizes within the published limits are produced (see Table 13). It is important not to overheat the medium.

2. Cool the medium to 45-50 °C and pour into the plates. Allow to set on a level surface, to a depth of approximately 4 mm. A 9-cm plate requires approximately 25 ml of medium.

3. When the agar has solidified, dry the plates for immediate use for 10 - 30 minutes at 35 °C, by placing them in the upright position in the incubator with the lids tilted.

4. Any unused plates may be stored in a plastic bag, which should be sealed and placed in the refrigerator. Plates stored in this way will keep for 2 weeks.

To ensure that the zone diameters are sufficiently reliable for testing susceptibility to sulfonamides and co-trimoxazole, the Mueller-Hinton agar must have low concentrations of the inhibitors thymidine and thymine. Each new lot of Mueller-Hinton agar should therefore be tested with a control strain of Enterococcus faecalis (ATCC 29212 or 33186) and a disc of co-trimoxazole. A satisfactory lot of medium will give a distinct inhibition zone of 20 mm or more that is essentially free of hazy growth or fine colonies.

Antibiotic discs

Any commercially available discs with the proper diameter and potency can be used. Stocks of antibiotic discs should preferably be kept at - 20°C; the freezer compartment of a home refrigerator is convenient. A small working supply of discs can be kept in the refrigerator for up to 1 month. On removal from the refrigerator, the containers should be left at room temperature for about 1 hour to allow the temperature to equilibrate. This procedure reduces the amount of condensation that occurs when warm air reaches the cold container. If a disc-dispensing apparatus is used, it should have a tight-fitting cover and be stored in the refrigerator. It should also be allowed to warm to room temperature before being opened.

Table 13. Zone diameter limits for control strainsa



Diameter of zone of inhibition (mm)

Antibiotic

Disc potency

S. aureus
(ATCC 25923)

E. coli
(ATCC 25922)

P. aeruginosa
(ATCC 27853)

amikacin

30 mg

20-26

19-26

18-26

ampicillin

10 mg

27-35

16-22

-

benzylpenicillin

10 IU

26-37

-

-

cefalotin

30 mg

29-37

17-22

-

ceftriaxone

30 mg

22-28

29-35

17-23

cefuroxime

30 mg

27-35

20-26

-

chloramphenicol

30 mg

19-26

21-27

-

ciprofloxacin

100 mg

22-30

30-40

25-33

clindamycin

2 mg

24-30

-

-

co-trimoxazole

25 mg

24-32

24-32

-

erythromycin

15 mg

22-30

8-14

-

gentamicin

10 mg

19-27

19-26

16-21

nalidixic acid

30 mg

-

22-28

-

nitrofurantoin

300 mg

18-22

20-25

-

norfloxacin

10 mg

17-28

28-35

22-29

oxacillin

1 mg

18-24

-

-

piperacillin

100 mg

-

24-30

25-33

sulfonamideb

300 mg

24-34

18-26

-

tetracycline

30 mg

19-28

18-25

-

tobramycin

10 mg

19-29

18-26

19-25

trimethoprim

5 mg

19-26

21-28

-

a National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disc susceptibility tests. Tentative standard. 4th ed. Villanova, PA, USA, NCCLS, 1988.

b Sulfafurazole.

Turbidity standard

Prepare the turbidity standard by pouring 0.6 ml of a 1% (10 g/litre) solution of barium chloride dihydrate into a 100-ml graduated cylinder, and filling to 100 ml with 1% (10 ml/litre) sulfuric acid. The turbidity standard solution should be placed in a tube identical to the one used for the broth sample. It can be stored in the dark at room temperature for 6 months, provided it is sealed to prevent evaporation.

Swabs

A supply of cotton wool swabs on wooden applicator sticks should be prepared. They can be sterilized in tins, culture tubes, or on paper, either in the autoclave or by dry heat.

Procedure

To prepare the inoculum from the primary culture plate, touch with a loop the tops of each of 3 - 5 colonies, of similar appearance, of the organism to be tested.

What factors affect disk diffusion method?

Figure

Transfer this growth to a tube of saline.

What factors affect disk diffusion method?

Figure

When the inoculum has to be made from a pure culture, a loopful of the confluent growth is similarly suspended in saline.

