Objectives: Four Enterococcus gallinarum isolates, all highly resistant to vancomycin, were studied in order to investigate their relationship and to gain insight into the molecular events responsible for their acquired resistance.
Methods: Extensive molecular analysis was performed to compare the four E. gallinarum isolates and their Tn1546-like elements.
Results: The four strains had very similar random amplified polymorphic DNA (RAPD) patterns and different but related PFGE profiles. Genotypic analysis demonstrated that all carried both vanC-1 and vanA genes. Using a vanA probe, no hybridization was detected to plasmid DNA, whereas hybridization to different SmaI fragments of the four strains was obtained with total DNA. Amplification and sequencing experiments showed that all four strains carried a Tn1546-like element that contained the orf2, vanR, vanS, vanH, vanA and vanX genes and was flanked at both ends by oppositely oriented IS1216V sequences. On the left side of the vanA cluster, all lacked IRL, and all had, upstream from orf2, 1029 bp of the 3′ end of orf1. On the right side, one of the strains lacked vanY, vanZ and IRR, whereas in one of the other three there was an IS1542 element inserted within the vanZ gene. In one strain, an additional IS1216V element was inserted in the intergenic region vanX–vanY.
Conclusions: This is the first study providing a molecular analysis of chromosomal Tn1546-like elements (possibly composite transposons) associated with high-level vancomycin resistance in human and animal strains of E. gallinarum. These molecular findings, together with those from PFGE and RAPD, suggest that the four E. gallinarum isolates are related and might have a common ancestor.
Received 19 May 2003; returned 16 June 2003; revised 9 August 2003; accepted 15 August 2003
Among vancomycin-resistant enterococci, the motile species Enterococcus gallinarum and Enterococcus casseliflavus/flavescens, only sporadically recovered from clinical specimens, represent the VanC phenotype, which expresses intrinsic, species-specific, low-level resistance to vancomycin.1 The VanA phenotype, which expresses inducible, high-level vancomycin resistance, is mostly detected in Enterococcus faecium and Enterococcus faecalis, i.e. the species most frequently isolated from humans.1 The vanA cluster, carried by Tn1546-like elements, is usually formed by seven van genes—two (vanR and vanS) with regulatory functions, three (vanH, vanA and vanX) essential for expression of resistance and two (vanY and vanZ) controlling accessory functions—located immediately downstream from orf1 and orf2, two genes associated with transposition functions.1 However, considerable heterogeneity may exist among these elements, largely resulting from the presence of insertion sequences (ISs) or from deletions in non-essential genes and intergenic regions. In the vanC cluster, at least two species-specific vanC genes have been described: vanC-1 in E. gallinarum and vanC-2/3 in E. casseliflavus/flavescens.1
We have recently described a clinical strain of E. gallinarum expressing the VanA phenotype and carrying both vanC-1 and vanA genes, the latter harboured by a Tn1546-related element located on the chromosome and deleted at both ends (i.e. lacking orf1, vanY and vanZ).2 In this study, the same strain was analysed further, together with three other isolates of E. gallinarum highly resistant to vancomycin, in order to assess their relationship and gain insight into the molecular events responsible for acquired resistance.
Materials and methods
Bacterial strains and susceptibility tests
Four E. gallinarum isolates, with glycopeptide susceptibilities inconsistent with the species-specific VanC phenotype, were isolated in different areas of Italy during 1996–1999: three (EP9, E14 and E32) from clinical specimens (blood cultures) and one (EA65) from food (raw poultry meat). The isolation and preliminary characterization of strain E32 has been reported elsewhere.2 Six unrelated strains of the same species regularly exhibiting the VanC phenotype (four isolated from food and two from human intestinal carriers in different areas of Italy) were used as controls. Vancomycin and teicoplanin MICs were determined using the Etest method. E. faecium BM4147 was used as the vanA prototype strain.
