Transfer of the methicillin resistance genomic island among staphylococci by conjugation.

Methicillin resistance creates a major obstacle for treatment of Staphylococcus aureus infections. The resistance gene, mecA, is carried on a large (20 kb to > 60 kb) genomic island, staphylococcal cassette chromosome mec (SCCmec), that excises from and inserts site-specifically into the staphylococcal chromosome. However, although SCCmec has been designated a mobile genetic element, a mechanism for its transfer has not been defined. Here we demonstrate the capture and conjugative transfer of excised SCCmec. SCCmec was captured on pGO400, a mupirocin-resistant derivative of the pGO1/pSK41 staphylococcal conjugative plasmid lineage, and pGO400::SCCmec (pRM27) was transferred by filter-mating into both homologous and heterologous S. aureus recipients representing a range of clonal complexes as well as S. epidermidis. The DNA sequence of pRM27 showed that SCCmec had been transferred in its entirety and that its capture had occurred by recombination between IS257/431 elements present on all SCCmec types and pGO1/pSK41 conjugative plasmids. The captured SCCmec excised from the plasmid and inserted site-specifically into the chromosomal att site of both an isogenic S. aureus and a S. epidermidis recipient. These studies describe a means by which methicillin resistance can be environmentally disseminated and a novel mechanism, IS-mediated recombination, for the capture and conjugative transfer of genomic islands.

Transcription of the gene mediating methicillin resistance in Staphylococcus aureus (mecA) is corepressed but not coinduced by cognate mecA and beta-lactamase regulators.

Resistance to beta-lactam antibiotics in staphylococci is mediated by mecA and blaZ, genes encoding a penicillin-binding protein (PBP2a) with low beta-lactam affinity and beta-lactamase, respectively. The mec and bla regulators, mecR1-mecI and blaR1-blaI, respectively, encode inducer-repressors with sufficient amino acid homology to suggest that they could coregulate PBP2a production. In order to test this hypothesis, plasmids containing mec and bla regulatory sequences were introduced into Staphylococcus aureus containing a chromosomal mecA-lacZ transcriptional fusion. Corepression was confirmed by demonstrating a gene dosage-dependent reduction in beta-galactosidase activity by either MecI or BlaI and additive repression when both were present. Both MecI-MecI and BlaI-BlaI homodimer and MecI-BlaI heterodimer interactions were demonstrated in the yeast two-hybrid assay, and purified MecI and BlaI protected the same mec promoter-operator sequences. However, MecI was approximately threefold more effective at mecA-lacZ transcriptional repression than was BlaI. While MecI and BlaI displayed similar activity as repressors of mecA transcription, there was a marked difference between MecR1 and BlaR1 in the rate and specificity of induction. Induction through BlaR1 by a beta-lactam was 10-fold greater than through MecR1 at 60 min and was 81% of maximal by 2 h, while induction through MecR1 never exceeded 20% of maximal. Furthermore, complementation studies showed that MecI- or BlaI-mediated mecA transcriptional repression could be relieved by induction through homologous but not heterologous sensor-inducer proteins, demonstrating the repressor specificity of induction.

Role of penicillin-binding protein 4 in expression of vancomycin resistance among clinical isolates of oxacillin-resistant Staphylococcus aureus.

It has been reported that penicillin-binding protein 4 (PBP4) activity decreases when a vancomycin-susceptible Staphylococcus aureus isolate is passaged in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible S. aureus (VISA) clinical isolates and found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, and HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visible in five vancomycin-susceptible, oxacillin-resistant S. aureus (ORSA) isolates. The nucleotide sequences of the pbp4 structural gene and flanking sequences did not different between the VISA and vancomycin-susceptible isolates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isolates produced a two- to threefold decrease in vancomycin MICs. Inactivation of pbp4 by allelic replacement mutagenesis in three vancomycin-susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in vancomycin susceptibility, an increase in highly vancomycin-resistant subpopulations, and decreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded pbp4 restored the vancomycin MIC and increased cell wall cross-linking. These data suggest that alterations in PBP4 expression are at least partially responsible for the VISA phenotype.

