JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2011, p.

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0095-1137/11/$12.00 doi:10.1128/JCM.05524-11
Copyright 2011, American Society for Microbiology. All Rights Reserved.

Vol. 49, No. 12

Detection of an Unusual van Genotype in a Vancomycin-Resistant

Enterococcus faecium Hospital Isolate
Jeanette W. P. Teo,1* Prabha Krishnan,2 Roland Jureen,1 and Raymond T. P. Lin1
Department of Laboratory Medicine, National University Hospital, Singapore 119074, Republic of Singapore,1 and Department of
Laboratory Medicine, Tan Tock Seng Hospital, Singapore 308433, Republic of Singapore2
Received 22 August 2011/Returned for modification 18 September 2011/Accepted 7 October 2011

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.
izationtime of flight mass spectrometry (MALDI-TOF MS)
(Bruker Daltonik GmbH, Leipzig, Germany), and species-specific PCR targeting D-alanine-D-alanine ligase (ddlE. faecium) (3)
all confirmed that it was an E. faecium isolate. Susceptibility
testing was performed using the Etest (bioMerieux) 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
37C, was unchanged in either plate, suggesting that vancomycin was not an inducer of teicoplanin resistance.
As part of the hospitals 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 vanHM gene.
This amplification was performed using primers vanHMF (5CAGCGTGGGGCACAAGTCTGA-3) and vanHMR (5-T
GCCGTACGCCAACACGTGA-3). PCR amplification followed by direct sequencing of the open reading frames (ORFs)
spanning the vanM locus from the IS1216-like element to
vanXM (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 vanRM 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, gel-

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-D-glucopyranoside. It tested positive with raffinose. Vitek 2
Gram-positive (GP) identification card no. 21342 (bioMerieux,
Marcy lEtoile, France), matrix-assisted laser desorption ion* Corresponding author. Mailing address: Department of Laboratory Medicine, National University Hospital, Singapore 119074, Republic of Singapore. Phone: 6567724856. Fax: 6567751757. E-mail:
jeanette_teo@nuhs.edu.sg.

Published ahead of print on 12 October 2011.

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NOTES

atinase, 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 variablenumber 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

J. CLIN. MICROBIOL.

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.
This work was supported by a Health Service Development Programme Grant provided by the Ministry of Health, Singapore (grant
HSDP06/X04).
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