Plasmid analysis, PFGE and Southern hybridization
Plasmid analysis was performed as described elsewhere.2 Genomic DNA for PFGE was prepared following standard procedures. The chromosomal fragments obtained after restriction with SmaI (Roche Diagnostics, Milan, Italy) were separated using a CHEF Mapper system (Bio-Rad Laboratories, Milan, Italy). PFGE profiles were analysed with Diversity Database, version 2 for Macintosh (Bio-Rad). Comparison of the banding patterns was performed by the unweighted pair group method with arithmetic averages, on the basis of the Dice coefficient. Following PFGE, the SmaI-restricted DNA was transferred onto nylon membranes and hybridized with a vanA probe as described elsewhere.2 The S1 nuclease (Ambion-Celbio, Milan, Italy)–PFGE method was used to detect possible large plasmids, as described previously.3
Oligonucleotide primers and amplification experiments
Primers targeting Tn1546 sequences were as reported by Palepou et al.4 [inverted repeats (IRs)] and Biavasco et al.2 (orf1, orf2, vanA, vanY and vanZ). Primers targeting IS1216V were as described by Jensen.5 DNA amplification and electrophoresis of PCR products were carried out as described elsewhere.2 Random amplified polymorphic DNA (RAPD) analysis was performed by a standard procedure. Long PCR of Tn1546-like elements was as described by Palepou et al.4 using IR, ORF2D/VANZ4 and ORF2D/VANX2 primers and the Ex Taq system (TaKaRa Bio, Shiga, Japan).
Inverse PCR and DNA sequence analysis
Five primers (INV1, INV2, INV3, INV4 and VANZ5) were designed from the published sequence of Tn1546 (GenBank accession no. M97297, base positions 3077–3053, 3174–3195, 8578–8596, 8427–8448 and 10781–10800, respectively). Total DNA was restricted with ClaI or Csp6I (Roche) to analyse the regions upstream from orf1 and downstream from vanX, respectively, and circularized fragments were amplified using the primer pairs INV1/INV2 and INV3/INV4. With the same primers, and others designed on the basis of the first sequences obtained, amplicon sequencing was performed bidirectionally using ABI Prism (Perkin-Elmer Italia, Monza, Italy) with dye-labelled terminators, and sequences were analysed using the sequence navigator software package (Perkin-Elmer).
Conjugal transfer was performed on membrane filters. The four vanA-carrying E. gallinarum test strains were used as donors. E. faecalis JH2-2 was used as the recipient.
Phenotype and genotype of glycopeptide resistance
Vancomycin MICs for all four E. gallinarum test strains were ≥128 mg/L, whereas teicoplanin MICs ranged from borderline susceptible or intermediate (8 and 16 mg/L for strains E32 and EA65, respectively) to highly resistant (≥128 mg/L for strains EP9 and E14).
In all four strains, positive PCR reactions and PCR products of the appropriate sizes were yielded by primer pairs targeting the vanC-1 and the vanA gene sequences.
RAPD and PFGE typing
RAPD profiles of the four E. gallinarum carrying vanA were very similar, but clearly different from those yielded by the VanC control strains.
Following PFGE experiments (Figure 1a), analysis of banding profiles revealed two distinct clusters of isolates, one included the four vanA-carrying strains and the second, which had fewer similarities than the former, included the six controls (Figure 1c).
Plasmid analysis and vanA location
Plasmids of <10 kb were detected in all four vanA-carrying strains: one in strain EP9, two in strains E32 and EA65 (identical profile), and six in strain E14. No hybridization to plasmid DNA extracts was detected by Southern analysis with a vanA probe. S1 nuclease–PFGE assays ruled out that vanA was carried by large plasmids migrating with the chromosomal DNA. Using total DNA, the vanA probe hybridized to different SmaI fragments in the four strains (Figure 1b). The molecular mass of the hybridization bands was in the range 23–95 kb.
Analysis of Tn1546-like elements and sequencing of flanking regions
The Tn1546-like element of strain E32, previously reported to contain orf2, vanR, vanS, vanH, vanA and vanX and to lack orf1, vanY and vanZ,2 was further investigated by inverse PCR to analyse the sequences upstream from orf2 and downstream from vanX. This procedure led to the detection, upstream from orf2, of the intergenic region orf1–orf2 and of 1029 bp corresponding to the 3′ end of the orf1 gene; this truncated orf1 was preceded by a sequence corresponding to an IS1216V element. Downstream from vanX, we found 382 bp of the intergenic region vanX–vanY, followed by another IS1216V element oriented in the opposite direction.
