ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2005, p. 3256–3263 0066-4804/05/$08.00⫹0 doi:10.1128/AAC.49.8.3256–3263.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 49, No. 8

Comparison of Four Methods for Determining Lysostaphin Susceptibility of Various Strains of Staphylococcus aureus Caroline M. Kusuma and John F. Kokai-Kun* Biosynexus Incorporated, Gaithersburg, Maryland 20877 Received 14 December 2004/Returned for modification 23 February 2005/Accepted 22 May 2005

Lysostaphin is an endopeptidase that cleaves the pentaglycine cross-bridges of the staphylococcal cell wall rapidly lysing the bacteria. Recently, lysostaphin has been examined for its potential to treat infections and to clear Staphylococcus aureus nasal colonization, requiring a reliable method for determining the lysostaphin susceptibility of strains of S. aureus. We compared four methods for determining the lysostaphin susceptibility of 57 strains of methicillin-sensitive S. aureus, methicillin-resistant S. aureus, vancomycin intermediately susceptible S. aureus (VISA), mupirocin-resistant S. aureus, and various defined genetic mutants of S. aureus. Three reference lysostaphin-resistant S. aureus variants were also included in the assays as negative controls. The assays examined included turbidity, MIC, minimum bactericidal concentration (MBC), and disk diffusion assays. All of the strains of S. aureus tested, including a VISA strain which had previously been reported to be lysostaphin resistant, were susceptible to lysostaphin by all four methods. The three reference lysostaphinresistant variants were resistant by all four methods. The disk diffusion assay was the simplest method to differentiate lysostaphin-susceptible S. aureus strains from lysostaphin-resistant variants, while the MBC assay could be used as a follow-up assay if required. In the disk diffusion assay, all strains of S. aureus tested revealed zones of inhibition of >11 mm using a 50-␮g lysostaphin disk, while the three reference lysostaphin-resistant S. aureus variants had no zones of inhibition. In MBC assays, concentrations of lysostaphin ranging from 0.16 ␮g/ml to 2.5 ␮g/ml were found to cause a 3 log or greater drop from the initial CFU of S. aureus within 30 min for all strains tested. hardt employed a turbidity assay for quantitative analysis of lysostaphin (34). In this study, we compared this turbidity assay with three more conventional methods for determining the susceptibility of various strains of S. aureus to lysostaphin, including MIC, minimum bactericidal concentration (MBC), and disk diffusion assays. These strains included methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), vancomycin intermediately susceptible S. aureus (VISA), mupirocin-resistant S. aureus, and several defined genetic mutants of S. aureus. We also included three in vitro-isolated lysostaphin-resistant S. aureus variants as negative controls in the various assays. We determined that the most simple and reproducible method for determination of lysostaphin susceptibility was the disk diffusion assay.

Lysostaphin, an antimicrobial enzyme first identified in a single strain of Staphylococcus simulans (34), is a 27-kDa glycylglycine endopeptidase that is capable of cleaving the pentaglycine cross-bridges of the staphylococcal cell wall leading to rapid lysis of the bacteria (3). The continuing emergence of antibiotic-resistant S. aureus infections (4, 16, 37) has spurred the need for new antimicrobial agents to treat these infections. Previous studies from the 1960s and 1970s demonstrated that lysostaphin is a potent anti-staphylococcal agent and has potential as a therapeutic agent against S. aureus infections (10, 13, 14, 32, 33, 36, 45). Study of lysostaphin as an anti-staphylococcal agent was discontinued, however, due to lack of homogeneous preparations of lysostaphin and the availability of other effective antibiotic treatments. With the rapidly decreasing effectiveness of current antibiotics for treatment of S. aureus infections and the availability of recombinant lysostaphin (30), studies investigating lysostaphin as a therapeutic agent for staphylococcal infections have reemerged (5–7, 19, 26). As lysostaphin continues to be studied as a possible therapy for S. aureus, there is a need for a reliable method for determining lysostaphin susceptibility that can be used clinically to differentiate lysostaphin-susceptible strains of S. aureus from lysostaphin-resistant variants, should they ever emerge. Lysostaphin is a highly active enzyme that rapidly lyses S. aureus and acts differently from many conventional antibiotics, thus, traditional methods of determining antibiotic susceptibility may not be the most appropriate. In 1964, Schindler and Schu-

MATERIALS AND METHODS Staphylococcus aureus strains. Fifty-seven S. aureus strains consisting of various MSSA, MRSA, VISA, mupirocin-resistant S. aureus, and genetically defined mutant strains of S. aureus were used in these studies and are listed in Tables 1 and 2. Three reference lysostaphin-resistant S. aureus variants isolated in vitro were also included in these assays as negative controls. The identity of some bacteria as S. aureus was confirmed by latex agglutination assay (Staphyloslide; BD, Sparks, MD) and api Staph strip (bioMerieux, Hazelwood, MO). Isolation of reference lysostaphin-resistant variants. Lysostaphin-resistant variants of three S. aureus strains, SA5-Lab, Col, and MBT 5040, were isolated in vitro by exposure to sub-MIC concentrations of lysostaphin as previously described (5). Following this initial isolation, the resistant variants were sequentially exposed to greater concentrations of lysostaphin up to 32 ␮g/ml in trypticase soy broth (TSB; BD) to ensure the stability of the lysostaphin-resistant phenotype. Materials and chemicals. Recombinant homogenous lysostaphin was produced by Biosynexus Incorporated. Other materials and chemicals used were from various commercial sources as noted.

