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Antimicrobial Agents and Chemotherapy, February 2007, p. 475-482, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00786-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Lysostaphin-Resistant Variants of Staphylococcus aureus Demonstrate Reduced Fitness In Vitro and In Vivo{triangledown}

Caroline Kusuma,{dagger} Anna Jadanova,{ddagger} Tanya Chanturiya,§ and John F. Kokai-Kun*

Biosynexus Incorporated, Gaithersburg, Maryland 20877

Received 29 June 2006/ Returned for modification 1 August 2006/ Accepted 31 October 2006


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ABSTRACT
 
Lysostaphin is under development as a therapy for serious staphylococcal infections. During preclinical development, lysostaphin-resistant Staphylococcus aureus variants have occasionally been reported in vitro and in vivo. The acquisition of resistance to this drug, however, leads to a significant increase in ß-lactam antibiotic susceptibility, rendering methicillin-resistant S. aureus (MRSA) strains functionally methicillin susceptible. In this study, we have demonstrated that the development of lysostaphin resistance by two strains of MRSA also led to a loss of fitness in the variants. Consistent with the mutations found in previously reported lysostaphin-resistant S. aureus variants, these two variants had mutations in their femA genes, resulting in nonfunctional FemA proteins and, thus, monoglycine cross bridges in the peptidoglycan. The diminished fitness of the lysostaphin-resistant variants was reflected by (i) a reduced logarithmic growth rate, with the variants being outcompeted in cocultures by their wild-type parental strains; (ii) increased susceptibility to elevated temperatures; and (iii) at least fivefold less virulence of the lysostaphin-resistant variants than their wild-type strains in a mouse kidney infection model, with the lysostaphin-resistant variants being outcompeted in coinfections with their wild-type parental strains. During a 14-day serial passage without selective pressure, the lysostaphin-resistant variants failed to develop compensatory mutations which restored their fitness. These results suggest that should lysostaphin resistance due to an alteration in the FemA function emerge in S. aureus during therapy with lysostaphin, the resistant variants would be less fit and less virulent, and, in addition, infections with these strains would be easily treatable with ß-lactam antibiotics.


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INTRODUCTION
 
As the incidence of antibiotic-resistant Staphylococcus aureus, including strains resistant to frequently used antibiotics such as vancomycin (4, 22, 35), continues to increase, there has been an effort to identify novel therapies for S. aureus infections. One antistaphylococcal agent for which there has been a recent renewed interest as a therapy for serious S. aureus infections is lysostaphin (5-7, 16, 18, 19, 28). Lysostaphin is a 27-kDa glycylglycine endopeptidase which cleaves between the third and fourth glycines (33) of the pentaglycine cross bridge of the staphylococcal cell wall, destabilizing the cell wall and, thus, rapidly lysing both actively growing and quiescent staphylococci (41), including staphylococci in biofilms (40).

Resistance to lysostaphin has been documented (6, 36), and this resistance generally results from mutations in the femA gene, which encodes the factor responsible for addition of the second and third glycines to the pentaglycine bridge of the cell wall (9). Inactivation of the FemA function results in monoglycine cross bridges, thus eliminating the target for lysostaphin cleavage (36). In addition to the reduction of the glycine content in the cell wall of femA mutants, these mutants have been characterized as having reduced cross-linkage and cell wall turnover, aberrant cell septum formation, and retardation of cell separation (36). It is also well documented that methicillin-resistant S. aureus (MRSA) strains that become resistant to lysostaphin lose their methicillin resistance phenotype (5, 20, 21, 31, 32, 36).

The development of resistance to antibiotics often brings with it a fitness cost to the bacteria (1). These fitness costs are likely, however, ameliorated by subsequent evolution in the form of compensatory mutations (23). As each new class of antibiotics is introduced, evolution selects for the mutants which can survive the antibiotic challenge. These mutants, however, are often less fit than their ancestral lineage, and thus, there is a second round of evolution which can occur to select for mutants which have compensated for the fitness deficit.