Mechanism and suppression of lysostaphin resistance in oxacillin-resistant Staphylococcus aureus.

The potential for the development of resistance in oxacillin-resistant Staphylococcus aureus (ORSA) to lysostaphin, a glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus, was examined in vitro and in an in vivo model of infection. Following in vitro exposure of ORSA to subinhibitory concentrations of lysostaphin, lysostaphin-resistant mutants were idenitifed among all isolates examined. Resistance to lysostaphin was associated with a loss of resistance to beta-lactams and a change in the muropeptide interpeptide cross bridge from pentaglycine to a single glycine. Mutations in femA, the gene required for incorporation of the second and third glycines into the cross bridge, were found following PCR amplification and nucleotide sequence analysis. Complementation of lysostaphin-resistant mutants with pBBB31, which encodes femA, restored the phenotype of oxacillin resistance and lysostaphin susceptibility. Addition of beta-lactam antibiotics to lysostaphin in vitro prevented the development of lysostaphin-resistant mutants. In the rabbit model of experimental endocarditis, administration of a low dose of lysostaphin for 3 days led predictably to the appearance of lysostaphin-resistant ORSA mutants in vegetations. Coadministration of nafcillin with lysostaphin prevented the emergence of lysostaphin-resistant mutants and led to a mean reduction in aortic valve vegetation counts of 7.5 log(10) CFU/g compared to those for untreated controls and eliminated the isolation of lysostaphin-resistant mutants from aortic valve vegetations. Treatment with nafcillin and lysostaphin given alone led to mean reductions of 1.35 and 1.65 log(10) CFU/g respectively. In ORSA, resistance to lysostaphin was associated with mutations in femA, but resistance could be suppressed by the coadministration of beta-lactam antibiotics.

Phenotypic expression of oxacillin resistance in Staphylococcus epidermidis: roles of mecA transcriptional regulation and resistant-subpopulation selection.

The MICs for many oxacillin-resistant (OR) Staphylococcus epidermidis (ORSE) strains are below the Staphylococcus aureus methicillin or oxacillin resistance breakpoint. The difficulty detecting the OR phenotype in S. epidermidis may be due to extreme heterotypy in resistance expression and/or transcriptional repression of mecA, the OR gene, by MecI. To determine the role of these factors in the phenotypic expression of ORSE, 17 geographically diverse mecI(+) ORSE isolates representing 14 distinct pulsed-field gel electrophoresis pulse types (>3 band differences) were investigated. Thirteen of the 14 types contained mecI and mecA promoter-operator sequences known to be associated with maximal mecA repression, and in all isolates, mecA transcription was repressed. All 17 were heterotypic in their resistance expression. Oxacillin MICs ranged from 1 to 128 microg/ml and increased for 16 of 17 isolates after beta-lactam induction. Allelic replacement inactivation of mecI in three isolates similarly resulted in a four- to sevenfold increase in MIC. In the two of these three isolates producing beta-lactamase, mecA transcription was regulated by both mecI and beta-lactamase regulatory sequences. Heterotypic expression of resistance in these three isolates was unaffected by either beta-lactam induction or mecI inactivation. However, prolonged incubation in concentrations of oxacillin just sufficient to produce a lag in growth (0.5 to 1.0 microg/ml) converted the population resistance expression from heterotypic to homotypic. Homotypic conversion could also be demonstrated in microtiter wells during MIC determinations in one isolate for which the MIC was high. We conclude that the phenotypic expression of S. epidermidis OR in broth can be affected both by mecA transcriptional regulation and by subpopulation resistance expression.

Combinations of vancomycin and beta-lactams are synergistic against staphylococci with reduced susceptibilities to vancomycin.