* Corresponding author. Mailing address: Biosynexus Incorporated, 9119 Gaither Rd., Gaithersburg, MD 20877. Phone: (301) 987-1172. Fax: (301) 990-4990. E-mail: [email protected]. 3256

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TABLE 1. Lysostaphin susceptibility testing of various S. aureus strains S. aureus strain designation

MSSA ATCC 49521

TOD50 ratio of sample to referencea

MIC (␮g/ml)

Disk diffusion (mm)

MBC (␮g/ml)

Reference(s) or sourceb

11.8 ⫾ 1.7 min to TOD50 1.07 ⫾ 0.16 NAe 0.86 ⫾ 0.11 0.91 ⫾ 0.05 1.13 ⫾ 0.15 1.94 ⫾ 0.51 2.82 ⫾ 0.53 2.74 ⫾ 0.22 1.04 ⫾ 0.03 1.29 ⫾ 0.58 2.25 ⫾ 0.21 1.32 ⫾ 0.42 1.13 ⫾ 0.27 0.86 ⫾ 0.22 1.73 ⫾ 0.20 1.16 ⫾ 0.18 0.76 ⫾ 0.29 1.10 ⫾ 0.33 1.63 ⫾ 0.82 1.29 ⫾ 0.27

0.016

18 ⫾ 0.0

0.004 ⬎32 0.004 0.002 0.031 0.008 0.250 0.008 0.004 0.004 0.004 0.004 0.016 0.002 0.008 0.004 0.002 0.004 0.004 0.002

13 ⫾ 0.3 0 ⫾ 0.0 14 ⫾ 0.6 14 ⫾ 0.3 17 ⫾ 0.3 18 ⫾ 0.5 19 ⫾ 0.0 16 ⫾ 0.0 14 ⫾ 0.0 15 ⫾ 0.0 16 ⫾ 0.0 16 ⫾ 0.6 11 ⫾ 0.0 11 ⫾ 0.0 17 ⫾ 0.0 18 ⫾ 0.0 15 ⫾ 0.0 20 ⫾ 0.0 11 ⫾ 0.0 18 ⫾ 0.0

0.16 ⬎100 0.16 0.16 0.16 0.16 0.25 0.16 0.16 0.16 0.16 ND 0.32 0.16 ND ND ND 0.16 ND ND

Biosynexus Isolated in vitro Biosynexus Biosynexus ATCC Biosynexus ATCC 12 12 12 12 12 Biosynexus Clinical Clinical Clinical Clinical Clinical Clinical Clinical

MRSA Col (NRS100) Col-LysoR BK161Y BK161W BK2352 BK2454 MBT5040 MBT5040-LysoR MRSA 12/12

1.47 ⫾ 0.19 NA 1.00 ⫾ 0.29 1.48 ⫾ 0.31 1.68 ⫾ 0.06 1.05 ⫾ 0.24 0.88 ⫾ 0.33 NA 0.54 ⫾ 0.25

0.008 ⬎32 0.016 0.008 0.016 0.008 0.004 ⬎32 0.002

17 ⫾ 0.0 0 ⫾ 0.0 15 ⫾ 0.6 15 ⫾ 0.0 16 ⫾ 0.0 16 ⫾ 0.0 17 ⫾ 0.0 0 ⫾ 0.0 14 ⫾ 0.0

0.16 ⬎100 0.16 0.16 ND ND 0.63 ⬎100 ND

NARSA Isolated in vitro G. Archer G. Archer G. Archer G. Archer Clinical Isolated in vitro Clinical

VISA ATCC 700698 HIP5827 NRS79 HIP5836 HIP6297

2.20 ⫾ 0.12 1.34 ⫾ 0.13 1.01 ⫾ 0.04 1.97 ⫾ 0.11 2.21 ⫾ 0.24

0.016 0.008 0.125 0.031 0.008

19 ⫾ 0.0 16 ⫾ 0.0 16 ⫾ 0.0 17 ⫾ 1.2 15 ⫾ 0.6

0.32 2.5 0.16 ND 0.16

Mupirocin resistant SA3865-MupR SA4236-MupR

0.80 ⫾ 0.03 0.96 ⫾ 0.43

0.004 0.016

19 ⫾ 0.6 16 ⫾ 0.0

0.16 0.16

G. Archer G. Archer

Stable small-colony variants III30 Schultz

0.43 ⫾ 0.04 0.67 ⫾ 0.01

0.002 0.001

24 ⫾ 0.0 24 ⫾ 0.6

0.63 0.16

R. Proctor R. Proctor

SA5 Lab SA5-LysoRd SA5 USU SA5 SAM ATCC 12605 SA8 SAM ATCC 33862 A890097 A960420 A920007 A980592 (NRS157) LY-19991509 XS MBT4532 MBT5017 MBD5547 MBD5608 M8379 M8394 M8437

