High-Level Potentiation of Lysostaphin Anti-Staphylococcal Activity by Lysozyme

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1982, p /82/ $02.00/0 Vol. 21, No. 4 High-Level Potentiation of Lysostaphin Anti-Staphylococcal Activity by Lysozyme GABRIELLA CISANI,* PIETRO E. VARALDO, GRAZIA GRAZI, AND ORNELLA SORO Istituto di Microbiologia dell'universitd di Genova, 16132, Genova, Italy Received 8 October 1981/Accepted 12 January 1982 The purpose of this study was to determine whether lysostaphin would enhance its anti-staphylococcal efficacy in combination with lysozyme. Minimum inhibitory concentrations (MICs) of lysostaphin and lysozyme were separately determined for 41 strains belonging to 10 different species of human staphylococci. Lysozyme was virtually inactive and showed MICs of mg/ml. On the contrary, all strains were susceptible to lysostaphin and showed MICs ranging from 2.5 to 60 jig/ml for the different Staphylococcus species. When the MIC of lysostaphin was determined in media containing submultiples of the MIC of lysozyme, the values obtained were much lower. The reduction of the lysostaphin MIC ranged from 16- to 200-fold in the different species tested. In Staphylococcus aureus, in particular, the combination of lysostaphin with 1.5 mg of lysozyme per ml reduced the MIC of lysostaphin by 25-fold. The activities of two combinations of the two enzymes were evaluated: one combination was expected to be active on S. aureus only, and the other combination was expected to inhibit all Staphylococcus strains. The first combination (0.5,ug of lysostaphin plus 0.5 mg of lysozyme per ml) was inhibitory to all of the 84 S. aureus strains tested, whereas 137 of 1 strains of other Staphylococcus species were unaffected. On the contrary, all of the 235 Staphylococcus strains tested were inhibited by the second combination (4,ug of lysostaphin plus 5 mg of lysozyme per ml). The possible mechanisms of lysostaphin potentiation by lysozyme are considered, and the potential use of a lysostaphin-lysozyme combination for topical therapy of staphylococcal infections resistant to other antibiotics is discussed. Lysozyme and lysostaphin are two well-characterized enzymes known to inhibit and lyse Micrococcaceae. The former enzyme is highly active on micrococci but only slightly active on staphylococci, whereas the latter inhibits staphylococci but is generally inactive on micrococci (7, 17, 19). Lysozyme acts mainly as an endo-p-muramidase (2), whereas in lysostaphin, three different enzymatic activities are present: an endopeptidase, which is the most active fraction, an endo-p-glucosaminidase, and an amidase (1, 6, 21). The therapeutic treatment of staphylococcal infections is sometimes difficult because of the relatively high number of infections which are caused by antibiotic-resistant strains. However, important pathogenic processes caused by staphylococci, such as surgical or accidental wound infections or certain infections involving bones or joints, can benefit by treatment with drugs suitable for topical use. Lysostaphin has been already successfully used in humans as a topical anti-staphylococcal agent (4, 11). Moreover, it has been shown that lysozyme can facilitate the penetration of several substances in bacteria, thereby potentiating the action of certain antimicrobial agents (13). We have considered the possibility that the two enzymes could potentiate each other and that their association could result in a strong anti-staphylococcal activity. This paper shows that lysozyme potentiates the anti-staphylococcal activity of lysostaphin to a variable extent (up to 200-fold) depending on the different staphylococcal species. 531 MATERIALS AND METHODS Bacterial strains. Of the 235 Staphylococcus strains tested in this study, 210 isolates were isolated from clinical material in our clinical bacteriology laboratory (Istituto di Microbiologia dell'universita di Genova) and were identified by two recently developed test systems (9, 20). The other 25 strains were obtained from the Czechoslovak Collection of Microorganisms or the American Type Culture Collection. Eight Micrococcus strains of different species were also used. All were from our institute's collection. Enzymes and evaluation of their MICs. Hen egg white lysozyme was from Societa Prodotti Antibiotici, Milano, Italy, and lysostaphin was purchased from Sigma Chemical Co., St. Louis, Mo. Stock solutions Downloaded from on August, 2018 by guest