Since lysostaphin is under development as therapy for staphylococcal infections and resistance to lysostaphin could possibly occur during lysostaphin therapy, we sought to systematically determine the fitness of in vitro-selected lysostaphin-resistant variants of two MRSA strains and to determine whether passage of these variants without selective pressure could restore their fitness. We took advantage of the mutually exclusive nature of ß-lactam resistance versus lysostaphin resistance (5) to allow selection of each phenotype following competitive cultures both in vitro and in vivo in a mouse model of infection.


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MATERIALS AND METHODS
 
Bacterial strains. Two strains of MRSA and their lysostaphin-resistant variants were used in these studies (Table 1). The two MRSA strains were Col (27) and MBT 5040 (18, 19). Lysostaphin-resistant variants of these two MRSA strains (strains Col-LysoR and MBT 5040-LysoR, respectively) were selected in vitro as described previously (19). Col is a well-characterized MRSA strain, while S. aureus MBT 5040 is a clinical isolate from Walter Reed Army Medical Center. Strain MBT 5040 was spa typed (34) as WGKAOMQ, which is associated with sequence type 239, which makes it a member of clonal complex 8.


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  		TABLE 1. Bacterial strains and antibiotic resistance

Sequencing of the femA genes of lysostaphin-resistant variants. Genomic DNA for PCR amplification was released by lysostaphin (Biosynexus Inc., Gaithersburg, MD) (26) treatment of the parental S. aureus strains (10 µg/ml, 10 min) or by boiling of the lysostaphin-resistant variants for 5 min. The nucleotide sequences of the femA genes of strains Col, MBT 5040, Col-LysoR, and MBT 5040-LysoR were determined by direct sequencing of specified amplified PCR products obtained from each strain's genomic DNA template. The entire femA genes of strains MBT 5040 and MBT5040-LysoR (1,263 bp) were amplified by PCR with an iCycler thermal cycler (Bio-Rad, Hercules, CA) and the following primers: FPCR (5'-GGGAAATAGAAAAACTGCAAATACGG-3') and RPCR (5'-ATTCCCTTCC-TAAAAAATTCTGTC-3'). Sequencing of the femA gene was performed by the dideoxy chain termination procedure on a CEQ 8000 genetic analysis system (Beckman Coulter, Palo Alto, CA) automated sequencer with CEQ DCTS quick start mix (Beckman Coulter). The following primers were used for sequencing: IntF1 (5'-CGTTGTCTATACCTACATATCGAT-3'), IntF2 (5'-GCTTTTGCTGATCGTGATGACA-3'), IntF3 (5'-GTTGTTTAT-TATGCTGGTGG-3'), IntR1 (5'-GCATTACCTGTAATCTCGCC-3'), and IntR2 (5'-GC-TAAAGGTACTAACACACGGTCT-3'). The femA genes of strains Col and Col-LysoR were amplified with the following primers: Col FPCR (5'-A-ATTAACGAGAGACAAATAGGAG-3') and Col RPCR (5'-CATGTTTTGATAAT-TCCCTTCC-3'). The primers used for sequencing were as follows: Col IntF1 (5'-GGTAATGC-TGGTAATGGTTGG-3'), Col IntF2 (5'-TAAAGAACTAAATGAAGAGCGTG-3'), Col IntR1 (5'-GCATTACCTGTAATCTCGCC-3'), and Col IntR2 (5'-CTAAAGGC-ACTAACACACGG-3'). Amplification and sequencing of femA were repeated three times for MBT 5040-LysoR to ensure that the single base pair insertion was real and not a sequencing artifact.

Bacterial culture. The rates of conversion of MRSA strains to the lysostaphin resistance phenotype and the lysostaphin-resistant variants to the MRSA phenotype were determined by plating 10-fold concentrated resuspensions and serial 10-fold dilutions of overnight tryptic soy broth (TSB; BD, Sparks, MD) cultures of the various S. aureus strains on Trypticase soy agar (TSA; BD) containing either 10 µg/ml lysostaphin or 10 µg/ml nafcillin (Sigma, St. Louis, MO). The conversion rate experiments were conducted with duplicate samples and were repeated four times. The MICs of both lysostaphin and nafcillin were determined as described previously (19).