0.16c

ATCC

15 40 2 and NARSA 37, 40 31

a

All strains were tested at least three times and compared to the reference strain ATCC 49521 ATCC, American Type Culture Collection; Biosynexus, Biosynexus strain collection; Clinical, clinical isolate; NARSA, Network on Antimicrobial Resistance in S. aureus; G. Archer, Gordon Archer, Virginia Commonwealth University; R. Proctor, Richard Proctor, University of Wisconsin. c Lowest dose of lysostaphin tested in this assay. d Lysostaphin resistant variant isolated in vitro as described in Materials and Methods e ND, not determined. NA, not applicable b

Turbidity assay. The procedure used was similar to the one employed by Schindler and Schuhardt (34) with modifications. S. aureus cells in an 18-h trypticase soy broth culture were pelleted and washed once with phosphatebuffered saline (PBS; BioWhittaker, Walkersville, MD). The cells were then resuspended in a volume of PBS such that the starting absorbance of the resuspended bacteria, as determined using a SmartSpec 3000 (Bio-Rad, Hercules, CA) at 650 nM, gave an absorbance reading of 1.55 ⫾ 0.04. The turbidity assay was performed by adding 4 ␮g/ml or 5 ␮g/ml of lysostaphin to the S. aureus suspension and then determining the optical density at 650 nm (OD650) at 30-s intervals (30 min total duration). The time to reach half starting absorbance (TOD50) of the bacterial suspension was determined for each strain. A reference

strain of S. aureus (ATCC 49521) was run in each assay and served to provide comparability between assays. Each S. aureus strain was assayed at least three times, and the mean ratio (sample TOD50/reference TOD50) and standard deviation were calculated for each strain. MIC assay. MIC assays were determined by the broth dilution method in a modification of standards of the NCCLS (23). The concentrations of lysostaphin used ranged from 0.25 ␮g/ml to 0.00025 ␮g/ml. Twofold dilutions of lysostaphin were performed in cation-adjusted Mueller Hinton broth (BD) supplemented with 2% NaCl (EM Science, Gibbstown, NJ) and 0.1% bovine serum albumin (BSA; Sigma, St. Louis, MO). Wells of a 96-well polystyrene plate (Corning Incorporated, Corning, NY) were inoculated with ⬃5 ⫻ 105 CFU/ml S. aureus

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TABLE 2. Lysostaphin susceptibility testing of various S. aureus strains and their defined genetic mutants S. aureus strain

Genetic mutation

TOD50 ratio of sample to referencea

MIC (␮g/ml)

Disk diffusion (mm)

MBC (␮g/ml)

Reference or source

ATCC 35556 ATCC 35556 ⌬dltA ATCC 35556 ⌬tagO ATCC 35556 ⌬ypfP Newman SKM3 SKM12 SKM7 SKM14 SH1000 SH1001 SH1002 SH1000 hla-lacZ 8325-4 Wood OS-2 RN 7736 RN 7857

Wild type dltA-KO tagO-KO ypfp-KO Wild type srtA-KO srtA-KO srtB-KO srtA-B-KO Wild type agr-KO sar-KO hla-lacZ fusion ␴B deficient Low protein A spa-KO spa-KO spa and agr-KO

0.54 ⫾ 0.13 0.78 ⫾ 0.05 1.07 ⫾ 0.13 0.58 ⫾ 0.05 1.14 ⫾ 0.21 1.20 ⫾ 0.16 1.22 ⫾ 0.12 1.18 ⫾ 0.03 1.34 ⫾ 0.17 0.77 ⫾ 0.24 0.67 ⫾ 0.02 3.27 ⫾ 0.96 0.65 ⫾ 0.08 0.79 ⫾ 0.08 0.51 ⫾ 0.05 0.91 ⫾ 0.10 0.76 ⫾ 0.17 0.76 ⫾ 0.15

0.004 0.002 0.004 0.004 0.004 0.008 0.008 0.004 0.004 0.004 0.004 0.031 0.004 0.001 0.001 0.002 0.002 0.001

15 ⫾ 0.00 17 ⫾ 0.00 17 ⫾ 0.58 14 ⫾ 0.00 16 ⫾ 0.00 16 ⫾ 0.00 16 ⫾ 0.00 16 ⫾ 0.00 16 ⫾ 0.00 13 ⫾ 1.53 16 ⫾ 0.00 15 ⫾ 0.00 14 ⫾ 0.58 18 ⫾ 0.00 18 ⫾ 0.58 16 ⫾ 0.00 14 ⫾ 0.00 14 ⫾ 0.00

