Species. Penicillins Against Escherichia coli and Proteus. In Vitro Activity of Cephalothin and Three

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1973, p Copyright 1973 American Society for Microbiology Vol. 4, No. 3 Printed in U.S.A. In Vitro Activity of Cephalothin and Three Penicillins Against Escherichia coli and Proteus Species ARTHUR L. BARRY AND PAUL D. HOEPRICH Section of Infectious and Immunologic Diseases, Department of Internal Medicine, School of Medicine, University of California, Davis, California Received for publication 3 May 1973 The susceptibility of clinical isolates of Escherichia coli (67) and Proteus species (58) to cephalothin, ampicillin, benzyl penicillin, and phenoxymethyl penicillin was determined in vitro by using broth dilution and disk diffusion tests. The data were correlated by using a four-category scheme for interpreting minimal inhibitory concentrations (groups 1 to 4) as recommended by a subcommittee of an international collaborative study of susceptibility testing. With cephalothin and ampicillin, groups 1 (susceptible) and 2 (moderately susceptible) were susceptible by the disk test, and with benzyl penicillin, similar results were observed when the interpretive zone standards were changed. Strains categorized as group 4 (very resistant) were resistant by the disk method, but group 3 (moderately resistant) strains were not adequately distinguished by disk testing. Group 3 susceptibility to benzyl and phenoxymethyl penicillins can be predicted by extrapolating results from tests with ampicillin disks. At relatively high concentrations, the penicillins are active against many of the enteric previously described by Ericsson et al. (4). Four laborative study (5) and approved the concept bacilli. The closely related cephalosporins are categories of susceptibility (groups 1 to 4) were generally active against Escherichia coli and defined as follows. "Group 1 should include Proteus mirabilis, but not against the indolepositive species of Proteus. With the standard in vivo response probable when mild to moder- high degrees of bacterial sensitivity that makes disk method of Bauer et al. (2) as outlined by ately severe systemic infections are treated with the Food and Drug Administration (6), most usual dosages of antibiotic." This would be the strains of E. coli and of P. mirabilis would be oral route when applicable (e.g., ampicillin). considered susceptible to ampicillin, but not Group 1 can be defined as "sensitive" without susceptible to benzyl penicillin (penicillin G). further qualification. Group 1 might include However, if massive doses are applied in therapy, it is possible to obtain such high concentration (MIC) no greater than.25 gg of benzyl strains with a minimal inhibitory concentrations in the blood and other body fluids that penicillin per ml, 2 Ag of ampicillin per ml, or 4 successful treatment of infections caused by,gg of cephalothin per ml. "Group 2 should those enteric bacilli which are only moderately include degrees of sensitivity which make in susceptible to the penicillins may result (8). vivo response probable in systemic infections Lower-urinary-tract infections caused by enteric bacilli that are resistant to concentrations to the limits of toxicity." Group 2 might include when the antibiotic is given in high dosage or up ordinarily attained in the blood may respond to strains with MICs as great as 16,ug of benzyl treatment with either benzyl or phenoxymethyl penicillin or ampicillin per ml or 32 gg of penicillin, because these drugs are excreted into cephalothin per ml. Group 3 might include the urine, often attaining very high concentrations (7). Consequently, a reevaluation of the ml for all three drugs-that is, "degrees of bacteria with MICs no greater than 128 yg per standards for interpretating in vitro susceptibility tests is appropriate. ble in the treatment of localized infections at sensitivity,which make in vivo response proba- Recently, a subcommittee on interpretation sites where the agent can be concentrated by reviewed the subject for an international col- physiological processes or local application." 354 Downloaded from on June 3, 218 by guest

