J Med Microbiol 55 (2006), 1237-1243; DOI: 10.1099/jmm.0.46560-0
© 2006 Society for General Microbiology
ISSN 1473-5644
Detection of clonally related vanB2-containing Enterococcus faecium strains in two Spanish hospitals
Carmen Torres1,
Susanna Escobar2,
Aránzazu Portillo1,3,
Luis Torres2,
Antonio Rezusta2,
Fernanda Ruiz-Larrea1,
Maria José Revillo2,
Carmen Aspiroz4 and
Myriam Zarazaga1
1 Área Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, Logroño 26006, Spain
2 Hospital Universitario Miguel Servet, Zaragoza, Spain
3 Área de Enfermedades Infecciosas, Hospital de La Rioja, Logroño, Spain
4 Hospital Royo Villanova, Zaragoza, Spain
Correspondence
Carmen Torres
carmen.torres{at}daa.unirioja.es
Received 8 February 2006
Accepted 17 May 2006
The aim of this study was to characterize the resistance mechanism in four clinical and five intestinal vancomycin-resistant Enterococcus faecium strains with VanB phenotype recovered from unrelated patients confined in two Spanish hospitals and to determine their clonal relationships. MIC values for vancomycin and teicoplanin were 16–32 and 0.5 µg ml–1, respectively. The mechanism of vancomycin resistance, as well as the genetic environment of the implicated gene, was analysed by PCR and sequencing. The vanB2 gene was detected in all nine E. faecium strains and the intergenic vanSB–YB region showed the characteristic mutations of the vanB2 subtype. Two possibly related PFGE patterns, A (seven strains) and B (two strains), were distinguished among these enterococci. The vanXB–ORFC intergenic region was amplified in the nine strains and two amino acid changes were detected in the protein encoded by the vanXB gene in strains of pattern A with respect to those of pattern B. The vanB2 gene cluster was integrated into Tn5382 in all nine strains, being pbp5 gene-linked to this transposon. The ant(6')-Ia, aph(3')-IIIa and erm(B) genes were also detected in all of the strains. Both isolates with PFGE pattern B contained the esp gene. In summary, vanB2-containing E. faecium strains with indistinguishable PFGE patterns were recovered from seven patients from two Spanish hospitals.
Abbreviations: ICU, intensive care unit; VREF-VBP, vancomycin-resistant Enterococcus faecium strains with VanB phenotype.
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INTRODUCTION
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Different mechanisms of vancomycin resistance have been detected in enterococci (vanA, vanB, vanC, vanD, vanE and vanG) (Cetinkaya et al., 2000; Gholizadeh & Courvalin, 2000; Murray, 1997; Perichon et al., 1997; Woodford, 2001). The vanB genotype is characterized by acquired inducible resistance to various levels of vancomycin and susceptibility to teicoplanin (Quintiliani et al., 1993). It is determined by a cluster of genes, vanRB, vanSB, vanYB, vanW, vanHB, vanB and vanXB. Three different subtypes of the vanB gene have been documented, vanB1, vanB2 and vanB3, based on sequence variability in the vanB ligase gene (Dahl et al., 1999). The vanB2 subtype has been found in human enterococcal strains from different countries (Dahl et al., 1999, 2000; Gold et al., 1993; Granlund et al., 2006; Kawalec et al., 2001; Lee et al., 2001; Lu et al., 2001, 2005; Mato et al., 1996; McGregor & Young, 2000; McGregor et al., 2001; Simonsen et al., 1998). In Spain, there has been one previous report on the detection of a vanB2-containing Enterococcus faecium clinical strain (Lorenzo-Díaz et al., 2004) and one on the detection of a vanB2-containing Enterococcus hirae strain from a faecal sample of a healthy pig (Torres et al., 2003). Recently, the vanB2 gene cluster associated with a Tn5382-like transposon has been found in a Clostridium strain from human faeces in Canada (Domingo et al., 2005).
Over a 1 year period, a series of clinical and intestinal vancomycin-resistant E. faecium strains with VanB phenotype (VREF-VBP) were recovered from two Spanish hospitals. The aim of this study was to characterize the resistance mechanisms of these strains and to determine their clonal relationships.