0.16b NDc ND ND 0.32 0.63 0.16 ND ND ND ND ND ND ND 0.16 0.16 0.16 0.16

43 43 43 20 44 44 44 44 44 17 17 17 24 17 24 35 R. Novickd R. Novick

a

All strains were tested at least three times and compared to the reference strain ATCC 49521. Lowest dose of lysostaphin tested in this assay. ND, not determined; KO, knockout. d Richard Novick, New York University. b c

per well diluted from an overnight culture of the bacteria grown in TSB. A positive control for growth containing no lysostaphin was included in each assay. MIC determinations were performed in the presence of 0.1% BSA to inhibit nonspecific lysostaphin adherence to the polystyrene plate as previously reported (6). Microtiter plates were incubated at 37°C with shaking (200 rpm) for 24 h. The endpoint for this assay was complete inhibition of growth (MIC-0) at 24 h as determined by measuring the absorbance at 650 nM using a microplate reader. Following determination of the absorbance at 650 nM, excess lysostaphin was added to some plate wells with growth. A concentrated solution of lysostaphin (10 mg/ml) in PBS was added to select wells to equal a final concentration of 200 ␮g/ml. The microplate was then further incubated for 3 h at room temperature with slow rotation, and the absorbance at 650 nM was again determined. Each S. aureus strain was assayed at least twice. Minimum bactericidal concentration (MBC) assay. MBCs for lysostaphin were determined by a modification of the NCCLS standards (22). Briefly, twofold dilutions of lysostaphin ranging from 10 ␮g/ml to 0.16 ␮g/ml were made in PBS plus 0.1% BSA. S. aureus from an overnight TSB culture was diluted to a final inoculum of ⬃106 CFU/ml in each lysostaphin dilution tube. A tube containing PBS plus 0.1% BSA but no lysostaphin was included as a control. The dilution tubes were incubated for 30 min at room temperature with vigorous shaking. At the end of 30 min incubation, an equal volume of 10 mg/ml Proteinase K (Sigma) in PBS was added to each tube to neutralize the remaining lysostaphin (19). A volume of each sample (100 ␮l) was plated on a blood agar plate (Remel, Lenexa, KS) to enumerate the surviving S. aureus. The minimum bactericidal concentration was defined as the dose of lysostaphin which led to a 3 log or greater drop from the starting bacterial concentration (99.9% killing of the initial inoculum). Each S. aureus strain was assayed at least twice. Disk diffusion assay. Sterile 6-mm filter paper disks (Whatman No.1; Whatman International Ltd.) were each impregnated with between 0.005 to 50 ␮g of lysostaphin in PBS. Disks were allowed to dry at room temperature overnight and then stored at ⫺20°C in a sealed container until used. To perform a pilot study, S. aureus strain ATCC 49521 from an overnight TSB culture was spread evenly on a cation-adjusted Mueller Hinton agar (BD) supplemented with 2% NaCl (CAMHA⫹) using a sterile swab, and then various disks containing between 0.005 and 50 ␮g of lysostaphin were placed on the agar surface. The CAMHA⫹ plate was incubated for 20 h at 37°C. Following the incubation, the diameter of zones of inhibition around the various lysostaphin disks were measured. To perform the disk diffusion assay for comparison of the various S. aureus strains, each S. aureus strain from an overnight TSB culture was spread evenly on CAMHA⫹ or brain heart infusion (BHI) agar (BD), and then a 20-␮g (Hardy Diagnostics, Santa Maria, CA) or 50-␮g (prepared for this study) lysostaphin disk was placed on the agar. The plates were incubated for 20 h at 37°C. Following the incubation, the diameter of zones of inhibition around the lysostaphin disks were measured. Each S. aureus strain was assayed at least three

times, and the mean zone of inhibition and standard deviation were calculated for each strain.

RESULTS Lysostaphin susceptibility determined by the turbidity assay. The turbidity assay measures the rapid drop in optical absorbance of a suspension of S. aureus when exposed to lysostaphin (34, 45). The time to reach half the starting absorbance (TOD50, equivalent to one-half log drop in initial viable bacteria) with concentrations of lysostaphin of 4 ␮g/ml or 5 ␮g/ml was determined, and the ratio of TOD50 sample to TOD50 of a reference strain of S. aureus (ATCC 49521) for the specific concentration of lysostaphin was calculated (Tables 1 and 2). S. aureus ATCC 49521 was included in each assay to serve as the reference standard to allow comparability between assays. There was some assay-to-assay variability in the TOD50 for the reference strain ATCC 49521 (mean TOD50 ⫽ 11.8 min, range ⫽ 9 to 15 min), but the ratios of sample strain to reference strain remained constant despite these differences in actual TOD50s (data not shown). Of the 57 strains of S. aureus tested, all S. aureus strains, except for the three reference lysostaphin-resistant variants, demonstrated a 50% or greater reduction in turbidity within 30 min with 4 or 5 ␮g/ml lysostaphin (see Fig. 1 for a sample of the data). The ratios of TOD50 of the sample strain to that of the reference strain were found to range from 0.51 to 3.27, and while this demonstrated heterogeneity in lysostaphin susceptibility as determined by this assay, this heterogeneity is relatively restricted over a sixfold range. Lysostaphin susceptibility determined by MIC assay. The MIC of lysostaphin for the various strains of S. aureus which resulted in no measurable bacterial growth, as determined by a variation of NCCLS standards, ranged from 0.001 to 0.250 ␮g/ml (Tables 1 and 2). The MIC at which 50% of the strains tested were inhibited (MIC50) was ⬍0.008 ␮g/ml, and the MIC at which 90% of the strains tested were inhibited (MIC90) was