2 532 CISANI ET AL. TABLE 1. Evaluation of the MICs of lysostaphin and lysozyme for strains of different staphylococcal species Lysostaphin MIC' Lysozyme MIC' Species (no.) (pg/mi) (mg/ml) S. aureus (5) 2.5 S. capitis (3) 60 S. cohnii (4) 8 S. epidermidis (5) 16 S. haemolyticus (5) 50 S. hominis (3) 60 S. saprophyticus (5) 16 S. simulans (3) 4 S. warneri (4) 40 S. xylosus (4) 14 a The values reported indicate concentrations inhibiting all strains tested (100% MIC). of lysozyme and lysostaphin were prepared in phosphate-buffered saline or 0.05 M Tris buffer containing M NaCl, respectively. The minimum inhibitory concentration (MIC) of each enzyme was determined with the agar-dilution method. Plates of LB medium (Bacto tryptone [Difco Laboratories, Detroit, Mich.], 10 g; Bacto yeast extract [Difco], 5 g; agar, g; and NaCl, 10 g per liter) containing either lysozyme or lysostaphin at various concentrations were inoculated with 0.01 ml of bacterial suspensions containing 104 colony-forming units of the various test strains. The MIC was defined as the lowest concentration of each enzyme that, after an 18- to 20-h incubation, completely inhibited visible growth of each particular strain tested. Preparation of lysostaphin-imbibed paper disks or strips. Whatman no. 1 paper disks (6-mm diameter) were sterilized in a dry oven at 170 C for 30 min. The disks were divided into groups of 100, and every group was flooded with 1.2 ml of a suitable lysostaphin solution. When disks flooded with a lysostaphin solution at the same concentration as the respective MIC were used, 9-mm-diameter zones of inhibition of bacterial growth were obtained. Therefore, we regarded as MICs of lysostaphin in lysozyme-containing media those lysostaphin concentrations which, when applied to the disks, produced an inhibition zone of equal size. Paper strips (6 by 85 mm) were also used in some cases with S. aureus strains. The strips were imbibed with a 0.5-,ug/ml solution of lysostaphin. Evaluation of the MIC values of lysostaphin-lysozyme combinations. Plates of LB medium containing various lysozyme concentrations below the MIC were prepared. The plates were inoculated with the strain to be tested by spreading 0.1 ml of an overnight broth culture on their surfaces. Paper disks or strips each containing a different dose of lysostaphin were then deposited on the surface of the inoculated plates. The amount of lysostaphin contained in the paper disks was below the enzyme MIC. Tests for synergy. The character of the interaction between lysostaphin and lysozyme was tested on four strains of each staphylococcal species, and an isobologram was plotted from these results (). The concentration of lysostaphin formed the ordinate, and the concentration of lysozyme formed the abscissa. Syn- ANTIMICROB. AGENTS CHEMOTHER. ergy was recorded when the curves bowed downward toward the ordinate with respect to the straight line joining the lysostaphin MIC to the lysozyme MIC. Moreover, the combination was considered significantly synergistic if less than one-fourth of the MIC of each enzyme when used separately produced inhibition of bacterial growth when combined (10). In preliminary trials carried out with fewer strains, a typical checkerboard agar-dilution procedure (10) was also used to assess lysostaphin-lysozyme combinations. RESULTS Evaluation of susceptibility of staphylococci to lysostaphin and lysozyme. Since it was shown by others that the various Staphylococcus species have different degrees of susceptibility to lysostaphin (5, 8), the MIC of lysostaphin was evaluated for several strains of each different species. Table 1 shows that all strains were susceptible to lysostaphin, although the MIC values varied significantly from species to species. A 24-fold difference was observed among the most susceptible species (S. aureus) and the most resistant ones (S. hominis and S. capitis). Table 1 also confirms the known resistance of staphylococci to lysozyme, showing that mg of this enzyme per ml is needed to inhibit growth of the various staphylococcal species. Evaluation of the possible potentiation of the anti-staphylococcal activity of lysostaphin by lysozyme. We then evaluated the MIC of lysostaphin for three to five strains of the different staphylococcal species in the presence of various noninhibitory concentrations of lysozyme. Figure 1 shows the effect on the growth of a S. aureus strain of paper strips containing a dose of lysostaphin five times