Compare the tube with the turbidity standard and adjust the density of the test suspension to that of the standard by adding more bacteria or more sterile saline.

What factors affect disk diffusion method?

Figure

Proper adjustment of the turbidity or the inoculum is essential to ensure that the resulting lawn of growth is confluent or almost confluent.

Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess inoculum by pressing and rotating the swab firmly against the side of the tube above the level of the liquid.

What factors affect disk diffusion method?

Figure

Streak the swab all over the surface of the medium three times, rotating the plate through an angle of 60 ° after each application. Finally, pass the swab round the edge of the agar surface. Leave the inoculum to dry for a few minutes at room temperature with the lid closed.

What factors affect disk diffusion method?

Figure

The antibiotic discs may be placed on the inoculated plates using a pair of sterile forceps. It is convenient to use a template (see Fig. 10) to place the discs uniformly.

What factors affect disk diffusion method?

Figure

A sterile needle tip may also be used to place the antibiotic discs on the plate.

What factors affect disk diffusion method?

Figure

Alternatively, an antibiotic disc dispenser can be used to apply the discs to the inoculated plate.

What factors affect disk diffusion method?

Figure

A maximum of seven discs can be placed on a 9-10 cm plate. Six discs may be spaced evenly, approximately 15 mm from the edge of the plate, and 1 disc placed in the centre of the plate. Each disc should be gently pressed down to ensure even contact with the medium.

The plates should be placed in an incubator at 35 °C within 30 minutes of preparation. Temperatures above 35 °C invalidate results for oxacillin/meticillin.

Do not incubate in an atmosphere of carbon dioxide.

After overnight incubation, the diameter of each zone (including the diameter of the disc) should be measured and recorded in mm. The results should then be interpreted according to the critical diameters shown in Table 14.

The measurements can be made with a ruler on the under-surface of the plate without opening the lid.

What factors affect disk diffusion method?

Figure

If the medium is opaque, the zone can be measured by means of a pair of calipers.

What factors affect disk diffusion method?

Figure

A template (see Fig. 9) may be used to assess the final result of the susceptibility tests.

What factors affect disk diffusion method?

Figure

The endpoint of inhibition is judged by the naked eye at the edge where the growth starts, but there are three exceptions:

· With sulfonamides and co-trimoxazole, slight growth occurs within the inhibition zone; such growth should be ignored.

· When b-lactamase-producing staphylococci are tested against penicillin, zones of inhibition are produced with a heaped-up, clearly defined edge; these are readily recognizable when compared with the sensitive control, and regardless of size of zone of inhibition, they should be reported as resistant.

· Certain Proteus species may swarm into the area of inhibition around some antibiotics, but the zone of inhibition is usually clearly outlined and the thin layer of swarming growth should be ignored.

Interpretation of the zone sizes

· Using a template. When the zone sizes are compared with the template (see Fig. 9) the result - susceptible, resistant, or intermediate - can be read at once: “susceptible”, when the zone edge is outside the black circle; “resistant”, when there is no zone, or when it lies within the white circle; and “intermediate”, when the edge of the zone of inhibition lies on the black circle.

· Using a ruler. When the zone sizes are measured in mm, the results should be interpreted according to the critical diameters given in Table 14.

Direct versus indirect susceptibility tests

In the standardized method outlined above, the inoculum is prepared from colonies on a primary culture plate or from a pure culture. This is called an “indirect sensitivity test”. In certain cases, where a rapid answer is important, the standardized inoculum may be replaced by the pathological specimen itself, e.g., urine, a positive blood culture, or a swab of pus. For specimens of urine, a microscopical examination of the sediment should first be made in order to see if there is evidence of infection, i.e., the presence of pus cells and/or organisms. The urine may then be used as the inoculum in the standard test. Likewise, susceptibility tests may be performed on incubated blood cultures showing evidence of bacterial growth, or a swab of pus may be used as a direct inoculum, when a Gram-stained smear shows the presence of large numbers of a single type of organism. This is called a “direct susceptibility test”; its advantage over the indirect test is that a result is obtained 24 hours earlier. The main disadvantage is that the inoculum cannot be properly controlled. When the susceptibility plate shows too light or too heavy growth, or when the culture is a mixture, the results should be interpreted with caution, and the test repeated on pure cultures.

