Detection of an Unusual van Genotype in a Vancomycin-Resistant Enterococcus faecium Hospital Isolate

Abstract

We highlight the detection of a rare vanM genotype in Enterococcus faecium. This isolate exhibited a VanB phenotype, with high levels of resistance to vancomycin (MIC, >256 mg/liter) and susceptibility to teicoplanin (MIC, 1 mg/liter). It was, however, vanB negative by PCR. Further screening for other van loci revealed the presence of a complete vanM operon.

TEXT

Glycopeptide resistance in enterococci is mediated by van gene clusters, and to date, eight operons (vanA, vanB, vanC, vanD, vanE, vanG, vanL, and vanM), each with distinguishing resistance characteristics, have been described (1, 13, 14). Isolates harboring vanA, vanB, and vanM are resistant to high levels of vancomycin (>128 mg/liter). Additionally, vanA and vanM isolates are typically resistant to high levels of teicoplanin, although exceptions have been noted (9, 13). The vanB gene cluster, on the other hand, produces little or no resistance to teicoplanin (MIC, <1 mg/liter). vanD strains are resistant to moderate levels of vancomycin (MIC, 16 to 128 mg/liter) and susceptible to teicoplanin, while vanC, vanE, vanL, and vanG strains exhibit low-level resistance to vancomycin (1, 13, 14). Clinically, the vanA and vanB operons are the two most dominant and relevant vancomycin resistance factors (14).

Locally, infrequent outbreaks of vancomycin-resistant enterococci (VRE) have been traced to a dominant vanB Enterococcus faecium clone (8). This is in contrast to the other Asian countries like South Korea (10) and Taiwan (2, 7), where vanA is the predominant genotype. We report the detection of an uncommon van genotype, vanM, which has thus far been reported only from China with a handful (n = 6) of characterized isolates (13).

A vancomycin-resistant E. faecium strain was isolated from a screening rectal swab of a 64-year-old female who had been a contact of another patient carrying VRE with a vanA genotype. She had no travel history. The patient was admitted to the orthopedic unit with a fractured femur with poorly controlled diabetes mellitus and hypertension. She had been on vancomycin for a long period for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis of the right foot but was not treated for the VRE.

The isolate was nonmotile and nonpigmented. Biochemically, it was unable to ferment sorbitol, arabinose, and methyl-α-d-glucopyranoside. It tested positive with raffinose. Vitek 2 Gram-positive (GP) identification card no. 21342 (bioMérieux, Marcy l'Etoile, France), matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik GmbH, Leipzig, Germany), and species-specific PCR targeting d-alanine-d-alanine ligase (ddl_{E. faecium}) (3) all confirmed that it was an E. faecium isolate. Susceptibility testing was performed using the Etest (bioMérieux) method with breakpoints defined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The isolate was highly resistant to vancomycin (MIC, >256 mg/liter) and sensitive to teicoplanin (MIC, 1 mg/liter), indicative of a VanB phenotype. The isolate was also sensitive to daptomycin, linezolid, and quinupristin-dalfopristin with MICs of 1, 1.5, and 0.75 mg/liter, respectively, but resistant to ampicillin, ciprofloxacin, and rifampin with MICs of >32 mg/liter. Inducibility of teicoplanin resistance was tested by inoculating cultures onto Muller-Hinton agar plates without vancomycin or containing a subinhibitory concentration of vancomycin (5 mg/liter) (6), and a teicoplanin Etest strip was overlaid on the plate. The Etest MIC, determined after a 48-h incubation at 37°C, was unchanged in either plate, suggesting that vancomycin was not an inducer of teicoplanin resistance.

As part of the hospital's infection control policy, all VRE isolates are PCR screened for vanA and vanB, for which the isolate tested negative. Further screening for vanC, vanD, vanE, vanL, and vanG ligases (1, 4, 5) was performed. The isolate was positive for a 377-bp fragment of the vanH_M gene. This amplification was performed using primers vanHMF (5′-CAGCGTGGGGCACAAGTCTGA-3′) and vanHMR (5′-TGCCGTACGCCAACACGTGA-3′). PCR amplification followed by direct sequencing of the open reading frames (ORFs) spanning the vanM locus from the IS1216-like element to vanX_M (nucleotide positions 55 to 6503 of the vanM operon [GenBank accession no. FJ349556.1]) revealed a 100% identity to the GenBank sequence except for a 173-bp insertion 171 bp upstream of the vanR_M ORF. The insertion event has been similarly reported by Xu et al. (13) in one of their isolates exhibiting a high level of vancomycin resistance (>256 mg/liter) and teicoplanin sensitivity (0.75 mg/liter). Since their remaining isolates had high levels of both vancomycin and teicoplanin resistance, it was speculated that the insertion might have caused the loss of teicoplanin resistance (13).

PCRs for the detection of five virulence genes, asa1, gelE, cylA, esp, and hyl (12), coding for aggregation substance, gelatinase, cytolysin, enterococcal surface protein, and hyaluronidase, respectively, were performed. The isolate was positive only for the esp gene.

Multilocus sequence typing (MLST) using the protocol available at http://efaecium.mlst.net/misc/info.asp indicated that the isolate belonged to sequence type (ST) 78. This was also the dominant strain type detected among four out of the six (67%) Chinese vanM isolates (13). Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) was performed (http://www.umcutrecht.nl/subsite/MLVA/) and revealed that the isolate was MLVA type (MT) 12. Collectively, genotyping results, resistance to ampicillin and quinolones, and the presence of an enterococcal surface protein indicate that the isolate belongs to clonal complex 17 (CC17). E. faecium CC17 has been associated with epidemic nosocomial outbreaks and infections (2, 11).

Southern blot hybridization analysis of plasmid and genomic DNA was performed using the digoxigenin (DIG) DNA labeling and detection kit (Roche Diagnostics, Mannheim, Germany). Probing with the DIG-labeled amplicon derived from nucleotide positions 5513 to 5823 of the vanM operon (FJ349556.1) suggested that vanM was chromosomally encoded. vanM has been previously found to be plasmid localized with the demonstration of conjugal transfer of the vanM cluster to an antibiotic-susceptible E. faecium recipient (13). Xu et al. (13) speculate that the transfer may have been facilitated by an IS1216 transposition event. The association of vanM with the insertion sequence (IS) element may also similarly mediate dissemination in our isolate. However, due to the unavailability of a suitable E. faecium recipient, we were not able to demonstrate transferability of vanM.

VRE isolated at the hospital during the study period and from the previous year were of either vanA or vanB genotype, and this is the first instance of vanM detection. This report serves to highlight the existence of unusual van genotypes in hospitals whose detection may be missed. Commercial molecular assays such as the Cepheid GeneXpert vanA/vanB assay (Cepheid, Sunnyvale, CA) or the BD GeneOhm VanR assay (BD GeneOhm, San Diego, CA) specifically target the vanA and/or vanB genes, which have no more than 83% nucleotide identity with vanM. Hence, we believe that it is unlikely that the vanM genotype would be picked up by such assays. Given the rare occurrence of vanM, we are currently uncertain about the clinical significance of vanM in Singapore.