Evidence of synergism between combinations of vancomycin and beta-lactam antibiotics against 59 isolates of methicillin-resistant staphylococci (Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus) for which vancomycin MICs ranged from 1 to 16 microg/ml were tested by broth microdilution checkerboard, disk diffusion, agar dilution, and time-kill antimicrobial susceptibility tests. The combination of vancomycin and oxacillin demonstrated synergy by all test methods against 30 of 59 isolates; no antagonism was seen. Synergy with vancomycin was also found by modified disk diffusion testing for ceftriaxone, ceftazidime, cefpodoxime, and amoxicillin-clavulanate but not for aztreonam. Evidence of synergy correlated directly with vancomycin MICs. The efficacy of vancomycin given alone and in combination with nafcillin was tested in the rabbit model of experimental endocarditis caused by three clinical isolates of glycopeptide-intermediate-susceptible S. aureus (GISA) (isolates HIP5827, HIP5836, and MU50). Two of the GISA isolates (isolates MU50 and HIP5836) were extremely virulent in this model, with 27 of 42 (64%) animals dying during the 3-day trial. Therapy with either vancomycin or nafcillin given as a single agent was ineffective for animals infected with HIP5827 or MU50. However, the combination of vancomycin and nafcillin resulted in a mean reduction of 4.52 log10 CFU/g of aortic valvular vegetations per g compared to the reduction for controls for animals infected with HIP5827 and a reduction of 4. 15 log10 CFU/g for animals infected with MU50. Renal abscesses caused by HIP5827 were sterilized significantly better with the combination of vancomycin and nafcillin than by either treatment alone. We conclude that the combination of vancomycin and beta-lactams with antistaphylococcal activity is an effective regimen for the treatment of infections with clinical strains of staphylococci which demonstrate reduced susceptibility to glycopeptides.

Lysostaphin treatment of experimental aortic valve endocarditis caused by a Staphylococcus aureus isolate with reduced susceptibility to vancomycin.

The rabbit model of endocarditis was used to test the effectiveness of vancomycin and two different lysostaphin dosing regimens for the treatment of infections caused by a Staphylococcus aureus strain with reduced susceptibility to vancomycin (glycopeptide-intermediate susceptible S. aureus [GISA]). Vancomycin was ineffective, with no evidence of sterilization of aortic valve vegetations. However, rates of sterilization of aortic valve vegetations were significantly better for animals treated with either a single dose of lysostaphin (43%) or lysostaphin given twice daily for 3 days (83%) than for animals treated with vancomycin. Rabbits given a single dose of lysostaphin followed by a 3-day drug-free period had mean reductions in aortic valve vegetation bacterial counts of 7.27 and 6.63 log10 CFU/g compared with those for untreated control rabbits and the vancomycin-treated group, respectively. We conclude that lysostaphin is an effective alternative for the treatment of experimental aortic valve endocarditis caused by a clinical VISA strain.

Lysostaphin treatment of experimental methicillin-resistant Staphylococcus aureus aortic valve endocarditis.

The emergence of clinical isolates of methicillin-resistant Staphylococcus aureus with reduced susceptibility to vancomycin has prompted a search for new and novel therapeutic agents active against S. aureus. Lysostaphin, a peptidase produced by Staphylococcus simulans, specifically cleaves the glycine-glycine bonds unique to the interpeptide cross-bridge of the S. aureus cell wall. The effectiveness of various regimens of dosing with intravenous lysostaphin was compared to that of vancomycin in the rabbit model of aortic valve endocarditis caused by a clinical methicillin-resistant S. aureus isolate. All animals were treated for a total of 3 days. The most active regimen, lysostaphin given three times daily, produced sterile vegetations in 10 of 11 treated rabbits, with a mean reduction in vegetation bacterial counts of 8.5 log10 CFU/g compared to the counts in the untreated controls. In contrast, vancomycin given twice daily sterilized no vegetations and reduced vegetation bacterial counts by only 4.8 log10 CFU/g. Lysostaphin given once daily was less effective, reducing mean vegetation bacterial counts by only 3.6 log10 CFU/g, but the combination of lysostaphin once daily and vancomycin twice daily reduced the mean vegetation bacterial density by 7.5 log10 CFU/g, a result that was significantly better than that for either regimen alone (P < 0.05). Lysostaphin was well tolerated by the rabbits, with no evidence of immunological reactions following up to 9 weeks of intravenous administration. We conclude that lysostaphin given alone or in combination with vancomycin is more effective in the treatment of experimental methicillin-resistant S. aureus aortic valve endocarditis than vancomycin alone.

