Primary structure and biological features of a thermostable nuclease isolated from Staphylococcus hyicus.

The nucH gene, encoding a thermostable nuclease (TNase), was isolated from the cellular DNA of Staphylococcus hyicus strain E80 and sequenced. NucH, the 169-amino-acid (aa) protein encoded by this gene, contains, at its N-terminus, a signal peptide which appears to be cleaved at the same site in S. hyicus and Escherichia coli, yielding a mature protein which is exported extracellularly from S. hyicus, but not from E. coli. The aa sequence of NucH is highly homologous with that of the TNase from S. intermedius strain LRA076, whereas significant similarities are observed with the TNase from S. aureus, as well as with three other bacterial proteins of which only one has been shown to exhibit DNase activity. As seen in a multiple sequence alignment, the invariant residues are mostly located in the regions involved in the biological activity of the S. aureus TNase. The ability of crude cell extracts of E. coli strains carrying nucH to degrade various forms of nucleic acids with or without Ca2+ supplementation was studied. Under our experimental conditions, the enzyme encoded by nucH was active at 37 degrees C on both DNA and RNA, had the potential to act as an endonuclease, and functioned in the presence of Ca2+. Moreover, activity was retained after heating at 100 degrees C, suggesting that the enzyme could undergo reversible unfolding.

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.

Sequence of a staphylococcal gene, vat, encoding an acetyltransferase inactivating the A-type compounds of virginiamycin-like antibiotics.

The Staphylococcus aureus plasmids, pIP680 and pIP1156, which confer resistance to A-type compounds of virginiamycin-like antibiotics (Vml: streptogramin A, pristinamycin IIA, virginiamycin M) and to synergistic mixtures of the A and B compounds of Vml antibiotics, were shown to direct the modification of A-type compounds by acetylation. The vat gene, encoding the acetyltransferase modifying A-type compounds, was isolated from plasmid pIP680 and sequenced. This gene potentially encodes a 219-amino-acid (aa) protein, VAT, of 24 330 Da showing at least 38% aa identity with two chloramphenicol acetyltransferases encoded by cat genes isolated from Escherichia coli and Agrobacterium tumefaciens. Resistance to A-type compounds of Vml antibiotics conferred to S. aureus by vat was not expressed in E. coli, although a protein having a M(r) similar to that encoded by this gene was detected in E. coli minicells. The vat gene was detected by the polymerase chain reaction in two chromosomally located staphylococcal conjugative elements and in the conjugative plasmid, pIP1156, conferring resistance to A-type compounds.

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.

An epidemiological assessment of coagulase-negative staphylococci from an intensive care unit.

Detection of an unusual combination of four resistance markers among coagulase-negative staphylococci (CNS) isolated in the same intensive care unit led to the undertaking of an epidemiological assessment. Seventeen CNS isolates from the same unit and 38 epidemiologically unrelated Staphylococcus epidermidis isolates were typed by eight methods, including analysis of immunoblot patterns and hybridisation patterns (HP) obtained with three probes. The probes comprised plasmids carrying the genes encoding 16S rRNA (pBA2), aacA-aphD (pSF815A), and aacA-aphD with part of IS256 (pIP1307). Immunoblot patterns and HP with pIP1307 indicated that 14 of the 17 CNS isolates from the same unit resulted from the spread of an epidemic strain.

Epidemiological study of Staphylococcus aureus resistance to new quinolones in a university hospital.

During a 14-month period, from December 1984 to February 1986, 630 Staphylococcus aureus isolates were identified at Broussais Hospital. Thirty-eight isolates (6%), from 35 patients, were found to be pefloxacin-resistant S. aureus (PRSA) with minimal inhibitory concentrations greater than or equal to 8 mg l-1. PRSA isolates were tested for susceptibility to 35 antibiotics, including nine quinolones, and heavy metal ions. Phage-type was determined. Out of the 38 PRSA isolates, 35 (92%) were methicillin- and multiply-resistant; however, all PRSA isolates were sensitive to vancomycin and coumermycin. Fifteen isolates (39%) had similar phage-type and identical antibiotic susceptibility pattern with high level resistance to pefloxacin (MICs equal to 64 mg l-1); they were isolated from the same surgical unit. The 23 remaining PRSA isolates differed by their phage and susceptibility patterns. Pefloxacin MICs ranged from 8 to 512 mg l-1 with a bimodal distribution; cross-resistance was observed with the eight other quinolones tested. Only nine PRSA isolates (24%), including four 'epidemic' isolates, were obtained from patients who had been treated with quinolones. From these data there is apparently no direct relationship between quinolone administration and selection of PRSA in infected patients.

Diversity among the gram-positive acetyltransferases inactivating streptogramin A and structurally related compounds and characterization of a new staphylococcal determinant, vatB.

A gene encoding an acetyltransferase inactivating streptogramin A (SgA) and structurally similar compounds was isolated from a staphylococcal plasmid and sequenced. This gene, designated vatB, potentially encodes a 212-amino-acid protein, VatB, of 23,320 Da with 47.4 and 58.4% amino acid identities with two other enzymes with the same activity, Vat and SatA, respectively, which are encoded by a staphylococcal plasmid and an enterococcal plasmid, respectively. The C-terminal parts of these three enzymes share significant homology with the C-terminal parts of 10 other acetyltransferases modifying various substrates. A pair of degenerate primers representing the conserved motifs shared by VatB, Vat, and SatA was designed to detect the three genes encoding these SgA acetyltransferases. Five of 12 clinical SgAr Staphylococcus aureus isolates tested carried neither these genes nor the gene vga, which confers resistance to SgA by a different mechanism, suggesting that another gene(s) and possibly another mechanism of resistance to SgA in staphylococci remains to be characterized.

