INTRODUCTION

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Most gram-positive bacteria are naturally susceptible to glycopeptide antibiotics (vancomycin and teicoplanin). Resistance to these compounds is an intrinsic property of some occasional human pathogens such as lactobacilli, leuconostoc, pediococci, Erysipelothrix rhusiopathiae, Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens (22). Acquisition of glycopeptide resistance was first described in enterococci in 1988 (9, 20) and is mediated by three classes of related gene clusters that confer inducible resistance to high levels of vancomycin and teicoplanin (vanA), inducible resistance to various levels of vancomycin (vanB), or resistance to vancomycin and low levels of teicoplanin (vanD) (1, 4, 12, 14, 21).

VanA enterococci harbor a Tn3-like transposon (the prototype was designated Tn1546) which carries a cluster of seven genes (vanR, vanS, vanH, vanA, vanX, vanY, and vanZ) required for the phenotypic expression of resistance and which is generally located on large plasmids (1). The vanB cluster is carried by large conjugative elements and less frequently by plasmids (17). Transfer of genetic elements carrying vanA and vanB genes to susceptible enterococcal strains and, in the case of vanA, to strains of nonenterococcal species such as Streptococcus sanguis, Streptococcus lactis, Streptococcus pyogenes, Bacillus thuringiensis, Staphylococcus aureus, and listeriae of different species has been achieved under laboratory conditions (2, 3, 8, 10, 13). However, the natural spread of vancomycin resistance determinants among gram-positive bacteria other than enterococci has seldom been reported. To date, this phenomenon has been documented by the isolation from clinical specimens of one vancomycin-resistant strain of Oerskovia turbata and one strain of Arcanobacterium (Corynebacterium) haemolyticum, both of which harbor the vanA gene, and of one Streptococcus bovis strain that harbors the vanB gene (15, 16).

Recently, we described a glycopeptide-resistant strain of Bacillus circulans (a species naturally susceptible to these antibiotics) isolated from a catheter tip as part of routine activities at the Clinical Microbiology Laboratory of the Verona University Hospital (6). Since the natural dissemination of vancomycin resistance determinants among gram-positive bacteria is a matter of serious public health concern, we studied the mechanism of vancomycin resistance in our clinical isolate of B. circulans. We found that this strain harbored a vanA-like gene cluster with a high degree of homology with that of enterococci but was borne by the chromosome and was apparently carried by a genetic element other than Tn1546.

	MATERIALS AND METHODS

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Bacterial strains. B. circulans VR0709 is a motile, catalase-positive, aerobic, spore-forming, gram-positive rod and was identified by the API CH and API 20E System biochemical profiles (bioMèrieux, Marcy l'Etoile, France). B. circulans ATCC 4513 was obtained from the American Type Culture Collection, Rockville, Md. Enterococcus faecium BM4147 carrying Tn1546 in plasmid pIP816 was used as the reference strain. Escherichia coli DH5 was purchased from Stratagene (Cambridge, United Kingdom). Bacteria were routinely grown in brain heart infusion (BHI) broth and agar (Difco, Detroit, Mich.) at 37°C.

Susceptibility test. The MICs of vancomycin and teicoplanin were determined on Mueller-Hinton broth (MHB) or Mueller-Hinton agar (MHA) (Difco) by standard dilution techniques (11).

Inducibility of vancomycin resistance. Cultures of strain VR0709 were grown overnight in MHB at 37°C in the presence of subinhibitory concentrations of vancomycin (8 µg/ml) and were diluted to an A₆₄₀ of 0.1 in fresh MHB with and without the same concentrations of vancomycin. The cultures were grown at 37°C with shaking, and A₆₄₀ values were measured at regular intervals on a Pharmacia spectrophotometer.

Primers and digoxigenin-labeled probes. Primers A1 and A2 (vanA), B1 and B2 (vanB), C1 and C2 (vanC1), and D1 and D2 (vanC2, vanC3), selected by Dutka-Malen et al. (5), were used to specifically amplify the van genes. The primer sets used for amplification of ORFs 1 and 2 and the van genes of Tn1546 were as described previously (7). When necessary, primers were also derived from the B. circulans DNA by sequencing. The primers were synthesized by Boehringer Mannheim, Mannheim, Germany. To be used as hybridization probes, the PCR products were labeled directly with digoxigenin (DIG)-dUTP by using a PCR DIG-labeling mix (Boehringer Mannheim). The reaction was run in a 100-µl volume under the PCR conditions described below.

Preparation of DNA. Plasmid DNA was prepared by a rapid alkaline lysis method (18). For preparation of total DNA, 3 ml of bacterial culture in BHI broth (37°C, 18 to 48 h) was centrifuged (14,000 × g, 10 min). After washing with 0.85% sterile NaCl, the bacteria were suspended in 400 µl of sucrose solution (6.7%), 25 µl of lysozyme (10 mg/ml) was added, and the mixture was incubated for 30 min at 37°C. Fifty microliters of 20% sodium dodecyl sulfate (SDS) was then added, and the mixture was incubated for 30 min and digested with 5 µl of proteinase K solution (20 mg/ml) at 37°C for 30 min. Genomic DNA was extracted twice with 1 volume of phenol-chloroform-isoamyl alcohol. After adding 0.1 volume of 3 M sodium acetate, DNA was precipitated in ethanol, air dried, and dissolved in 80 µl of sterile water.

