Vol. 93, No. 2

JOURNAL OF BACTERIOLOGY, Feb., 1967, p. 520-524 Copyright ( 1967 American Society for Microbiology

Printed in U.S.A.

Lysostaphin in Experimental Renal Infections EDWARD F. HARRISON AND WALTER A. ZYGMUNT Department of Microbiology and Natural Products Research, Mead Johnson Research Center, Evansville, Indiana

Received for publication 8 September 1966 ABSTRACT

By use of a renal staphylococcal infection model in mice, single intravenous doses of lysostaphin ranging from 1.56 to 50 mg/kg were effective in: (i) controlling the staphylococcal population of kidneys, (ii) reducing the mortality rate, and (iii) clearing high numbers of kidneys of infection. Semisynthetic penicillins and other antistaphylococcal antibiotics given in the same manner did not have significant activity. Only by the administration of a long-acting, depot form of penicillin (Bicillin) could results comparable to those seen with lysostaphin be obtained. The results of this study suggest that lysostaphin may be useful in staphylococcal septicemias in preventing the establishment of new foci of infection.

Lysostaphin is an extracellular bacterial enzyme with highly specific antistaphylococcal

MATERIALS AND METHODS

Test animals. Male albino mice (Swiss-Webster activity primarily against coagulase-positive strain) ranging from 25 to 30 g in body weight were strains. Its in vitro antistaphylococcal activity used. The mice were caged in groups of five animals has been studied extensively (2, 10). Subsequently, each and received food and water ad lib. Infection model. Staphylococcus aureus Giorgio comparisons were made in vitro with other antistaphylococcal antibiotics and semisynthetic strain, kindly supplied by R. M. McCune, Jr., Cornell penicillins (4, 12, 13). All of these investigations University Medical School, was used to produce the renal abscesses. A 24-hr culture grown in Trypticase substantiate the fact that lysostaphin is an ex- Soy (BBL) at 37 C was centrifuged, washed tremely potent antistaphylococcal antibiotic once,Broth and to its original volume in which offers a novel and potentially useful physiologicalresuspended saline. The suspension was diluted with to approach chemotherapy. saline so that each mouse received approximately 108 Schuhardt and Schindler (11) demonstrated viable staphylococci when 0.5 ml of the inoculum that lysostaphin is effective in vivo against experi- was injected into the tail vein. At 1 hr after infection, mental staphylococcal peritonitis when given by the kidneys contained approximately 104 viable either the intraperitoneal or subcutaneous routes. staphylococci per milliliter as determined by standard This observation was confirmed in our laboratory. plate counts. A total of 40 mice were infected with drug tested, so that groups of 10 mice could be Using other in vivo models, Harrison and Cropp each from the test and examined at intervals of (Can. J. Microbiol., in press) found that lysosta- withdrawn 11, 18, and 21 days after infection and medication. phin is capable of reducing the edema produced 4, The chemotherapeutic effects of the antistaphyloby a localized staphylococcal infection in the coccal agents were determined by use of procedures mouse leg, and that topical application of the previously reported (6, 7, 8). Each agent was adminantibiotic reduces the number of viable staphylo- istered as a single intravenous dose (in milligrams cocci in experimental rabbit ear infections. per kilogram) 1 hr after infection except for Bicillin Because of the high chemotherapeutic potency (benzathine penicillin G), which was given as a single of this antibiotic in each of the experimental in- intramuscular dose. At the time intervals designated, fection models that were tried, the studies re- groups of 10 mice were withdrawn from the test, and kidneys were examined for viable staphylococci ported here were undertaken to establish the the use of two different methods. Three or four aniby in of chemotherapeutic potential lysostaphin mals from each group were killed, and their kidneys staphylococcal renal abscesses produced experi- were removed. Separate homogenates were prepared mentally in mice. The infection model used is from the kidneys of each animal in sterile buffer. The similar to certain chronic staphylococcal ab- total staphylococcal populations of the kidneys were scesses seen in man which currently are not expressed as numbers of viable cells per milliliter of entirely amenable to therapy with clinically kidney as described by McCune and Tompsett (9). The volume of the kidney was taken into account in available antibiotics. 520

