Journal of Medical Microbiology (2007), 56, 71–76

DOI 10.1099/jmm.0.46788-0

In vitro activity of recombinant lysostaphin against Staphylococcus aureus isolates from hospitals in Beijing, China Xin-Yi Yang,13 Cong-Ran Li,13 Ren-Hui Lou,1 Yue-Ming Wang,1 Wei-Xin Zhang,1 Hui-Zhen Chen,1 Qing-Shan Huang,2 Yan-Xing Han,1 Jian-Dong Jiang1 and Xue-Fu You1 1

Laboratory of Pharmacology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China

Correspondence Xue-Fu You [email protected]

2

State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, PR China

Jian-Dong Jiang [email protected]

Received 17 June 2006 Accepted 8 September 2006

Lysostaphin is a glycylglycine endopeptidase. It cleaves the pentaglycine cross-bridge structure unique to the staphylococcal cell wall and is considered to be a potential drug for Staphylococcus aureus. In the present study, the in vitro activity of recombinant lysostaphin was investigated in 257 S. aureus isolates collected from hospital patients in Beijing, China, by determination of MIC and minimum bactericidal concentration (MBC) and a time–kill curve test. An agar dilution method was used for MIC determination in all of the isolates and a macrobroth dilution method was employed to verify MIC values for a subset of the isolates. All of the S. aureus strains were sensitive to the recombinant lysostaphin with MICs ranging from 0.03 to 2 mg ml”1 in the agar dilution assay. The antibacterial activity of lysostaphin was greater than that of vancomycin and other reference agents. For most of the isolates, the MICs from the agar dilution method were higher than those from the broth dilution method. The MBCs of lysostaphin in the test isolates were between 1- and 8-fold higher than their MIC values. Bactericidal activity (>99.9 % reduction) was observed after 2 h exposure of the isolates to lysostaphin at concentrations of ¢0.5 MIC. Lysostaphin showed a rapid bactericidal activity against the test strains of meticillin-susceptible S. aureus and meticillinresistant S. aureus. Its activity at ¢0.5 MIC was sustained for at least 6 h. These results will be informative for the clinical application and evaluation of lysostaphin.

INTRODUCTION Control of infections caused by meticillin-resistant Staphylococcus aureus (MRSA) is difficult because of the development of resistance to the drug (Aires de Sousa et al., 2003; Berger-Bachi & Rohrer, 2002). Over the past few decades, vancomycin has been employed as the drug of first choice against MRSA infections. However, MRSA with reduced susceptibility or full resistance to vancomycin has been reported (Srinivasan et al., 2002). Although full (or homogeneous) vancomycin-resistant MRSA has not been isolated from hospitals in China, clinical isolates with heterogeneous resistance to vancomycin have been documented (Wu et al., 2002). If vancomycin is continuously used widely as an empirical therapy in MRSA infections, 3These authors contributed equally to this work. Abbreviations: MBC, minimum bactericidal concentration; MRSA, meticillin-resistant Staphylococcus aureus; MSSA, meticillin-susceptible Staphylococcus aureus.

46788 G 2007 SGM

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staphylococcal isolates with full resistance to vancomycin will develop. Thus, new anti-staphylococcal agents with novel mechanisms are highly desirable. Lysostaphin is a 27 kDa endopeptidase that was first isolated from a culture of Staphylococcus simulans by Schindler & Schuhardt (1964). The enzyme kills the organism by hydrolysing a pentaglycine cross-bridge structure unique to the staphylococcal cell wall. As the cell wall cross-bridges of S. aureus are composed of a high proportion of pentaglycine, both proliferating and quiescent S. aureus cells are highly sensitive to lysostaphin (Kokai-Kun et al., 2003). Lysostaphin kills meticillin-susceptible S. aureus (MSSA) and MRSA equally well (Wu et al., 2003) and has been demonstrated to be a potent therapeutic agent for S. aureus infections in various animal studies (Dajcs et al., 2001; Goldberg et al., 1967; Harrison & Zygmunt, 1967; Patron et al., 1999). In the USA, two therapeutic products formulated with recombinant lysostaphin for topical use have entered clinical studies (McCoy, 2004; Mierau et al., 2005). 71

