INTRODUCTION

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Streptogramins and related antibiotics are produced by streptomycetes and are classified as A and B compounds, according to their basic primary structures (13). These compounds bind different targets in the peptidyltransferase domain of the 50S ribosomal subunit and inhibit protein elongation at different steps. A compounds provoke a conformational modification of the bacterial ribosome at the B compound binding site and decrease the dissociation constant of B compounds (14). Thus, A and B compounds, which are bacteriostatic when used separately, act synergistically when combined and may become bactericidal, mainly against gram-positive bacteria. Natural mixtures, such as pristinamycin, synergistin, virginiamycin, and mikamycin, are used orally and topically. Quinupristin-dalfopristin (Synercid), consisting of an injectable semisynthetic mixture of derivatives of streptogramins B and A, respectively, is now available in hospitals for the treatment of infections due to gram-positive cocci resistant to other antibiotics.

Staphylococcal resistance to synergistic mixtures of A and B compounds (pristinamycin MICs of 2 mg · liter¹) is always associated with resistance to A compounds (pristinamycin IIA MICs of 8 mg · liter¹) but not necessarily with resistance to B compounds (1, 16). To date, seven genes encoding resistance to A compounds have been isolated from staphylococcal and enterococcal plasmids. Genes vat (8) [now named vat(A) (28)], vat(B) (2), vat(C) (5), sat(A) (27) [renamed vat(D) (28)], and sat(G) (20, 32) [renamed vat(E) (28)] encode related proteins (50.4 to 60.1% identical amino acids) conferring resistance to streptogramin A and similar compounds by acetylation of the drugs. The degenerate primers M and N can be used to detect any of these genes by PCR experiments (1). The staphylococcal vga gene (6) [renamed vga(A)] and vga(B) (3) encode related ATP-binding proteins (58.8% identical amino acids) probably involved in the active efflux of A compounds (16).

We report here the cloning, sequencing, and distribution of a variant of the staphylococcal gene vga(A), which encodes an ATP-binding protein conferring resistance to A compounds.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The relevant characteristics of the strains and plasmids used are reported in Table 1. Two collections of strains resistant to streptogramin A were screened for the presence of the variant of vga(A). One collection consisted of 52 staphylococci belonging to five species (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus cohnii subsp. urealyticum, and Staphylococcus simulans) (1) and included the clinical isolates described in Table 1 and the S. cohnii strain harboring pIP1714, which carries vat(C) and vgb(B) (5). The other collection consisted of 51 Enterococcus faecium strains isolated from fecal samples from poultry, pigs, farmers, and suburban residents in The Netherlands (20).

Media. Staphylococci were grown in brain heart infusion (Difco Laboratories, Detroit, Mich.), and Escherichia coli cells were grown in Luria broth. Susceptibility to antibiotics was tested on Mueller-Hinton agar (Diagnostics Pasteur, Marnes-la-Coquette, France).

Susceptibility to antimicrobial drugs. Susceptibility to antibiotics was determined by a disk diffusion assay (12) with commercially available antibiotic disks (Diagnostics Pasteur) and disks prepared in our laboratory as described previously (19). MICs of pristinamycin IIA and pristinamycin (Rhône-Poulenc Rorer, Vitry, France) were determined with serial 1:2 dilutions of antibiotics in Mueller-Hinton agar (17).

DNA isolation and analysis. Total cellular DNA was isolated from staphylococcal strains and was purified using the QIAamp tissue kit from Qiagen (Hilden, Germany). Plasmid DNA was extracted and purified from E. coli using the QIAprep spin plasmid kit from Qiagen. Restriction endonucleases were obtained from Amersham-Pharmacia Biotech Inc. (Piscataway, N.J.) and were used according to the manufacturer's instructions. DNA fragments of less than 500 bp were separated by electrophoresis in 4% NuSieve GTG agarose gels (FMC BioProducts, Rockland, Maine). SmaI digestion and pulsed-field gel electrophoresis were performed as described previously (15).

Cloning and DNA sequencing. Fragments amplified by PCR were cloned using the TOPO TA cloning kit (Invitrogen, Groningen, The Netherlands) following the manufacturer's instructions. DNA restriction fragments were inserted into E. coli vectors using the ligase of the Fast-Link ligation kit (Epicentre Technologies Corporation, Madison, Wis.), and the recombinant plasmids were introduced by transformation into competent E. coli XL2-Blue cells (Stratagene, La Jolla, Calif.) following the manufacturer's instructions.

