1 Centre de Recherche en Infectiologie de l'Université Laval, CHUQ, Pavillon CHUL, 2705 boul. Laurier, Sainte-Foy, Québec, G1V 4G2; 2 Division de Microbiologie, Faculté de Médecine, Université Laval, Sainte-Foy, Québec; 3 Infectio Diagnostic (I.D.I.) Inc., 2050 boul. René Levesque O., 4e étage, Sainte-Foy, Québec, G1V 2K8, Canada
Received 10 November 2004; returned 5 December 2004; revised 17 December 2004; accepted 22 December 2004
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Abstract |
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Methods: Strain CCRI-9842 was characterized by 16S rDNA sequencing and susceptibility testing. PCR mapping and sequencing of the vanB-containing element, as well as plasmid extraction and mating experiments, were carried out to investigate the genetic basis of vancomycin resistance in this strain.
Results: Strain CCRI-9842 was identified as a Clostridium species closely related to Clostridium bolteae (96.8% 16S rDNA identity). This strain was resistant to a high level of vancomycin (MIC of 256 mg/L), but was susceptible to teicoplanin and ampicillin. The complete sequence of the CCRI-9842 vanB gene exhibited 99.1% identity with that of vanB2. PCR mapping and sequencing showed that the genetic element carrying vanB2 was similar to transposon Tn5382/Tn1549. This Tn5382-like transposon forms circular intermediates and is flanked on the left and right ends by repeat sequences of at least 700 bp in the opposite direction. No plasmid was detected in this strain, suggesting that the Tn5382-like transposon was integrated into the chromosome. The vancomycin resistance was not transferable to enterococci.
Conclusions: Our report shows for the first time the presence of a Tn5382-like transposon carrying vanB2 in a Clostridium species of the human intestinal flora. This suggests that the vanB2 Tn5382-like transposon is an important vector for the spread of vancomycin resistance in several bacterial species.
Keywords: vancomycin resistance , anaerobes , intestinal flora
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Introduction |
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The origin of vanB genes is still unknown and, in addition to enterococci, the glycopeptide-resistance vanB genes have also been described in a Streptococcus mitis strain isolated from blood,19 in a Streptococcus bovis isolate,20 in an Eggerthella lenta-related strain and in three unidentified Clostridium species,21 all of which were obtained from human faecal samples. The vanB gene has also been identified in three faecal veal calf isolates, two Streptococcus gallolyticus and one Streptococcus lutetiensis.22,23 These observations suggest that the intestinal flora may serve as a reservoir for glycopeptide resistance genes, which seems to represent a favourable environment for horizontal gene transfer between different genera, as described for other antibiotic resistance genes.24,25
A surveillance programme of VRE carriers at the Montreal General Hospital (Montréal, Québec, Canada), using PCR detection of vanA and vanB genes from faecal samples, was undertaken in 2001. During this surveillance programme, several vanB-positive faecal samples were detected in the absence of culturable VRE. One sample was shown to contain a Clostridium isolate carrying the vanB gene. In this study, we report the genetic characterization of the vanB-containing genetic element from this Clostridium species.
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Materials and methods |
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The origin and characteristics of the strains used in this study are described in Table 1. Using PCR detection of vanA and vanB genes, the anaerobic strain CCRI-9842 containing the vanB gene was isolated in 2001 from a faecal specimen obtained during hospital surveillance of patients suspected of VRE colonization at the Montreal General Hospital (Montréal, Québec, Canada). Identification of the strain CCRI-9842 was performed by methods previously described26
and with the rapid ID32A test kit (bioMérieux, Marcy l'Étoile, France). Partial sequencing of the gene encoding 16S rRNA was performed with primers SSU27 (AGAGTTTGATCMTGGCTCAG) and SSU1492 (TACGGYTACCTTGTTACGACTT).27
The strains used as positive controls for mapping and sequencing the CCRI-9842 vanB2 gene cluster were E. faecium C68 (vanB2-containing transposon Tn5382) (kindly provided by L. B. Rice, Louis Stokes Cleveland VA Medical Center, Cleveland, OH, USA) and E. faecalis 268-10 (vanB2-containing transposon Tn1549) (kindly provided by N. Woodford, Health Protection Agency, Colindale, London, UK). E. faecalis 268-10, which contains plasmid pIP834 (8090 kb), was also used as a control to verify the efficiency of the method for isolation of large plasmids. E. faecium CCRI-1453 (resistant to gentamicin) and E. faecalis JH2-2 (resistant to fusidic acid and rifampicin)28
were obtained from L. Jetté (Laboratoire de Santé Publique du Québec, Sainte-Anne de Bellevue, Québec, Canada) and from N. Woodford, respectively. Both strains were used as recipients in conjugation experiments. Escherichia coli TOP10 (Invitrogen, Burlington, Ontario, Canada) was used as a host for recombinant plasmids. PCR products were cloned in the vector pCR2.1 using the TOPO TA Cloning kit (Invitrogen, Burlington, Ontario, Canada).
