Cell wall thickening is a common feature of vancomycin resistance in Staphylococcus aureus.

We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant Staphylococcus aureus (VRSA) (equivalent to vancomycin-intermediate S. aureus and glycopeptide-intermediate S. aureus) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, <4 mg/liter) were obtained. However, all of the passage-derived strains except one (15 of 16) still possessed subpopulations that were resistant to vancomycin as judged by population analysis, and vancomycin-resistant mutant strains were selected from the passage-derived strains by one-step vancomycin selection with a frequency of 4.25 x 10(-6) to 1.64 x 10(-3). The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < 0.001) and between MICs of imipenem and oxacillin (r = 0.787; P < 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in S. aureus.

Staphylococcus caprae strains carry determinants known to be involved in pathogenicity: a gene encoding an autolysin-binding fibronectin and the ica operon involved in biofilm formation.

The atlC gene (1,485 bp), encoding an autolysin which binds fibronectin, and the ica operon, involved in biofilm formation, were isolated from the chromosome of an infectious isolate of Staphylococcus caprae and sequenced. AtlC (155 kDa) is similar to the staphylococcal autolysins Atl, AtlE, Aas (48 to 72% amino acid identity) and contains a putative signal peptide of 29 amino acids and two enzymatic centers (N-acetylmuramoyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase) interconnected by three imperfect fibronectin-binding repeats. The glycine-tryptophan (GW) motif found in the central and end part of each repeat may serve for cell surface anchoring of AtlC as they do in Listeria monocytogenes. The S. caprae ica operon contains four genes closely related to S. epidermidis and S. aureus icaA, icaB, icaC, and icaD genes (> or = 68% similarity) and is preceded by a gene similar to icaR (> or =70% similarity). The polypeptides deduced from the S. caprae ica genes exhibit 67 to 88% amino acid identity to those of S. epidermidis and S. aureus ica genes. The ica operon and icaR gene were analyzed in 14 S. caprae strains from human specimens or goats' milk. Some of the strains produced biofilm, and others did not. All strains carry the ica operon and icaR of the same sizes and in the same relative positions, suggesting that the absence of biofilm formation is not related to the insertion of a mobile element such as an insertion sequence or a transposon.

Characterization of a variant of vga(A) conferring resistance to streptogramin A and related compounds.

A variant of the vga(A) gene (1,575 bp), encoding an ATP-binding cassette protein conferring resistance to streptogramin A and related antibiotics, was cloned from the chromosome of a Staphylococcus aureus clinical isolate and sequenced. The sequence of the variant was similar to that of the vga(A) gene (83.2% identity). However, the G+C content of the variant (35.6%) was higher than that of vga(A) (29%) and there was no cross hybridization between vga(A) and the variant at high stringency (> or =60 degrees C), the highest temperature at which a signal was detected being 55 degrees C. Unlike previous reports for vga(A) and vga(B), the variant of vga(A) may be present in multiple copies in the genome. These copies are chromosomal in some isolates and both chromosomal and plasmid-borne in others. Nucleotide sequences hybridizing at 65 degrees C with the vga(A) variant were found in all the staphylococcal strains harboring plasmids carrying both vga(B) and vat(B), which also encode resistance to streptogramin A.

Screening for Staphylococcus epidermidis markers discriminating between skin-flora strains and those responsible for infections of joint prostheses.

Fifty-four Staphylococcus epidermidis strains responsible for infections of joint prostheses and 23 strains isolated from skin flora were studied for markers of virulence, to discriminate invasive strains from normal flora. They were screened for binding to polystyrene and matrix proteins and for the presence of staphylococcal genes involved in adhesion. The ica operon involved in biofilm formation was the only marker discriminating between these 2 categories of strains.

The value of rRNA gene restriction site polymorphism analysis for delineating taxa in the genus Staphylococcus.

A total of 101 staphylococcal strains were ribotyped using EcoRI and HindIII as restriction enzymes and plasmid pBA2 as the rDNA probe. Isolates from 10 newly described staphylococcal taxa were among those examined. All the ribotypes were added to our database, Staph DB, which now contains the sizes of the bands of 135 EcoRI and 120 HindIII ribotypes from 408 strains belonging to 42 staphylococcal taxa. The relatedness of ribotypes was evaluated by using the Dice coefficient. The ribotypes, and thus the strains, were clustered by the unweighted pair group method with averages (UPGMA). Separation into clusters correlated well with the delineation of the staphylococcal species but not with that of the different subspecies. No discrimination was possible between Staphylococcus vitulinus and Staphylococcus pulvereri. Ecovar-specific groups were evident within Staphylococcus intermedius and Staphylococcus hyicus. The data increase the usefulness of rRNA gene restriction site polymorphism analysis for staphylococcal taxonomy.