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METHODS
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Strains and patients.
Three VREF-VBP clinical strains (E. faecium C393, C395 and C461) were recovered from blood and urine samples from three patients from the Miguel Servet Hospital in Zaragoza, Spain (Table 1
). MIC values of vancomycin and teicoplanin for these strains (16 and 0.5 µg ml–1, respectively) were indicative of the VanB-resistant phenotype. They were detected among a series of 1036 clinical enterococcal isolates recovered over a 1 year period (February 2002 to January 2003) in that hospital (0.3 %). An additional VREF-VBP clinical strain (E. faecium C396) was recovered in March 2002 from the abdominal abscess of a patient from Royo Villanova Hospital, located 4 km away from Miguel Servet Hospital. Relationships among the patients from the two hospitals were not known.
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Table 1. Characteristics of the VREF strains included in this study and of the patients from whom they were recovered
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In order to detect intestinal colonization by VREF-VBP strains, a surveillance programme was carried out with all faecal samples (n=632) collected in Miguel Servet Hospital from February to June 2002. Faecal samples were diluted in sterile saline solution and seeded in bile aesculin agar plates supplemented with vancomycin (6 µg ml–1) and aztreonam (75 µg ml–1). Colonies with a typical enterococcal morphology were identified by classical biochemical methods and putative enterococci were verified using specific PCR assays (Table 2). Four of these 632 faecal samples (0.6 %) were shown to be colonized by VREF-VBP strains (E. faecium C397, C402, C404 and C406). In addition, from July 2002 to January 2003, sporadic faecal samples from particular patients [confined in the intensive care unit (ICU) and oncology wards, among others] were also analysed for this purpose in Miguel Servet Hospital and one additional VREF-VBP strain was collected (E. faecium C460) (Table 1
).
The nine VREF-VBP strains included in this study, implicated in infection processes (four strains) or in intestinal colonization (five strains), were recovered from nine different patients from these two Spanish hospitals (Table 1). All patients had severe diseases and six were or had been admitted to an ICU ward of the hospital. Five had been treated previously with glycopeptides, whilst two others had received broad-spectrum antibiotics, such as imipenem or ceftazidime. No information about previous antibiotic consumption was available for the remaining two patients.
Antibiotic susceptibility testing.
Determination of the MICs of different antibiotics (vancomycin, teicoplanin, streptomycin, gentamicin, kanamycin, ampicillin, erythromycin, ciprofloxacin, tetracycline and chloramphenicol) was carried out using the NCCLS agar dilution method (NCCLS, 2002). E. faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were used as control strains.
Characterization of the vancomycin resistance mechanism.
Vancomycin resistance mechanisms were analysed in VREF-VBP strains using specific primers for amplification of the vanA, vanC-1, vanC-2/3 and vanD genes (Table 2). E. faecium AR1 and E. faecalis SF300 (kindly supplied by E. Cercenado, Hosp. Gregorio Marañón, Madrid, Spain) were used as positive controls for the vanA and vanB PCR assays, respectively (Gold et al., 1993; Torres et al., 1994). Enterococcus gallinarum Z380 and Enterococcus casseliflavus Z406, from the collection of strains of the University of La Rioja, were used as positive controls for PCR detection of the vanC-1 and vanC-2/3 genes, respectively. vanB ligase genes, as well as the intergenic vanSB–vanYB region, were amplified by PCR. In order to determine whether the vanB2 operon of the vanB2-positive strains was associated with a Tn5382-like element, specific primers for the non-integrase (left) end of Tn5382 and for the vanXB–ORFC intergenic region and flanking coding sequences in Tn5382 were used for PCR assays. The same strains were examined for pbp5–Tn5382 linkage by PCR amplifying a 1079 bp region between pbp5 and Tn5382. Subsequent sequence analysis of PCR products was performed for all PCR assays mentioned above. PCR primers and conditions, as well as the relevant references, are given in Table 2.
Detection of other antibiotic resistance or virulence genes.