Inoculum density

If the inoculum is too light, the inhibition zones will be larger although the sensitivity of the organism is unchanged. Relatively resistant strains may then be reported as susceptible. Conversely, if the inoculum is too heavy, the zone size will be reduced and susceptible strains may be reported as resistant. Usually optimal results are obtained with an inoculum size that produces near confluent growth.

What factors affect disk diffusion method?

Fig. 9. Zone diameters for the determination of susceptibility with the standard disc diffusion method

Table 14. Interpretative chart of zone sizesa


Diameter of zone of inhibition (mm)

Antibiotic or chemotherapeutic agent

Disc potency

Resistant

Intermediate/
moderately susceptible

Susceptible

amikacin

30 mg

£ 14

15-16

³ 17

ampicillin when testing:





- Enterobacteriaceae

10 mg

£ 13

14-16

³ 17

- Enterococcus faecalis

10 mg

£ 16

-

³ 17

benzylpenicillin when testing staphylococci

10 IU

£ 28

-

³ 29

ceftriaxone

30 mg

£ 13

14-20

³ 21

cefuroxime sodium

30 mg

£ 14

15-17

³ 18

cefalotin

30 mg

£ 14

15-17

³ 18

chloramphenicol

30 mg

£ 12

13-17

³ 18

clindamycin

2 mg

£ 14

15-20

³ 21

co-trimoxazole

25 mg

£ 10

11-15

³ 16

erythromycin

15 mg

£ 13

14-22

³ 23

gentamicin

10 mg

£ 12

13-14

³ 15

nalidixic acid

30 mg

£ 13

14-18

³ 19

nitrofurantoin

300 mg

£ 14

15-16

³ 17

oxacillin when testing:





- staphylococci

1 mg

£ 10

11-12

³ 13

- pneumococci

1 mg

£ 19

-

³ 20

piperacillin when testing:





- Enterobacteriaceae

100 mg

£ 17

18-20

³ 21

- Pseudomonas

100 mg

£ 14

15-17

³ 18

sulfonamides

300 mg

£ 12

13-16

³ 17

tetracycline

30 mg

£ 14

15-18

³ 19

tobramycin

10 mg

£ 12

13-14

³ 15

trimethoprim

5 mg

£ 10

11-15

³ 16

a National Committee for Clinical Laboratory Standards. Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

Timing of disc application

If the plates, after being seeded with the test strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take place before the discs are applied. This causes a reduction in the zone diameter and may result in a susceptible strain being reported as resistant.

Temperature of incubation

Susceptibility tests are normally incubated at 35 °C for optimal growth. If the temperature is lowered, the time required for effective growth is extended and larger zones result. When a heterogeneous resistant strain of Staphylococcus aureus is being tested against meticillin (oxacillin), the resistant portion of the population can be detected at 35 °C. At higher temperatures the entire culture appears to be susceptible. At 35 °C or lower temperatures, resistant colonies develop within the zone of inhibition. These resistant colonies can be seen more easily if the plate is left for several hours at room temperature before the result is read. Such colonies should always be identified to check whether they are contaminants.

What factors affect disk diffusion method?

Fig. 10. Template for uniform placement of susceptibility discs on plates of 90 mm diameter

Incubation time

Most techniques adopt an incubation period of between 16 and 18 hours. In emergencies, however, a provisional report may be made after 6 hours. This is not recommended as a routine and the result should always be confirmed after the conventional incubation time.

Size of plate, depth of agar medium, and spacing of the antibiotic discs

Susceptibility tests are usually carried out with 9 - 10 cm plates and no more than 6 or 7 antibiotic discs on each plate. If larger numbers of antibiotics have to be tested, two plates, or one 14-cm diameter plate, is to be preferred. Excessively large inhibition zones may be formed on very thin media; the converse is true for thick media. Minor changes in the depth of the agar layer have negligible effect. Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or deformation near the edge of the plates (see Fig. 10).

Potency of the antibiotic discs

The diameter of the inhibition zone is related to the amount of drug in the disc. If the potency of the drug is reduced owing to deterioration during storage, the inhibition zone will show a corresponding reduction in size.