2 VOL. 4, 1973 Group 4 includes bacteria with MICs greater than 128 ug per ml, i.e., "... a degree of resistance which makes in vivo response improbable." The interpretive standards for categorization of susceptibility to phenoxymethyl penicillin (penicillin V) should be about the same as that for benzyl penicillin, but the upper limit dividing groups 3 and 4 might be greater, because a given oral dose of phenoxymethyl penicillin will yield a concentration in the urine at least twice that obtained with the same oral dose of benzyl penicillin (3). The current study was undertaken for two purposes. The first was to compare, in vitro, the relative activity of cephalothin, ampicillin, benzyl penicillin, and phenoxymethyl penicillin against recent clinical isolates of E. coli and Proteus species. For this purpose, the drugs were diluted so as to permit comparison on an equimolar basis; bactericidal as well as bacteriostatic end points were determined. Secondly, disk diffusion tests were performed to determine whether different levels of susceptibility could be distinguished by using commercially available disks. It was found that zones around disks containing 1,g of ampicillin could be used to predict group 3 susceptibility to benzyl penicillin and phenoxymethyl penicillin, whereas disks containing 1 U (6.3 gg) of benzyl penicillin were unsatisfactory for this purpose. The interpretation of in vitro tests with benzyl penicillin was reexamined, and new interpretive zone standards are proposed for gram-negative bacilli. MATERIALS AND METHODS The clinical isolates used for testing included 67 E. coli, 37 P. mirabilis, 7 Proteus vulgaris, 7 Proteus rettgeri, and 7 Proteus morganii strains. Disk diffusion tests were performed with the agar overlay technique (1), as recommended by the Food and Drug Administration (6), by using commercially available (Pfizer) disks containing 1 U (6.3 Mg) of benzyl penicillin, 1 Ag of ampicillin; and 3,gg of cephalothin. Dilution tests were carried out in Mueller-Hinton broth with a microbroth dilution technique by using a Mini-titer apparatus (Canalco Co., Rockville, Md.) to prepare doubling dilutions in.5-ml volumes. The dilution of 1 Mmol per ml (i.e., Ag [521.7 U] of benzyl penicillin, 35.4 Ag of phenoxymethyl penicillin, gg of ampicillin, and ug of cephalothin). Molar definition of concentrations was selected because the differences in molecular weights ranged from a low with benzyl-phenoxymethyl penicillins (phenoxymethyl is 4.8% heavier than benzyl penicillin) to a high with benzyl penicillin-cephalothin (cephalothin is 18.5% heavier than benzyl penicillin). The inoculum consisted of.5 ml of a 1-4 dilution of an ovemight culture in Mueller-Hinton broth. The IN VITRO ACTIVITY OF CEPHALOTHIN 355 MIC was determined after overnight incubation at 35 C, and the minimal lethal concentration was determined by subculturing approximately.1 ml (hand-held replicator) to Mueller-Hinton agar without antimicrobics. The minimal lethal (bactericidal) concentration was taken as the lowest concentration which failed to produce any colonies within 24 h after subculturing in this manner. RESULTS Broth dilution tests. The in vitro activity of the four drugs under study is summarized in Fig. 1. Against E. coli and P. mirabilis, benzyl penicillin was more active than phenoxymethyl penicillin, but both penicillins were relatively inactive against the indole-positive species of Proteus. Ampicillin was significantly more active than the other penicillins against E. coli and all four species of Proteus. A few indolepositive Proteus species were inhibited by relatively low concentrations of the penicillins (particularly ampicillin), but all required very high concentrations for a bactericidal effect. With E. coli and P. mirabilis all four drugs were lethal at concentrations generally equal to or one doubling dilution greater than the MIC. Against P. mirabilis, cephalothin had about the same activity as benzyl