New Method for Antibiotic Susceptibility Testing

ANTIMIROBIAL AGENTS AND HEMOTHERAPY, Aug. 1972, p. 51-56 opyright 1972 American Society for Microbiology Vol. 2, No. 2 Printed in U.S.A. New Method for Antibiotic Susceptibility Testing G. N. ROLINSON AND ELIZABETH J. RUSSELL Beecham Research Laboratories, hemotherapeutic Research entre, Betchworth, Surrey, England Received for publication 4 April 1972 The most widely used method for routine antibiotic susceptibility testing of clinical isolates of bacteria is the paper-disc method. This has the advantage of simplicity, but to obtain reliable results considerable care is required both in the standardization of the procedure and in the interpretation of the zones of inhibition. A new susceptibility test method is described in this report which retains the simplicity of the paper-disc method but which enables organisms to be tested directly for susceptibility to known concentrations of antibiotic in agar. Organisms may be tested against a single concentration of antibiotic or, altematively, a range of concentrations may be used to determine the minimal inhibitory concentration. The method utilizes stable pre-prepared materials and is not appreciably more time consuming than the conventional disc method. The two principal types of test used for routine antibiotic susceptibility testing of bacteria in clinical laboratories are the serial dilution test and the disc test. In the serial dilution test, doubling dilutions of antibiotic are prepared in a liquid or solid medium which is then inoculated with the test organism. After incubation, the minimal inhibitory concentration is determined to the nearest dilution used in the series. This test is generally recognized as being the most satisfactory method of determining the antibiotic susceptibility of bacteria, but since it is time-consuming most laboratories are unable to use it as a routine procedure. In the disc test, paper discs impregnated with antibiotic are placed on the surface of an agar plate inoculated with the test organism, and after incubation susceptibility is determined by observing the size of the resulting zone of inhibition surrounding each disc. This method has the advantage of simplicity, but for a given antibiotic disc the size of the zone of inhibition is markedly influenced by a number of variables including inoculum size, depth of agar, conditions of incubation, and medium composition. Unless these variables are carefully controlled, the results obtained may be misleading, and a number of surveys have shown that the results of routine susceptibility testing are quite frequently unreliable (2, 5, 6). Recommendations for the standardization and interpretation of disc susceptibility testing have been made, notably by Ericsson (3), Bauer et al. (1), and Ericsson and Sherris (4), but these recommendations involve such rigid control of the test conditions that the simplicity of the disc test is to some extent lost and many laboratories may be unable to adopt these recommendations in full. 51 In an attempt to overcome some of the limitations of the existing procedures, a new susceptibility test method is described in this report which combines the simplicity of the disc test with the quantitative results obtained by serial dilution. In this method, organisms are tested for susceptibility to known concentrations of antibiotic, thus avoiding completely the question of interpretation of a zone of inhibition. The antibiotic concentrations are achieved by placing papers impregnated with antibiotic onto the surface of small volumes of agar contained in a plastic tray and allowing the antibiotic to diffuse from the paper into the agar. The experimental work has shown that the antibiotic diffuses almost completely into the agar and becomes almost uniformly distributed throughout the agar in a few hours. After the diffusion period, the papers are removed, and the agar surface is inoculated with the test organism. After overnight incubation, bacterial growth or inhibition of growth can be observed. In this way, it is possible to test directly for susceptibility to particular concentrations of antibiotic by a procedure which is not significantly more time-consuming than the paper-disc method. MATERIALS AND METHODS Diffusion of antibiotic through agar. Experiments were carried out to determine the time required for different antibiotics to diffuse from paper and become uniformly distributed throughout a layer of agar 3 mm in depth. Squares of Whatman no. 1 filter paper, 10 by 10 mm, were impregnated with known amounts of antibiotic by applying 0.02-ml volumes of the appropriate antibiotic solutions. After drying at 37 for 30 min, the papers were placed on the upper surface of blocks of agar 10 by 10 by 3 mm (Blood Agar Base