In vitro growth curves of the various S. aureus strains were performed in triplicate. Nephalo flasks containing 20 ml prewarmed TSB were inoculated with 1:100 dilutions of overnight TSB cultures. The inoculated cultures were incubated with shaking at 250 rpm at 37°C. The optical densities at 650 nm were determined on a Spectronic D 20+ light spectrometer (Spectronic Unicam, Rochester, NY) at various times during the incubation. In some experiments, serial 10-fold dilutions in phosphate-buffered saline (PBS; Cambrex, Walkersville, MD) of various broth cultures were plated on either blood agar (Remel, Lenexa, KN) or TSA supplemented with either lysostaphin (10 µg/ml) or nafcillin (10 µg/ml).

In one set of experiments, broth cultures of the various strains were serially passaged for 14 consecutive days. TSB (3 ml) without antibiotics was inoculated with 30 µl of overnight TSB cultures from the previous day. Samples were taken on each day of the serial passage, diluted, and plated on various selective and nonselective media. The growth curves for the cultures serially passaged for 14 days were also determined as described above.

In another set of experiments to determine the temperature tolerances of the various strains, dilutions of bacteria (starting inoculum, ~2 x 106 CFU/ml) were incubated at various temperatures from 37°C to 55°C in PBS for 10 min in a water bath prior to plating of serial dilutions on blood agar to determine the numbers of surviving CFU/ml. The numbers of CFU recovered following 10 min of incubation at various temperatures were compared with the numbers of CFU recovered following 10 min of incubation at the control temperature of 37°C for each experiment. The data for each incubation temperature were expressed as a percentage of the CFU recovered in the matched control sample, which was arbitrarily set at 100%. These experiments were performed three separate times.

Triton X-100-induced autolysis. To determine whether there was a difference in the autolytic phenotype between the parental S. aureus strains and their lysostaphin-resistant variants, the susceptibilities of mid-log-phase cultures to Triton X-100-induced lysis were assessed by the method of Mani et al. (24). Briefly, S. aureus cultures were grown to mid-log phase (absorbance at 650 nm, ~0.700) in TSB medium. The cells were washed twice in ice-cold water and then resuspended in 0.05 M Tris-HCl (pH 7.2) containing 0.05% (vol/vol) Triton X-100 (Pierce, Rockford, IL). The samples were incubated at 30°C, and the optical density at 650 nm was measured every 15 min for 8 h.

Mouse kidney infection model. Six-week-old female CF-1 mice (HSD, Indianapolis, IN) were challenged with various doses of S. aureus via intravenous injection through the tail vein. At 7 days postchallenge, the animals were sacrificed and their kidneys were harvested. The organs were placed in 1 ml of PBS and disrupted by mechanical disruption. An aliquot (100 µl) of each supernatant was plated on the various agar media described above. Colony counts for the kidneys were enumerated after 24 to 48 h of incubation at 37°C. All experiments involving animals were carried out in accordance with the guidance of the Biosynexus Inc. Institutional Animal Care and Use Committee.


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RESULTS
 
The femA sequences of both lysostaphin-resistant variants contained mutations. The gene product of femA is well established as the enzyme responsible for addition of the second and third glycines of the pentaglycine bridge (31), and mutations of this gene are known to lead to monoglycine cross bridges and lysostaphin resistance (5, 9, 36). The femA genes of the two parental S. aureus strains as well as those of their lysostaphin-resistant variants were sequenced, and the sequences were compared to the annotated sequence for femA of strain Col (available at http://www.tigr.org). The sequences of the femA genes of both parental S. aureus strains, strains Col and MBT 5040, were found to match the genome sequence of S. aureus Col. As shown in Fig. 1, however, and consistent with previous reports (5, 21, 36), mutations were found in the femA genes of both lysostaphin-resistant S. aureus variants. Strain MBT 5040-LysoR had a single base insertion which led to a +1 frame shift and the formation of a stop codon at amino acid 133 of femA, while strain Col-LysoR had a 66-bp in-frame deletion in femA which would delete amino acids 101 to 122 of the FemA protein.