penicillin. When tested against ampicillin-susceptible E. coli, cephalothin had an activity about equal to that of ampicillin. Cephalothin was also effective against all but three of the ampicillin-resistant isolates included in this study. Cephalothin disk tests. The results of cephalothin disk diffusion and broth dilution tests are compared in Fig. 2. The interpretative zone standards established for the disk diffusion technique (6) classify microorganisms as being susceptible if the zone of inhibition is 18 mm or more; this correlates with MIC of 32,g per ml, which represents an appropriate break point between the moderately susceptible group 2 and moderately resistant group 3 bacteria. Most of the highly resistant strains in group 4 gave zones less than 14 mm, whereas most of the moderately resistant group 3 isolates gave zones greater than 14 mm. Consequently, the disk diffusion technique is capable of predicting the moderate susceptibility of E. coli and Proteus species to cephalothin, but does not adequately distinguish between group 2 and group 3. Alterations of the interpretive standards would not improve the correlation. The overall incidence of error with cephalothin disk tests is rather low (11 "errors" out of 127 tests). Ampicillin disk tests. Disk diffusion tests with 1-Mg ampicillin disk are compared with broth dilution tests in Fig. 3. Two distinct Downloaded from on June 3, 218 by guest

3 356 BARRY AND HOEPRICH a (m w I- n wlj Cl) - Cl) IL -J w I-. 4 a U MINIMAL CONCENTRATIONS INHIBITORY LETHAL Escherichia coli (67 isolates) Proteus species, indole +(21 isolates) 7 AMP x o----o CCEPH :-H - PEN G.. PEN V IF. I ^.-tsss*sf~~~ t co C o6 od If) X O O (D CD If) N N o - o o o ANTIMICROBIC CONCENTRATIONS, JAmole/mi FIG. 1. Relative activity of cephalothin (CEPH), ampicillin (AMP), benzyl penicillin (PEN G), and phenoxymethyl penicillin (PEN V) against Escherichia coli (67 isolates) and Proteus species (58 isolates). populations were demarcated by the 14 mm or greater zone size currently accepted (6) as indicating susceptibility (a MIC < 32 ug per ml). Those bacteria which fell into the susceptible groups 1 and 2 gave zones 14 mm in diameter or greater, whereas all but three of the resistant group 4 bacteria gave no zone of inhibition. Most of the isolates which would be considered only moderately resistant (group 3) were also susceptible by the disk diffusion technique. The accuracy of the disk test would PI I 1 I-~~ I1 1 ANTIMICROB. AG. CHEMOTHER. ll be improved if the upper limit dividing groups 2 and 3 were changed from 16 to 32 yg/ml, but such a change would not be justified by the data included in this report. Benzyl penicillin disk tests. Disks containing 1 U (6.3 1g) of benzyl penicillin were also tested, and the resulting zones were compared with broth dilution studies (Fig. 4). P. mnirabilis, with the only isolates that gave zones of inhibition that were 14 mm or greater, were all fairly susceptible (group 2). The current zone Downloaded from on June 3, 218 by guest

4 VOL. 4, 1973 IN VITRO ACTIVITY OF CEPHALOTHIN 357 i) Ag/ml Uimole/ml >396.4 > z S _ z _ _ T _ j _ X _ 1I 6 1 ZONE DIAMI FIG. 2. Relationship of d1isk diffusion and discs) broth both cases, group 1 and 2 gram-negative bacilli dilution tests with cephalot hin against Escherichia are included in the "susceptible" category. coli and Proteus spp. The reg. ression formula Y = MIC There was no inhibition with the moderately in log., Aglml (i.e., Y = 1.2!to.3x) was used. resistant group 3 bacteria that might respond to treatment when very high concentrations of the AMPICILLIN drug can be maintained at the site of infections. z Ag/mi.mole/nml B367 4 >IO C I z I_ u I _ of I Ia _ I _ z.7.2 F.4.1 _ LI.L...I j,g/mi Jimole/ml z>334.4 > 1. F _ l w CEPHALOTHIN 21 mm in diameter. This end point is * Escherichia coil (67) unsatisoprotu miroilis (39) factory, because it would cut through the center ProtMus sp., MM/ #,(2)/ of of 39 fairly susceptible (group 2) P. * & GROUP4 mirabilis. The interpretative zone standards ~~~GROUP 3 1GRO3 established for ampicillin