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Brain heart infusion (BHI) broth (Difco, Detroit, MI, USA) supplemented with vitamin K (0.001 mg/mL), haemin (0.005 mg/mL), and L-cystine (0.5 mg/mL) and named enriched BHI (eBHI) was used for cultures. MICs were determined by the Etest method (AB Biodisk, Sweden) using blood agar supplemented with vitamin K (0.001 mg/mL), haemin (0.005 mg/mL) and L-cystine (0.5 mg/mL), which was named enriched blood agar (eBA).
Mapping and sequencing of the vanB gene cluster
The PCR primers used in this study to map and sequence the vanB gene cluster from Clostridium sp. CCRI-9842 are listed in Table 2. PCR reactions were performed with standard conditions using purified genomic DNA and the Taq DNA polymerase (Promega, Mississauga, Ontario, Canada), as previously described.29 Genomic DNA was purified with the Gnome kit (Qbiogene Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. Purification of the amplification products and sequencing reactions were performed as previously described.29 Comparison with known sequences was carried out using the programs from the GCG package (version 10.3; Accelrys, San Diego, CA, USA).
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The DNA sequence flanking the Tn5382-like transposon from Clostridium sp. CCRI-9842 located upstream from orf13 and downstream from the int gene was determined by using the DNA Walking SpeedUp kit (Bio/Can Scientific, Mississauga, Canada). PCRs were carried out using orf13-specific primers (P527, P476 and P317) and int-specific primers (P33228 and P33386) (Table 2) in combination with universal primers provided by the manufacturer. The PCR conditions recommended by the manufacturer were modified by using 2.5 mM MgCl2 in the PCR mixture and by increasing the length of the extension step to 180 s. Both strands of the PCR products obtained from the left and right ends of the transposon were sequenced as previously described.29
Circular form of the vanB-containing genetic element
To find out if the vanB-containing genetic element produces a circular form, PCR was performed by using purified genomic DNA as a template and primers P476 and P33386 directed outward of orf13 and the int gene of transposon Tn1549,18 respectively (Table 2). PCR products were cloned into vector pCR2.1 using the TOPO TA Cloning kit following the manufacturer's instructions. Plasmid DNA extraction from 15 clones was performed using the QIAGEN Plasmid Mini kit (QIAGEN, Mississauga, Ontario). The inserts from these recombinant plasmids were sequenced using the M13 forward and M13 reverse primers.
Filter mating
To study the transfer of the vanB2 operon, mating on filters was performed as described previously.30 E. faecalis JH2-2 and E. faecium CCRI-1453 recipient cells, as well as Clostridium sp. CCRI-9842 donor cells, were grown on eBA plates. Colonies from an 18 h old pure culture were grown in eBHI for recipient cells and eBHI supplemented with vancomycin (64 mg/L) for donor cells Donor cells were grown overnight at 37°C under anaerobic conditions (SHELLAB Bactron Anaerobic/Environmental chamber; Sheldon Manufacturing Inc., Cornelius, OR, USA), whereas both strains of recipient cells were grown for 4 h under aerobic conditions. An aliquot of 100 µL of each culture was washed with 1 mL of eBHI. Cultures of donor and recipient were mixed in 50 µL of eBHI and spread onto nitrocellulose filters (0.45 µm pore-size) placed onto the surface of a blood agar plate. The bacterial counts were 1 x 109 cfu/mL for the donor CCRI-9842, 3 x 107 cfu/mL for the recipient JH2-2 and 6 x 106 cfu/mL for the recipient CCRI-1453. The filter-containing blood agar plates were incubated for 18 h at 37°C under anaerobic conditions. After the mating period, the filters were removed from the agar plates, placed in 15 mL bottles containing 1 mL of eBHI broth and vortexed for 1020 s. An aliquot of 250 µL was then plated onto selective blood agar plates containing vancomycin (20 mg/L), rifampicin (20 mg/L) and fusidic acid (10 mg/L) for mating experiments with JH2-2 and vancomycin (16 mg/L) alone for mating experiments with CCRI-1453. The plates were incubated for 5 days at 37°C under aerobic conditions and examined each day for appearance of colonies.