The presence of other resistance genes, such as aac(6')-Ie-aph(2'')-Ia, aph(3')-IIIa, ant(6)-Ia, erm(A), erm(B) and erm(C), was analysed by specific PCRs using primers and conditions reported previously (Table 2). In addition, the presence of the esp and hyl genes (encoding an enterococcal surface protein and hyaluronidase, respectively) was also studied and positive amplicons were sequenced for confirmation. Positive and negative controls from our collection of strains were included in all PCR assays.
Mating experiments.
The transferability of vancomycin resistance genetic determinants was tested by conjugation using a filter-mating method (Dunny et al., 1979), with E. faecalis strain JH2-2 as recipient (rifampicin- and fusidic acid-resistant, vancomycin-susceptible) and two VREF-VBP strains (C393 and C461) as donors (fusidic acid-susceptible, vancomycin-resistant). Vancomycin-resistant transconjugants were selected on brain heart infusion agar plates supplemented with fusidic acid (25 µg ml–1) and vancomycin (5 µg ml–1).
PFGE analysis of VREF-VBP strains.
The clonal relationships of the nine vanB2-containing E. faecium strains were studied by PFGE by analysing their genomic DNA after digestion with SmaI (New England Biolabs), as reported previously (Turabelidze et al., 2000). This technique was performed using clamped homogeneous electric fields (CHEF DRII; Bio-Rad). The running parameters were as follows: voltage, 6 V cm–1; block 1: run time 12 h; initial switch time 3.5 s; final switch time 25 s; block 2: run time 8 h; initial switch time 1 s; final switch time 5 s. A bacteriophage 
ladder (Bio-Rad) was used as a size marker. PFGE banding patterns were analysed and compared visually. Isolates were classified as indistinguishable, closely related, possibly related or unrelated by following previously defined criteria for bacterial strain typing (Tenover et al., 1995).
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RESULTS AND DISCUSSION
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All nine of the VREF-VBP strains of clinical and intestinal origin included in this study showed low-level vancomycin resistance (MIC 16–32 µg ml–1) and susceptibility to teicoplanin (MIC 0.5 µg ml–1). Positive PCR assays were obtained with the consensus vanB primers for the nine strains, showing negative results for the remaining vanA, vanC-1, vanC-2/3 and vanD PCR tests. Nucleotide sequences of the vanB amplicons revealed the presence of the vanB2 subtype in all nine strains (Table 1) and they were 100 % identical to that previously reported for the vanB2 gene in human and animal enterococcal isolates (Garnier et al., 2000; Gold et al., 1993; Torres et al., 2003).
According to the PFGE analysis, two possibly related PFGE patterns, A (seven strains) and B (two strains), could be distinguished (Table 1 and Fig. 1). Curiously, strains of PFGE pattern A were recovered at the beginning of the study (from seven patients of the two hospitals from February to May 2002), whilst strains belonging to PFGE pattern B were isolated at the end of the study (from two patients of Miguel Servet Hospital in December 2002 and January 2003).
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Fig. 1. PFGE of SmaI-digested genomic DNA from the nine vanB2 E. faecium strains. Lanes: 1 and 11, PFGE DNA marker; 2, E. faecium C393; 3, E. faecium C395; 4, E. faecium C396, 5, E. faecium C397; 6, E. faecium C402; 7, E. faecium C404; 8, E. faecium C406; 9, E. faecium C460; 10, E. faecium C461.