X.-Y. Yang and others

The present study is to the best of our knowledge the first evaluation of the in vitro antibacterial activity of recombinant lysostaphin and was carried out in 257 clinical strains of S. aureus collected from hospitals in Beijing, China. As many antibiotics have either lost or are losing their activity against MRSA in Chinese hospitals, we consider recombinant lysostaphin to be a potential alternative therapy for S. aureus infection in China.

METHODS Organisms. All isolates of S. aureus used in this study were collected from hospitals in Beijing between 2000 and 2003. The isolates comprised 168 strains of MSSA and 89 strains of MRSA. S. aureus ATCC 29213 served as a control strain. The MRSA isolates were detected by the oxacillin-salt agar screening test and/or the latex agglutination test for PBP2a in hospital laboratories and were further confirmed using the standard oxacillin agar dilution method as recommended by the National Committee for Clinical Laboratory Standards (now called the Clinical and Laboratory Standards Institute) in our laboratory. Antimicrobial agents and culture medium. Cefotaxime, pipera-

cillin, clarithromycin, vancomycin and ciprofloxacin were used as references and obtained from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Recombinant lysostaphin from Bacillus subtilis was provided by Shanghai Hi-Tech Bioengineering. Lyophilized lysostaphin contained 1000 U mg21. Mueller–Hinton (MH) agar and MH broth were purchased from Difco. Bivalent cations in the MH broth were adjusted with Ca2+ and Mg2+ based on procedures recommended by the National Committee for Clinical Laboratory Standards (2003a). Determination of MIC. MIC is defined as the lowest drug concen-

tration that inhibits the visible growth of micro-organisms after overnight incubation. Following the standard method recommended by the National Committee for Clinical Laboratory Standards (2003a), we used an agar dilution method to determine the MICs of lysostaphin and the reference antimicrobial agents in the staphylococcal isolates. All of the S. aureus strains were tested at a final inoculum concentration of 104 c.f.u. per spot using a multipoint inoculator (Denley Instruments) and incubated at 35 uC for 18 h. The in vitro sensitivity of the test strains to lysostaphin was obtained based on the interpretive criteria (16 mg ml21) presented by Climo et al. (2001). The drug sensitivity of the test strains was judged based on the interpretive criteria provided by the National Committee for Clinical Laboratory Standards (2003b). MIC50 and MIC90 were determined as the concentrations at which 50 and 90 % of the S. aureus isolates were inhibited, respectively. As the agar dilution assay used for determining lysostaphin susceptibility of staphylococci has rarely been reported previously, a macrobroth dilution assay was performed on a subset of the strains to compare with the results from agar dilution. Twenty-five isolates of S. aureus were randomly selected for the broth dilution assay and vancomycin was used as a control agent. The macrobroth dilution test was performed in cation-adjusted MH (CAMH) broth using the procedures provided by the National Committee for Clinical Laboratory Standards (2003a). S. aureus ATCC 29213 was included in the broth assay as a control to compare with the results of the agar dilution assay. MIC assays were carried in duplicate for each S. aureus strain. Determination of minimum bactericidal concentration. A

minimum bactericidal concentration (MBC) assay was employed to determine the lowest concentration of the agents lethal to the test 72