Composition of the medium

The medium influences the size of the zone by its effect on the rate of growth of the organism, the rate of diffusion of the antibiotic, and the activity of the agent. It is essential to use the medium appropriate to the particular method.

The many factors influencing the zone diameters that may be obtained for the same test organism clearly demonstrate the need for standardization of disc diffusion methods. Only if the conditions laid down in a particular method are closely followed can valid results be obtained. Alteration of any of the factors affecting the test can result in grossly misleading reports for the clinician.

The precision and accuracy of the method should be monitored by establishing the quality control programme described below. Variations can then be immediately investigated and corrective action taken to eliminate them.

Quality control

The need for quality control in the susceptibility test

The final result of a disc diffusion test is influenced by a large number of variables. Some of the factors, such as the inoculum density and the incubation temperature, are easy to control, but a laboratory rarely knows the exact composition of a commercial medium or the batch-to-batch variations in its quality, and it cannot take for granted the antimicrobial content of the discs. The results of the test must therefore be monitored constantly by a quality control programme, which should be considered part of the procedure itself.

The precision and accuracy of the test are controlled by the parallel use of a set of control strains, with known susceptibility to the antimicrobial agents. These quality control strains are tested using exactly the same procedure as for the test organisms. The zone sizes shown by the control organisms should fall within the range of diameters given in Table 13. When results regularly fall outside this range, they should be regarded as evidence that a technical error has been introduced into the test, or that the reagents are at fault. Each reagent and each step in the test should then be investigated until the cause of the error has been found and eliminated.

Standard procedure for quality control

The quality control programme should use standard reference strains of bacteria that are tested in parallel with the clinical cultures. They should preferably be run every week, or with every fifth batch of tests, and, in addition, every time that a new batch of Mueller-Hinton agar or a new batch of discs is used.

Standard strains for quality control

Staphylococcus aureus (ATCC 25923)
Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC 27853)

These cultures can be obtained from national culture collections. They are commercially available in the form of pellets of desiccated pure cultures.

Cultures for day-to-day use should be grown on slants of nutrient agar (tryptic soy agar is convenient) and stored in the refrigerator. They should be subcultured on to fresh slants every 2 weeks.

Preparing the inoculum

The cultures may be inoculated into any type of broth, and incubated until the broth is turbid. Each broth should be streaked on to an agar plate and incubated overnight. Single colonies should then be picked off and submitted to susceptibility tests as described on “Procedure”.

Placing antimicrobial discs

After the inoculum has been streaked on to the plates, as described on “Procedure”, the appropriate discs should be applied. The discs to be selected for each control strain are listed in Table 13.

Reading the plates

After 16 to 18 hours’ incubation, the diameters of the inhibition zones should be measured with a ruler and recorded, together with the date of the test, on a special quality control chart. This chart should display data for each disc - strain combination. The chart is labelled in millimetres, with an indication of the range of acceptable zone sizes. An example of such a chart is shown in Fig. 11. When the results consistently fall outside the acceptable limits, action should be taken to improve the quality of the test.

Grossly aberrant results, which cannot be explained by technical errors in the procedure, may indicate contamination or sudden changes in the susceptibility or growth characteristics of the control strain. If this occurs, a fresh stock-strain should be obtained from a reliable source.

What factors affect disk diffusion method?

Fig. 11. Quality control chart for antimicrobial susceptibility testing

Control disc: Ampicillin disc 10 mg. Control strain: E. coli ATCC 25922


Page 6

Inoculum density

If the inoculum is too light, the inhibition zones will be larger although the sensitivity of the organism is unchanged. Relatively resistant strains may then be reported as susceptible. Conversely, if the inoculum is too heavy, the zone size will be reduced and susceptible strains may be reported as resistant. Usually optimal results are obtained with an inoculum size that produces near confluent growth.

What factors affect disk diffusion method?