Labeling of DNA probes, blotting, and hybridization. Plasmid DNA was labeled with [-³²]dCTP (110 TBq mmol¹) by random priming using the Megaprime DNA labeling system (Amersham).

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PCR. DNA was amplified by PCR using the Ready-To-Go kit (Amersham) according to the manufacturer's instructions in a Crocodile III apparatus (Appligène, Illkirch, France). The following primers were used: A, 5'-AAYTAYWCNAAYTAYRTNGARCARAARGA-3' [nucleotide (nt) 1386 to nt 1414 in vga(A), accession no. M90056]; B, 5'-NACRTTYTCNARNATNGAYTT-3' [nt 1967 to nt 1947 in vga(A), accession no. M90056]; C, 5'-CTTCAATTGGGATCCTCAGGATAGG-3' [nt 40 to nt 64 in the vga(A) variant, accession no. AF186237; BamHI site is underlined]; D, 5'-GTTATGGTACCTTCTTGTTAGG-3' [nt 1866 to nt 1845 in vga(A) variant, accession no. AF186237; BamHI site is underlined]; E, 5'-CTCTTTGTACGAGTATATGG-3' [nt 612 to nt 631 in vga(A) variant, accession no. AF186237]; and F, 5'-GTTTCTTAGTAGCTCGTTGAGC-3' [nt 809 to nt 788 in vga(A) variant, accession no. AF186237]. PCR experiments with primers A and B were carried out at low stringency (initial cycle of 5 min at 95°C followed by 35 cycles of 30 s at 40°C, 30 s at 72°C, and 30 s at 95°C with a final extension step of 4 min at 40°C and 10 min at 72°C), and those with primers C and D, and E and F, were carried out at high stringency (initial cycle of 5 min at 95°C and 2 min at 55°C followed by 35 cycles of 1 min at 72°C, 30 s at 95°C, and 1 min at 55°C and a final extension step of 5 min at 72°C).

Nucleotide sequence accession number. The nucleotide sequences of the vga(A) variant gene and the flanking regions (19 nt upstream and 305 nt downstream) have been submitted to GenBank under accession no. AF186237.

	RESULTS AND DISCUSSION

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Filter mating experiments. Each of the four S. aureus isolates (BM3249, BM3250, BM3252, and BM3327 [Table 1]) resistant to pristinamycin IIA (MICs of 64 to 128 mg · liter¹) and to related antibiotics was crossed on a membrane filter with the S. aureus recipient strain ISP1127 (26). No transcipients were obtained by selection on pristinamycin IIA (frequency < 10¹⁰ transcipients/donor CFU).

Hybridization experiments with SgA^r gene probes at various temperatures. The total cellular DNA of the four S. aureus isolates cited above does not hybridize at high stringency (65°C) with the vat(A), vat(B), vat(C), vat(D), vat(E), vga(A), or vga(B) probes (1, 3). We repeated this experiment at 42°C, the lowest temperature at which no nonspecific signals were observed with the chromosomal DNA of the S. aureus strain RN4220 (22), susceptible to all antibiotics. At 42°C, a single 0.57-kb HindIII fragment hybridizing with the vga(A) probe was detected in the DNA of each of the four S. aureus clinical isolates (results not shown). A hybridizing HindIII fragment of the same size was detected in the DNA of the S. aureus clinical isolate, BM3385, harboring pIP1156 (60 kb), which carries vga(B) and vat(B), which are contiguous and are located in a 7-kb HindIII fragment. In the S. aureus transductant, BM3093, harboring pIP680 (4), the vga(A) probe hybridized at 42°C only with a HindIII fragment of 5.6 kb carrying vga(A), confirming the absence of nonspecific hybridization at 42°C.

PCR experiments with degenerate primers, A and B, which encode conserved motifs in vga(A) and vga(B). Conserved motifs in the peptide sequences deduced from vga(A) and vga(B) were chosen outside the regions containing the Walker motifs A and B, as they are widespread in ATP-binding cassette proteins. Primers A (coding strand) and B (complementary strand) (Materials and Methods) were expected to amplify 582- and 579-bp DNA fragments from within the vga(A) and vga(B) genes, respectively.

