J Clin Pathol: first published as 10.1136/jcp.30.1.35 on 1 January 1977. Downloaded from J. clin. Path., 1977, 30, 35-39

Susceptibility of the 'penicillinase-resistant' and to penicillinase of aureus

R. W. LACEY AND ANNE STOKES From the Department of Bacteriology, North Cambridgeshire Hospital, Wisbech, Cambridgeshire

SUMMARY The activities of some semisynthetic penicillins and cephalosporins have been tested against clinical strains of . The apparent activity in vitro varies with the method of testing used. Determination of MICs using light inocula fails to detect the destructive effect of penicillinase on the . This was, however, demonstrated reproducibly by the use of a technique in which a heavy inoculum was pre-incubated for two hours before application of antibiotic to wells. This method of testing probably represents most of the clinical situations in which the drugs are used since both in vitro and in vivo a growing culture is exposed to an antibiotic gradient. was inactivated by penicillinase considerably more than either , cloxacil- lin, or . was the most vulnerable of the cephalosporins. Cephazolin, cepha- copyright. lothin, and cephalexin were intermediate. Cephradine was the least hydrolysed by staphylococcal penicillinase. It is recommended that the activities of all penicillins and cephalosporins against staphylococci should be tested by diffusion at 37°C with pre-incubation of the culture for two hours at this temperature. http://jcp.bmj.com/ Determination of minimum inhibitory concentra- These enzymes are released into the media with little tions (MIC) has become accepted as the ideal method residual protein adherent to the cell. In this category by which laboratories express antibiotic sensitivity the enzyme that is of paramount clinical importance and resistance. This method has the advantage that is the ,B-lactamase which, to varying degrees, most of the conditions of the test can be controlled hydrolyses all the penicillins and cephalosporins (see accurately and reproduced easily, so that different below). Although a large proportion of the fl-lacta-

laboratories can, by reference to standard organisms mase synthesised by Gram-negative bacteria is still on October 1, 2021 by guest. Protected and , obtain confidence in the accuracy of cell-associated after subculture of the organism to their sensitivity testing. fresh media, the amount of lactamase retained by the In most clinical situations the MIC level obtained staphylococcal cell is small. Thus, MIC methodology in vitro is thought to correlate well with the outcome may fail to detect much ofthe effect ofstaphylococcal of antibacterial therapy in vivo. That laboratories do lactamase on penicillins and cephalosporins. not express routinely many results as MICs is an This phenomenon was well recognised in the 1940s indication of the length of time that this method and 1950s when resistance to benzyl in takes compared to diffusion methods. vitro was usually identified by diffusion testing, since There is probably, however, one serious drawback determination of MIC was thought to give a falsely in the use of MIC methods to report antibiotic optimistic view of the activity of this drug against sensitivity. Under certain conditions (particularly penicillin-resistant strains of Staphylococcus aureus. when a dilute inoculum is used) the assessment of However, with the arrival of the 'penicillinase- sensitivity obtained by MIC methods will fail to take resistant' penicillins and cephalosporins throughout into account the production of certain enzymes. the 1960s and 1970s, this shortcoming of MIC assessment has apparently been forgotten. The Received for publication 27 May 1976 activity of each new penicillin or has 35 J Clin Pathol: first published as 10.1136/jcp.30.1.35 on 1 January 1977. Downloaded from 36 R. W. Lacey and Anne Stokes been assessed primarily by MICs. The possibility that incubated aerobically for 18 hours at 37°C and the the drug may be significantly hydrolysed by end point was taken as no visible growth. staphylococcal penicillinase has usually been dis- counted, at least in the promotional literature. DIFFUSION TESTING There is, however, mounting evidence, derived These experiments were performed using a standard both from the chemotherapy of experimentally surface seeding technique with a neat overnight induced in animals and from clinical culture in peptone water that was applied to the experience in man, that certain of these new 'penicil- surface ofplates containing