Figure 1
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  		FIG. 1. Mutations detected in the femA genes of strains Col-LysoR and MBT 5040-LysoR compared to the sequences of their wild-type parental strains. The femA gene of Col-LysoR had a 66-bp deletion (as indicated on the figure) which extended from base 302 to base 368 of the femA open reading frame and which eliminated amino acids 101 to 122 of the resulting FemA protein. The deletion is in frame and reforms the tyrosine residue at position 123 (indicated in italics). The femA gene of MBT 5040-LysoR had a single adenosine insertion at base pair 337 (A, as indicated by the arrow), which shifted the reading frame +1 (the altered amino acid sequence is in italics) and which led to a stop codon (indicated in boldface) at amino acid 133 (*). The numbers correspond to amino acids of the wild-type FemA protein, while the dashes indicate missing base pairs or amino acids; the dots are space holders.

Phenotypic observations. Several phenotypic differences were notable between the lysostaphin-resistant variants and their wild-type counterparts. Both lysostaphin-resistant variants grew as smaller colonies than their parental strains on blood agar; and while both the parental MRSA strains and the lysostaphin-resistant variants were only weakly hemolytic following 24 or 48 h incubation at 37°C, both of the lysostaphin-resistant variant strains displayed pronounced hemolysis following overnight refrigeration of the blood agar plates (data not shown). The parental strains remained weakly hemolytic after a similar incubation. Upon microscopic observation of Gram-stained broth cultures, the parental strains were observed to have the clustered morphology typical of staphylococci, but as previously described for other fem mutants (36), the two lysostaphin-resistant variants in this study both displayed altered morphologies. The bacterial cells appeared to grow as short chains rather than clusters, and larger cells with incomplete septation were also observed within these chains (data not shown).

The lysostaphin-resistant variants demonstrated a reduced rate of exponential growth compared to that of their wild-type counterparts. Serial dilutions of overnight (18-h) TSB cultures of both wild-type MRSA strains and their lysostaphin-resistant variants were plated on blood agar to determine the numbers of CFU. The numbers of CFU/ml of the overnight cultures for both the wild-type MRSA strains and their lysostaphin-resistant variants were similar at ~2 x 109 CFU/ml. Figure 2A and B shows the growth curves for the wild-type strains compared with those for their lysostaphin-resistant variants when equal volumes of overnight cultures of each strain were inoculated into fresh media. Both lysostaphin-resistant variants had reduced rates of logarithmic growth. This reduced rate was more pronounced for strain MBT 5040-LysoR (Fig. 2A). The difference in logarithmic growth was reproducible and very similar in three separate experiments, as shown in Fig. 2. Serial dilution and plating of 5-h broth cultures of wild-type and lysostaphin-resistant variants confirmed that the wild-type strains had grown more at that point than their lysostaphin-resistant variants. A 5-h broth culture of MBT 5040 contained 6 x 107 CFU/ml, while MBT 5040-LysoR had 2.8 x 107 CFU. Col at 5 h had 5.9 x 107 CFU/ml, while Col-LysoR had 1.4 x 107 CFU/ml at 5 h.


Figure 2
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  		FIG. 2. Growth curves of wild-type S. aureus strains compared with those of their lysostaphin-resistant variants. The optical density of the bacterial suspension measured at 650 nm (OD650) versus time (in minutes) in prewarmed TSB with aeration at 37°C for strain MBT 5040 (A and C) or Col (B and D) prior to serial passage (A and B) and after 14 days of serial passage without selective pressure (C and D) are shown for the wild-type parental strains (solid lines) and their lysostaphin-resistant variants (dashed lines). These are mean data from three separate repetitions of each comparison, with standard deviations as indicated. Points without error bars had a standard deviation too small to depict.