Staphylococcus aureus: a well-armed pathogen.

Staphylococcus aureus is a virulent pathogen that is currently the most common cause of infections in hospitalized patients. S. aureus infection can involve any organ system. The success of S. aureus as a pathogen and its ability to cause such a wide range of infections are the result of its extensive virulence factors. The increase in the resistance of this virulent pathogen to antibacterial agents, coupled with its increasing prevalence as a nosocomial pathogen, is of major concern. The core resistance phenotype that seems to be most associated with the persistence of S. aureus in the hospital is methicillin resistance. Methicillin resistance in nosocomial S. aureus isolates has been increasing dramatically in United States hospitals and is also associated with resistance to other useful antistaphylococcal compounds. Possible ways to decrease the incidence of nosocomial S. aureus infections include instituting more effective infection control, decreasing nasal colonization, developing vaccines, and developing new or improved antimicrobials.

Determination of the chromosomal relationship between mecA and gyrA in methicillin-resistant coagulase-negative staphylococci.

mecA, the gene that mediates methicillin resistance, and its accompanying mec locus DNA, insert near the gyrA gene in Staphylococcus aureus. To investigate whether there is a similar relationship between mecA and gyrA in coagulase-negative staphylococci (CNS), mecA- and gyrA-specific DNA fragments were used to probe methicillin-resistant isolates of Staphylococcus epidermidis (MRSE) (n = 11) and Staphylococcus haemolyticus (MRSH) (n = 11). The gyrA probe hybridized to the same SmaI DNA fragment as the mecA probe in all strains tested. However, since the size of the SmaI fragments containing mecA and gyrA varied from 73 to 600 kb, the distance between the two genes was determined more precisely. Cloned mecA or gyrA fragments plus vector sequences each containing a SmaI site were introduced into the chromosome of three isolates each of MRSE and methicillin-resistant S. aureus (MRSA), and the sizes of the generated SmaI fragments were determined by pulsed-field gel electrophoresis. The distance between gyrA and mecA was found to be between 38 and 42 kb in both MRSE and MRSA, and the two genes were in the same relative orientation in all strains. Restriction fragment length polymorphism (RFLP) patterns around the gyrA gene in CNS were identical, but species specific, for all 10 MRSE and 10 MRSH isolates examined. In contrast, 8 of 11 methicillin-susceptible S. epidermidis isolates and 7 of 7 methicillin-susceptible S. haemolyticus isolates had different gyrA RFLP patterns. These data show that mecA is site and orientation specific, relative to gyrA, in both MRSE and MRSA. In addition, the local environment around gyrA in methicillin-resistant CNS, in contrast to methicillin-susceptible isolates, is similar, suggesting clonality or the requirement for specific DNA sequences with which the mec complex must interact for chromosomal integration to occur.

Interaction of native and mutant MecI repressors with sequences that regulate mecA, the gene encoding penicillin binding protein 2a in methicillin-resistant staphylococci.