Phenotypic and molecular typing of nosocomial methicillin-resistant Staphylococcus aureus strains susceptible to gentamicin isolated in france from 1995 to 1997.

Methicillin-resistant strains susceptible to gentamicin (Gm(s) MRSA) have emerged since 1993 in several French hospitals. To study whether particular clones have spread in various French cities and whether some clones are related to gentamicin-resistant (Gm(r)) MRSA strains, various methods (antibiotyping, phage typing, determination of SmaI macrorestriction patterns before and after hybridization with IS256 transposase and aacA-aphD probes) were used to compare 62 Gm(s) MRSA strains isolated from 1995 to 1997 in nine cities and 15 Gm(r) MRSA strains. Eighteen major SmaI genotypes were identified, of which 11 included only Gm(s) MRSA strains and 5 included only Gm(r) MRSA strains. Each of the Gm(r) MRSA strains contained 6 to 13 SmaI fragments hybridizing with the insertion sequence IS256, of which a single band also hybridized with the aacA-aphD gene. No such hybridizing sequences were detected in 60 of the 62 Gm(s) MRSA strains. Thus, the divergence between Gm(r) and Gm(s) MRSA strains is revealed, not only by their distributions in distinct SmaI genotypes but also by the differences in hybridization patterns. Two of the 62 Gm(s) MRSA strains had the uncommon feature of carrying several SmaI bands hybridizing with IS256, suggesting that they are possibly related to the Gm(r) MRSA strains grouped in the same SmaI genotype. Five of the 11 SmaI genotypes including only Gm(s) MRSA strains contained strains from diverse cities, isolated during different years and with different antibiograms, suggesting that some clones have spread beyond their cities of origin and persisted.

satG, conferring resistance to streptogramin A, is widely distributed in Enterococcus faecium strains but not in staphylococci.

A gene almost identical to satG was isolated from an Enterococcus faecium strain. This gene was transferred to a Staphylococcus aureus recipient strain where it conferred resistance to streptogramin A. satG was found to be widely distributed among E. faecium strains but not detected among staphylococci.

Comparative analysis of staphylococcal plasmids carrying three streptogramin-resistance genes: vat-vgb-vga.

Several staphylococcal plasmids (26-45 kb) carry all three streptogramin-resistance (Sg(R)) genes, vat, vgb, and vga. Seven such plasmids harbored by independent strains belonging to three taxa (Staphylococcus aureus, S. simulans, and S. cohnii subsp. urealyticum) were compared and the deleted derivative of one of them, pIP680 (11.3 kb), carrying the three streptogramin-resistance genes was sequenced. The seven native plasmids had in common a 12.1-kb part cocarrying the three Sg(R) genes. Sequence analysis of pIP680 revealed that the simultaneous presence of these three genes has probably resulted from cointegration of two plasmids: (i) a pAMbeta1-like plasmid harboring vat-vgb and whose replication gene has been inactivated by an IS257 insertion and (ii) a functional vga plasmid whose replication is similar to that of two staphylococcal plasmids, pSX267 and pSK41.

Transposition of IS1181 in the genomes of Staphylococcus and Listeria.

The recombinant plasmid pIP1713 was constructed to analyse the transpositional activity of the insertion sequence IS1181 in Staphylococcus aureus RN4220, Staphylococcus carnosus TM300 and Listeria monocytogenes EGD. This 11.3-kb plasmid contains two genetically different elements: (i) a pE194ts-derived replicon, the ermC gene of which confers resistance to erythromycin in Gram-positive bacteria of several species, and (ii) a copy of IS1181, cloned from S. aureus BM3121, in which the tetracycline resistance gene, tet(T), has been inserted between the transposase-encoded gene and the downstream inverted repeat. When introduced by electroporation into the three bacterial hosts, pIP1713 delivered IS1181 omega tet(T) to various chromosomal sites. Cointegrate structures between pIP1713 and the host chromosome were occasionally detected. Transposition was associated with 8-bp repeats at the insertion sites. IS1181 omega tet(T) could be used for random mutagenesis in Gram-positive bacteria.

Phenotypic and genomic variation among Staphylococcus epidermidis strains infecting joint prostheses.