52 ROLINSON AND RUSSELL ANTIMIROB. AG. HEMOTHER. no. 1, Oxoid). At suitable time intervals over a period of several hours at toom temperature, the papers were removed from two replicate blocks, and the distribution of the antibiotic was determined as follows. Residual antibiotic in the papers after removal from the agar blocks was assayed by placing the papers on agar seeded with a suitable test organism and measuring the inhibition zones obtained after incubation. Standard lines were obtained by applying known amounts of antibiotic to 10-mm squares of paper and placing these on the same assay plates. After incubation, regression lines were prepared by plotting the size of the inhibition zones against the quantity of drug, and from these lines the residual amounts of antibiotic in the papers could be calculated. The antibiotic concentrations at the upper and lower surfaces of the agar block were determined by applying 10-mm squares of drug-free Whatman no. 1 paper to both surfaces of the block and allowing them to absorb liquid from the agar surface. By this means, a standard volume of liquid was obtained in which the antibiotic concentration was representative of that present at the agar surface. After 15 sec of contact, the papers were removed from the blocks and placed on an agar assay plate seeded with a suitable test organism. After incubation, the resulting zones of inhibition were used to calculate the antibiotic concentration by reference to regression lines. These were obtained by placing 10-mm squares of filter paper on the surface of similar blocks of agar containing known concentrations of antibiotic, transferring these to the assay plate and plotting the size of the resulting zones of inhibition against drug concentration. In the experiments shown in Table 1, papers containing j,g of antibiotic were used. The assay organisms used were Bacillus subtilis for benzylpenicillin, ampicillin, methicillin, cloxacillin, novobiocin, vancomycin, fusidic acid, and cephaloridine; Staphylococcus aureus for tetracycline, streptomycin, erythromycin, gentamicin, lincomycin, andkanamycin; and Bordetella bronchiseptica for polymyxin. Staphylococcus aureus and Sarcina lutea were used to assay the diffusion of benzylpenicillin when applied at 10 and 1,ug, respectively, and Bacillus cereus was used to assay the diffusion of 10 Ag of tetracycline. Minimal inhibitory concentrations. Minimal inhibitory concentrations of antibiotics were determined by the conventional method of serial dilution in agar, and the results were compared with the values obtained by use of the diffusion method described in this report. At the time these particular experiments were carried out, a suitable plastic tray of the type shown in Fig. 2-5 was not available; to obtain wells of a suitable size which could be filled with agar, a sheet of sterile silicone rubber 3 mm thick containing 25 holes 13 mm in diameter was pressed firmly onto the surface of a sterile plate of glass cut to fit inside a 10-cm square plastic petri dish. The wells so formed were then filled with agar flush with the surface of the rubber. Paper discs containing known amounts of antibiotic were placed on the surface of the agar wells, and diffusion was allowed to take place at 37 for 3 hr. The discs were prepared by dropping 0.02-ml volumes of antibiotic solutions of appropriate concentration onto sterile 12-mm discs of Whatman no. 1 filter paper which were then dried at 37 for 30 min. The quantity of antibiotic on each disc was that required to give the desired agar concentration, assuming complete distribution throughout the agar after the period of diffusion. Experimental work had shown that diffusion of the antibiotic from the paper into the agar was almost complete, and, since the volume of agar in the wells was known, the appropriate quantity of antibiotic on the disc could readily be calculated. After the period of diffusion, the discs of paper were removed and the agar surface was inoculated by use of a swab. In the serial dilution test, the minimal inhibitory concentrations were determined by adding 2-ml volumes of the appropriate concentrations of antibiotic to 18-ml volumes of cooled molten agar which were then poured into petri dishes. The plates were dried at 37 and inoculated with the test organisms by use of an automatic replicator delivering an inoculum of approximately 0.003 ml. In both the serial dilution test and the diffusion test, Blood Agar Base (Oxoid) was used, except in the case of sulfamethoxazole, for which D.S.T. agar (Oxoid) containing 5% lysed horse blood was employed. In both tests, overnight nutrient broth cultures of the test organisms were used as inoculum, diluted as indicated in Table 2. RESULTS AND DISUSSION Results are shown in Fig. 1 for the rate of diffusion of benzylpenicillin in a block of agar 3 mm thick at room temperature, when paper impreg- 0) 0) (3 1ooo0 05 1.0 1-5 2-0 2-5 Time (hours) _ 80 a 60 E 0) c 40-2 0 20 20 *1 FIG. 1. Diffusion of benzylpenicillin from paper into agar 3 mm in depth. Squares of filter paper (I cm) containing,.g of benzylpenicillin were applied to the surface of blocks of agar I by I cm and 3 mm deep. After intervals of time, the papers were removed and the antibiotic concentration present at the upper and lower surfaces of the agar block was assayed as described in Materials and Methods. The amount of antibiotic retained in the papers was also determined. Symbols: *, concentration of penicillin in the upper surface of agar; 0, concentration of penicillin in the lower surface of agar; O, penicillin remaining in the filter paper.