Plasmid extraction from Clostridium sp. CCRI-9842
To find out if the Tn5382-like transposon of the Clostridium sp. CCRI-9842 strain was located on a plasmid, the modified plasmid extraction alkaline lysis method previously reported was used.31
Nucleotide sequence accession number
The GenBank accession numbers of the vanB2 cluster sequence of Clostridium sp. CCRI-9842 and the left and right ends of the Tn5382-like transposon are AY772783, AY772782 and AY772784, respectively.
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Results |
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The anaerobic bacterial strain CCRI-9842, containing the vanB gene that was isolated from a human faecal specimen, was identified as Clostridium sp. by phenotypic identification methods. Analysis of the partial sequence of its 16S rDNA revealed that this Clostridium strain was most similar (98.8% identity) to an uncultured bacterium HuCC34 of human colonic flora (GenBank accession number AJ315486),32 to an uncultured Clostridium strain N6 (97.3% identity) from human faeces (GenBank accession number AJ582080), to an uncultured Clostridium strain DR6A (96.7% identity) from the rumen of red deer (GenBank accession number Y10028) and to Clostridium bolteae (96.8% identity) isolated from human faeces (GenBank accession number AJ508452).33 Clostridium sp. CCRI-9842 was resistant to vancomycin (MIC of 256 mg/L) and susceptible to teicoplanin (MIC of 1.5 mg/L) and ampicillin (MIC of 0.38 mg/L).
Characterization of the vanB operon from Clostridium sp. CCRI-9842
The vanB gene cluster from Clostridium sp. CCRI-9842 was characterized to determine its genetic organization. Mapping with primers specific to both the vanB1 operon of E. faecalis V58310 and the vanB2 operon of E. faecalis 268-1018 showed that the order of the vanB gene cluster from Clostridium sp. CCRI-9842 was similar to those of known vanB gene clusters (Figure 1). The complete DNA sequence of the Clostridium vanB operon (from vanRB to vanXB, 6431 bp) exhibited 95.2% identity with that of vanB1 and 98.9% identity with that of vanB2. The nucleotide and deduced amino acid sequences of each gene of the vanB operon from Clostridium sp. CCRI-9842 were compared with those of the vanB1 and vanB2 operons (Table 3). The deduced amino acid sequences of VanRB, VanSB, VanYB, VanWB, VanHB, VanB and VanXB exhibited the highest identities (96%100%) with those of the vanB2 operon.
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To characterize the genetic element carrying the vanB operon in CCRI-9842, the presence of the two conjugative elements Tn1547 and Tn5382/Tn1549, which usually carry vanB gene clusters, was studied. No PCR product was obtained when the primers used to amplify the region upstream from vanRB or downstream from vanXB were specific to the corresponding regions of the vanB1 gene cluster from E. faecalis V583.10 Also, no PCR product was generated with primers specific to the IS16 and IS256-like insertion sequences from E. faecalis BM4281 containing Tn1547.16 PCR amplification using several primers specific to the Tn1549 transposon targeting the region starting from the left inverted repeat (IRL) to vanRB, as well as the region located downstream of vanXB and extending to the int gene, showed that the genetic element carrying vanB in CCRI-9842 was similar to Tn1549 and contained homologous orfs (Figure 1). Analysis of the complete DNA sequences of orf13, orf14 and orf15 and the partial sequences of orf16 and orf30 from CCRI-9842 located at the left of the vanB2 gene cluster showed that they each exhibited 100% identities with their counterparts in Tn1549. The complete DNA sequences of orf7, orf8 and int and the partial sequence of the xis gene from the CCRI-9842 transposon located at the right of the vanB2 gene cluster exhibited 99%, 99.1%, 99.8% and 100% identities with their counterparts in Tn1549, respectively.
Location of the vanB-containing genetic element
To determine the chromosomal or plasmid location of the vanB-containing genetic element, plasmid extraction was performed from Clostridium sp. CCRI-9842. No plasmid DNA could be detected from this strain, whereas plasmid extraction was efficient for the control strain 268-10 carrying the large pIP834 plasmid (data not shown).
