Rate of Penicillin Killing of Staphylococcus aureus and

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1982, p. 27-274 95-1137/82/227-5$2./ Vol. 15, No. 2 Rate of Penicillin Killing of Staphylococcus aureus and Autobac 1 Susceptibility Test Results JO-ANN HARRIS' AND DOLORES FURTADO2* Departments of Pediatrics1 and Microbiology,2 University of Kansas School of Medicine, Kansas City, Kansas 6613 Received 9 April 1981/Accepted 3 August 1981 A clinical isolate of Staphylococcus aureus interpreted as resistant to penicillin by the Autobac 1 susceptibility testing method (i.e., light-scattering index of.77) was found to be susceptible to penicillin by both the disk diffusion and broth dilution techniques. The growth rate of the clinical isolate during a 4-h incubation interval was similar to that of a known sensitive reference strain (S. aureus ATCC 25923) used as a control organism for the Autobac test. The bactericidal effect of penicillin was evaluated by measuring the rate of killing over a 4-h interval. The percentages of organisms surviving exposure to 5. or 2.5 U of penicillin G per ml (number of organisms recovered at 3 h/number of organisms introduced as inoculum) were 68 and 76%, respectively, for the clinical isolate and 15 and 21%, respectively, for the reference strain. After 24 h of incubation, penicillin was bactericidal for both strains. The need to increase the time of incubation for those S. aureus isolates resistant to pencillin after 3 h of standard incubation time in the Autobac system is discussed. Recently, a rapid automated system for the determination of antimicrobial susceptibilities, Autobac 1 (Pfizer, Inc., New York, N.Y.), has been put into use in clinical microbiology laboratories. The proposed advantages of this new system as compared with the more conventional methods of susceptibility testing, disk diffusion, and tube dilution include greater efficiency, greater uniformity of results, and greater rapidity with which these results can be obtained, i.e., 3 to 5 h versus 18 to 24 h, respectively. Although Thornsberry et al. in their collaborative study (11) found a good correlation (92%) between the Autobac and the more standard susceptibility tests, he as well as others (3, 6, 1) have also noted some major discrepancies. These discrepancies occur with certain antibiotic and bacterial combinations, including the susceptibility of the staphylococci to the penicillins. We obtained an isolate of Staphylococcus aureus from the blood of a 4-year-old patient who developed acute bilateral pyelonephritis with abscess formation. By Autobac testing, this organism was resistant to penicillin and susceptible to nafcillin. The susceptibility to nafcillin was also confirmed by the tube dilution method. Because of the discrepancies reported with the Autobac method (3, 6, 1), we measured the susceptibility of this isolate to penicillin G by both disk diffusion and broth dilution techniques. The results obtained by these conventional methods indicated that the organisms were susceptible to penicillin. 27 In this study, the results of the growth rate and rate of killing by penicillin are compared for the clinical isolate and for S. aureus (ATCC 25923), which is susceptible to penicillin by the Autobac technique. MATERIALS AND METHODS Baterial species. S. aureus recovered from a blood specimen was designated S. aureus KU. S. aureus ATCC 25923, interpreted as susceptible to penicillin by the Autobac method, was used as the control organism. Stock cultures were maintained on blood agar slants and stored at 4 C. Media. Trypticase soy broth (TSB), brain heart infusion broth, and Mueller-Hinton broth media (BBL Microbiology Systems, Cockeysville, Md.) were prepared in the standard manner. Penicillin susceptibility tests. (i) Autobac 1. The clinical laboratory at the University of