Methicillin resistance in staphylococci is mediated by PBP2a, a penicillin binding protein with low affinity for beta-lactam antibiotics. The gene encoding PBP2a, mecA, is transcriptionally regulated in some clinical isolates by mecR1 and mecI, genes divergently transcribed from mecA that encode a signal transducer and repressor, respectively. The biochemical basis of MecI-mediated mecA transcriptional repression was investigated by using purified MecI. In DNase I protection studies, MecI protected a 30-bp palindrome encompassing the predicted mecA -10 and the mecR1 -35 promoter sequences. The larger palindrome contained 15 bp of dyad symmetry within which was a smaller 6-bp palindrome. Electrophoretic mobility shift assays established a requirement for the entire 15-bp half-site for initial repressor binding. Fragments containing the 30-bp palindrome and the entire mecA-mecR1 intergenic region were retarded in gels as multiple discrete bands varying in molecular size, characteristic of cooperative DNA binding. Glutaraldehyde cross-linking confirmed oligomerization of repressor in solution. A naturally occurring MecI mutant (MecI*; D39G) repressed mecA transcription sixfold less well than the wild type in vivo. Although MecI* protected the same target sequences and exhibited similar gel shift patterns to MecI, 5- to 10-fold more protein was required. MecI* exhibited defective oligomerization in solution, suggesting that the MecI amino terminus is important in protein-protein interactions and that protein oligomerization is necessary for optimum repression.

Comparison of the in-vitro and in-vivo efficacy of FK037, vancomycin, imipenem and nafcillin against staphylococcal species.

The activity of methicillin, oxacillin, vancomycin, imipenem and FK037 against 106 isolates of staphylococci was assessed using a microbroth dilution method with low (10(5) cfu/mL) and high (10(7) cfu/mL) inoculum sizes. Overall, FK037, an oxime-type cephem antibiotic, was as active as imipenem, but less active than vancomycin (MIC90s 25, 25 and 6.25 mg/L, respectively) at the lower inoculum. Efficiency of plating experiments were also performed to characterize phenotypic expression of resistance to FK037, imipenem and methicillin. Five of 24 isolates and 18 of 24 isolates contained subpopulations resistant to FK037 and imipenem, respectively. In a rabbit model of endocarditis, FK037 was equally effective as other antibiotics tested in the treatment of methicillin-sensitive Staphylococcus aureus infection. In the treatment of endocarditis due to a homotypic methicillin-resistant S. aureus, FK037 and vancomycin were the most active antibiotics. The presence of subpopulations resistant to imipenem and FK037, as demonstrated by efficiency of plating and high inoculum MIC testing, did not correlate with antibiotic effectiveness in the rabbit model of endocarditis. Cultures of vegetation material following treatment with imipenem and FK037 demonstrated a lower frequency of organisms resistant to FK037 when compared with imipenem. Thus FK037 shows in-vitro and in-vivo activity against some methicillin-resistant staphylococcal species.

Role of mecA transcriptional regulation in the phenotypic expression of methicillin resistance in Staphylococcus aureus.

The gene required for methicillin resistance in staphylococci, mecA, encodes the low-affinity penicillin-binding protein 2a (PBP2a). Transcriptional regulation of mecA is accomplished in some isolates by mecR1 and mecI, cotranscribed chromosomal genes that encode a putative signal transducer and a transcriptional repressor, respectively. Two Staphylococcus aureus strains that have identical mecR1-mecI nucleotide sequences, BMS1 and N315P, both exhibit low-level, heterotypic expression of methicillin resistance and contain no beta-lactamase coregulatory sequences. mecR1-mecI was amplified from BMS1 by PCR and was shown to be functional on a high-copy-number plasmid when introduced into an S. aureus strain with a deleted mecR1-mecI locus. Cloned mecR1-mecI repressed phenotypic expression of methicillin resistance, mecA transcription and PBP2a production and mediated PBP2a induction in response to certain beta-lactam antibiotics. However, mecR1-mecI had different regulatory activities in its native chromosomal location in N315P compared with those in BMS1. Uninduced mecA transcription was markedly repressed in N315P, and mecI inactivation increased mecA transcription and PBP2a production 5- and 40-fold, respectively. Furthermore, the N315P phenotype changed from low-level, heterotypic resistance with intact mecI to high-level, homotypic resistance in strains with disrupted mecI. In contrast, uninduced BMS1 produced abundant mecA transcript and PBP2a, while the disruption of mecI had no effect on phenotype and little effect on mecA transcription or PBP2a production. Thus, mecI-mediated repression of mecA appears to be dysfunctional in BMS1 because of the presence or absence of additional regulatory cofactors. Furthermore, heterotypic resistance expression in this strain is independent of mecA transcriptional regulation.