We studied the SmaI and SstII macrorestriction patterns of 54 Staphylococcus epidermidis strains isolated from 14 patients infected following the implantation of joint prostheses. Multiple strains from pus and infected tissue specimens of each patient were selected on the basis of different colony morphologies and drug resistance patterns. The same criteria were used to select 23 S. epidermidis strains from hand swabs of eight healthy individuals. For 10 of the 14 patients, all the intrapatient strains appeared to be closely or possibly related, whereas related strains were detected in the skin flora of only one of the eight healthy individuals. This observation suggests that, in most cases, the patients were infected by a single S. epidermidis clone which subsequently underwent rearrangements that yielded derivatives with divergent phenotypes and, occasionally, divergent macrorestriction patterns. The four patients whose specimens contained unrelated S. epidermidis strains were probably infected with several polyclonal strains.

Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics.

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation.

Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study.

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.

Staphylococcal resistance to streptogramins and related antibiotics.

Streptogramin and related antibiotics are mixtures of two compounds, A and B (e.g. Dalfopristin and Quinupristin), particularly against Gram-positive bacteria. Staphylococci resistant to these mixtures are always resistant to the A compounds but are not necessarily resistant to the B compounds. Resistance to A compounds and to the mixtures is conferred by acetyltransferases or ATP-binding proteins via unknown mechanisms. Several genes encoding each of the two categories of protein have been characterized and regularly detected on plasmids. Genes encoding lactonases, which inactivate B compounds, have been occasionally detected on these plasmids. Staphylococci which harbour plasmids conferring resistance to A compounds should not be treated with the mixtures even if they appear susceptible in vitro. Indeed, susceptibility to the mixtures of staphylococci carrying resistance to A compounds has often been attributed to partial loss of the plasmids conferring this resistance. When staphylococci are constitutively resistant to B compounds, the in vitro activities of the mixtures should be evaluated, because they are better correlated than MICs with their efficacy in therapy.

Multiplicity of the genes and plasmids conferring resistance to pristinamycin in staphylococci selected in an Algerian hospital.

In an Algerian hospital where pristinamycin (Pt) was extensively used for the treatment of chronic osteomyelitis and for prophylaxis in bone surgery, the prevalence of pristinamycin-resistant (PtR) staphylococci during a five-month period (20%) was higher than that among staphylococci isolated elsewhere in Algeria (4.5%). Analysis of 13 PtR staphylococci isolated in this hospital revealed a diversity of plasmids and genes conferring resistance to Pt and to related antibiotics. Most of the PtR staphylococci were unrelated: they belonged to either different taxa or types. Nevertheless, some of the unrelated staphylococci harboured structurally related plasmids carrying streptogramin resistance genes. Thus, these plasmids may have contributed to the dispersion of these genes.

Contribution of a typing method based on IS256 probing of SmaI-digested cellular DNA to discrimination of European phage type 77 methicillin-resistant Staphylococcus aureus strains.

The incidence of infections with phage type 77 methicillin-resistant Staphylococcus aureus (MRSA) strains increased in France in 1987. These strains are widespread in numerous European hospitals. The SmaI restriction profiles of total DNA extracted from 74 phage type 77 MRSA strains isolated from 1987 to 1994 in 10 hospitals in eight European cities (in France, Belgium, and Spain) were analyzed. Hybridization with a probe containing a 468-bp DNA fragment from within the transposase gene of the insertion sequence IS256 was also examined. Forty-three SmaI profiles were detected. Twenty major genotypes were identified, and each genotype contained strains with the same profile or profiles which differed by no more than three bands. Strains isolated in different countries and at several-year intervals were often grouped within the same genotype. A larger number of genotypes could be discriminated by analysis of the patterns of hybridization with the IS256 probe. SmaI restriction fragments with the same apparent electrophoretic mobility could, in some cases, be distinguished by the presence or the absence of nucleotide sequences hybridizing with IS256. The strains that grouped within the same genotype after hybridization with IS256 were mostly those isolated in the same hospital and at less than 12-month intervals. Consequently, the IS256 probe that we used improved restriction profile analysis for discrimination between the intrahospital, outbreak-related phage type 77 MRSA strains and the endemic strains disseminated in various cities and countries.

Mapping the regions carrying the three contiguous antibiotic resistance genes aadE, sat4, and aphA-3 in the genomes of staphylococci.

Tn5405 (12 kb) is a staphylococcal composite transposon delimited by two inverted copies of IS1182, one of which contains IS1181. The internal part of this transposon carries three antibiotic resistance genes, aphA-3, aadE, and sat4, and three open reading frames (ORFs), orfx, orfy, and orfz, of unknown function. The dispersion of Tn5405 and the genes and ORFs included in this transposon were investigated in 50 epidemiologically unrelated staphylococci carrying aphA-3. Twenty-three maps, distinguishable by the presence or absence of the investigated genes or ORFs and/or by the sizes of the restriction fragments carrying them, were identified. Four isolates carried Tn5405, and 15 other isolates contained a Tn5405-related element. IS1182 was not detected in the aphA-3 regions mapped in 31 isolates which carried the following combinations: orfx, orfy, aadE, sat4, and aphA-3 +/- orfz; orfy, aadE, sat4, and aphA-3 +/- orfz; and aadE, sat4, aphA-3, and orfz. In all isolates, the genes and ORFs investigated were in relative positions similar to those in Tn5405. Thus, the internal part of Tn5405 appeared to be partially conserved with the maintenance, in all of the isolates, of at least the three antibiotic resistance genes.