Kansas Medical Center (Kansas City) performed this test by standard procedures at the time the organisms were isolated from the blood of the patient. After several laboratory passages, the Autobac susceptibility test was repeated on the isolate. (ii) Disk diffusion test. The standard disk diffusion method as recommended by the National Committee for Clinical Laboratory Standards (7) was used in this study. Both a commercial disk containing 1 U of penicillin (BBL) as well as a sterile.25-in (.635-cm) blank disk (Difco Laboratories, Detroit, Mich.) to which 1 U of penicillin G (lot no. 392983A, Parke, Davis & Co., Detroit, Mich.) had been added were tested. The inoculum contained 9.2 x 16 colonyforming units. (iii) Broth dilution test. A freshly prepared stock of penicillin G (Parke, Davis & Co.) was serially diluted

VOL. 15, 1982 AUTOBAC 1 SUSCEPTIBILITY TESTING OF S. AUREUS 271 in sterile physiological saline at a starting concentration of 12 ijg/.1 ml. For each determination, 15 tubes were used. To each tube,.1 ml of sterile physiological saline was added, and then twofold serial dilutions of penicillin prepared as described above were made in the first 14 tubes, the 15th tube being a control without penicillin. To each tube,.8 ml of TSB was added, followed by the addition of.1 ml of log-phase organisms (1.1 x 1' colony-forming units). The inoculum for the test was prepared by transferring a loopful of stock organisms to a 15-ml tube of TSB and incubating.n n o overnight at 37 C. One milliliter was transferred to a _ E flask containing 45 ml of TSB and placed on a shaker water bath at 37 C for 3 h. The log-phase culture was E mq diluted with physiological saline, and bacterial counts. c were made by the standard surface plating technique. The 15 tubes were incubated overnight at 37 C, and that concentration of antibiotic which inhibited turbid.e'r= growth was defined as the minimal inhibitory concentration. To determine the minimal bactericidal concen- X _ tration, a.1-ml sample from each nonturbid tube was inoculated on Trypticase soy agar plates and mixed C with 1,25 U of penicillinase (Penase, lot no. AOE-.2 LYQ; BBL) contained in.1 volume (to inactivate any i Xc residual active penicillin) before spreading on the Xo. surface of the plate. The antibiotic concentration lead- X Ec ing to -99.9% killing of the inoculum was recorded as X to the minimal bactericidal concentration..: * c In vitro growth studies. A 1.-ml inoculum from a 3-h, o log-phase culture was introduced into a flask contain- E t a co o ing 45 ml of TSB, mixed, and incubated in a 37 C Z - CZ J shaker water bath..-. - Samples (1. ml) were removed at X c S times, 1, 2, 3, and 4 h. Serial 1-fold dilutions in. sterile physiological saline were made, and samples i E were plated by using a standard surface plating technique (4). 'A4o Studies on penicillin killing. Penicillin G, 225 or 112.5 E. X X 83 U in.5 ml, was added to 45 ml of TSB, providing a. final concentration of 5. or 2.5 U/ml. To the flask, 1. Ao ml of an overnight culture of organisms was then 4A added, and the mixture was incubated at 37 C in a shaker water bath. Samples were removed at times, c Oi 1, 2, 3, and 4 h and serially diluted in physiological X saline containing 1, U of penicillinase per ml for quantitative analysis. C Animal survival studies. Midcontinent white female., mice weighing 2 to 25 g each were used. Animals > were housed in air-conditioned quarters in cages and D) fed lab chow ad libitum. -e A.5-ml volume of an appropriate dilution of log- _o phase cultures was inoculated into a lateral tail vein as A E previously described (4). Mortality over a 7-day obser- c., vation period was recorded. On day 7, a.5-ml CA orbital sinus blood specimen was obtained from ani- m. mals just before sacrifice and quantitatively analyzed :53 for bacteria, using the surface plating technique. Ani- < no Z ou mals were then sacrificed by cervical dislocation. The V-- kidneys were examined for gross abscesses, aseptical- 4- 'C ly removed, and homogenized in glass grinding tubes 2. o containing 1.8 ml of sterile saline. Samples of the v homogenate were diluted and plated to determine the number of bacteria recovered per pair of kidneys. U, U < X RESULTS Penicillin susceptibility tests. The results of the Autobac, disk diffusion, and broth dilution