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LYSOSTAPHIN IN EXPERIMENTAL RENAL INFECrIONS

preparing the homogenates for determining the colony counts. Mannitol Salt Agar (Difco) was inoculated with appropriate dilutions of the tissue homogenates, and colony counts of the viable staphylococci were made after 2 days of incubation at 37 C. The data obtained from the counts were used to calculate the mean number of viable staphylococci per milliliter of kidney. The mean of the logarithms of the kidney staphylococcal populations was plotted for each drug as a function of time in days. The kidneys from the animals remaining in each group were examined qualitatively for the presence of viable staphylococci. The kidneys were removed aseptically and bisected with sterile scissors. A streak plate was made by passing the inner surface of the kidney over the surface of a Mannitol Salt Agar plate several times. After incubation, the plates were examined for colonies of staphylococci. Kidneys that failed to produce typical staphylococcal colonies were considered to be cleared of the infection. Since the data on cleared kidneys obtained by homogenate plate counts were in general agreement with that obtained by the qualitative streak plate method, both sets of data were included in the final tabulation of results. Antibiotics. The following antibiotics were used: kanamycin, oxacillin, methicillin, and ampicillin (Bristol Laboratories, Syracuse, N.Y.); vancomycin and propicillin (Eli Lilly & Co., Indianapolis, Ind.); cloxacillin and dicloxacillin (Ayerst Laboratories, Rouses Point, N.Y.); nafcillin and Bicillin-mixture of benzathine, procaine, and potassium penicillin G (Wyeth Laboratories, Philadelphia, Pa.); lincomycin (The Upjohn Co., Kalamazoo, Mich.); penicillin G, potassium salt (USP) (Nutritional Biochemicals Corp., Cleveland, Ohio); cephaloridine (Glaxo Laboratories, Greenford, Middlesex, England); and lysostaphin (Mead Johnson & Co., Evansville, Ind.). The lysostaphin used in this study was a lyophilized preparation which had a potency of 200 units of lytic activity per mg, and which was approximately 95% protein when assayed by the method of Lowry et al. (5) when lysozyme was used as a standard. All drugs were dissolved in physiological saline except lysostaphin, which was dissolved in 0.05 M tris(hydroxymethyl)aminomethane (Tris) in physiological saline, pH 7.5. Sensitivity tests. The antibiotic sensitivity of the S. aureus Giorgio, expressed as the minimal inhibitory concentration (MIC), was determined by the conventional twofold tube dilution method (3) with Trypticase Soy Broth. The level of inoculum used was 105 organisms per milliliter.

RESULTS AND DIscussIoN The MIC values for the test culture obtained in our laboratory (Table 1) agree with those reported by McCune (6) for oxacillin and methicillin, although the sensitivity to penicillin G was greater in our experiments (0.095 ,ug/ml compared with 1.6 to 3.1 Ag/ml). The culture was virulent as evidenced by the overall high rate of kidney infection in the nonmedicated control

521

mice (93%). The 3-week mortality rate in the nonmedicated groups ranged from 55 to 80% (60% average) in five different experiments. The bacterial census was adequate for evaluating the chemotherapeutic effect of the antistaphylococcal agents. Single intravenous doses of lysostaphin, ranging from 1.56 to 50 mg/kg, produced significantly lower bacterial populations in the kidney (Fig. 1). Lysostaphin decreased the staphylococcal census by 5 log units as compared with the nonmedicated control kidneys. Similar observations have been made by W. Schaffner, M. A. Melly, and M. G. Koenig (Clin. Res. 14:343, 1966) using a comparable test model. At 0.5 mg/kg, elevated bacterial counts were observed at 4 and 11 days; however, the 18- and 21-day kidney populations were markedly decreased. Thus, a single dose of lysostaphin at this low level was apparently sufficient to control the staphylococcal population to the point where the normal host defense mecha-

nisms could function. High numbers of kidneys were cleared of the infection by the single-dose therapy of lysostaphin (Table 2). The percentage of kidneys cleared ranged from 46 to 85% for lysostaphin, in contrast to 7 % for the nonmedicated control animals. Likewise, low mortality rates were obtained with all of the drug concentrations when lysostaphin was used for medication. The mortality rate of 55 to 80% for the nonmedicated control animals was lowered significantly by single-dose therapy of lysostaphin (0 to 15 %). Single intravenous doses of lysostaphin as low as 1.56 mg/kg demonstrated a significant therapeutic effect, whereas numerous semisynthetic penicillins and other antibiotics administered in a similar manner at levels up to 25 mg/kg were essentially inactive. Only by the administration of a long-acting, depot form of penicillin (Bacillin) TABLE 1. In vitro senisitivity of Staphylococcus aureus Giorgio to various antibiotics Antibiotics

MIC

Lysostaphin ........................

isgImi 0.095

Propicillin............................0.0 Penicillin G. 0.095 0.095 Cephaloridine . Ampicillin........................... 0.19 Cloxacillin ......................... 0.19 Dicloxacillin ..........................0.19 Nafcillin ........................... 0.39 Oxacillin ........................... 0.78 0.78 Lincomycin. 1.56 Vancomycin ........................ 3.12 Methicillin. 6.25 Kanamycin .........................