Identification and characterization of the origin of conjugative transfer (oriT) and a gene (nes) encoding a single-stranded endonuclease on the staphylococcal plasmid pGO1.

The genes mediating the conjugative transfer of the 52-kb staphylococcal plasmid pGO1 are within a 14.4-kb gene cluster designated trs. However, a clone containing trs alone cannot transfer independently and no candidate oriT has been found within or contiguous to trs. In this study, we identified a 1,987-bp open reading frame (ORF) 24 kb 3' and 13 kb 5' to trs that was essential for conjugative transfer: transposon insertions into the ORF abolished transfer and a plasmid containing the ORF could complement these transposon-inactivated pGO1 mutants for transfer. Analysis of the nucleotide sequence of this ORF revealed significant homology between the amino terminus of its predicted protein and those of several single-stranded endonucleases. In addition, a 12-bp DNA sequence located 100 bp 5' to the ORF's translational start site was identical to the oriT sequences of the conjugative or mobilizable plasmids RSF1010, pTF1, R1162, pSC101, and pIP501. The ability of the ORF, designated nes (for nicking enzyme of staphylococci), to generate a single-stranded nick at the oriT was demonstrated in Escherichia coli by alkaline gel and DNA sequence analysis of open circular plasmid DNA. Plasmids that could be converted to the open circular form by the presence of oriT and nes could also be mobilized at high frequency into Staphylococcus aureus recipients with a second plasmid containing only trs. We propose that the 14.4 kb of trs and the approximately 2.2 kb of the oriT-nes region, coupled with an origin of replication, make up the minimal staphylococcal conjugative replicon.

Characterization of IS1272, an insertion sequence-like element from Staphylococcus haemolyticus.

We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.

Characterization of a conjugative staphylococcal mupirocin resistance plasmid.

We studied conjugative plasmids encoding high-level mupirocin resistance. These plasmids were found in Staphylococcus aureus isolates from two geographic locations in the United States. Transfer genes on three mupirocin resistance plasmids with different restriction endonuclease profiles were indistinguishable by DNA hybridization from those on pG01, a conjugative aminoglycoside resistance plasmid representative of similar plasmids that are prevalent in the United States. One mupirocin resistance plasmid, pG0400 (34 kb), was smaller than pG01 (52 kb) because of the absence from pG0400 of DNA, found on pG01, that contained genes encoding resistance to aminoglycosides, trimethoprim, and quaternary ammonium compounds flanked by directly repeated copies of the insertion sequence (IS)-like element IS431-IS257. The plasmids pG0400 and pG01 were otherwise indistinguishable except for the presence in pG0400 of a 4.5-kb HinDIII fragment encoding mupirocin resistance. The added mupirocin resistance gene was flanked by two directly repeated copies of IS431/257. The nucleotide sequence of DNA contiguous to the outside of the IS elements, as well as those of the elements themselves, was identical in both pG01 and pG0400, and there were no target site duplications flanking either copy of the element. We conclude that the mupirocin resistance gene was added to an existing conjugative plasmid in conjunction with the deletion of other resistance genes by recombination at IS elements. The construction of conjugative plasmids carrying a mupirocin resistance gene may be a model for the mobility of other resistance genes newly acquired by staphylococci.