272 HARRIS AND FURTADO methods for testing the susceptibility to penicillin of both the clinical isolate, S. aureus KU, and the strain susceptible by the Autobac technique, S. aureus ATCC 25923, are summarized in Table 1. Both S. aureus KU and S. aureus ATCC 25923 were considered susceptible to penicillin G by the disk diffusion and broth dilution techniques (1). However, by using the established Autobac light-scattering index (LSI) tables (11), in which an LSI of..9 is interpreted as susceptible, our clinical isolate, with an LSI of.77, was interpreted as resistant. The control organism, on the other hand, had an LSI of..9, the defined LSI reading for penicillin susceptibility. A technical consideration which became apparent when comparing the results obtained by these various methods was the variation in incubation time for the tests; that is, the Autobac process was completed in 3 h, whereas the more conventional tests were read after 18 to 24 h of incubation. Since the effectiveness of penicillin is based on an ability to interfere with active cell wall synthesis, a slower growth rate could mask the bactericidal effectiveness of penicillin in a short incubation time. This could be the basis for the Autobac test result interpretation as resistant. To assess whether our clinical isolate did in fact grow more slowly than the reference strain, the in vitro growth rates were compared. In vitro growth studies. There was no appreciable increase in the numbers of organisms per milliliter after 2 or 3 h with either S. aureus KU or S. aureus ATCC 25923 (Fig. 1). Because the medium used has been described to be an important consideration in measuring the antibiotic susceptibility of bacteria (8), the growth studies done in TSB were repeated, using Mueller- Hinton and brain-heart infusion broths. The clinical isolate showed a 2- or 3-h lag period in all media (Fig. 2). The discrepant results of the Autobac test with these two strains could not be attributed to a difference in growth rate. The next parameter we studied was the rate of killing by penicillin within a 4-h interval. In vitro killing by penicillin. The addition at time zero of either 2.5 or 5 U of penicillin G per ml to the flask containing S. aureus KU did not reduce the numbers of surviving organisms in the first 2 h (Fig. 3). The number of viable organisms recovered after 3 h with 2.5 U/ml was reduced by 24% (76% survived), and the number recovered with 5. U/ml was reduced by 32% (68% survived). By 4 h, 66% of the organisms had survived exposure to 2.5 U of penicillin per ml, as compared with 52% which had survived exposure to 5 U/ml. However, with the strain susceptible by the Autobac technique, killing by 2.5 or 5 U of penicillin per ml was apparent within 1 h. At 1 h, only 51 and 45% of the inocula survived, and the Mi 1 7 o1 z U9 1 HOURS 3 4 FIG. 1. In vitro growth of S. aureus KU () and S. aureus ATCC 25923 () in TSB. number per milliliter continued to decline to 21 and 15% by 3 h. All flasks were incubated overnight at 37 C and analyzed after 24 h. There were no viable organisms recovered from either test flask, indicating that penicillin was bactericidal for both strains. Z 1 9 1 ILl8 1Q.6. t J. CLIN. MICROBIOL. 6 1 1 3 4 HOURS FIG. 2. In vitro growth of S. aureus KU in TSB (), Mueller-Hinton broth (), and brain heart infusion (A) culture media.