DST agar (Oxoid). These linase-resistant' penicillins are indeed vulnerable to were drained for 10 minutes and then allowed to dry staphylococcal penicillinase in vivo. for a further 10 minutes. Solutions of antibiotics Thus, cephaloridine fares poorly in the treatment were applied in wells, 8 mm diameter, either im- of induced staphylococcal in rabbits, mediately or after incubation of the seeded plates at probably because ofits destruction by staphylococcal 37°C for various intervals, usually two hours. The penicillinase (Norden and Dickens, 1973). Burgess diameters of the inhibition zones were measured after and Evans (1966) found that cephaloridine failed in incubation at 37°C for a further 18 hours. In general, the treatment of staphylococcal endocarditis in eight replicas of each antibiotic solution for each seemingly adequate dose, again due to the production test organism were allocated randomly to the plates. of staphylococcal penicillinase. Relative antibiotic activities for each strain were We have found that flucloxacillin was unable to calculated by reference to a standard curve. This was eliminate penicillinase-producing staphylococci in obtained by measuring the inhibition zones resulting two patients (Lacey and Lewis, 1975, 1976). In each from various concentrations of each antibiotic and a instance detailed evidence was presented which 'standard' penicillinase-negative culture. This organ- indicated that the therapeutic failure was due to ism was used throughout. The reproducibility of the destruction of flucloxacillin in vivo by staphylococcal technique was established as follows. were for activity against penicillinase. Six antibiotics assessed copyright. A large number of authors have described an four different strains. The experiments were then 'inoculum effect' with cephaloridine, ie, the MIC for repeated 'blind' after randomising of strains, and the a particular strain rises with increasing numbers of absolute activities were calculated on each occasion. bacteria in the inoculum. We have found that such an The standard error between the two sets of results effect occurs only with penicillinase-producing was 30%. strains. Controls included the checking of the variation in These observations suggest that both cephalo- the surface seeding, examination for possible ridine and flucloxacillin are significantly inactivated differences in the growth rate of the inoculum oncehttp://jcp.bmj.com/ in vivo by staphylococcal penicillinase. applied to the plate, and measurement of inhibition The purpose of this paper is to identify the condi- zones obtained with other antibiotics, including tions in vitro under which this inactivation occurs, and gentamicin. It was concluded that and to assess which of the penicillins and cephalo- variations in zone size attained were unlikely to be sporins are likely to be least vulnerable to staphylo- due to factors other than variation in the ability of coccal penicillinase in vivo. the culture to produce penicillinase. Penicillinase was demonstrated both chemically (starch-iodine Material and methods method) and by the effect on benzyl penicillin in disc on October 1, 2021 by guest. Protected testing. STRAINS Strains of Staph. aureus were isolated consecutively Results from lesions ofinpatients and outpatients at Wisbech and King's Lynn District Hospitals during 1975. In The MICs of 'single cell' inocula (ie, an inoculum of addition, three pairs of strains isolated in Bristol in about 50-200 discrete colony-forming units when 1971 were investigated; the latter were constructed applied to the surface of antibiotic-free medium) are so that they were isogenic except for variation in their shown in Table 1. For each antibiotic the range of ability to produce penicillinase (see results). MIC was narrow among the 60 strains and was not affected by the ability of the culture to produce SENSITIVITY TESTING penicillinase. As many other workers have found, MICs were determined by inoculating dilutions of methicillin was the least potent of the penicillins. Of overnight peptone water (Oxoid) cultures on to the the cephalosporins, cephaloridine was the most surface of agar (Oxoid blood base No. 2) containing active and cephalexin and cephradine the least. appropriate levels of antibiotic. The plates were Since several reports have indicated that the MICs J Clin Pathol: first published as 10.1136/jcp.30.1.35 on 1 January 1977. Downloaded from Susceptibility ofthe 'penicillinase-resistant'penicillins and cephalosporins