organism in vitro. The MBCs of lysostaphin were measured using the guidelines provided by the National Committee for Clinical Laboratory Standards (1999). MBCs were determined by a followup assay of the macrobroth dilution assay. Briefly, a series of broth tubes containing test bacteria (initial inoculum of 56105 c.f.u. ml21) were pre-treated with different concentrations of lysostaphin (0.001–4 mg ml21) at 35 uC for 24 h, as in the standard macrobroth dilution procedure of the MIC assay described above. A sample of broth (0.1 ml) was taken from the tubes and mixed with sufficient proteinase K (Sigma) to neutralize the residual lysostaphin, as described by Kokai-Kun et al. (2003) and Kusuma & Kokai-Kun (2005). The mixture was transferred on to a lysostaphin-free MH agar plate and subcultured for 24 h at 35 uC. Each S. aureus strain was assayed in duplicate. The number of colonies formed was counted for each plate and the mean count was calculated for each strain. The MBC value was the lowest concentration of the antibacterial agent that caused a reduction in colony count of at least 3 logs compared with the initial bacterial concentration. The sensitivity of the colony formation assay in this study was 10 c.f.u. ml21. MBC50 and MBC90 were determined as the concentrations at which 50 and 90 % of the S. aureus isolates were killed, respectively. Determination of time–kill curve. A time–kill curve was deter-

mined to analyse the kinetics of lysostaphin bactericidal activity in vitro. An overnight broth culture of S. aureus was diluted with fresh CAMH broth to a final inoculum concentration of ~106 c.f.u. ml21 in a 500 ml glass flask. After the broth had stabilized for 30 min at 35 uC, a sample was taken as the pre-treatment control and subincubated on to lysostaphin-free agar in duplicate using serial 10-fold dilutions. At the end of the 30 min incubation, the broth was divided equally into five 50 ml glass flasks. Each sample was treated with or without lysostaphin at a final concentration of 0, 0.25, 0.5, 1, 2 and 4 MICs in the glass flasks at 35 uC, where the MIC was 0.06 mg ml21. Broth without lysostaphin served as a control. A 0.1 ml sample was removed from each flask after 0, 2, 4, 6 and 8 h of treatment. Serial 10-fold dilutions were made with 0.9 ml sterile saline and subincubated on to lysostaphin-free agar in duplicate. After overnight incubation at 35 uC, colony numbers were counted and the mean count was calculated for each strain. The time–kill curve was scaled as log10 reduction in bacterial counts within a specific time period. Samples were considered bactericidal at a concentration that resulted in the original inoculum being reduced by over 3 log10 (99.9 % reduction) during the assay.

RESULTS AND DISCUSSION Determination of MICs In the agar dilution test, all of the S. aureus strains were sensitive to the recombinant lysostaphin with MICs ranging from 0.03 to 2 mg ml21 (Table 1). MIC50 and MIC90 values were 0.25 and 0.5 mg ml21, respectively. There was no significant difference between the sensitivity of MSSA and MRSA to lysostaphin. Most of the MRSA strains and few of the MSSA strains were resistant to cefotaxime, piperacillin, clarithromycin and ciprofloxacin. The organisms resistant to these drugs were all susceptible to the recombinant lysostaphin, suggesting that lysostaphin did not cross-react with these antimicrobial agents. The antibacterial activity of lysostaphin against the 257 S. aureus isolates was greater than that of vancomycin and the other reference agents with a fourfold reduction in MIC50 and MIC90 values compared with the activity of vancomycin. Although lysostaphin showed promising results in the agar dilution assay in all of Journal of Medical Microbiology 56

In vitro activity of lysostaphin against S. aureus

Table 1. Antibacterial activity of recombinant lysostaphin and other agents in the 257 clinical isolates of S. aureus Organism

No. of strains

Antibacterial agent*

MIC range (mg ml”1)

MSSA

168

MRSA

89

rLspn CTX VAN PIP CLR CIP rLspn CTX VAN PIP CLR CIP

0.03 to 2 1 to 128 0.5 to 2 0.25 to >128 ¡0.03 to 128 0.06 to 64 0.03 to 2 4 to >128 0.5 to 2 0.5 to >128 0.06 to >128 0.06 to 128