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FIG. 1. An example of data from a turbidity assay for several S. aureus strains, including ATCC 49521 (reference strain), MBT5040 LysoR (in vitro isolated lysostaphin-resistant variant), SA3865 (MupR), MBT5040 (MRSA), XS (MSSA), and HIP5827 (VISA), as indicated on the figure. The time to 50% of the starting OD (shown as “OD50 average” on the figure) was determined for each strain as indicated on the figure by the vertical lines.

⬍0.031 ␮g/ml. S. aureus ATCC 33862 had the highest MIC of the susceptible strains tested at 0.250 ␮g/ml, while the three reference lysostaphin-resistant variants all had MICs greater than 32 ␮g/ml, the highest dose of lysostaphin used in these assays. After 24 h of incubation in the presence of lysostaphin, some wells were observed to have bacterial growth above the apparent MIC level for some of the S. aureus strains (Fig. 2A). The bacteria in these wells were found to be S. aureus by microbiologic methods (data not shown) and were further tested for lysostaphin susceptibility by adding an additional 200 ␮g/ml lysostaphin in a small volume of PBS to all wells with visible growth. Wells with growth that were not originally exposed to lysostaphin (control wells) or wells below the actual lysostaphin MIC had a dramatic drop in absorbance over 3 h of incubation following the addition of excess lysostaphin as the lysostaphin lysed the S. aureus in the wells (Fig. 2B). “Resistance-outgrowth” was defined as wells that displayed no drop in absorbance following the 3-h incubation with excess lysostaphin (Fig. 2B). This “resistance-outgrowth” phenomenon did not occur for every strain of S. aureus and did not even occur in every assay with any particular strain of S. aureus. Lysostaphin susceptibility determined by MBC assay. Thirty-seven S. aureus strains underwent testing for antimicrobial susceptibility to lysostaphin by MBC assay. The MBCs for the various S. aureus strains tested ranged from ⬍0.16 ␮g/ml to 2.5 ␮g/ml. The MBC at which 90% of the strains tested were susceptible (MBC90) was ⬍0.16 ␮g/ml (Tables 1 and 2). There were no significant reductions in bacterial counts for any of the three reference lysostaphin-resistant variants after 30 min of incubation even at 100 ␮g/ml lysostaphin. Lysostaphin susceptibility determined by disk diffusion assay. A pilot study was conducted with S. aureus ATCC 49521

and various concentration lysostaphin disks (0.005 to 50 ␮g/ disk) to determine the optimum amount of lysostaphin per disk. The ideal disk concentration is one that provides zones of inhibition with diameters between 15 and 25 mm for most susceptible strains, with only small or no zone of inhibition diameters with resistant strains (21). The following zones of inhibition were determined for ATCC 49521 after 20 h incubation: 0.005- to 0.50-␮g disks resulted in no zone of inhibition, while a 5-␮g disk gave a 10-mm zone of inhibition, and a 50-␮g lysostaphin disk gave an 18-mm zone of inhibition. Thus, 50-␮g lysostaphin disks were chosen to be used in subsequent studies. The three reference lysostaphin-resistant S. aureus variants did not have any zone of inhibition around 50-␮g lysostaphin disks, while all the other strains tested had zones of inhibition after 20 h of incubation at 37°C with 50-␮g lysostaphin disks which ranged from 11 to 20 mm (Tables 1 and 2). Commercially available 20-␮g lysostaphin disks were also tested on several S. aureus strains. These 20-␮g disks showed proportionally smaller zones of inhibition for lysostaphin-sensitive strains, as expected (ranging from 9 to 14 mm following 20 h of incubation in 37°C). For some of the disk assays conducted with 20-␮g lysostaphin disks, however, it was difficult to distinguish lysostaphin-susceptible strains from lysostaphin-resistant variants due to the very small zones of inhibition. Occasionally, very small colonies were present within the lysostaphin zones of inhibition for some S. aureus strains (data not shown) on CAMHA⫹. These small colonies were further tested for lysostaphin resistance on trypticase soy agar containing 10 ␮g/ml lysostaphin and found to be susceptible to lysostaphin, as no growth was seen (data not shown). It was suspected that these small colonies may be small-colony variants (29), and when BHI agar was substituted for CAMHA⫹, no small colonies were observed in the zones of inhibition around