VOL. 15, 1982 13 r 12o 11 1 4%9~~~~ >8 X\ 7-~~~~~~~~~~ 6 \ - z 5.,4 W3QO 2" 1 2 3 4 HOU R S FIG. 3. Effect of penicillin G on in vitro survival of 2.5 (@-4) and 5. (-) U of S. aureus KU and 2.5 (O---O) and 5. (O---) U of S. aureus ATCC 25923. The 1.-ml inocula added to the 45-ml flask contained 8.5 x 18 and 4.3 x 18 colony-forming units. It is important to note that these observations correspond to the discrepancies noted in the penicillin susceptibility test results. The Autobac, which has a short incubation period, interpreted the clinical strain as resistant, whereas the more conventional methods, which have incubation times of 18 to 24 h, interpreted it to be susceptible. A possible way to avoid such a discrepancy might be to incubate for an additional 1 to 2 h those Autobac tests which are interpreted as resistant at 3 h. Animal virulence. In addition to the above in vitro tests, we undertook in our laboratory studies to determine the renal virulence in mice of TABLE 2. AUTOBAC 1 SUSCEPTIBILITY TESTING OF S. AUREUS 273 this particular isolate in anticipation that this model could then be used to investigate the in vivo treatment of S. aureus infection with penicillin G. We compared the virulence of the clinical isolate, S. aureus KU to that of the reference strain by using a previously defined mouse model (4, 5). A total of 17 organisms of S. aureus KU was lethal for 25% of the mice and produced renal abscesses in 1% of the mice sacrificed on day 7 (Table 2). In these animals, 6 of 15 mice (4%) had at least 13 organisms per ml of blood at sacrifice. Reducing the challenge dose to 16 organisms resulted in septic death in 4 of 31 mice (13%) and renal disease in 27 of 27 mice. Bacteremia lasting for 7 days was observed in 7 of 28 mice (3%). An inoculum of 17 organisms of S. aureus ATCC 25923 resulted in the deaths of 3 of 22 mice (14%) within 7 days. Animals sacrificed on day 7 also had renal disease in 1% of the kidneys. However, bacteremia was detected in only 3 of 19 mice (16%). On the basis of the greater number of septic deaths as well as the production of persistent bacteremia, S. aureus KU was ranked by us as slightly more virulent than S. aureus ATCC 25923. Studies are under way in our laboratory, comparing the efficacy of antibiotic treatment in preventing septic deaths and in the treatment of pyelonephritis produced by these two strains. DISCUSSION The Autobac 1 system for antimicrobial susceptibility testing is an efficient, rapid, and accurate method, and, for more than 9% of organism-antibiotic combinations, its results correlate well with those of more conventional susceptibility testing methods (3, 6, 11). However, there have been some discrepancies noted between certain organisms and antibiotics, including S. aureus and penicillin; methicillinresistant S. aureus and methicillin, erythromycin, or clindamycin; Escherichia coli and ampicillin; and Proteus mirabilis and ampicillin (3, 6, 1). While investigating the discrepancies Morbidity and mortality after intravenous inoculation of S. aureus in mice No. (%) with recovered organisms at sacrifice on day 7 No. of colony- No. of deaths/ from: S. aureus strain forming units total no. of mice per ml of (%)b Renal homogenates (.1' Blood (-13 organisms inoculuma organisms per pair) per ml)" KU 17 5/2 (25) 15/15 (1) 6/15 (4) KU 16 4/31 (13) 27/27 (1) 7/28 (3) ATCC 25923 17 3/22 (14) 19/19 (1) 3/19 (16) a Lateral tail vein injection of a.5-ml volume. b Number of deaths within 7 days after injection. c A.5-ml blood sample was obtained by orbital sinus bleeding.