VOL. 2, 1972 ANTIBIOTI SUSEPTIBILITY TESTING 53 nated with the antibiotic was placed on the upper surface of the agar. It can be seen that diffusion of the antibiotic into the agar was rapid, and after only 1 hr approximately 95%/ of the antibiotic had passed from the paper into the agar. Diffusion of the antibiotic within the agar was also rapid, and assay of the antibiotic concentration at the upper and lower surfaces of the agar block showed that distribution of the antibiotic was essentially uniform after 1.5 to 2 hr. The time taken for the concentration of benzylpenicillin at the lower surface (L) to reach 95% of the concentration at the upper surface (U), subsequently referred to as the diffusion time, was determined by plotting L/U against time and was found to be 110 min. As would be expected, the diffusion time was independent of the amount of penicillin in the paper applied to the agar, over the range of 1 to,g. Similarly, the diffusion time with 10,g of tetracycline was found to be the same as that required when Ag was applied. The diffusion time for a number of different antibiotics is shown in Table 1. It can be seen that diffusion of the penicillins and cephaloridine was rapid, with a diffusion time of approximately 2 hr. With fusidic acid, streptomycin, kanamycin, gentamicin, andlincomycin a period of approximately 3 hr was required to achieve a 95% distribution, whereas in the case of erythromycin, tetracycline, novobiocin, and vancomycin 4 to 5 hr were required. Approximately 8 hr of diffusion time was required for polymyxin to become uniformly distributed throughout the agar. However, the concentration at the upper surface differed from the final uniform concentration by less than a factor of two after only 3 hr of diffusion, and in practice the results of susceptibility tests (Table 2) with polymyxin have shown a 3-hr diffusion period to be adequate to obtain valid results. Results are also shown in Table 1 for the antibiotic concentrations present in the agar at the end of the diffusion period when essentially uniform distribution in the agar had been achieved. These antibiotic concentrations are expressed as a percentage of the concentration which would have been achieved if all of the antibiotic on the paper had been uniformly distributed throughout the agar in the well. It can be seen that the concentrations achieved ranged from 81 to 103% of the theoretical value calculated from the known volume of agar and the quantity of antibiotic present on the paper. Allowing for some experimental error, the results show that under these conditions diffusion of the antibiotic into the agar is virtually complete. In the case of certain antibiotics, results are also shown in Table 1 for the residual amount of antibiotic remaining in the paper at the end of TABLE 1. Rate of diffiusioni of anttibiotics in agara Antibiotic ephaloridine... Benzylpenicillin... Ampicillin... Methicillin... loxacillin... Fusidic acid. Streptomycin... Kanamycin... Gentamicin... Lincomycin... Erythromycin... Tetracycline... Novobiocin... Vancomycin... Polymyxin... Diffusion time (min)b 110 120 130 140 150 160 160 1 200 220 230 270 300 480 Residual Antibiotic cnn antibiotic on paper'4 coacn in 103 89 85 94 81 87 89 a Pieces of Whatman no. 1 paper, 1 cm square, impregnated with mg of antibiotic were placed on the upper surface of blocks of Blood Agar Base (Oxoid), 1 by 1 cm and 3 mm thick, and the diffusion of the antibiotic into the agar at room temperature was measured as described in Materials and Methods. b Diffusion time is the time taken for the concentration of antibiotic at the lower surface of the agar to reach 95% of the concentration at the upper surface, i.e., the time taken to achieve essentially uniform distribution. c Antibiotic concentration in agar at end of diffusion period expressed as a percentage of the theoretical concentration which would have been present if all of the antibiotic on the paper had been uniformly distributed throughout that particular volume of agar. d Residual amount of antibiotic in the paper at the end of the diffusion period expressed as a percentage of the initial quantity. the diffusion period. These ranged from 3 to 9% of the initial amount. Results are shown in Table 2 for the minimal inhibitory concentrations of different antibiotics when determined by the method described in this report, with the use of a diffusion period of 3 hr, compared with the results obtained by the conventional serial dilution procedure. The minimal inhibitory concentrations obtained by these two procedures were in close agreement for all of the antibiotics tested, including polymyxin, and the differences in results between the two tests did not exceed one dilution step. These results serve to confirm that the antibiotic concentrations achieved in agar by the diffusion procedure are valid. The results in Table 2 also show that, in the diffusion test described here, a varia- 4 3 4 7 4 8 9