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VOL. 93, 1967

523

LYSOSTAPHIN IN EXPERIMENTAL RENAL INFECIIONS

TABLE 2. Chemotherapeutic effect of lysostaphin and other antistaphylococcal drugs on renal infection and mortality rate in mice Antibiotic Antibiotic

Lysostaphin

Bicillin

Cephaloridine Ampicillin Propicillin Penicillin G Nafcillin Vancomycin Kanamycin Lincomycin Dicloxacillin Cloxacillin Oxacillin Methicillin Nonmediated control b

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on

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days postinfection

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Mortality

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85 64 76 75 85 65 46 93 65 24 18 14 11 11 11 10 9 5 3 3 3 7

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Number of kidneys cleared of staphylococci per number of kidneys examined. Administered intramuscularly.

could results comparable to those observed with lysostaphin be obtained. Best clearance of the renal infections was demonstrated by penicillins having benzyl or phenoxy side chains (penicillin G, ampicillin, propicillin) of the several penicillins tested. Penicillin G produced a low mortality rate, but it was not impressive in the total clearance or bacterial-census evaluation. Cephaloridine, the only cephalosporin tested, produced good clearance of the infection, but the kidney bacterial populations remained elevated until the terminal plating. The remaining semisynthetic penicillins kanamycin, vancomycin, and lincomycin were essentially inactive. This study indicates that lysostaphin may have a role in systemic therapy of certain chronic staphylococcal lesions and their accompanying septicemias. Lysostaphin, because of its enzymatic mode of action (1), has the advantage of bringing about a rapid decrease in the staphylococcal titers, whereas most conventional antistaphylococcal antibiotics merely inhibit bacterial multiplication and, consequently, require much longer time periods to elicit marked decreases in staphylococcal counts in the blood. Lysostaphin has the added advantage of lysing coagulase-

positive staphylococci readily regardless of their metabolic state, whereas all penicillin-like antibiotics inhibit only those bacteria which are actively multiplying and presumably building new cell wall structures. From the data presented in this study, it appears that lysostaphin warrants further consideration as a potential antistaphylococcal agent for systemic use alone or as an adjunct to therapy with the semisynthetic penicillins. ACKNOWLEDGMENTS We wish to thank C. B. Cropp, M. E. Fuquay, R. L. Stratman, and D. E. Thomas for their technical assistance during the course of this investigation. LITERATURE CITED 1. BROWDER, H. P., W. A. ZYGMUNT, J. R. YOUNG. AND P. A. TAVORMINA. 1965. Lysostaphiri. enzymatic mode of action. Biochem. Biophys. Res. Commun. 19:383-389. 2. CROPP, C. B., AND E. F. HARRISON. 1964. The in vitro effect of lysostaphin on clinical isolates of Staphylococcus aureus. Can. J. Microbiol. 10:823-828. 3. ENGLISH, A. R., AND P. C. GELWICKS. 1951. The

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in vitro microbial resistance pattern to terramycin. Antibiot. Chemotherapy 1:118-124. 4. HARRISON, E. F., AND C. B. CRoPP. 1965. Comparative in vitro activities of lysostaphin and other antistaphylococcal antibiotics on clinical

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isolates of Staphylococcus aureus. Appl. Microbiol. 13:212-215. 5. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,

10.

AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol.

Chem. 193:265-275. 6. MCCUNE, R. 1963. Influence of oxacillin on staphylococcal populations in mouse kidneys. Antimicrobial Agents and Chemotherapy1962, p. 107-113. 7. McCuNE, R. M., JR., P. A. DINEEN, AND J. C. BATrEN. 1956. The effect of antimicrobial drugs on an experimental staphylococcal infection in mice. Ann. N.Y. Acad. Sci. 65:91-102. 8. McCuNE, R., P. DINEEN, AND J. C. BATTEN. 1960. The influence of antimicrobial agents on total

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populations of staphylococci in animal tissues. J. Immunol. 85:447-464. MCCUNE, R. M., AND R. TOMPSETr. 1956. Fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. J. Exptl. Med. 104:737-762. SCHINDLER, C. A., AND V. T. SCHUHARDT. 1964. Lysostaphin: a new bacteriolytic agent for the staphylococcus. Proc. Natl. Acad. Sci. U.S. 51:414-421. SCHUHARDT, V. T., AND C. A. SCHINDLER. 1964. Lysostaphin therapy in mice infected with Staphylococcus aureus. J. Bacteriol. 88:815-816. ZYGMUNT, W. A., H. P. BROWDER, AND P. A. TAVORMINA. 1966. In vitro effect of lysostaphin, neomycin, and bacitracin on Staphylococcus aureus. Can. J. Microbiol. 12:204-206. ZYGMuNT, W. A., E. F. HARRISON, AND H. P. BROWDER. 1965. Microbiological activities of lysostaphin and penicillins against bacteriophage 80/81 strains of Staphylococcus aureus. Appl. Microbiol. 13:491-493.