MIC50 (mg ml”1) 0.25 2 1 4 2 0.5 0.25 >128 1 >128 128 16

MIC90 (mg ml”1) 0.5 4 2 8 >128 4 0.5 >128 2 >128 >128 128

Sensitivity ratio (%) (susceptible/total) 100 (168/168) 98.8 (166/168) 100 (168/168) 90.5 (152/168) 53.0 (89/168) 88.1 (148/168) 100 (89/89) 16.9 (15/89) 100 (89/89) 20.2 (18/89) 15.7 (14/89) 30.3 (27/89)

*rLspn, Recombinant lysostaphin; CTX, cefotaxime; VAN, vancomycin; PIP, piperacillin; CLR, clarithromycin; CIP, ciprofloxacin.

the S. aureus strains tested, the actual MIC results required verification, as lysostaphin does not readily diffuse through the agar in this assay (Kusuma & Kokai-Kun, 2005). Therefore, 25 S. aureus strains tested in the agar dilution assay were also tested in a macrobroth dilution assay. In addition, a number of the strains were used in the subsequent MBC study. Higher MICs for lysostaphin against the S. aureus strains were obtained in the agar dilution assay compared with those in the broth dilution assay; however, the MICs for vancomycin did not show significant differences between the two methods (Table 2). In comparison studies, the MICs for vancomycin for most of the test strains remained invariable between the two dilution assays. The ratio of agar MIC to broth MIC for vancomycin was between 0.5 and 4 in all of the strains, with a median value of 1. The MIC values for vancomycin for the control strain ATCC 29213 were identical in the two assays. As the MIC variability of vancomycin in the test strains was within an acceptable range between the agar dilution and broth dilution assays, it was feasible to compare the agar dilution MICs of lysostaphin with the broth dilution MICs. In contrast to the results for vancomycin, the MICs of lysostaphin for most of the test strains were higher in the agar dilution assay than in the broth dilution assay. As shown in Table 2, the MICs of lysostaphin for the test strains in the macrobroth dilution assay ranged from 0.004 to 0.125 mg ml21. The MICs of lysostaphin determined by the agar dilution method were 2- to 16-fold higher than those of the broth dilution method in most of the strains with three exceptions. Accordingly, the MIC50 and MIC90 values of lysostaphin from the agar dilution method were higher than those from the broth dilution method in the S. aureus strains. Ratios of agar MICs to broth MICs for lysostaphin for the strains were between 1 and 16, with a median value of 4. Although there were differences in MICs between the two methods, the results here showed that S. aureus isolates from Beijing hospitals are highly susceptible to lysostaphin. http://jmm.sgmjournals.org

Differences in the MICs for various test strains between the two assays were not uniform. As lysostaphin is a 27 kDa molecule that has some difficulty diffusing in agar (Kusuma & Kokai-Kun, 2005), it is possible that the lysostaphin molecules in the molten agar medium were not distributed equally in the plate, causing variations in the final MIC results. Thus, caution should be used to interpret the lysostaphin MIC results derived from the agar dilution method. MBC and time–kill curves After 24 h incubation in the presence of lysostaphin in the macrobroth MIC assay, 17 strains of S. aureus underwent an additional MBC assay. The MBCs for the S. aureus isolates ranged from 0.016 to 1 mg ml21 (Table 2). The MBC50 and MBC90 values were 0.06 and 0.25 mg ml21, respectively. The ratios of MBCs to broth MICs for lysostaphin were between 1 and 8, with a median value of 4. A comparison between the MICs and MBCs of lysostaphin indicated that these S. aureus isolates from hospitals in Beijing were fairly sensitive to lysostaphin. The time–kill curves of lysostaphin in two representative strains of S. aureus (one MSSA and one MRSA) were similar and are shown in Fig. 1(a, b). Bactericidal activity (>99.9 % reduction) was observed after a 2 h exposure of the isolates to lysostaphin at concentrations of 0.5, 1, 2 and 4 MIC. A bactericidal activity of lysostaphin of 0.5 MIC or higher was sustained in CAMH broth for at least 6 h. Lysostaphin showed a dose-dependent and rapid bactericidal activity against the test MSSA and MRSA as shown in time–kill curves. The activity of lysostaphin has been well documented in western countries (Climo et al., 1998; Polak et al., 1993) and lysostaphin has entered clinical trials in the USA (McCoy, 2004; Mierau et al., 2005). As a new recombinant 73