The right and left ends and flanking regions of the Tn5382-like transposon
By using a DNA PCR walking method, the left and right ends as well as the DNA flanking the Tn5382-like transposon from Clostridium sp. CCRI-9842 were amplified and sequenced. By using primers targeting the orf13 in combination with universal primers, an 1400 bp amplification productcorresponding to the left extremity of the Tn5382-like transposon and flanking regionwas produced. The 1024 bp sequence of this PCR product suitable for DNA analysis showed that the first 32 nucleotidesstarting from the orf13 primercorrespond to the 3' end of orf13. The next 251 nucleotides located upstream from orf13 exhibited 100% identity with the left ends of Tn1549. The left end of the Tn5382-like transposon from CCRI-9842 comprised an (11 bp) imperfect IR sequence, which is 100% identical to that of Tn5382/Tn1549 (Figure 2). The DNA sequence flanking the right end of the Tn5382-like transposon from CCRI-9842 was also determined using primers specific to the int gene in combination with universal primers. Sequence analysis of 1091 nucleotides of the
1400 bp amplification productcorresponding to the right extremity of the Tn5382-like transposon and flanking regionshowed that the first 176 bp, starting from the int primer, corresponded to the 5' end of int. The next 174 bp located downstream of int exhibited 100% identity with the right ends of Tn5382/Tn1549. The right end of the Tn5382-like transposon from CCRI-9842 comprised an (11 bp) imperfect IR sequence, which is 100% identical with that of Tn5382/Tn1549 (Figure 2).
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To verify if the Tn5382-like transposon from Clostridium sp. CCRI-9842 could form circular intermediates, PCR amplification using primers specific to orf13 and to the int gene and directed outward from the ends of the Tn5382-like transposon was performed. The 900 bp PCR product obtained was cloned and sequenced. Sequence analysis of 15 different recombinant plasmids revealed PCR product inserts that exhibited 100% nucleotide sequence identity among the 15 recombinants, except for the joint region. The two termini of the Tn5382-like transposon were found in an orientation consistent with the formation of a circular molecule for the 15 recombinants (Figure 4). The two ends of the transposon were separated by: (i) a 5-nucleotide GCTAT sequence in seven recombinants; (ii) a 5-nucleotide ATAGC sequence in one recombinant; and (iii) a 6-nucleotide CCGGGG sequence in seven recombinants (Figure 4). These sequences represent the point at which the two ends of the transposon are joined. The joint sequences of the distinct circular forms of the Tn5382-like transposon from Clostridium sp. CCRI-9842 are different from those of transposon Tn5382 from E. faecium C6817
(data not shown).
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We performed mating experiments between Clostridium sp. CCRI-9842 and strains E. faecalis JH2-2 and E. faecium CCRI-1453 to determine whether the Tn5382-like transposon identified in Clostridium sp. CCRI-9842 was conjugative. No transfer to either enterococcal strain was observed in several mating experiments.
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Discussion |
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Genetic analysis revealed that strain CCRI-9842 carries a vanB gene cluster closely related to the vanB2 gene cluster of enterococci. Mapping and sequencing of the region upstream and downstream from vanRB and vanXB, respectively, showed that the CCRI-9842 vanB2 gene cluster was carried by a genetic element similar to transposons Tn5382 and Tn1549,17,18 and hence was designated Tn5382-like.
The Tn5382-like transposon from strain CCRI-9842 seems to be integrated into the chromosome since no plasmid was detected in this strain. Chromosomal location of transposon Tn5382 harbouring the vanB2 gene cluster was described in E. faecium C68,17
whereas the similar transposon Tn1549 (Tn5382-like) also harbouring the vanB2 gene cluster was found on plasmid pIP834 in E. faecalis 268-10.18
The Tn5382-like transposon of CCRI-9842 is flanked by 741-nucleotide sequences, which are identical and orientated in opposite directions. These flanking sequences differ from that of E. faecium C68 and E. faecalis 268-10.17,18
Indeed, in E. faecium C68, transposon Tn5382 is integrated into the bacterial chromosome in the region downstream of the pbp5 gene, whereas the similar transposon Tn1549 is integrated into two different sites of the pAD1 plasmid in E. faecalis 268-10 and 654-6.18
This may be explained by the ability of Tn5382 to insert into different sites in different hosts as well as in different sites in the same host.17,18,23
The DNA G + C content of the identical sequences flanking the Tn5382-like transposon was higher (56 mol%) than those of the clostridia (30 mol%) and enterococci (
40 mol%) genomes, suggesting another origin for these sequences. These identical sequences could represent insertion sequences (IS) present at both ends of this transposon. Indeed, IS sequences were previously described within the vanB2 gene cluster as well as within the Tn5382 transposon of several enterococcal isolates.35,36
However, the sequences flanking both ends of the Tn5382-like transposon did not show any significant homology with any known IS sequence or transposase. Such non-coding regions surrounding the Clostridium perfringens enterotoxin gene (cpe) were previously described in several C. perfringens strains,37,38
but the role of these non-coding regions remains unclear.