TABLE 2. omparison of minimal inhibitory concentrations determined by the method described in this report and by conventional serial dilution in agara Minimal inhibitory concn (,ug/ml) Drug and organism onventional serial dilution method Test described in this report 0 1: 1:1,000 0 1: 1:1,000 Benzylpenicillin Staphylococcus aureus... 0.05 0.05 Methicillin S. aureus... 1.0 0.5 0.5 0.5 loxacillin S. aureus... 0.25 0.1 0.25 0.25 Ampicillin S. aureus... 0.05 0.1 Escherichia coli... 2.5 2.5 2.5 2.5 Proteus mirabilis.... 5.0 2.5 5.0 5.0 arbenicillin E. coli... 5.0 5.0 5.0 5.0 Pseudomontas aeruginosa...1 50 25 50 25 Enterobacter cloacae... 5.0 5.0 5.0 5.0 P. mirabilis... 5.0 5.0 5.0 2.5 ephaloridine E. coli...... 5.0 2.5 2.5 2.5 P. mirabilis....... 10 5.0 10 5.0 Shigella sonnei... 5.0 5.0 5.0 5.0 Tetracycline E. coli...... 5.0 5.0 5.0 5.0 P. morganii.... 2.5 2.5 2.5 2.5 S. sonnei... 5.0 5.0 5.0 5.0 Erythromycin S. aureus...0...0.25 0.1 0.25 0.1 Novobiocin S. aureus.... 0.25 0.25 0.5 0.5 Vancomycin S. aureus i 2.5 2.5 hloramphenicol 2 2.5 Salmonella typhi 1.0 1.0 1.0 1.0 E. coli... 2.5 2.5 2.5 2.5 Streptomycin E. coli... 1.0 1.0 2.5 1.0 S.aureus.... 2.5 2.5 2.5 2.5 Kanamycin E. coli......... 2.5 2.5 2.5 2.5 S. aureus...... 2.5 1.0 2.5 2.5 P. morganii... 2.5 1.0 1.0 1.0 Gentamicin P. aeruginosa... 5.0 5.0 5.0 5.0 S. aureus... 0.1 0.1 0.1 0.1 Polymyxin P. aeruginosa... 10 5.0 10 10 Sulfamethoxazole E. coli...l... 2.5 2.5 S. aureus 2.5. 2.5 Klebsiella aerogenes... 2.5 2.5 a Minimal inhibitory concentrations were determined by the method described in this report with the use of Whatman no. 1 papers containing the appropriate amounts of antibiotics. Diffusion was allowed to take place for 3 hr at 37 and the plates were inoculated by use of a swab. In the serial dilution tests, petri dishes containing doubling dilutions of antibiotic were inoculated with an automatic replicator delivering approximately 0.003 ml. In both tests, Blood Agar Base (Oxoid) was used, except in the case of sulfamethoxazole, for which D.S.T. agar (Oxoid) containing 5% lysed horse blood was employed. Overnight broth cultures were used as inoculum in both tests, diluted as indicated at the top of each column.

VOL. 2, 1972 ANTIBIOTI SUSEPTIBILITY TESTING 55 tion in inoculum size from undiluted overnight broth culture to a dilution of 1 : had little effect on the results obtained. (3-Lactamase-producing organisms, however, form a special case, and, with those penicillins and cephalosporins which are labile, the inoculum size may have a very marked effect on the results obtained, as indeed is the case with the conventional serial dilution test. For routine susceptibility testing by the method described in this report, it is envisaged that a plastic tray would be made available commercially, containing prepoured agar ready for use. A suitable design of plastic tray is shown in Fig. 2. This plate provides four rows of eight wells, each 10 by 15 mm and 3 mm in depth, with a closefitting lid to prevent excessive evaporation during incubation. Inoculation of the plate may be carried out by use of a swab soaked in a suitable dilution of a suspension of the test organism, the swab being streaked across the plate from one side to the other. arry-over of antibiotic from one well to another has proved to be insufficient to influence the results. This has been established by streaking a swab of bacteria across wells containing antibiotic and onto adjacent wells containing drug-free agar. The antibiotics used were carbenicillin (,ug/ml), ampicillin (8,g/ml), gentamicin (4,g/ml), and tetracycline (4,ug/ml). With the Oxford staphylococcus, for which the minimal inhibitory concentrations of these antibiotics were 1.25, 0.02, 0.1 and 0.5,ug/ml, respectively, growth was not inhibited on any of the wells containing drug-free agar. In addition to a plastic tray prepoured with agar, it is envisaged that papers impregnated with the appropriate quantities of antibiotic would also be supplied commercially, as is currently the case with susceptibility discs. For use in the test described here, however, the antibiotic papers are required in the form of strips 1 cm wide, and these are applied to the plate in the vertical direction along each column of four wells as shown in Fig. 3. Each paper strip is impregnated uniformly with one particular antibiotic so that the correct quantity diffuses into each of the four wells of agar. In the direction in which the papers are applied, the agar wells are 15 mm in length and the distance separating each well (2 mm) can be ignored. Each vertical column of wells is thus impregnated with the same antibiotic, and the adjacent columns are impregnated in the same way with different antibiotics or different concentrations. The plate shown in Fig. 2-5 enables four organisms to be tested against seven antibiotics with one column containing drug-free agar for control growth. After the period of diffusion, the antibiotic papers are removed and the plate is inoculated as shown in Fig. 4. After overnight incubation, bacterial growth or inhibition of growth can be seen directly, as shown in Fig. 5. The bacteria used in FIG. 3. Paper strips impregnated being applied to the agar surface. with antibiotic FIG. 2. Plastic tray containing four rows of eight wells, each 10 by 15 mm and 3 mm in depth. The wells are filled with agar ready for use. FIG. 4. Inoculation of the plate with a swab.