lower than its MIC when tested on plates containing lysozyme at concentrations 10 or 5 times lower than its MIC. Growth inhibition around the paper strips was clearly evident under both conditions and increased with lysozyme concentration. Lysozyme enhanced the lysostaphin inhibitory effects toward the strains of all staphylococcal species and the interaction between the two drugs was synergistic, although large differences in the degree of synergy emerged among the various species (Fig. 2). We have reported the lysozyme concentrations that gave the strongest enhancement of the lysostaphin activity in the different staphylococcal species (Table 2). It is evident that the strongest enhancing effect was observed with the S. cohnii strains. Particularly strong enhancements were also observed for S. epidermidis and, to a lesser extent, for S. simulans. It should also be noted that in some species a strong synergistic action required a relatively high concentration of lysozyme (7.5 mg/ml), whereas with other species, a relatively low concentration was sufficient (1.5 mg/ml). Downloaded from on August, 2018 by guest

3 VOL. 21, LYSOSTAPHIN POTENTIATION BY LYSOZYME 533 The method used to assess lysostaphin-lysozyme synergy was a combination of a strip diffusion and an agar-dilution procedure, which was preferred to more standard techniques, mainly because of the smaller amount of lysostaphin required. However, a typical checkerboard agar-dilution assay was carried out for one representative strain of each Staphylococcus species. The results obtained correlated very well with those reported above. For example, "& v_o B lysozyme concentration (mg/m1) FIG. 2. Isobolograms of the lysostaphin-lysozyme interactions investigated with four different strains from each of the following species: (A) 0, S. aureus; 0, S. xylosus; *, S. saprophyticus; a, S. cohnii; and A, S. simulans; and (B) 0, S. haemolyticus; 0, S. hominis; *, S. capitis; El, S. epidermidis; and A, S. warneri. Downloaded from on August, 2018 by guest FIG. 1. Growth of S. aureus strains ATCC as influenced by the interaction of lysostaphin and lysozyme at doses below their MICs. Lysostaphin was contained in the paper strips, which were imbibed with a solution of this substanee at a concentration of 0.5 jig/ml. Lysozyme was contained in the agar media shown on the right half of each plate (left halves were controls without lysozyme) at concentrations of 1.5 (A) or 3 mg/ml (B). potentiation rates of lysostaphin by lysozyme of 32- and 16-fold were obtained for S. aureus and S. epidermidis, respectively. Selection of concentrations of lysostaphin and lysozyme inhibitory for most staphylococcal strains. We then tested the susceptibility of a total of 235 strains (most of which were isolated from clinical specimens), including representatives of all staphylococcal species, to two different lysostaphin-lysozyme combinations: one combination was expected to inhibit chiefly S. aureus strains and the other was expected to inhibit all staphylococci. In the presence of 0.5

4 534 CISANI ET AL. TABLE 2. Evaluation of potentiation of the antistaphylococcal activity of lysostaphin by lysozyme Potentiation Lysozyme Lysostaphin rate of Species (no.) concn MICa lysostaphin (mg/ml) (pug/ml) activity (fold) S. aureus (50) S. capitis (4) S. cohnii (6) S. epidermidis (48) S. haemolyticus (6) S. hominis (5) S. saprophyticus (9) S. simulans (6) S. warneri (5) S. xylosus (5) a The values reported refer to lysostaphin concentrations inhibiting all strains tested (100% MIC) in the presence of lysozyme at the concentration indicated. plg of lysostaphin plus 0.5 mg of lysozyme per ml, all of the 84 S. aureus strains tested were inhibited, whereas among the other species only 14 strains of the 1 strains tested were inhibited (Table 3). On the contrary, in the presence of 4,ug of lysostaphin and 5 mg of lysozyme per ml, all strains were inhibited. Analysis of the possible potentiation of the antimicrococcal activity of lysozyme by lysostaphin. To test the possible potentiation of the antimicrococcal activity of lysozyme by lysostaphin, we evaluated the lysozyme MIC of eight different micrococci and their susceptibility to lysostaphin. All strains were inhibited by 0.1,ug of lysozyme per ml and were resistant to 100,ug of lysostaphin per ml. However, when the susceptibility of micrococci to lysozyme was tested in the presence of 100,ug of lysostaphin per ml, no evident enhancement of the