would be more appro- priate for testing benzyl penicillin disks against 1^ GROUP28 the enteric bacilli; i.e., susceptible is defined as ^-*ne<s C 14 mm or larger and resistant as 11 mm or less. I- -I--- - With this change, the group 2 isolates would be l CD reported to be susceptible to benzyl penicillin GROUP by the disk method. Such a redefinition of Il t susceptibility would yield results more consist- E5ERS IN ent with those observed with ampicillin; i.e., in Nmm 13 jig * Eschorichia coti (67) For this the 1-U benzyl penicillin disk Prot*us s,irahi/is (39) a Prote/s sp., Indole *(2/) is unsatisfactory. GROUP 4 Because disks with a higher content of benzyl penicillin and disks. containing phenoxymethyl I * GROUP 3 penicillin are not available commercially, a -- practical altemative method was sought for GROUP 2 predicting high-level susceptibility to benzyl.,} and phenoxymethyl penicillins. A direct com- -o parison between MIC values for ampicillin and GROUP benzyl penicillin demonstrated an excellent 6 1 IS ~ 3 ZONE IAMWETERS IN (IOAig discs) correlation, although MICs for ampicillin tended to be about two doubling dilutions lower FIG. 3. Relationship of diisk diffusion and broth than those for benzyl penicillin. Accordingly, dilution tests with ampicillin against Escherichia coli the data were examined to determine if it would and Proteus species. The regr ession formula Y = MIC be feasible to predict susceptibility to benzyl or in log2, ug/ml (i.e., Y = 1. to.35x) was used. phenoxymethyl penicillins, or both, by using disks containing the more active ampicillin. 8ENZYL PENICILLIN Prediction of susceptibility to benzyl peni- * Escherichia co/i (67) cillin and phenoxymethyl penicillin Proteus reirohilis (39) Protes Sp.; /do/ + (2/) by using 1-.g ampicillin disks. In Fig. 5, broth dilution tests with benzyl penicillin and with phenoxymethyl penicillin are compared with diameters I* GROUP 3 of zones of inhibition obtained with 1-,ug am- > _ Fe picillin disks. The moderately resistant, group 3 We E. coli that gave no zones of inhibition with z o benzyl penicillin disks gave zones greater than a mm with ampicillin disks. The moderately z.65.2 susceptible group 2 P. mirabilis gave much I I! 2I 1O larger zones (21 mm or 15 greater) ZONE DIAMETERS IN mm (1 unit discs) Phenoxymethyl penicillin was less active in FIG. 4. Relationship of diisk diffusion and broth vitro, and a 14-mm zone around a 1-t.g amdilution tests with benzyl pienicillin against Esche- picillin disk correlated with an MIC of 218,ug richia coli and Proteus specie?s. per ml, whereas a 21-mm zone correlated with an MIC of 96,ug per ml. If the break point between group 3 and group 4 were placed at size standards for benzyl Ipenicillin (6) consider 256,ug per ml, most strains which are suscepti- to be suscep- ble to ampicillin by the disk method (14 mm or bacteria other than staphsylococci tible if the zone diameter iis 22 mm or more and more) should be moderately resistant to phelity if the zone is 12 to noxymethyl penicillin (group 3). The intermediate in susceptibi: highly Downloaded from on June 3, 218 by guest

5 358 BARRY AND HOEPRICH ANTIMICROB. AG. CHEMOTHER. jig/ml >334.4 cn z *<, 83.6 Z N o 1.4 o a] 5.2 o 2.6 I Z >35 J 35 )Lmole/ml > > * Escherichio co/i (67) o Proteus mirabi/is (39).1i6 Proteus sp., Indole +.21) 16 \ ^GROUP 4 s 1X* -o 1 UstI _ WI 3 6 GROUP3 - *1 _.3 6 o _- o o 88 ~~~~~~ oso _ 8 R GROUP 2 I o o X > I I I I >3 ZONE DIAMETERS IN mm (IoAxg AMPICILLIN discs) FIG. 5. Prediction of the susceptibility of Escherichia coli and Proteus species to benzyl and phenoxymethyl penicillins by using ampicillin disks. resistant group 4 strains give no zone around ampicillin disks. DISCUSSION Once the etiologic agent has been identified, the most appropriate antimicrobial agent for treatment can be selected by considering the severity and location of the infection and the pharmacological properties of the various potentially useful drugs. Data of the sort presented in this report can be used to estimate the approximate concentration of drug that would be