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.

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.

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.

Rearrangements in the staphylococcal beta-lactamase-encoding plasmid, pIP1066, including a DNA inversion that generates two alternative transposons.

The plasmid plP1066, harboured by by a methicillin-resistant Staphylococcus aureus strain isolated in France, carries genes specifying beta-lactamase. This plasmid undergoes numerous rearrangements. One of these was insertion, between the genes binR and sin encoding resolvases, of a 16 kb element which displayed the characteristic features of a transposon. This putative transposon, named Tn5404, carried genes encoding proteins involved in its transposition, as well as a resolution system, which were indistinguishable from those of the S. aureus transposon Tn552. These were: p480 encoding a probable transposase, p271 encoding a putative ATP-binding protein, binL encoding a resolvase, and a resolution site, resL. In addition, Tn5404 carried aminoglycoside-resistance genes (aphA, str) and the insertion sequence IS1181. Tn5404 contained at its termini 116 bp imperfect inverted repeats, similar to those of Tn552, and was flanked by 6 bp direct repeats. Insertion of Tn5404 close to resR and to the structural and regulatory beta-lactamase genes (blaZ, blal, blaR1) of pIP1066, generated a 3.5 kb invertible segment flanked by inversely repeated resolution sites (resR, resL). This invertible segment, which carried p480, p271 and binL, generated in Tn552 or Tn5404, depending on its orientation. Thus, these two transposons share their transposition and resolution systems.

Isolation and characterization of IS1181, an insertion sequence from Staphylococcus aureus.

The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181. It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches. The three copies of IS1181 isolated from distinct parts of the chromosome of S. aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence. IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis. IS1181 was detected in at least two to eight copies in 41 of the 52 S. aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.

Thermonuclease gene as a target nucleotide sequence for specific recognition of Staphylococcus aureus.

DNA fragments, 450 bp in length, were amplified by polymerase chain reaction (PCR) from the thermonuclease gene (nuc) carried by seven epidemiologically independent Staphylococcus aureus isolates. Sequencing of the PCR products led us to characterize 210 bp strictly conserved. A 186 bp piece from within this conserved region was cloned into pUC18. The resulting recombinant plasmid, pIP1608, was used as a probe against the cellular DNA of 360 staphylococcal isolates belonging to 28 species. Only the 146 S. aureus isolates, including four which were not thermonuclease producers, had DNA that hybridized with pIP1608. Among the 214 non-S. aureus staphylococci, 55 exhibited a thermonuclease activity. For 32 of these, the enzymatic activity was inhibited by a commercially available polyclonal antiserum directed against the thermonuclease of an S. aureus strain. These results are in favour of the use of pIP1608 as a probe to specifically recognize S. aureus. Furthermore, we propose a method based on colony blot hybridization and potentially useful to enumerate S. aureus cells in biological samples.

Multiple copies of IS256 in staphylococci.

EcoRI-digested cellular DNAs of 150 staphylococcal clinical isolates were probed with a plasmid containing a DNA piece from within the open reading frame of IS256. Most of the Gmr staphylococcal isolates tested contained more copies of IS256 than those associated with Tn4001 carried by these isolates. Three distinct copies of IS256 together with their flanking DNA were isolated by cloning from an EcoRI digest of the cellular DNA of the Gmr isolate, BM3121. These three copies of IS256 were shown to be intact. The results from DNA sequencing revealed that one of the three copies is flanked by 8-bp direct repeats, and it is suggested that this may be the result of insertion of the IS256 at this site by autonomous movement of the IS element. The insert DNA of all three clones hybridized to the cellular DNA prepared from a Staphylococcus aureus strain that carries neither IS256 nor the aacA-aphD gene. Two of the clones hybridized to several EcoRI fragments of the cellular DNA of this strain, indicating that in these cases IS256 is flanked by DNA present as several copies on the chromosome.

Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes.

Most of the aminoglycoside resistant Staphylococcus aureus strains isolated in France are resistant to all the antibiotics belonging to this family. Two aminoglycoside-modifying enzymes were detected in the wild-type strains studied: an APH3'III and an AAC6'-APH2". These strains also carry two types of streptomycin resistance: high-level resistance due to chromosomal mutation(s) affecting ribosome affinity and low-level resistance, the mechanism of which was not characterized. All the aminoglycoside resistance genes were located on the chromosome. DNA fragments of 1.5 and 1.95 kb carrying the aphA and aacA genes, respectively, were isolated, by cloning, from the cellular DNA of a clinical isolate. When these genes were introduced into Escherichia coli and Bacillus subtilis strains, the enzymes synthesized were indistinguishable from those produced by the S. aureus strains. When the cellular DNAs of wild-type and resistant strains were hybridized with the cloned fragments, sequences homologous to the fragment carrying the aphA gene were found to be located at the same chromosomal site, while those hybridizing with the fragment carrying the aacA gene were at different chromosomal sites.

Nucleotide sequence of a staphylococcal plasmid gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin-like antibiotics.

The nucleotide sequence of a 1883 bp fragment isolated from a resistance plasmid harbored by a Staphylococcus aureus clinical isolate and carrying the gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin family has been determined. The sequence contains one open reading frame which extends from the ATG codon at nt 641 to a TGA codon at nt 1537 and which potentially codes for a protein of 33.035 Da. This value is in agreement with the apparent size (33 kDa) of the protein observed, in minicell extracts. Inactivation of the B components of the virginiamycin antibiotics as well as resistance to these antibiotics were expressed in a virginiamycin sensitive mutant of Escherichia coli recipient containing the gene on a high copy number plasmid.

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.

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.

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.

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.