Fig. 9. Zone diameters for the determination of susceptibility with the standard disc diffusion method

Table 14. Interpretative chart of zone sizesa


Diameter of zone of inhibition (mm)

Antibiotic or chemotherapeutic agent

Disc potency

Resistant

Intermediate/
moderately susceptible

Susceptible

amikacin

30 mg

£ 14

15-16

³ 17

ampicillin when testing:





- Enterobacteriaceae

10 mg

£ 13

14-16

³ 17

- Enterococcus faecalis

10 mg

£ 16

-

³ 17

benzylpenicillin when testing staphylococci

10 IU

£ 28

-

³ 29

ceftriaxone

30 mg

£ 13

14-20

³ 21

cefuroxime sodium

30 mg

£ 14

15-17

³ 18

cefalotin

30 mg

£ 14

15-17

³ 18

chloramphenicol

30 mg

£ 12

13-17

³ 18

clindamycin

2 mg

£ 14

15-20

³ 21

co-trimoxazole

25 mg

£ 10

11-15

³ 16

erythromycin

15 mg

£ 13

14-22

³ 23

gentamicin

10 mg

£ 12

13-14

³ 15

nalidixic acid

30 mg

£ 13

14-18

³ 19

nitrofurantoin

300 mg

£ 14

15-16

³ 17

oxacillin when testing:





- staphylococci

1 mg

£ 10

11-12

³ 13

- pneumococci

1 mg

£ 19

-

³ 20

piperacillin when testing:





- Enterobacteriaceae

100 mg

£ 17

18-20

³ 21

- Pseudomonas

100 mg

£ 14

15-17

³ 18

sulfonamides

300 mg

£ 12

13-16

³ 17

tetracycline

30 mg

£ 14

15-18

³ 19

tobramycin

10 mg

£ 12

13-14

³ 15

trimethoprim

5 mg

£ 10

11-15

³ 16

a National Committee for Clinical Laboratory Standards. Voluntary consensus standards for clinical laboratory testing. Villanova, PA, NCCLS, 1990.

Timing of disc application

If the plates, after being seeded with the test strain, are left at room temperature for periods longer than the standard time, multiplication of the inoculum may take place before the discs are applied. This causes a reduction in the zone diameter and may result in a susceptible strain being reported as resistant.

Temperature of incubation

Susceptibility tests are normally incubated at 35 °C for optimal growth. If the temperature is lowered, the time required for effective growth is extended and larger zones result. When a heterogeneous resistant strain of Staphylococcus aureus is being tested against meticillin (oxacillin), the resistant portion of the population can be detected at 35 °C. At higher temperatures the entire culture appears to be susceptible. At 35 °C or lower temperatures, resistant colonies develop within the zone of inhibition. These resistant colonies can be seen more easily if the plate is left for several hours at room temperature before the result is read. Such colonies should always be identified to check whether they are contaminants.

What factors affect disk diffusion method?

Fig. 10. Template for uniform placement of susceptibility discs on plates of 90 mm diameter

Incubation time

Most techniques adopt an incubation period of between 16 and 18 hours. In emergencies, however, a provisional report may be made after 6 hours. This is not recommended as a routine and the result should always be confirmed after the conventional incubation time.

Size of plate, depth of agar medium, and spacing of the antibiotic discs

Susceptibility tests are usually carried out with 9 - 10 cm plates and no more than 6 or 7 antibiotic discs on each plate. If larger numbers of antibiotics have to be tested, two plates, or one 14-cm diameter plate, is to be preferred. Excessively large inhibition zones may be formed on very thin media; the converse is true for thick media. Minor changes in the depth of the agar layer have negligible effect. Proper spacing of the discs is essential to avoid overlapping of the inhibition zones or deformation near the edge of the plates (see Fig. 10).

Potency of the antibiotic discs

The diameter of the inhibition zone is related to the amount of drug in the disc. If the potency of the drug is reduced owing to deterioration during storage, the inhibition zone will show a corresponding reduction in size.

Composition of the medium

The medium influences the size of the zone by its effect on the rate of growth of the organism, the rate of diffusion of the antibiotic, and the activity of the agent. It is essential to use the medium appropriate to the particular method.

The many factors influencing the zone diameters that may be obtained for the same test organism clearly demonstrate the need for standardization of disc diffusion methods. Only if the conditions laid down in a particular method are closely followed can valid results be obtained. Alteration of any of the factors affecting the test can result in grossly misleading reports for the clinician.

The precision and accuracy of the method should be monitored by establishing the quality control programme described below. Variations can then be immediately investigated and corrective action taken to eliminate them.