274 HARRIS AND FURTADO seen with S. aureus and penicillin, Funnel and Guiness (3) found that, among beta-lactamasenegative S. aureus isolates, 36% had an LSI of between.6 to.9 and were therefore interpreted as resistant to penicillin by the Autobac technique. When the cuvettes were reincubated for 3 min to allow more time for antibioticorganism interaction, the LSI reading was.9, and the Autobac results were interpreted as susceptible to penicillin. It could be inferred from the work of Funnel and Guiness (3) that these particular strains of S. aureus may have had different growth rates which were overcome by extending the incubation period for an additional 3 min. Our results support the suggestion that the incubation time for testing penicillin killing of S. aureus by the Autobac method may need to be extended even more, that is, for at least 6 to 9 min longer. The increased incubation time is needed not because of a discernible difference in the growth rates, but rather because of a delayed onset in killing by penicillin. Altered penicillin killing of S. aureus has also been reported as a new type of penicillin resistance called tolerance (2, 9). Tolerant strains of S. aureus are inhibited but not killed by conventional levels of penicillin when measured by the broth dilution susceptibility testing method. Sabath et al. (9) hypothesized that tolerant S. aureus may be deficient in autolytic enzyme activity, which is necessary for rapid bactericidal effect of penicillin. Bradley et al. (2), while investigating the characteristics of tolerant strains of S. aureus, showed a 18 to 24-h delay in killing by penicillin. In contrast to Bradley's tolerant strains of S. aureus, our clinical isolate showed a 2-h delay in penicillin killing, followed by complete loss of viability within 18 to 24 h. Penicillin tolerance did not explain the Autobac results. The different results could simply be a reflection of the Autobac's shorter incubation time of 3 h as compared with 18 to 24-h incubation times for the conventional tube dilution and disk diffusion methods. The difference in the killing rates was less apparent at 4 h, suggesting that the Autobac test reliability would improve if read after 4 or 5 h of incubation. The Autobac remains an efficient, rapid, and accurate method for testing the susceptibility of S. aureus isolates to penicillin. It is only for J. CLIN. MICROBIOL. those strains in which the LSI reading is between.6 and.9 that the cuvettes should be incubated for a longer time than 3 h. The delay in reporting the Autobac results would be minimum. Treatment with penicillin, a less toxic and less expensive drug, would permit physicians to reserve the beta-lactamase-resistant penicillins for treating infections caused by beta-lactamaseproducing strains of S. aureus. ACKNOWLEDGMENTS The significant advice and assistance of Norma Lindsey, Clinical Laboratories, University of Kansas Medical Center, is gratefully acknowledged. This investigation was supported by a research grant from the Kansas Heart Association, Topeka. LITERATURE CITED 1. Barry, A. L. 1976. The antimicrobic susceptibility test: principles and practices, p. 134-141. Lea and Febiger, Philadelphia, Pa. 2. Bradley, J. J., C. G. Mayhall, and H. P. Dalton. 1978. Incidence and characteristics of antibiotic-tolerant strains of Staphylococcus aureus. Antimicrob. Agents Chemother. 13:152-157. 3. Funnel, G. R., and M. D. G. Guiness. 1979. Australian evaluation of Autobac 1 with suggested interpretive and technical modifications. Antimicrob. Agents Chemother. 16:225-261. 4. Furtado, D. 1971. Effect of diuresis on Staphylococcus aureus kidney infections in mice. Infect. Immun. 14:742-746. 5. Gorrill, R. H. 1958. The establishment of staphylococcal abscesses in the mouse kidney. Br. J. Exp. Pathol. 39:23-212. 6. Mogyoros, M., J. R. Morgan, and J. A. Smith. 1977. Evaluation of the Autobac 1 susceptibility testing system in a clinical diagnostic laboratory. Antimicrob. Agents Chemother. 11:75-752. 7. National Committee for Clinical Laboratory Standards. 1979. Performance standards for antimicrobic disc susceptibility tests, second ed., NCCLS approved standard ASM-2. National Committee for Clinical Laboratory Standards. 8. Peterson, L. R., D. N. Griding, W. H. Hall, and E. A. Schierl. 1978. Medium-dependent variation in bactericidal activity of antibiotics against susceptible Staphylococcus aureus. Antimicrob. Agents Chemother. 13:665-668. 9. Sabath, L. D, N. Wheeler, M. Laverdiere, D. Blazevic, and B. J. Wilkinson. 1977. A type of penicillin resistance of Staphylococcus aureus. Lancet i:443-447. 1. Stubbs, K. G., and K. Wicher. 1977. Laboratory evaluation of an automated antimicrobial susceptibility system. Am. J. Clin. Pathol. 68:769-777. 11. Thornsberry, C., T. L. Gavan, J. C. Sherris, A. Balows, J. M. Matsen, L. D. Sabath, F. Schoenknecht, L. D. Thrupp, and J. A. Washington II. 1975. Laboratory evaluation of a rapid, automated susceptibility testing system: report of a collaborative study. Antimicrob. Agents Chemother. 7:466-48.