antibacterial activity of lysozyme was observed. ANTIMICROB. AGENTS CHEMOTHER. DISCUSSION Different hypotheses can be presented to explain the mechanism by which lysozyme potentiates the antibacterial activity of lysostaphin. One possibility is that lysozyme, which is unable to hydrolyze the glycosidic bonds of the intact staphylococcal cell wall, could split the glycosidic bonds after lysostaphin has interrupted the integrity of peptidoglycan. Potentiation is mostly observed in the presence of high doses of lysozyme. This could be due to the need for the glycosidic bonds to be split at kinetic rates which are fast enough to result in a significant increase in the overall peptidoglycan degradation and bacterial growth inhibition. Since lysozyme is also known to bind to the walls of bacteria resistant to its enzymatic action (18), it is possible that by binding to the staphylococcal cells, lysozyme facilitates the interaction of lysostaphin with its specific peptidoglycan target. It has been known for a long time that lysozyme can trigger autolysis of bacteria, in which the cell walls are resistant to lysozyme hydrolysis (3, 14). Furthermore, since lysostaphin is an enzyme complex excreted by some staphylococcal strains (16), it is quite likely that it has an important role in the autolytic system of these strains and possibly other staphylococci. Enhancement of the anti-staphylococcal activity of lysostaphin could then be just a special aspect of the ability of the lysozyme to stimulate bacterial autolysins. The fact that lysostaphin does not potentiate the anti-micrococcal activity of lysozyme could simply be due to the fact that, with lysozyme being capable of digesting the micrococcal peptidoglycan very rapidly, doses much higher than those used here could be needed to cause an accelerated digestion. This obviously implies that the specificity of lysostaphin for the chemical bonds of micrococcal peptidoglycans is low compared with that of lysozyme. On the other hand, lysozyme potentiates the anti-staphylococcal activity of lysostaphin only if present at high concentrations. It therefore cannot be excluded that concentrations of lysostaphin higher than 100 jig/ml, i.e., the highest dose tested, potentiate the anti-micrococcal activity of lysozyme. The high cost of lysostaphin discouraged us from testing the effect of lysostaphin concentrations higher than 100,ug/ml. Lysostaphin is a powerful anti-staphylococcal drug already used topically in humans (4, 11). We have shown here that, when combined with lysozyme, lysostaphin is potentiated by 16- to 200-fold and appears to be one of the most active anti-staphylococcal agents reported in the litera- TABLE 3. Susceptibility of 235 strains from different staphylococcal species to two lysostaphinlysozyme combinations No. of strains inhibited by: Species (no.) (0.5 pig/mi) (4 jig/mi) + + lysozyme lysozyme (0.5 mg/ml) (5 mg/ml) S. aureus (84) S. capitis (8) 0 8 S. cohnii (6) 4 6 S. epidermidis (70) 0 70 S. haemolyticus (14) 0 14 S. hominis (14) 0 14 S. saprophyticus (12) 0 12 S. simulans (13) S. warneri (7) 0 7 S. xylosus (7) 0 7 Species (no.) (Lysostaphin Lysostaphin Downloaded from on August, 2018 by guest

5 VOL. 21, 1982 ture. This enzyme combination might prove suitable for topical use in the treatment of staphylococcal infections resistant to other antibiotics. Possibly because of the fact that lysostaphin is formed by three different enzyme activities (6, 21), resistance to this antibiotic seems to be rare among staphylococci (22). Furthermore, the lysostaphin-lysozyme combination inhibits bacterial growth through the digestion of the bacterial cell wall and is bactericidal and equally active on rapidly growing and nongrowing cells. These last properties may prove particularly useful in the treatment of torpid chronic staphylococcal infections. Special attention should be paid to the lysostaphin-lysozyme combination which was found to inhibit virtually only S. auteus strains. Its topical use in cases of skin and mucosal relapsing infections caused by S. aureus could be particularly successful in selectively removing the causative agent without interfering with nonpathogenic staphylococci inhabiting the surface near the infected area. This could favor subsequent colonization by nonpathogenic staphylococci and other saprophytic organisms and be helpful in preventing reinfection. It should be noted that lysostaphin alone, although much less active than in combination