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FIG. 2. An example of a MIC assay plate for S. aureus ATCC 49521 and the phenomenon termed “resistant outgrowth.” (A) MIC plate after 24 h of incubation, the MIC for this strain was 0.016 ␮g/ml (labeled as “16” on the figure). The concentrations of lysostaphin used ranged from 0.25 ␮g/ml to 0.00025 ␮g/ml. (B) The same MIC plate following several hours of incubation with excess lysostaphin. Wells with growth at 0.008 ␮g/ml (labeled as “8” on the figure) and above were tested for lysostaphin resistance outgrowth by adding an extra 200 ␮g/ml lysostaphin to these wells and then incubating the plate at room temperature for several additional hours. All of the wells initially at 0.008 ␮g/ml lysostaphin were clear after incubation with excess lysostaphin, which indicated that this was growth of normal S. aureus at that concentration of lysostaphin. The wells above 0.008 ␮g/ml indicated with arrows remained cloudy and were due to outgrowth of lysostaphin-resistant S. aureus.

50-␮g lysostaphin disks (data not shown). As a follow up, stable small-colony variants were obtained and tested for susceptibility to lysostaphin by various assays. The two stable small-colony variants tested were susceptible to lysostaphin by all methods used (Table 1). DISCUSSION The promising prospect of lysostaphin as a therapeutic agent against S. aureus requires appropriate methods to test the susceptibility of S. aureus isolates to lysostaphin. We have

examined four methods for determining the lysostaphin susceptibility of S. aureus, and while it appeared that there was not consistent correlation regarding the degree of lysostaphin susceptibility from method to method for individual S. aureus strains as demonstrated by comparing the log 2 MICs with the zones of inhibition as determined by disk diffusion assay (Fig. 3), there was good correlation between all four assays in terms of determining lysostaphin-susceptible strains versus lysostaphin-resistant variants. All S. aureus strains tested in the study, except for the three reference lysostaphin-resistant vari-

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FIG. 3. A graphic comparison of the MIC (log 2) values of lysostaphin (in micrograms/milliliter) with the zones of inhibition (in millimeters) around 50-␮g lysostaphin disks for 31 strains of S. aureus.

ants, were susceptible to lysostaphin in all four assays. The lack of correlation of degrees of susceptibility among the four assays (the best R2 value for interassay comparison was 0.23 obtained by comparison of turbidity assay data with MIC data; data not shown) may be due to different environmental conditions in which the assays were performed. Bacteria express different factors in liquid media than on solid media, e.g., growth conditions such as the culture media have been previously shown to influence capsular polysaccharide production in S. aureus (8, 25, 28, 39). The environmentally induced capsular polysaccharide production of different S. aureus strains may limit the access of lysostaphin to the pentaglycine bridge in the peptidoglycan cell wall and therefore result in the apparently different levels of susceptibility of various strains of S. aureus to lysostaphin depending on the assay used. There appears to be two distinct populations of S. aureus in these studies. Most of the S. aureus strains tested were exquisitely sensitive to lysostaphin by all assays, while the three lysostaphin-resistant variants were insensitive to lysostaphin under all assay conditions. Only two S. aureus strains had MICs above 0.031 ␮g/ml, one of which was ATCC 33862, a beta-toxinproducing strain of S. aureus. While this strain had both a MIC and an MBC of 0.25 ␮g/ml, it also had one of the larger zones of inhibition on disk diffusion analysis (Table 1). There were no S. aureus strains that were consistently less sensitive to lysostaphin in all four assays (other than the lysostaphin-resistant variants), and these findings may be due to the known mechanisms of lysostaphin resistance. The best-studied mechanism of resistance to lysostaphin involves mutations that affect femA, the gene responsible for addition of the second and third glycines to the pentaglycine cross bridge. Mutations that render this gene nonfunctional result in monoglycine crossbridges and render the S. aureus completely resistant to lysostaphin as the enzymatic target for lysostaphin is no longer present (11, 38). A second less likely mechanism could involve acquisition of the gene for the lysostaphin immunity factor (lif), which is found on the pACK1 plasmid which also encodes lysostaphin in S. simulans biovar staphylolyticus (41). This factor is also called the endopeptidase resistance gene (epr) (9) and results in substitution of serines for glycines in the pentaglycine cross-bridges when introduced into S. aureus on a shuttle vector. These serine substitutions render the recipient strain resistant to lysostaphin and could result in a strain of S.