Wild-type strains outcompete the lysostaphin-resistant variants in cocultures. TSB was inoculated with equal volumes of overnight cultures of wild-type parental strains and their corresponding lysostaphin-resistant variants. The starting inocula of each strain were determined and found to be equal, as reported above. Taking advantage of the fact that lysostaphin-resistant variants of MRSA strains are functionally methicillin susceptible, aliquots of mixed cultures were serial diluted and plated on blood agar and TSA supplemented with either lysostaphin or nafcillin at 5 and 24 h. Table 2 shows that the wild-type parental strains of both Col and MBT 5040 outcompeted their lysostaphin-resistant variants in these mixed cultures. Approximately four- to fivefold more wild-type bacteria were present in the mixed culture at both 5 and 24 h. These experiments were repeated six times, and the fold difference between the amount of the parental strain and the amount of the lysostaphin-resistant variant recovered from the coculture at each time point was significant (Table 2).


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  		TABLE 2. Fold difference in CFU/ml of wild-type versus lysostaphin-resistant variant S. aureus strains recovered from coculture experiments

The phenotypes of the various S. aureus strains remained stable during repeated culturing without selective pressure. Antibiotic-resistant mutants that initially display a fitness cost often accumulate secondary mutations that are compensatory for these fitness deficiencies (23). In order to determine whether the lysostaphin-resistant variants that demonstrated an in vitro fitness deficiency would develop compensatory mutations that would make up for these deficiencies, the parental strains and the lysostaphin-resistant variants were serially passaged for 14 days in TSB without selective pressure. On each day of passage, serial dilutions of each culture were plated on TSA-lysostaphin and TSA-nafcillin. The lysostaphin-resistant variants remained lysostaphin resistant over the 14 days, and no nafcillin-resistant revertant of a lysostaphin-resistant variant was seen during this period (data not shown).

When the in vitro growth curves of the serially passaged lysostaphin-resistant variants were compared to the growth curves of the serially passaged wild-type parental strains, a pattern of growth similar to that observed prior to serial passage was seen (Fig. 2C and D). In fact, a more pronounced difference was noted for the lysostaphin-resistant variants than for the parental strains following the 14-day serial passage, further suggesting that compensatory mutations did not occur.

Temperature tolerance of wild-type bacteria versus those of lysostaphin-resistant bacteria. In order to determine whether the lysostaphin-resistant variants were any more susceptible to temperature stress than their wild-type counterparts, aliquots of overnight cultures were incubated at various elevated temperatures for 10 min, followed by serial dilution and plating to determine the numbers of surviving bacteria. As shown in Fig. 3, the wild-type parental strains were more temperature tolerant than their lysostaphin-resistant variants, and this was especially true for strain MBT 5040 and its lysostaphin-resistant variant. Furthermore, wild-type strain MBT 5040 appeared to be generally more temperature tolerant than wild-type strain Col.


Figure 3
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  		FIG. 3. Relative temperature tolerances of wild-type parental strains and their lysostaphin-resistant variants. The numbers of CFU recovered following 10 min of incubation at various temperatures (as noted) were compared with the numbers of CFU recovered from identical control samples incubated at 37°C for 10 min. The numbers of CFU recovered from the control samples were arbitrarily set at 100%, and the data are expressed as a percentage of that arbitrary 100%. *, significant difference (P < 0.05) by a two-tailed test for paired samples between the numbers of CFU recovered following incubation at the specified temperature and the numbers of CFU recovered from the control samples for that particular strain; **, significant difference (P < 0.05) by a two-tailed test for paired samples between the percentage of CFU recovered at a specific temperature for a lysostaphin-resistant variant and the percentage of CFU recovered for the matched wild-type parental strain. The data are the means of three separate experiments, with standard deviations as indicated.

Triton X-100-induced autolysis. The autolytic phenotypes of the wild-type parental strains were compared to those of the lysostaphin-resistant variants by subjecting mid-log-phase samples of the various strains to treatment with Triton X-100. Upon four repetitions of this experiment, very similar patterns of autolysis were seen for all four S. aureus strains, both the wild-type strains and the lysostaphin-resistant variants (data not shown), suggesting that the variants were not defective for autolysis.