to penicillinase ofStaph. aureus 37 Table 1 MICs ( Fg/ml) ofpenicillins and cephalosporin clinical situation in which an antibiotic is admin- for penicillinase-positive andpenicillinase-negative strains istered to a patient with a developing bacterial ofStaph. aureus . Pre-incubation beyond two hours gave more marked differences but the zones were less Antibiotic Penicillinase-positive Penicillinase negative strains (44): strains (14): clearly defined and difficult to read. mean (range) mean (range) ACTIVITIES OF METHICILLIN, , Methicillin 2-38 1-75 (1-05-0) (1-02-5) NAFCILLIN, AND FLUCLOXACILLIN AGAINST 50 Cloxacillin 0-28 0-24 PENICILLINASE-PRODUCING STAPHYLOCOCCI (0 25-0 5) (0 1-0-25) Flucloxacillin 0-25 0-23 IN DIFFUSION TESTING (0 1-0 5) (0 1-0-25) Initially, 50 consecutively isolated strains were Nafcillin 0-38' 0 25' (0-25-0-5) (0.25-0-25) examined; many of these produced low levels of Cephaloridine 0-071 0-039 penicillinase. The activities of each strain were (0-05-0 1) (0-025-0-1) to the of that of a Cephalexin 1-84 1-64 expressed as relative activities (1-0-5-0) (1-05-0) constant standard penicillinase-negative culture Cephradine 1-45 1 11 (Table There was considerable variation in the (1-02-0) (0-5-2 0) 2). Cephalothin 0-27 0-23 (0-25-0-5) (0 1-0 5) activities some Cephazolin 0 34 0-26 Table 2 Relative of semi-synthetic (0-25-0-5) (0 1-0 5) penicillins to 50 consecutively isolated strains of Staph. aureus: activities expressed as % of the standard in 115 only tested diffusion testing Antibiotic Penicillinase-positive Penicillinase-negative strains (38) strains (12) of cephaloridine may be increased if a heavy inoculum is used, this was investigated for 10 Mean Range Mean Range penicillinase-positive and six penicillinase-negative Methicillin 78-1 38 0-200 0 76-2 21-0-122-0 copyright. of neat and diluted Nafcillin 58-4 2-5-123-0 49-5 9-0- 90-0 cultures by placing drops serially Cloxacillin 39-2 4-8-141-0 71-6 12-0-141-0 (10-fold dilutions) cultures onto the surface of plates Flucloxacillin 30-0 3-3- 76-0 88-6 54-330-0 containing varying levels of cephaloridine. With each of the penicillinase-producing cultures a marked inoculum effect was demonstrated. Indeed, five of sensitivity of these isolates to each of these anti- the cultures that produced penicillinase became 50 biotics, although, in general, those strains that were times more resistant when a neat culture was applied particularly active against one drug were also active to the agar rather than a single cell inoculum. In against the others. The relative resistance of the http://jcp.bmj.com/ contrast, no inoculum effect was demonstrated with drugs to penicillinase was in the following order: penicillinase-negative cultures. methicillin (most resistant), nafcillin, cloxacillin, and flucloxacillin (most vulnerable). SENSITIVITY OBTAINED BY DIFFUSION TESTING Preliminary experiments indicated that the zones of ACTIVITIES OF CEPHALOSPORINS AGAINST inhibition obtained in diffusion testing were variable, PENICILLINASE-POSITIVE CULTURES and many factors contributed to this. Of particular In preliminary experiments, the activities of on October 1, 2021 by guest. Protected importance was the temporal relationship of the cephaloridine, cephazolin, cephalothin, cephalexin, application of antibiotics to the growth phase of the and cephradine were tested in a way similar to that culture. Thus, cultures in exponential phase when of the penicillins. Cephradine was the least vulner- first exposed to the drug gave smaller zones of able to penicillinase and cephaloridine the most. inhibition than those in stationary phase, particularly One surprising finding from these experiments was if that culture produced penicillinase. The following that the antibiotics cephalexin and cephradine, experiments were performed with a standardised although structurally almost identical, had appar- pre-incubation time of two hours at 37°C before ently significant differences in their resistance application of antibiotic to the wells in the agar. Two to penicillinase. This was confirmed as follows: hours' incubation was selected because the difference The activities of 60 strains, for which the MIC data between the activity of the antibiotics under test was had been obtained, were each examined against markedly and reproducibly different between penicil- these antibiotics by diffusion testing. The activities linase-positive and -negative cultures. It was felt that of the two drugs against 13 penicillinase-negative two hours was sufficient time for the culture to be strains from these cultures were, as expected, almost in early exponential phase and would resemble the identical; the mean relative activity of cephalexin J Clin Pathol: first published as 10.1136/jcp.30.1.35 on 1 January 1977. Downloaded from 38 R. W. Lacey and Anne Stokes was 122% and that of cephradine 120% of the resistance identical with the wild strain were con- standard (t = 0-13; p > 0 50). In contrast, there sidered to possess plasmids intact except for their were marked differences between the activities of the inability to cause the cells to synthesise penicillinase. 