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aureus that is of intermediate susceptibility to lysostaphin (9, 41). This gene, however, has not been found in S. aureus outside of the laboratory. No other mechanisms have been reported that result in reduced susceptibility of S. aureus to lysostaphin. An example of in vitro selection of lysostaphin-resistant variants was seen in the phenomenon of “resistance outgrowth” during MIC testing of some strains of S. aureus. Spontaneous DNA mutations that affect femA during overnight growth of the starter inoculum cultures could lead to a minute subpopulation of lysostaphin-resistant variants present in the starter culture and thus in some wells of the MIC assay. The conversion rate to lysostaphin resistance has been reported as being between 4 ⫻ 10⫺1 and 7.6 ⫻ 10⫺7 for seven MRSA strains (5), while in our hands, conversion rates of various strains of S. aureus to lysostaphin resistance ranged from 3 ⫻ 10⫺6 to ⬍3 ⫻ 10⫺9 (unpublished data). Lysostaphin-resistant variants of S. aureus strains are less fit and slower growing than their wildtype counterparts (38) and would normally be rapidly outgrown by the healthier wild-type bacteria. During the lysostaphin MIC assay, however, the wild-type S. aureus in the initial inoculum would be quickly killed if sufficient concentrations of lysostaphin are present in a certain well, thus allowing the outgrowth of any lysostaphin-resistant variant (if present) in the well. This phenomenon was not seen with every strain of S. aureus or even reproducibly with every assay of a particular strain. Resistance outgrowth in MIC testing does not necessarily reflect in vivo events inasmuch as lysostaphin-resistant variants are more frequently isolated in vitro (5) than in vivo (6, 19, 26). Furthermore, while the MIC assay is commonly used for the determination of antibiotic susceptibility, it may not be the most appropriate assay for a rapidly acting lytic enzyme like lysostaphin, since the MIC assay measures the growth inhibition activity of an antimicrobial agent while lysostaphin would likely kill the initial inoculum. The disk diffusion assay was the most simple and reproducible assay to differentiate lysostaphin-susceptible from lysostaphin-resistant strains of S. aureus. All strains tested, except for the three reference lysostaphin-resistant variants, had zones of inhibition of ⱖ11 mm, and this is in agreement with a study by von Eiff et al. (42). Despite lysostaphin being very staphylocidal, 50 ␮g of lysostaphin per disk was required to produce a usable zone of inhibition for assay purposes. Furthermore, the zones of inhibition for increasing concentrations of lysostaphin on disks did not appear proportional as might have been expected. Lysostaphin is a highly charged 27-kDa protein (3), while most conventional antibiotics are fairly small (⬍500 Da) molecules; it is likely that lysostaphin does not easily diffuse through agar compared to smaller antibiotics, thus, higher concentrations of lysostaphin are required for effective testing using disks on agar. The lack of proportionality of the zones of inhibition noted with lower concentrations of lysostaphin are likely due to the nature of the lysostaphin molecule. This theory was supported by the observation that prolonged incubation (more than 20 h) of agar plates with 50-␮g lysostaphin disks led to growing zones of secondary lysis which continued to expand over time as the lysostaphin continued to diffuse through the agar and lyse S. aureus; this observation was also made by von Eiff et al. (42). During disk diffusion testing with lysostaphin on CAMHA⫹,

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very small colonies were observed growing within the zone of inhibition around the 50-␮g lysostaphin disk for some strains of S. aureus. These colonies were suspected to be small-colony variants (29), a conclusion which was supported by the finding that these small colonies did not appear around lysostaphin disks when the assay was conducted on BHI agar. The auxotrophic small-colony phenotype can be reversed by supplying certain nutrients available in a rich medium like brain heart infusion (BHI) agar (29). As a follow-up, two stable smallcolony variants were obtained and found to be susceptible to lysostaphin by all four assays (Table 1). This study also included a number of defined genetic mutants of S. aureus from various sources (Table 2). Many of these strains had mutations that affected the outer surface of the bacteria (e.g., ATCC 35556 ⌬tagO, which does not make wall teichoic acid [43], or SKM 14, in which both sortases A and B have been disabled, leading to reduced trafficking of proteins to the bacterial surface [44]), while other mutants were disrupted for various regulatory systems (e.g., SH1001 [17] and SH2 1002 [17]). All mutants tested remained susceptible to lysostaphin by all four test methods, suggesting that many mutations that might occur naturally in S. aureus would not render the resulting mutants resistant to lysostaphin. There was some variability in lysostaphin susceptibility when comparing mutants with their wild types depending on which assay was used, but this was not consistent across all four assays, which further supports the theory that each of these assays actually measures the interaction of lysostaphin with S. aureus under different conditions and that while relative lysostaphin susceptibility may vary from assay to assay, all four assays can differentiate lysostaphin-susceptible strains of S. aureus from lysostaphin-resistant variants. There have been reports in the literature that some VISA strains may be less susceptible to lysostaphin (2, 18, 27) than their parental strains. Two of these studies were conducted with in vitro passage-selected VISA strains which may not reflect in vivo selection of vancomycin-intermediate susceptibility (18, 27), and one of those studies (18) used a turbidity assay to assess lysostaphin susceptibility, and as demonstrated in our study, the lysostaphin turbidity assay may only reflect part of the interaction of lysostaphin with a particular strain of S. aureus. The VISA strain (NRS79) with reported resistance to lysostaphin from the third study (2) was examined in our study, and while this strain did have a somewhat higher lysostaphin MIC, it had similar lysostaphin susceptibility to the other S. aureus strains by the other three assays (Table 1). Indeed, NRS79 was a strain of S. aureus which consistently displayed lysostaphin “resistance outgrowth” upon MIC testing (data not shown), which may have lead to the mischaracterization of this strain as lysostaphin resistant, since it was characterized as lysostaphin resistant by MIC testing in the original study (2). Furthermore, our study also included several other clinical VISA isolates (Table 1) which were all susceptible to lysostaphin in all of the assays used. While the changes in VISA strains that are believed to be responsible for the reduced susceptibility to glycopeptides (1) may appear to affect lysostaphin susceptibility of some strains depending on the assays used, it is clear from this study that lysostaphin retains the capacity to kill clinical VISA isolates (Table 1) and thus may provide alternative therapy for VISA infections. Further-