Wild-type strains outcompeted their less virulent lysostaphin-resistant variants in mixed infections in mice. CF-1 mice were challenged intravenously with ~2 x 107 cells of S. aureus MBT 5040, MBT 5040-LysoR, Col, or Col-LysoR to determine the virulence of the strains. Most of the animals challenged with this dose of either wild-type strain of S. aureus had infected kidneys (Table 3) when they were sacrificed on day 7, but none of the animals challenged with the lysostaphin-resistant variants had infected kidneys when they were challenged with that dose (data not shown). It was determined that a dose of ~108 of either lysostaphin-resistant variant was required to cause consistent kidney infections in mice (Table 3). To assess the competitive nature of a mixed infection with the wild type and the lysostaphin-resistant variant, a ratio of ~1:5 wild type to variant was used for coinfection challenge experiments. Five CF-1 mice each were challenged with MBT 5040, MBT 5040-LysoR, Col, or Col-LysoR or the appropriate wild type-variant mixture thereof. At 7 days postchallenge, the mice were sacrificed and kidney infection was determined by plating the disrupted tissue on agar containing either lysostaphin or nafcillin. Table 3 shows that at 7 days postchallenge most animals had kidneys infected with S. aureus at the doses administered. Challenge with the two wild-type parental strains, even at one-fifth the dose used for challenge with the lysostaphin-resistant variant strains, led to the recovery of increased numbers of CFU from the kidneys of the infected animals. In the mixed challenges, only wild-type MRSA was recovered from the infected kidneys of seven of eight animals, while the kidney of only one animal from a mixed challenge group was infected with a lysostaphin-resistant variant strain. No lysostaphin-resistant S. aureus strains were recovered from the kidneys of an animal challenged with wild-type MRSA, and no methicillin-resistant revertants were recovered from the kidneys of animals challenged with a lysostaphin-resistant variant.


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  		TABLE 3. Kidney infection of mice at 7 days postchallenge with various individual and mixed S. aureus strains


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DISCUSSION
 
The use of lysostaphin as a therapeutic drug (6, 7, 18, 28) could potentially lead to the in vivo selection of variants that are resistant to lysostaphin. While the use of combination therapy of lysostaphin with a ß-lactam would preclude this occurrence (5, 20, 21, 31, 32, 36), conditions may arise in which lysostaphin-resistant variants are selected in vivo. This study demonstrated that two in vitro-selected, lysostaphin-resistant variants were less fit and less virulent than their wild-type parental strains. The lysostaphin-resistant variants were outcompeted in vitro by their wild-type S. aureus strains in coculture and had a reduced capacity to tolerate temperature stress compared to that of the wild-type strains. In vivo, the lysostaphin-resistant variants were less virulent than the wild-type strains and were outcompeted by the wild-type bacteria in mixed-infection experiments, even when the challenge ratio was 5:1 in favor of the variant.

Consistent with the findings previously reported for lysostaphin-resistant S. aureus variants (5, 9, 36), these two lysostaphin-resistant variants were both found to have mutations in their femA genes that would render the resulting gene products inactive and that would also be expected to result in monoglycine cross bridges (5, 9). An artificial substrate consisting of five glycines has previously been reported to be a target for lysostaphin (17). One variant (Col-LysoR) had a 66-bp deletion within the femA gene, while the other variant (MBT 5040-LysoR) had a single base insertion which led to a frame shift and an in-frame stop codon (Fig. 1) which would truncate the resulting protein at about one-quarter of its full size.