Nucleotide sequence of the Staphylococcus aureus transposon, Tn5405, carrying aminoglycosides resistance genes.

Tn5405 is a 12 kb staphylococcal composite transposon delimited by two inverted copies of the insertion sequence IS1182. This transposon carries two aminoglycosides resistance genes, aphA-3 and aadE, an altered gene similar to sat4 from Campylobacter coli BE/G4, and three open reading frames of unknown functions.

Characterization of a new staphylococcal gene, vgaB, encoding a putative ABC transporter conferring resistance to streptogramin A and related compounds.

The Staphylococcus aureus plasmid gene, vgaB, conferring resistance to streptogramin (SgA) and related compounds (PIIA, virginiamycin M, mikamycin A, synergistin A, Dalfopristin) was cloned and sequenced. This gene potentially encodes a 552-aa protein, VgaB, of 61,327 Da, which exhibits a significant similarity with the ATP-binding domains of numerous proteins. VgaB has two ATP-binding domains containing each of the A and the B motifs described by Walker et al. [Walker, J.E., Saraste, M., Runswick, M.J., Gay, N.J., 1982. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J., 1, 945-951], but does not include TM hydrophobic domains. The 155-amino-acid sequence between the two ATP-binding domains of VgaB is richer in Glu than the rest of the protein. The vgaB gene was found in 21 of the 52 SgA(R) and independent wt staphylococci investigated. In each of the 21 staphylococci, vgaB was carried on a plasmid of 50-90 kb also harboring the vatB gene encoding an acetyltransferase inactivating SgA. In all plasmids, vgaB and vatB have the same relative positions.

Distribution of genes encoding resistance to streptogramin A and related compounds among staphylococci resistant to these antibiotics.

The levels of resistance to pristinamycin (Pt) and to its major constituents, pristinamycin IIA and IB (PIIA and PIB, respectively; classified as streptogramins A and B, respectively) were determined for 126 staphylococcal isolates. The results suggest tentative susceptibility breakpoints of < or = 2, < or = 8, and < or = 0.5 microgram/ml for PIIA, PIB, and Pt, respectively. Fifty-six isolates that were inhibited by > or = 4 micrograms of PIIA per ml were investigated for the presence of staphylococcal genes encoding resistance to PIIA (vga, vat, and vatB) and PIB (vgb). None of these genes was found in the 4 isolates inhibited by 4 micrograms of PIIA per ml or in 4 of the other 52 isolates tested. The remaining 48 isolates harbored plasmids carrying vatB and vga or combinations of genes (vga-vat-vgb or vga-vat). The absence of any known PIIA resistance gene from the four Staphylococcus aureus isolates inhibited by > or = 8 micrograms of PIIA per ml suggests that there is at least one PIIA resistance mechanism in staphylococci that has not yet been characterized.

Characterization of a Staphylococcus aureus transposon, Tn5405, located within Tn5404 and carrying the aminoglycoside resistance genes, aphA-3 and aadE.

A new staphylococcal composite transposon, designated Tn5405, carrying the genes aphA-3 and aadE, which encode resistance to aminoglycosides, was partially characterized. The transposon is 12 kb long and is flanked by inverted repeated sequences displaying the characteristic features of an insertion sequence, named IS1182. This insertion sequence is 1864 bp long and has 23/33-bp imperfect inverted repeats at its ends. One of the IS1182 copies delimiting Tn5405 contains a copy of IS1181 flanked by 8-bp direct repeats. Tn5405 was found in the chromosome of MRSA clinical isolate BM3121, within a Tn552-related transposon, Tn5404. Tn5404 was previously characterized following its transposition onto a beta-lactamase plasmid harbored by BM3121. Two forms of the recombinant beta-lactamase-encoding plasmid generated by the inversion of Tn5405 within Tn5404 were detected. IS1182 was not detected in the DNA of 4 of the 17 tested MRSA isolates containing aphA-3 and resistant to streptomycin. Thus, aphA-3 and aadE genes are not disseminated only by Tn5405 or related transposons delimited by IS1182.