PCR amplification. PCR was performed on a DNA thermal cycler (MiniCycler; GENENCO, Florence, Italy) in a final volume of 50 µl containing 250 ng of DNA as template, 0.5 µM (each) primer, 200 µM (each) deoxynucleoside triphosphate, and 2 U of Taq high-fidelity DNA polymerase (Boehringer Mannheim) in a 1× amplification buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl₂). The PCR mixtures were denatured (2 min at 94°C) and were then subjected to 35 cycles of amplification (1 min of annealing at 55°C, 1 min of elongation at 72°C, and 1 min of denaturation at 94°C) and a final elongation step of 72°C for 10 min. PCR products were resolved by electrophoresis in a 1% agarose gel stained with ethidium bromide.

DNA-DNA hybridization. Genomic and plasmid DNAs were digested with restriction enzymes as recommended by the manufacturer (Boehringer Mannheim) and were electrophoresed through a 0.8% agarose gel. The DNA restriction fragments were electroblotted onto a Hybond-N⁺ nylon membrane (Amersham International, Amersham, United Kingdom) and were hybridized with the DIG-dUTP-labeled PCR products. Hybridizations were performed as follows: prehybridization and hybridization were carried out for 1 and 18 h, respectively, at 68°C in 5× SSC (1× SSC is 0.15 M sodium chloride plus 0.015 M sodium citrate)-0.02% SDS-1% (wt/vol) blocking reagent for nucleic acid hybridization (Boehringer Mannheim)-0.1% N-lauryl-sarcosine, followed by two washings in 0.2× SSC-0.1% SDS at 65°C for 15 min.

DNA cloning and sequencing. The sequence from vanR to vanY of the B. circulans chromosome was determined on cloned PCR fragments obtained by amplification of B. circulans DNA with primers derived from the sequence of Tn1546 or based on the sequence obtained from fragments of B. circulans DNA. The region upstream from vanR was sequenced by cloning and sequencing first the 1.5-kb HindIII fragment containing vanR (Fig. 1) and then the fragment produced by inverse PCR carried out with a primer (3' to 5') based on the 5' sequence of the 1.5-kb HindIII fragment and a primer (5' to 3') based on the 3' end sequence of the 7.6-kb EcoRI fragment (Fig. 1). The sequence downstream from vanZ of B. circulans was determined by cloning and sequencing the 1.7-kb HindIII fragment containing vanZ. The amplification products were blunt ended and phosphorylated by standard procedures (18) and were then ligated into SmaI-digested, dephosphorylated SK plasmid. Fragments derived from enzymatic digestion were cloned into pUC18. M13 universal and reverse primers were used to sequence both strands of the inserts. Sequencing was performed with three independent PCR clones by the dideoxynucleotide termination method (19) with an ALF Sequencer and fluorescent dATP (Pharmacia, St. Albans, United Kingdom).

View larger version (8K): [in this window] [in a new window]   FIG. 1.   Restriction map of the 9.2-kb EcoRI-HindIII fragment of B. circulans VR0709 containing the vanAbc cluster. EI, EcoRI; EV, EcoRV; HI, HindIII; P, PstI; X, XbaI.

Nucleotide sequence accession numbers. The nucleotide sequences of the genes of the B. circulans vanA (vanA_bc) cluster have been assigned EMBL accession no. Y15704 (vanA_bc), Y15705 (vanH_bc), Y15706 (vanR_bc), Y15707 (vanS_bc), Y15708 (vanX_bc), Y17303 (vanY_bc), Y17304 (vanZ_bc), and Y17305 (ORF).

	RESULTS AND DISCUSSION

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B. circulans VR0709 properties. B. circulans VR0709 is a motile, catalase-positive, aerobic, spore-forming, gram-positive rod identified as B. circulans by conventional biochemical methods. On BHI agar or MHA it formed ovoid colonies which swarmed after only a few days of incubation. Morphologically, the isolate showed the characteristic shape of aerobic bacilli. It formed rods (0.8 to 1 µm in width and 2 to 5 µm in length) that mostly contained a central or subterminal spore. Interestingly, the spore-forming ability was greatly reduced for bacteria grown in the presence of vancomycin at sub-MICs.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Growth of B. circulans VR0709 in MHB in the absence of vancomycin (diamonds) and in the presence of 8 µg of vancomycin per ml after pregrowth in MHB with (black circles) and without (black triangles) the antibiotic. OD640, optical density at 640 nm.

Identification and localization of vancomycin resistance determinant of B. circulans VR0709. The presence of the van genes was investigated in VR0709 by PCR carried out with primers from the vanA, vanB, and vanC genes of enterococci (5). A positive reaction was obtained with vanA primers only, which gave the expected 800-bp amplification product. No amplification of B. circulans ATCC 4513 DNA was obtained with the same sets of primers.

Characterization of the van gene cluster of B. circulans VR0709. The presence of other genes of the vanA cluster in strain VR0709 was investigated by PCR performed with primers from ORFs 1 and 2 and from all the seven genes of the enterococcal cluster. If amplification products of the expected size were not obtained, the presence of a gene was investigated by probing the total DNA with DIG-labeled amplicons of the corresponding enterococcal gene. By using this strategy the presence of sequences homologous to all seven genes of the cluster but not to ORFs 1 and 2 was demonstrated.

View larger version (75K): [in this window] [in a new window]   FIG. 3.   Nucleotide sequence of the regions flanking the vanA gene cluster contained in the 9.2-kb EcoRI-HindIII fragment of B. circulans DNA. The left (IRL) and right (IRR) terminal IRs of the hypothetical transposon-like element containing the vanAbc cluster are boxed. The deduced amino acid sequence of the ORF is shown below the nucleotide sequence. Ribosome-binding sites (RBS) and translation initiation codons are underlined. The IRs in the ORF-vanRbc intergenic region and at the left extremity of the fragment are indicated by arrows.