S. aureus strains with mutated femAB have been described as "pseudomulticellular with thickened cell walls and abnormal septa due to impaired cell wall turnover" and to have poorly cross-linked cell walls (31). Furthermore, while these mutants become resistant to lysostaphin (5, 9, 36), they completely lose resistance to methicillin (5, 36) and become hypersusceptible to many unrelated antibiotics (21). The two parental strains in this study (strains Col and MBT 5040) are both MRSA stains, but the development of lysostaphin resistance has led to the expected reversion to a methicillin-susceptible phenotype for their lysostaphin-resistant variants (Table 1), which is consistent with loss of the FemA function (5, 21, 36). It has been demonstrated that the modified penicillin binding protein 2 (PBP 2a), which is encoded by the mecA gene and which confers ß-lactam resistance on MRSA strains, is unable to perform its function in the absence of the five-membered interpeptide chains (13, 31). That is, while the low-affinity PBP 2a is still produced in MRSA strains that become lysostaphin resistant due to mutations to femA (20, 31), PBP 2a can no longer function as a transpeptidase for monoglycine interpeptide chains. The normal PBP 2, however, that is required for PBP 2a to be fully functional can use these monoglycine interpeptides, but since PBP 2 can be inhibited by ß-lactam antibiotics, the resultant phenotype is methicillin susceptible (13). It has previously been documented that complementation of nonfunctional femA with a functional copy of femA delivered in trans restores the pentaglycine cross bridges and also restores lysostaphin susceptibility and methicillin resistance in MRSA strains (5, 21).

Another mechanism of resistance for staphylococci to glycylglycine proteolytic factors like lysostaphin can occur through the incorporation of other amino acids, like serines, into polyglycine cross bridges (10, 14, 37, 38). This incorporation disrupts the glycine-glycine target for proteolytic cleavage and can occur through specific immunity factors like lif (lysostaphin immunity factor), found on the plasmid which carries the lysostaphin gene in Staphylococcus simulans bv. staphylolyticus (38); epr (endopeptidase resistance), found on the plasmid which carries the gene for ALE-1, a lytic agent similar to lysostaphin, found in Staphylococcus capitis EPK1 (37); or an epr-like gene found on a plasmid which expresses an N-acetylmuramyl-L-alanine amidase-like gene in Staphylococcus sciuri strain DD 4747 (14). Coagulase-negative staphylococci can also randomly incorporate serine and alanine into their pentaglycine cross bridges to various degrees, and the degree of incorporation is directly reflected in the lysostaphin susceptibilities of the various species or strains (8, 10, 16, 30, 37). S. aureus appears to lack the capacity to randomly incorporate other amino acids like serines into its cross bridge without the introduction of exogenous immunity factors like lif or epr (8, 37). Thus, this mechanism of lysostaphin resistance would not be expected to be found in our naturally selected lysostaphin-resistant S. aureus variants.

Recently, it has been reported that disruption of SAV2335, now called lyrA, for lysostaphin resistance A, leads to reduced lysostaphin susceptibility in S. aureus (12). The LyrA protein appears to have a polytopic membrane domain with a predicted protease domain, but its role in cell wall assembly or function remains unclear. Disruption of this gene leads to a fourfold increase in the lysostaphin MIC and a slower reduction in the optical density in response to lysostaphin compared to those of the wild-type strains. How disruption of lyrA actually leads to reduced lysostaphin susceptibility, as well as the virulence and fitness of the lyrA mutants and their relevance in terms of selection in vivo, remains to be assessed.

The lysostaphin-resistant variants evaluated in this study were also less tolerant of heat stress than their wild-type parental strains (Table 2), perhaps as a result of their altered cell wall structure. Interestingly, there did not appear to be a significant difference in Triton X-100-inducible autolysis (24) between the wild-type strains and the lysostaphin-resistant variant strains (data not shown), suggesting that the autolytic function of the variants remained intact and that this was not the cause of the reduced growth rate (Fig. 2) seen for the lysostaphin-resistant variants.