Circular intermediates harbouring three different joint sequencesrepresented by nucleotides ATAGC, GCTAT or CCGGGGwere found in the Tn5382-like transposon from Clostridium sp. CCRI-9842. These circular intermediates were detected in a purified genomic DNA preparation, which had been stored at 4°C, indicating that these intermediates were stable. Variable nucleotide sequences connecting the two ends of circular intermediates were previously reported in different strains for transposons Tn916, Tn5381 and Tn5382.39,40 The 5 or 6 nucleotide joint sequences found in the circular intermediates of the Tn5382-like transposon from CCRI-9842 differ from those found in Tn5382 from E. faecium C68 represented by either TTTGT or TTTGTA.17 In Tn5382 from E. faecium C68, as well as in the Tn5381 and Tn916 transposons, the joint nucleotide sequences of the circular intermediates are found at the left or right end of the target junction sequence of these transposons.17,39,40 However, the joint nucleotide sequences of the circular intermediates of the CCRI-9842 transposon were not found at the left or right end of the transposon's target junction. The meaning of this observation is not clear, but it suggests a more complex mechanism of transposon excision and integration in CCRI-9842, as compared with previously described conjugative transposons.4143 Nevertheless, these results provided evidence that the products of the transposon-encoded int and xis genes had retained the ability to excise the Tn5382-like transposon as a circular molecule and are therefore functional in this Clostridium strain. The formation of circular and stable forms of the Tn5382-like transposon has important implications in the possible movement and transfer of the vanB2 gene cluster among human bowel bacteria by any gene transfer mechanism. In the related conjugative transposons Tn916 and Tn5382, circular forms were shown to be the transposition intermediates.17,39,40 However, transfer of the vanB2 gene cluster from Clostridium sp. CCRI-9842 to E. faecalis JH2-2 and E. faecium CCRI-1453 recipients was not detected. Several reasons can explain the failure of vancomycin resistance transfer in these experiments. It is possible that the E. faecalis and E. faecium strains are inefficient recipients in mating with the genus Clostridium, or that the conditions of mating were not appropriate. It may also be explained by the absence of natural systems for transfer or expression in Clostridium. It is also possible that the transfer functions of the Tn5382-like transposon are not expressed in Clostridium or that a mechanism other than conjugative transposition was responsible for the resistance transfer. Another possibility is that the transfer of the Tn5382-like transposon from CCRI-9842 falls below the limit of detection under the experimental conditions used as reported for S. gallolyticus.23
The vanB2 gene cluster is predominant in enterococci, especially in E. faecium, and is responsible for high-level vancomycin resistance in hospital settings.35,4447 Non-enterococcal bacteria containing vanB2 genes isolated from human and veal calves faecal specimens have been described. These reports identified S. bovis, S. gallolyticus, S. lutetiensis, E. lenta-related strain and Clostridium species as vanB2 gene carriers.20,21,23 For some of these bacterial species, the genetic element carrying vanB2 has been studied and shown to be related to Tn5382.23,48 Although the presence of vanB2 genes in Clostridium species has been documented previously,21 our report is the first descriptive study of a conjugative Tn5382-like transposon in the genus Clostridium. The Clostridium strain described in this study belongs to the phylogenetic cluster XIVa of Clostridium. Clostridium species belonging to this cluster have been shown to be one of the major constituents of human faecal flora based on 16S rDNA sequence analysis.32,49,50 On this basis, it may not be surprising to find a Clostridium species belonging to this cluster carrying a vanB2 Tn5382-like transposon in the human faecal flora. The high density of Clostridium species in the human colon would most likely favour horizontal vancomycin gene transfer events. Tn5382-like transposons have been shown to undergo conjugal transfer among enterococci and streptococci.17,18,23,35 The results of this study suggest that Tn5382, which is a member of the Tn916 class of conjugative transposon, has the potential to transfer to many different Gram-positive bacterial species of the bowel flora, which may serve as a reservoir of vancomycin-resistance genes. This has important clinical implications, because conjugative transposons are one of the major vectors involved in the spread of antibiotic resistance among bacterial pathogens.42,43
In conclusion, we have shown that a Clostridium strain of the human faecal flora carries a chromosomally integrated Tn5382-like transposon containing the vanB2 gene cluster. This transposon produces circular intermediate molecules, which were not transferable to E. faecalis and E. faecium. The failure of vancomycin resistance transfer from Clostridium sp. CCRI-9842 to enterococci suggests that the level of transposition in natural systems would be low.
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Acknowledgements |
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