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Sequencing of the vanSB–YB intergenic regions for the nine vanB2-containing strains showed the typical 11 point mutations and the 5 bp deletion of the vanB2 subtype (Dahl et al., 2000). All of the strains showed positive PCR results for Tn5382 and the sequences obtained showed 100 % identity with a partial sequence of the previously reported Tn5382 and Tn1549 transposons (GenBank accession nos AF175739 and AF192329, respectively). These sequences were also 100 % identical to that previously found by our group in a vanB2-containing E. hirae strain of animal origin (Torres et al., 2003). However, slight differences for the vanXB–ORFC amplicons of the nine strains were observed. The seven strains belonging to PFGE pattern A showed a sequence 100 % identical to that reported previously for vanB2-containing E. faecalis and E. faecium strains with the Tn5382 transposon (GenBank accession nos AF203411 and AF203417, respectively). Nevertheless, a different sequence with a deduced valine residue (codon GTG) at position 46 of the VanXB protein and an asparagine residue (codon AAT) at position 89 was obtained for the remaining two strains with the PFGE pattern B (E. faecium C460 and C461). This VanXB sequence was 100 % identical to that previously reported for other vanB2-containing E. faecium strains with Tn5382 (GenBank accession no. AF203412 and others) and also identical to that found in the vanB2 E. hirae strain of animal origin previously published (Torres et al., 2003). The impact of these amino acid changes on the activity of the VanXB protein remains to be investigated. According to our results, the vanB2 gene cluster could be included in a Tn5382-like element in our strains. It should be noted that Tn5382 and Tn1549 show a strong similarity at both ends of the transposons (Carias et al., 1998; Garnier et al., 2000; Umeda et al., 2002) and it has been proposed that both could be integrated in the Tn5382–Tn1549 family (Umeda et al., 2002).
According to a recent report, vanB2-containing E. faecium strains from Taiwan were found to harbour Tn5382 and to be associated with the pbp5 gene (Lu et al., 2005). In our study, the nine VREF-VBP strains showed ampicillin resistance and using PCR it was possible to detect the presence of pbp5–Tn5382 amplicons of the expected size in all of them. Two of these amplicons were sequenced (E. faecium C395 and C393), confirming that sequences of Tn5382 were located downstream from a pbp5 gene. The sequence of the C-terminal part of the protein encoded by pbp5 in these two isolates presented nine amino acid changes with respect to the deduced sequence of the protein encoded by pbp5 (GenBank accession no. X84860) (Jureen et al., 2003). The amino acid changes detected in the protein encoded by the pbp5 gene in both isolates were: 461Q
K, 470HQ, 485MA, 496NK, 499AT, 525ED, 586VL, 629EV and 667PS. Most of these amino acid changes have been detected previously in the protein encoded by the pbp5 gene of ampicillin-resistant E. faecium isolates (Jureen et al., 2003; Rice et al., 2004).
The transferability of genetic determinants implicated in vancomycin resistance was tested by conjugation using E. faecium C393 (pattern A) or C461 (pattern B) as donor and E. faecalis strain JH2-2 as recipient. Results were negative in both cases and no transconjugants were detected.
The presence of the esp and hyl genes encoding virulence factors (enterococcal surface protein and hyaluronidase, respectively) was investigated by PCR and sequencing. The esp gene was detected in the two vanB2-containing E. faecium strains belonging to PFGE pattern B, but not in those of pattern A. The hyl gene was not identified in our VREF-VRP strains. The clonal spread of ampicillin/vancomycin-resistant E. faecium carrying the esp and hyl genes in German hospitals has been reported recently (Klare et al., 2005).
All of the strains in this study showed high-level streptomycin and kanamycin resistance, as well as resistance to erythromycin and ciprofloxacin. The two strains included in the PFGE pattern B also showed tetracycline resistance. The ant(6')-Ia, aph(3')-IIIa and erm(B) genes were detected in all nine strains.
In conclusion, this study reports for the first time the persistence of clonally related vanB2-containing E. faecium strains affecting patients in two hospitals in the same city in Spain. A previous clinical vanB2-containing E. faecium strain was detected in a Spanish hospital by other authors (Lorenzo-Díaz et al., 2004) and very recently a nosocomial outbreak of vanB2 vancomycin-resistant E. faecium was reported in Sweden (Granlund et al., 2006). In most cases, our vancomycin-resistant enterococci were recovered from patients with severe diseases (the majority had been hospitalized in ICU wards) who had previously received glycopeptides and other broad-spectrum antibiotics. Special care should be taken in the future to ensure the rapid detection and control of this type of strain in order to avoid its dissemination within a hospital.
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ACKNOWLEDGEMENTS
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This work was supported in part by a grant from the Fondo de Investigaciones Sanitarias of Spain (FIS 01/973).
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INTRODUCTION
METHODS