Hybridization experiments using pIP1799 as a probe. Cellular DNA extracted from BM3249, BM3250, BM3252, BM3327, BM3093, and BM3385 was cleaved with HindIII and probed with pIP1799 at high stringency (65°C). Nucleotide sequences hybridizing with the probe were detected in all the strains, except BM3093 (results not shown). Each hybridization pattern contained two or four HindIII fragments, two of which (0.57 and 1.3 kb) were common to all the patterns; additional hybridizing fragments were detected in the patterns of BM3249 and BM3250 (3 kb), BM3252 (1.1 and 3 kb), and BM3385 (1.1 kb). These results suggest that the 580-bp insert of pIP1799, which hybridized with neither vga(A) nor vga(B) at high stringency, did not originate from either of these genes.

Cloning and sequencing of the putative new gene carried by the cellular DNA of BM3327. BM3327 was chosen because it carried only the two HindIII fragments of 0.57 and 1.3 kb hybridizing with pIP1799 and common to the hybridization patterns of all five S. aureus clinical isolates tested. Each of the two fragments was inserted separately into the HindIII site of pUC18 and sequenced. Each HindIII insert contained part of the amplicon from pIP1799; thus, they were contiguous. An open reading frame of 1,418 nt including the 0.57-kb HindIII insert (575 nt) and 843 nt of the 1.3-kb HindIII insert was identified. The region of the genome upstream from the 0.57-kb HindIII fragment was sequenced to obtain the part of the putative gene encoding the N terminus of the putative protein.

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View larger version (6K): [in this window] [in a new window]   FIG. 1.   Restriction map of the chromosomal region of BM3327 carrying the vga(A) variant (accession no. AF186237). The primers A, B, C, D, E, and F, indicated by arrows, are described in Materials and Methods, and the sizes of the amplicons are indicated below the map.

Analysis of the drug resistance pattern conferred by the vga(A) variant gene. An 1,827-bp DNA fragment including the vga(A) variant was amplified with primers C and D (Fig. 1) from the cellular DNA of BM3327 and inserted into the shuttle vector pRB374 (11). The resulting plasmid, pIP1810, was introduced by electroporation into the S. aureus recipient RN4220; it conferred resistance to pristinamycin IIA (MICs of 32 mg · liter¹), whereas RN4220 harboring the vector pRB374 was inhibited by 2 mg of pristinamycin IIA liter¹. Pristinamycin MICs were the same (0.06 mg · liter¹) for the recipient strain RN4220 and for transformants harboring pIP1810 or pRB374. The four S. aureus clinical isolates carrying the vga(A) variant only, which were inhibited by 1 mg of pristinamycin liter¹ (Table 1), may be considered intermediately resistant to pristinamycin, because, for pristinamycin-resistant S. aureus strains, the MICs are 2 mg · liter¹ (1). None of the antibiotic resistance markers carried by these wild-type clinical isolates was conferred by pIP1810.

Distribution and location of the vga(A) variant gene among two collections of isolates resistant to A compounds. The isolates were screened for the presence of the vga(A) variant by hybridization at 65°C with pIP1799. Sequences hybridizing with pIP1799 were found in the 20 S. aureus isolates carrying vga(B) and vat(B) (1) including BM3318 and BM3385 (Table 1); in one S. epidermidis isolate carrying vga(A) only (1); and in the four S. aureus isolates BM3249, BM3250, BM3252, and BM3327 (Table 1). The hybridizing sequences comigrated with the chromosomal DNA fragment in all isolates, and in 15 isolates an additional signal was detected in extrachromosomal DNA bands (40 kb) (results not shown).

View larger version (47K): [in this window] [in a new window]   FIG. 2.   Pulsed-field gel electrophoresis of SmaI-digested total DNA from clinical S. aureus strains resistant to streptogramin A. (A) SmaI macrorestriction patterns; (B) hybridization patterns with the vga(A) probe (pIP1799) at high stringency (65°C, 5× SSPE buffer). Lanes 1, 8, and 9, bacteriophage lambda DNA concatemers (Bio-Rad); lanes 2, BM3385; lanes 3, BM3250; lanes 4, BM3252; lanes 5, BM3249; lanes 6, BM3327; lanes 7, NCTC8325 used as standard; lanes 10, BM3318.