needed to effect a cure of infections caused by E. coli or Proteus species. For example, isolates of P. mirabilis are more susceptible to the penicillins than most E. coli; the three indole-positive species of Proteus are even more resistant. However, resistant strains of P. mirabilis are encountered often enough to warrant the performance of susceptibility tests with each isolate. This can be done most efficiently with the standard disk diffusion methods that classify bacteria into one of two categories (susceptible or resistant), with an intermediate category which includes tests with indeterminate or equivocal results. In practice, very few isolates will give zones in the equivocal range if * z a 175 3, 87.6 E 43.8 o 21.9 *I GROUP 4 I'* a -co * GROUP 3 IIt I o 8 o 88B - the zone size break points fall at loci between two distinct populations, i.e., when there is a clear bimodal distribution of zone diameters.. Such a bimodal distribution is not evident with the drugs and genera under discussion, and thus some equivocal results are to be expected with the disk test. The intermediate category actually serves as a "buffer" zone between the two categories and helps to minimize the importance of relatively minor variations in technique which could influence the zone size enough to alter interpretation from resistant or from susceptible to intermediate (equivocal) strains rather than from resistant to susceptible strains. With benzyl penicillin, the intermediate category has been assigned a uniquely different meaning: a rather wide zone limit has been established (12 to 21 mm) to include those strains which are neither truly resistant nor fully susceptible, i.e., group 2 strains which would not respond to dosages normally used for treating highly susceptible genera such as Streptococcus pneumoniae. Moderately resistant group 3 strains are not separated from the very resistant group 4 strains as is done with other antimicrobics-instead both are considered resistant. Ta3 o... Downloaded from on June 3, 218 by guest

6 VOL. 4, 1973 IN VITRO ACTIVITY OF CEPHALOTHIN 359 The interpretive zone standards for the disk test are established after examining the correlation between zone diameters and MIC values. The MICs, in turn, may be interpreted according to the approximate concentration of drug that can be maintained at the site of the infection with the dosage schedule normally used for the type of infection and type of organism being treated. Because of the latter qualification, the criterion for defining susceptibility of one genus may differ from that used to classify another genus when different dosage schedules are recommended for the two genera. Consequently, the division of susceptible organisms into groups 1 and 2 is not essential, because the level of susceptibility (dosage normally required for therapy) for a given genus is cons,idered in establishing the interpretive standards. For the gram-negative enteric bacilli, susceptibility to benzyl penicillin could be redefined as an MIC of 16 Ag/ml or less (zone of 14 mm or greater). With such a classification most strains of P. mirabilis would be susceptible, whereas other Proteus species and E. coli would be resistant. Because P. mirabilis infections appear to respond to treatment with benzyl penicillin in doses greater than those normally used for more susceptible organisms, such a reclassification would seem to be appropriate. The currently recommended (6) zone standards are unsatisfactory, because about half of our isolates of P. mirabilis would have been reported to be susceptible to benzyl penicillin and the other half intermediate in susceptibility, irrespective of MICs. At the same time, most strains of P. mirabilis would be reported to be fully susceptible to ampicillin, although appropriate doses of either drug would produce blood levels well above the expected MIC values. Certain strains which are normally considered resistant might respond to therapy under certain circumstances. For example, in the treatment of urinary tract infections, the concentration of certain