Beyond the demonstrated fitness loss, the lysostaphin-resistant variants also had reduced virulence (Table 3) which, beyond the fitness deficit of these variants, may also have been due in part to the lack of pentaglycine cross bridges which serve as anchors for sortase-mediated sorting proteins on the bacterial surface (31, 39). Without a functional sorting mechanism, key virulence factors can no longer be localized to the staphylococcal cell wall (33). This lack of surface-bound virulence factors may also be the cause of the reduced capacity for nasal colonization, which we have previously reported for MBT 5404-LysoR (18). The lysostaphin-resistant variants in this study also displayed an altered phenotype in terms of hemolysis on blood agar. Both variants had more pronounced hemolysis than the wild-type strains after storage overnight at 4°C. This enhanced hemolytic phenotype could be caused by the increased expression or the increased secretion of ß-hemolysin. This hemolysin is known to demonstrate hot-cold hemolysis (3), but its role in virulence in our model is unknown.

Mutations in S. aureus leading to antibiotic resistance, their associated fitness costs, and the rise of compensatory mutations that restore fitness have been documented. Mupirocin is a topical antibiotic which inhibits bacterial isoleucyl-tRNA synthetase. Lower-level mupirocin resistance can occur through mutations to the ATP binding domain of the Rossman fold of the enzyme (2). These first-step mutations, which lead to lower-level resistance, are not associated with a substantial fitness cost; but second-step mutants with higher levels of resistance to mupirocin can be selected, and these mutants are found to be unfit (15). Fitness can be restored by subculture of these second-step mutants in the absence of mupirocin as a result of compensatory mutations, but these compensatory mutations also suppress mupirocin resistance (15). There is also a fitness cost associated with methicillin resistance and the staphylococcal chromosomal cassette mec (SCCmec) (11). Transformation of the type I SCCmec into S. aureus leads to a reduced growth rate, and while faster-growing variants can be selected, these variants have reduced resistance levels. In nature, however, the fitness cost of SCCmec is likely compensated for in MRSA strains. S. aureus also has a phenotypic switching mechanism which circumvents permanent fitness costs due to antibiotic resistance (25). Exposure of S. aureus to gentamicin results in the formation of gentamicin-resistant small-colony variants (29). A rapid switch between this gentamicin-resistant small-colony variant and the sensitive parental phenotype has been demonstrated, and the fitness of revertants relative to stable gentamicin-resistant variants is greatly increased.

In our study, a 14-day serial passage experiment without selective pressure failed to induce compensatory mutations to overcome the fitness deficit of the lysostaphin-resistant variants (Fig. 2C and D). It has been suggested that mutations in femAB of S. aureus which result in monoglycine cross bridges are so detrimental that the only mutants that survive are those that quickly accumulate compensatory mutations (21). Thus, it is possible that the variants under examination in this study may already have one or more compensatory mutations. Even if these compensatory mutations are present, they have not fully restored the fitness of the variants, nor have they restored methicillin resistance; and additional compensatory mutations do not appear to accumulate even during extended passage without selective pressure.

The mutually exclusive nature between lysostaphin resistance and ß-lactam resistance coupled with the reduced fitness and virulence of lysostaphin-resistant variants suggests that resistance to lysostaphin during clinical use may not be a significant impediment to the development of lysostaphin as an effective therapy for serious staphylococcal infections.


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ACKNOWLEDGMENTS
 
We acknowledge Jeff Stinson and Luba Grinberg for assistance with sequencing and Julio Canas and Elizabeth Mendez for additional technical assistance, Gordon Archer for spa typing of S. aureus strain MBT 5040, and Jimmy Mond for helpful discussions and critical review of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Biosynexus Incorporated, 9119 Gaither Rd., Gaithersburg, MD 20877. Phone: (301) 987-1172. Fax: (301) 990-4990. E-mail: johnkun{at}biosynexus.com. Back

{triangledown} Published ahead of print on 13 November 2006. Back

{dagger} Present address: AFG Biosolutions, Gaithersburg, MD. Back

{ddagger} Present address: Hofstra University, Long Island, NY. Back

§ Present address: National Institutes of Health, Bethesda, MD. Back


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Antimicrobial Agents and Chemotherapy, February 2007, p. 475-482, Vol. 51, No. 2
0066-4804/07/$08.00+0     doi:10.1128/AAC.00786-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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