56 ROLINSON AND RUSSELL ANTIMIROB. AG. HEMOTHER., c: PG p Amn 05 8 8 a Ge T Ka 2 4 4 E. col BiI E.col i 10418 Kleb. aerogeqnes PseL domoo as FIG. 5. Inoculated plate after overniight incubation. Test organisms and antibiotics as indicated. Anitibiotic concentrations shown in micrograms per milliliter. PG = penicillin G; p = cephaloridine; Am = ampicillin; a = carbenicillin; Ge = gentamicin; T = tetracycline; Ka = kanamycini. the test shown in Fig. 5 were two strains of Escherichia coli, a strain of Klebsiella aerogenes, and a strain of Pseudomonas aeruginosa. The antibiotics used were benzylpenicillin (0.5 ug/ml), cephaloridine (8,ug/ml), ampicillin (8,ug/ml), carbenicillin (,g/ml), gentamicin (2 Ag/ml), tetracycline (4,g/ml), and kanamycin (4,ug/ml). These antibiotics, and the particular concentrations, are purely arbitrary and were chosen merely to illustrate the test. For general use, some agreement would be required as to what the antibiotic concentrations should be, but each laboratory could then choose a range of antibiotics best suited to the particular organisms under test. The antibiotic strips could provide seven different antibiotics or, if desired, more than one concentration of a particular antibiotic could be employed and these could be related to dosage of the drug, route of administration, and also the site of infection. Antibiotic combinations such as carbenicillin and gentamicin or benzylpenicillin and aminoglycosides could also be tested by using paper strips impregnated with both drugs. The test described in this report may be used for routine susceptibility testing against a range of different antibiotics, or it could be used to determine the minimal inhibitory concentration of a single antibiotic by using papers providing a range of twofold dilutions. The method described here for routine susceptibility testing is not significantly more timeconsuming than the conventional disc method and has certain advantages. First, the test determines susceptibility directly and avoids the need to interpret a zone of inhibition. Second, the results obtained by this method are influenced less by the test conditions, including inoculum size, than is the case with the disc test. This is because diffusion of the antibiotic and growth of the test organism do not proceed simultaneously as they do in the disc test. The main advantage of the method described here, however, is that organisms can be tested for susceptibility to known concentrations of antibiotic, and these concentrations can be chosen in relation to the levels of antibiotic likely to be achieved in the body. AKNOWLEDGMENTS We are indebted to Peter R. Watt for his help in suggesting and making equipment involved in this work, to R. Sutherland for helpful discussion, and to Douglas F. Lawson for the photographs. LITERATURE ITED 1. Bauer, A. W., W. M. M. Kirby, J.. Sherris, and M. Turck- 1966. Antibiotic susceptibility testing by a standardized single disk method. Amer. J. lin. Pathol. 45:493-496. 2. ollege of Pathologists of Australia. 1968. A survey of antibiotic sensitivity testing. Med. J. Aust. 2:171-172. 3. Ericsson, H. 1960. Rational use of antibiotics in hospitals. Scand. J. lin. Lab. Invest. 12:Suppl. 50. 4. Ericsson, H. M., and J.. Sherris. 1971. Antibiotic sensitivity testing. Acta Pathol. Microbiol. Scand. Suppl. 217. 5. Report. 1960. A survey of antibiotic sensitivity tests. J. Med. Lab. Technol. 17:133-143. 6. Report. 1965. Report on antibiotic sensitivity test trial organized by the Bacteriology ommittee of the Association of linical Pathologists. J. lin. Pathol. 18:1-5.