with lysozyme, was previously shown not only to reduce carriage rates in nasal carriers of S. aureus (4, 11), but also to favor reacquisition of normal flora (12). The MIC values of lysostaphin reported in this study for the different Staphylococcus species are lower than those reported by others (8). Such differences most likely depend on the different methods used and the different numbers of strains tested for each species. ACKNOWLEDGMENTS We thank G. Satta for his helpful suggestions and discussion throughout this work. We are also indebted to S. Raffanti for his help in the preparation of the manuscript. LYSOSTAPHIN POTENTIATION BY LYSOZYME 535 LITERATURE CITED 1. Browder, H. P., W. A. Zygmunt, J. R. Young, and P. A. Tavormina Lysostaphin: enzymatic mode of action. Biochem. Biophys. Res. Commun. 19: Chipman, D. M., and N. Sharon Mechanism of lysozyme action. Science 165: Ghuysen, J.-M Use of bacteriolytic enzymes in determination of wall structure and their role in cell metabolism. Bacteriol. Rev. 32: Harris, R. L., A. W. Nunnery, and H. I). Riley Effect of lysostaphin on staphylococcal carriage in infants and children, p Antimicrob. Agents Chemother Heddaeus, H., P. B. Heczko, and G. Pulverer Evaluation of the lysostaphin-susceptibility test for the classification of staphylococci. J. Med. Microbiol. 12: Iversen, 0. J., and A. Grov Studies on lysostaphin. Separation and characterization of three enzymes. Eur. J. Biochem. 38: Klesius, P. H., and V. T. Schuhardt Use of lysostaphin in the isolation of highly polymerized deoxyribonucleic acid and in the taxonomy of aerobic Micrococcaceae. J. Bacteriol. 95: Kloos, W. E., and K. H. Schliefer Isolation and characterization of staphylococci from human skin. II. Descriptions of four new species: Staphylococcus warneri, Staphylococcus capitis, Staphylococcus hominis, and Staphylococcus simulans. Int. J. Syst. Bacteriol. 25: Kloos, W. E., and K. H. Schleifer Simplified scheme for routine identification of human Staphylococcus species. J. Clin. Microbiol. 1: Krogstad, D. J., and R. C. Moellering, Jr Combinations of antibiotics, mechanisms of interactions against bacteria, p In V. Lorian (ed.), Antibiotics in laboratory medicine. The Williams & Wilkins Co., Baltimore, Md. 11. Martlin, R. R., and A. White. 1%7. The selective activity of lysostaphin in vivo. J. Lab. Clih. Med. 70: Martin, R. R., and A. WhIte The reacquisition of staphylococci by treated carriers: a demonstration of bacterial interference. J. Lab. Clin. Med. 71: Naranjo, P., and J. C. De Moreno Combined action of lysozyme with antibiotics and chemotherapeutic agents, p In Atti del 30 Symposium Internazionale sul Lisozima di Fleming, vol. 1, sect. 3. Societa Prodotti Antibiotici, Milano, Italy. 14. Pollock, J. J., V. J. Iacono, H. Goodman-Bicker, B. J. MacKay, L. I. Katona, and E. Thomas The binding, aggregation and lytic properties of lysozyme, p In H. M. Stiles, W. J. Loesche, and T. C. O'Brien (ed.), Proceedings: Microbial Aspects of Dental Caries (a special supplement to Microbiology Abstracts), vol. III. Information Retrieval, Inc., Washington, D.C.. Sabath, L. D Synergy of antibacterial substances by apparently known mechanisms, p Antimicrob. Agents Chemother Schindler, C. A., and V. T. Schuhardt Lysostaphin: a new bacteriolytic agent for the staphylococcus. Proc. Nati. Acad. Sci. U.S.A. 51: SchieUfer, K. H., and W. E. Kloos Separation of staphylococci from micrococci, p In J. Jeljaszewicz (ed.), Staphylococci and staphylococcal diseases. Gustav Fischer Verlag, Stuttgart. 18. Strominger, J. L., and D. J. Tipper Structure of bacterial cell walls: the lysozyme substrate, p In E. F. Osserman, R. E. Canfield, and S. Beychok (ed.), Lysozyme. Academic Press, Inc., New York. 19. Thomas, E. T., and V. T. Schuhardt Susceptibility of aerobic Micrococcaceae to lysostaphin. Bacteriol. Proc Varaldo, P. E., G. Grazi, 0. Soro, G. CIsani, and G. Satta Simplified lyogroup system, a new method for routine identification of staphylococci: description and comparison with three other methods. J. Clin. Microbiol. 12: Wadstrom, T., and 0. Vesterberg Studies on endo- P-N-acetylglucosaminidase, staphylolytic peptidase, and N-acetylmuramyl-L-alanine amidase in lysostaphin and from Staphylococcus aureus. Acta Pathol. Microbiol. Scand. Sect. B 79: Zygmunt, W. A., and P. A. Tavormina Lysostaphin: model for a specific enzymatic approach to infectious disease. Prog. Drug Res. 16: Downloaded from on August, 2018 by guest