In order to compare the three other E. gallinarum test strains with strain E32, the four isolates were subjected to amplification experiments using primers targeting the IRs, Tn1546 genes and the IS1216V element in different combinations. No amplification was observed with the primer targeting the IRs, whereas amplification products were obtained with primer pairs ORF1C–1/IS1216V1, targeting orf1 and IS1216V, and vanZ2/IS1216V1, targeting vanZ and IS1216V. These results showed the presence of two oppositely oriented IS1216V sequences flanking the ends of the Tn1546-like elements of E. gallinarum strains EP9, E14 and EA65, as demonstrated by inverse PCR in E. gallinarum E32. Moreover, the three strains showed PCR products consistent with the presence of the same 1029 bp 3′ end of orf1 as detected in E32 and of the orf2, vanR, vanS, vanH, vanA, vanX and vanY genes. Using the primer pair VANZ1/VANZ4, PCR products corresponding to those obtained with vanA prototype strain BM4147 were obtained with strains EP9 and E14, whereas a ∼1300 bp larger amplicon was obtained with strain EA65. The amplicon sequence, determined using the same primers as employed for the amplification, showed the insertion of an IS1542 element 98 bp downstream from the starting codon of vanZ. Using the primer pair INV3/VANY1, targeting vanX and vanY, an amplicon ∼800 bp larger than the one obtained with the Tn1546 prototype, due to the insertion of an IS1216V in the intergenic region vanX–vanY, was detected in the Tn1546-like element carried by strain EP9. To analyse better the right end of the Tn1546-like elements carried by strains EP9, E14 and EA65, the amplicons of ∼2050 bp obtained with the primer pair VANZ2/IS1216V1 were sequenced using primers VANZ5 and IS1216V1. In these three strains, an intergenic region vanZ–IRR and an IRR sequence corresponding to those of the Tn1546 prototype, followed by a 620 bp sequence still to be determined, were detected before the IS1216V element.
These results are summarized in the model shown in Figure 2.
In conjugation experiments, transconjugants were only detected starting from strain EP9 (transfer frequency, 6 × 10–9 per recipient cell).
With this study, a molecular analysis of the Tn1546-like elements associated with high-level vancomycin resistance in human and animal strains of E. gallinarum is provided for the first time. The four highly vancomycin-resistant E. gallinarum isolates investigated in this study all carried a chromosomally located Tn1546-like element flanked at both ends by oppositely oriented IS1216V sequences. In all isolates, the left IS1216V element was followed by a truncated orf1 gene. A certain degree of polymorphism, with the presence of ISs and deletions, was detected in the region downstream from the vanX gene. These findings, together with the data from PFGE and RAPD typing assays, suggest that the four E. gallinarum isolates are related and might have a common ancestor. Time and geographic distance may justify some variation in the Tn1546-like elements. The heterogeneity of the fragments hybridizing to the vanA probe in the four isolates could be due to intrachromosomal mobility of the Tn1546-like element, possibly facilitated by the presence of multiple copies of the IS1216V sequence in the genome of the species E. gallinarum (A. Pantosti and F. Biavasco, unpublished results). These sequences are also found in association with Tn1546-like elements in E. faecium,4,6 thus suggesting a possible role for them in the integration of mobile vancomycin resistance elements from E. faecium into the E. gallinarum genome.
Deletions on the left side of Tn1546 are a relatively common feature of VanA enterococci from both human and non-human sources.4,7,8 The occurrence of ISs, including IS1216V and IS1542, in the intergenic regions or inside transposition or accessory genes has been extensively documented in E. faecium.4–8 However, the presence in our strains of two IS1216V sequences with opposite orientation near the two putative termini of the Tn1546-like sequence suggests that we are dealing with a new element, possibly a composite transposon. Moreover, mating experiments suggest that the vanA carried by E. gallinarum strains could contribute to further dissemination of the resistance to other enterococci, possibly in relation to the specific integration site of the Tn1546-like element in the genome.
The apparent relationship of the four E. gallinarum strains studied, isolated from human and animal food sources in different areas of Italy, appears to be consistent with the clonal spread of a vancomycin-resistant strain. The species E. gallinarum has been reported to colonize more often the gastrointestinal tract of humans9 than of animals:10 thus, although food-to-human transmission cannot be excluded, the presence of a common source as well as a human origin of environmental contamination should be seriously considered.
We wish to thank Esther Manso and Pietro Martino for providing some clinical strains of E. gallinarum and Alfredo Caprioli for helpful discussion. This work was supported in part by grants from the European Union (contract no. QLK2-CT-2002–00843) and the Italian Ministry of Health (RF 1999 and RC IZSVE 005/00).
Corresponding author. Tel: +39-071-2204697; Fax: +39-071-2204693; E-mail: email@example.com
1Department of Microbiology and Biomedical Sciences, Università Politecnica delle Marche, Via Ranieri, Monte d’Ago, 60131 Ancona; 2Laboratory of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, 00161 Rome, Italy