47 penicillinase-positive cultures for cephradine Each of these plasmids was then transduced into a (89Y) and cephalexin (41 %) relative to that of the fresh clone of the corresponding original host cell. standard. Differences even greater than this could be Thus, three cultures were prepared that were detected if the antibiotics were added to a culture in apparently identical except for variation in their mid-exponential phase. ability to produce penicillinase. These six cultures were then tested for their resistance to penicillins ACTIVITIES OF PENICILLINS AND CEPHALO- and cephalosporins. As expected, no variation in SPORINS AGAINST 15 STRAINS OF Staph. aureus MIC was seen between members of each pair, but PRODUCING HIGH LEVELS OF PENICILLINASE in diffusion testing greatly reduced zones were seen Fifteen strains that produced high levels of penicil- around wells of most of the cephalosporins and also linase were then selected from another 50 cultures. flucloxacillin compared to their penicillinase- When tested against these strains, flucloxacillin was negative counterparts. found to be inactivated to a greater extent than the The zones of both penicillinase-positive and other semisynthetic penicillins, and of the cephalo- -negative cultures were similar to corresponding sporins cephaloridine was the most vulnerable, and types of strains described above. Thus, it appears cephradine the least (Table 3). Combined data for highly likely that it was the production of penicil- linase itself that caused the reduction in zone sizes Table 3 Relative activities (as % ofstandard sensitive in diffusion testing. strain) ofcephalosporins andpenicillins for 15 The reason for this reduction in zone size could penicillinase-positive strains ofStaph. aureus be due either to destruction of the antibiotic or to the presence of the antibiotic acting as an inducer of Antibiotic Mean Range enzyme production during diffusion. Attempts to Cephaloridine 0-12 0 01- 0-2 demonstrate the latter have been unsuccessful.copyright. Cephalexin 6-6 15 - 20-0 Cultures were pre-incubated in subinhibitory con- Cephradinc 12-2 2-4 - 44-2 Cephalothin 2-6 0-6 - 6-9 centrations of each drug singly before the diffusion Cephazolin 4-4 2-7 - 8-9 experiment was performed as before. No differences Methicillin 48-4 20-9 -118-0 Nafcillin 42-5 17-0 - 87-0 were observed. Cloxacillin 122-1 28-8 -398-0 Flucloxacillin 12-9 4-2 - 29-5 Discussion

the 38 strains previously examined, and the selected These results indicate that, while the activity of anyhttp://jcp.bmj.com/ 15, indicated that flucloxacillin (mean activity 25 %V) one of the penicillinase-resistant penicillins or was much more vulnerable to penicillinase than cephalosporins is almost uniform against staphylo- either methicillin (69 %O), nafcillin (54Y.), or cloxacil- cocci if tested by MIC methods, it is extremely lin (63Y). variable in diffusion testing. This variation has been demonstrated (most emphatically) by pre-incubation EVIDENCE THAT VARIATION IN ACTIVITY IS (for two hours) of the seeded plate before application DUE TO PRODUCTION OF PENICILLINASE of the drug. Evidence has been presented that this on October 1, 2021 by guest. Protected Three cultures of Staph. aureus that produced either variation is due to two main factors: (1) the pro- A, C, or D-type penicillinase (Rosdahl, 1973) were duction of penicillinase by some cultures, and treated (Lacey, 1971) with a mutagen, nitroso- (2) variation in the ability of the various antibiotics guanidine, and then plated on media containing to resist penicillinase. 