drugs at the site of infection might greatly exceed that normally found in the serum. Furthermore, in the treatment of systemic infections with relatively nontoxic drugs such as the penicillins, the average bloed levels can be exceeded by administering massive doses. Consequently, it would seem advisable to develop a test system which would distinguish those resistant strains which might respond under such circumstances (group 3) from the group 4 strains which are highly resistant, and thus not likely to respond. With ampicillin and cephalothin, the resistant group 4 bacteria were clearly resistant by the disk method, but the less resistant group 3 isolates presented some discrepancies. With these two drugs, the few strains which fell into the group 3 category gave zones in all three established ranges of susceptibility (fewest in the intermediate range). With benzyl penicillin disks, groups 3 and 4 gave no zone of inhibition and thus could not be distinguished. However, a 1-,Ag ampicillin disk would distinguish the slightly susceptible group 3 bacteria from the more resistant group 4 strains. Phenoxymethyl penicillin was the least active drug included in the present study and is probably inappropriate for treating gram-negative bacillary infections, except in situations where very high concentrations can be expected, e.g., in the urine. Thus, a group 3 category of susceptibility may be appropriate for the gram-negative bacilli. Those strains which are susceptible to ampicillin by the disk method will be slightly susceptible to phenoxymethyl penicillin and should respond in cases where the concentration can be expected to exceed 25jug/ml. On the other hand, ampicillin-resistant strains should not respond, even to these extremely high concentrations of phenoxymethyl penicillin. In actual practice, considerable information can be obtained from tests with ampicillin disks alone. Gram-negative bacilli that give zones greater than 21 mm in diameter are quite susceptible (groups 1 and 2) to ampicillin, moderately susceptible (group 2) to benzyl penicillin, and slightly susceptible (group 3) to phenoxymethyl penicillin. Strains that give zones between 14 and 2 mm are likely to be somewhat less susceptible to all three drugs. Isolates that give zones less than 11 mm in diameter around ampicillin disks are likely to be highly resistant to all three penicillins, but are often susceptible to the cephalosporins. ACKNOWLEDGMENTS The technical aid of Allen P. Adams is gratefully acknowledged. This study was supported in part by Public Health Service grants AI-8955 and AI-384 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Barry, A. L., F. Garcia, and L. D. Thrupp An improved single-disc method for testing the antibiotic susceptibility of rapidly growing pathogens. Amer. J. Clin. Pathol. 53: Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck Antibiotic susceptibility testing by a standardized single disc method. Amer. J. Clin. Pathol. 45: Cox, F., J. M. Colville, J. Truant, and E. L. Quinn Further observations on the use of large doses of oral Downloaded from on June 3, 218 by guest

7 36 BARRY AND HOEPRICH penicillin V. (phenoxymethyl penicillin). Antibiot. Annu : Ericsson, H., G. Tunevall, and K. Wickman The paper disc method for determination of bacterial sensitivity to antibiotics. Scand. J. Clin. Lab. Invest. 12: Ericsson, H. M., and J. C. Sherris Antibiotic sensitivity testing, report of an intemational collaborative study. Acta Pathol. Microbiol. Scand. Suppl. no. 217, sect. B: 3-9. ANTIMICROB. AG. CHEMOTHER. 6. Fine, S. D Antibiotic susceptibility discs; certification procedure. Fed. Reg. 37: (191). 7. Stamey, T. A., D. E. Govan, and J. M. Palmer The localization and treatment of urinary tract infections: the role of bactericidal urine levels as opposed to serum levels. Medicine 44: Weinstein, L., P. I. Lerner, and W. H. Chew Clinical and bacteriologic studies of the effect of "massive" doses of penicillin G and infections caused by gram negative bacilli. N. Engl. J. Med. 271: Downloaded from on June 3, 218 by guest