ANTIMICROB. AGENTS CHEMOTHER.

more, Patron et al. demonstrated that lysostaphin is an effective alternative therapy for experimental aortic valve endocarditis caused by clinical VISA strains (26). Based on the findings of this study, the disk diffusion assay appeared to be the most simple and reproducible method for differentiating lysostaphin-susceptible S. aureus strains from lysostaphin-resistant variants. The MBC assay can be used as a follow-up assay for questionable strains to determine actual lysostaphin susceptibility concentrations, since it measures the staphylocidal activity of lysostaphin. Assignment of in vitro susceptibility criteria for lysostaphin in accordance with NCCLS guidelines (21) will require more research and may require adaptations in the guidelines to accommodate this unique rapidly cidal protein. This study, however, lays the ground work for this continuing research and provides direction for the continuing development of lysostaphin as an antistaphylococcal agent. ACKNOWLEDGMENTS We thank the following researchers who provided various strains for this study: Martin Ottolini, Simon Foster, Andreas Peschel, Gordon Archer, Richard Proctor, Jerome Etienne, Olaf Schneewind, and Richard Novick. We also thank Jimmy Mond for helpful discussions and manuscript review. Some of the strains used in this study were obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA), which is supported by a grant from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Avison, M., P. Bennett, R. Howe, and T. Walsh. 2002. Preliminary analysis of the genetic basis for vancomycin resistance in Staphylococcus aureus strain Mu50. J. Antimicrob. Chemother. 49:255–260. 2. Boyle-Vavra, S., R. Carey, and R. Daum. 2001. Development of vancomycin and lysostaphin resistance in a methicillin-resistant Staphylococcus aureus isolate. J. Antimicrob. Chemother. 48:617–625. 3. Browder, H. P., W. A. Zygmunt, J. R. Young, and P. A. Travormina. 1965. Lysostaphin: enzymatic mode of action. Biochem. BioPhys. Res. Comm. 19:383–389. 4. Centers for Disease Control and Prevention. 2002. Staphylococcus aureus resistant to vancomycin-United States, 2002. Morb. Mortal. Wkly. Rep. 51:565–567. 5. Climo, M., K. Ehlert, and G. Archer. 2001. Mechanism and suppression of lysostaphin-resistance in oxacillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 45:1431–1437. 6. Climo, M. W., R. L. Patron, B. P. Goldstein, and G. L. Archer. 1998. Lysostaphin treatment of experimental methicillin-resistant Staphylococcus aureus aortic valve endocarditis. Antimicrob. Agents Chemother. 42:1355– 1360. 7. Dajcs, J. J., E. B. Hume, J. M. Moreau, A. R. Caballero, B. M. Cannon, and R. J. O’Callaghan. 2000. Lysostaphin treatment of methicillin-resistant Staphylococcus aureus keratitis in the rabbit. Investig. Opthalmol. Vis. Sci. 41:1432–1437. 8. Dassy, B., W. T. Stringfellow, M. Lieb, and J. M. Fournier. 1991. Production of type 5 capsular polysaccharide by Staphylococcus aureus grown in semisynthetic medium. J. Gen. Microbiol. 137:1155–1162. 9. DeHart, H., H. Heath, L. Heath, P. LeBlanc, and G. Sloan. 1995. The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus. Appl. Environ. Microbiol. 61:1475–1479. 10. Dixon, R. E., J. S. Goodman, and M. G. Koenig. 1968. Lysostaphin: an enzymatic approach to staphylococcal disease. III. Combined lysostaphinoxacillin therapy of established staphylococcal abscesses in mice. Yale J. Biol. Med. 41:62–68. 11. Ehlhart, K., W. Schroder, and H. Labschinski. 1997. Specificities of FemA and FemB for different glycine residues: FemB cannot substitute for FemA in staphylococcal peptidoglycan pentaglycine side chain formation. J. Bacteriol. 179:7573–7576. 12. Gillet, Y., B. Issartel, P. Vanhems, J. C. Fournet, G. Lina, M. Bes, F. Vandenesch, Y. Piemont, N. Brousse, D. Floret, and J. Etienne. 2002. Association between Staphylococcus aureus strains gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients. Lancet 359:753–759. 13. Goldberg, L. M., J. M. DeFranco, C. Watanakunakorn, and M. Hamburger.

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