S. aureus strain*

Journal of Medical Microbiology 56

MSSA (n=12) ATCC 29213 0-3 0-7 0-8 0-24 0-30 1-6 2-70 3-7 3-27 3-29 3-31 MRSA (n=13) 0-27 0-33 0-34 1-11 1-14 1-15 1-21 1-33 1-34 1-41 2-81 2-86 3-2

MIC for VAN (mg ml”1)

MICagar/MICbroth for VAND

MIC for rLspn (mg ml”1)

MBC/MICbroth for rLspn

Broth dilution

1 1 2 1 2 1 1 2 1 1 0.5 1

1 2 1 1 0.5 1 1 2 0.5 0.5 1 0.5

1 0.5 2 1 4 1 1 1 2 2 0.5 2

0.125 0.125 0.25 0.125 0.125 0.125 0.06 0.125 0.25 0.5 0.06 0.25

0.06 0.03 0.125 0.06 0.008 0.016 0.06 0.125 0.06 0.125 0.016 0.06

2 4 2 2 16 8 1 1 4 4 4 4

0.25 0.06 1 0.06 0.03 0.125 0.25

4 2 8 1 4 8 4

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

1 1 1 1 1 0.5 1 1 1 1 2 1 0.5

1 1 1 1 1 0.5 0.5 0.5 0.5 1 1 1 0.5

1 1 1 1 1 1 2 2 2 1 2 1 1

0.125 0.125 0.03 0.06 0.125 0.03 0.125 0.125 0.125 0.125 0.25 0.5 0.03

0.008 0.008 0.004 0.008 0.06 0.008 0.008 0.125 0.03 0.06 0.06 0.125 0.016

16 16 8 8 2 4 16 1 4 2 4 4 2

0.03 0.03 0.016 0.03 0.06 0.016 0.06 0.125 0.125 0.25

4 4 4 4 1 2 8 1 4 4

ND

ND

ND

ND

ND

ND

MBCrange=0.016–1, MBC50=0.06, MBC90=0.25

Range=1–8, median=4

MICrange=0.5–2, MIC50=1, MIC90=1

Range=0.5–4, median=1

MICrange=0.03–0.5, MIC50=0.125, MIC90=0.25

Broth dilution

MBC for rLspn (mg ml”1)

Agar dilution

MICrange=0.5–2, MIC50=1, MIC90=2

Agar dilution

MICagar/MICbroth for rLspn

MICrange=0.004–0.125, MIC50=0.06, MIC90=0.125

VAN, vancomycin; rLspn, recombinant lysostaphin; ND, not determined *ATCC 29213 obtained from ATCC; other test S. aureus strains were sourced from hospitals in Beijing. DMICagar, MIC value determined by the agar dilution method; MICbroth, MIC value determined by the broth dilution method.

Range=1–16, median=4

X.-Y. Yang and others

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Table 2. Comparison of MICs and MBCs for lysostaphin susceptibility tests of various S. aureus strains

In vitro activity of lysostaphin against S. aureus

bactericidal kinetics in the test isolates. The MSSA and MRSA strains showed similar susceptibility and bactericidal kinetics in which all of the test organisms were rapidly killed within 2 h when the lysostaphin concentration was close to the MIC. We consider this to be an important feature and of clinical significance, as, in many cases, severe infections such as post-burn skin infections can largely be controlled if the bacteria are eradicated quickly from the infection lesion.

9 (a) 8 7 6 5 4

_

log10(c.f.u. ml 1)

3 2 1 2

4

6

8

ACKNOWLEDGEMENTS The project was supported by the 10th five-year plan (2003AA2Z347D) and the 973 programme (2004CB518908) of the Ministry of Sciences and Technology, PR China.

9 (b) 8 7 6

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