10-4M cadmium acetate. (This step was essential to The reason why MIC methodology gives uniform select plasmid-positive cells. Nitrosoguanidine treat- results for any one of the drugs against a variety of ment produces loss of the entire penicillinase plasmid test cultures is that it fails to detect the production of at a frequency much greater than point mutations in penicillinase, since that enzyme is readily lost from that plasmid; since each of the plasmids employed the dormant cell. carried the genes for cadmium-ion resistance, this We believe that, in general, the diffusion/pre- plating ensured the retention of each plasmid.) The incubation method of sensitivity testing relates more resultant colonies were replica-plated onto media closely to the clinical situation than MIC methods. containing starch, and penicillinase-negative variants An exception to this may be the use of antibiotics were identified. These were examined for metal-ion prophylactically. It is of extreme practical im- resistance; those derivatives that showed metal-ion portance to assess the correctness of laboratory J Clin Pathol: first published as 10.1136/jcp.30.1.35 on 1 January 1977. Downloaded from Susceptibility of the 'penicillinase-resistant' penicillins and cephalosporins to penicillinase ofStaph. aureus 39 methods in sensitivity testing, since consistently resistance to staphylococcal penicillinase has been different results are obtained, depending on which found: cephaloridine (the most vulnerable), cephazo- method is used. Perhaps under ideal conditions the lin, cephalothin, cephalexin, and cephradine (the method should be selected that represents most most stable). It is impossible to draw many specific closely the context of each individual prescription. conclusions from this as these drugs differ in many Thus, if an antibiotic is used prophylactically it properties other than their resistance to penicillinase. should be selected on the basis of expected MIC of However, it would seem unwise to use cephaloridine the predicted pathogens. In the treatment of where a is suspected, and established infections, diffusion methods would be cephradine is preferable to cephalexin and probably preferable. Such refinement is obviously difficult to also cephazolin and cephalothin where a staphylo- achieve in a routine laboratory. coccal infection is a possibility. We believe, therefore, that the correct general policy for testing the activity of these penicillins and cephalosporins against staphylococci should be by This work was supported by a research grant from diffusion against actively growing inocula at 37°C. the East Anglian Regional Health Authority. Assuming that this method is valid, how do the various drugs fare against penicillinase-producing References staphylococci? Of the penicillins tested, methicillin, cloxacillin, and nafcillin were relatively resistant to Burgess, H. A. and Evans, R. J. (1966). Failure of cephalo- penicillinase, methicillin and cloxacillin being most ridine in a case of staphylococcal endocarditis. British resistant, whereas flucloxacillin was much less Medical Journal, 2, 1244. Lacey, R. W. (1971). Transfer of tetracycline-resistance resistant (approximately 10 times more susceptible between strains of Staphylococcus aureus in mixed cul- than cloxacillin against some strains). While tures. Journal of General Microbiology, 69, 229-237. flucloxacillin has an advantage over cloxacillin in Lacey, R. W. and Lewis, E. L. (1975). Further evolution of a that higher blood levels are obtained after oral strain of Staphylococcus aureus in vivo: evidence for significant inactivation of flucloxacillin by penicillinase. administration, it seems to us unlikely that this Journal of Medical Microbiology, 8, 337-347. copyright. would counteract the increased susceptibility to Lacey, R. W. and Lewis, E. L. (1976). Inactivation of penicillinase. It is, however, hazardous to predict the 'penicillinase-resistant' penicillins and cephalosporins by clinical outcome from such considerations, and more staphylococcal penicillinase; clinical and laboratory studies. III International Symposium on Staphylococci and clinical data than the two cases quoted by us would Staphylococcal Infections. Springer, Berlin. (In press.) be desirable. Meanwhile, in the absence of other Norden, C. W. and Dickens, D. R. (1973). Experimental considerations (eg, cost) and of other clinical data, oxteomyelitis. III. Treatment with cephaloridine. Journal we recommend the use of cloxacillin in these of Infectious Diseases, 127, 525-528.

Rosdahl, V. T. (1973). Naturally occurring constitutive http://jcp.bmj.com/ circumstances. ,-lactamase of novel serotype in Staphylococcus aureus. Of the cephalosporins, the following order of Journal of General Microbiology, 77, 229-231. on October 1, 2021 by guest. Protected