Characterization of an Enterococcus gallinarum Isolate Carrying a Dual vanA and vanB Cassette

Abstract

The ability of vancomycin resistance determinants to be horizontally transferred within enterococci species is a concern. Identification and characterization of vancomycin-resistant enterococci (VRE) in a clinical isolate have a significant impact on infection control practices. In this study, we describe a clinical isolate of Enterococcus gallinarum exhibiting high-level resistance to vancomycin and teicoplanin. The genetic characterization of this isolate showed the presence of vanA and vanB genes in addition to the naturally carried vanC gene. vanA was identified on pA6981, a 35,608-bp circular plasmid with significant homology to plasmid pS177. The vanB operon was integrated into the bacterial chromosome and showed a high level of homology to previously reported Tn1549 and Tn5382. To the best of our knowledge, this is the first report of E. gallinarum carrying both vanA and vanB operons, indicating the importance of identifying the vancomycin resistance mechanism in non-E. faecium and non-E. faecalis enterococcal species.

INTRODUCTION

Vancomycin-resistant enterococci (VRE) were initially detected during the 1980s in Europe, and the first case of VRE in Canada was described in 1993 (1, 2). Subsequently, VRE became one of the most important nosocomial pathogens causing severe infections in the world. In addition, numerous outbreaks in hospital settings have been reported, resulting in significant investments in infection control resources to prevent transmission among hospitalized patients. Enterococcus faecium is the most common species among VRE isolates found in human clinical specimens, followed by E. faecalis. As a result, identification of these species not only is important for patient care but also is essential for preventing transmission of VRE among other hospitalized patients. E. gallinarum and E. casseliflavus are also found in clinical specimens, although less frequently, and are generally not considered significant pathogens. However, in certain situations, such as in immunocompromised hosts, E. gallinarum has been shown as the cause of severe infections, including bacteremia, endocarditis, and meningitis, as well as several hospital-acquired outbreaks (3–7).

Nine different types of glycopeptide resistance operons (vanA, vanB, vanC, vanD, vanE, vanG, vanL, vanM, and vanN) have been described in enterococci, of which vanA- and vanB-mediated vancomycin resistances are the most clinically significant in the genus. VanA confers a high level of resistance to both vancomycin and teicoplanin, whereas VanB confers resistance to only vancomycin. Both vanA and vanB gene clusters are mainly located on the mobile elements Tn1546 (Tn3 family) and Tn1549/Tn5382 (conjugative), respectively. As such, glycopeptide resistances mediated by vanA and vanB are disseminated by direct acquisition of these mobile elements or through plasmids (8–10). The vanC gene cluster is a chromosomally encoded nontransferable operon found in E. gallinarum and E. casseliflavus, conferring low-level resistance to vancomycin (11).

Although vanA and vanB genes are mainly found in E. faecium and E. faecalis, there are a few reports that highlight other enterococcal species such as E. gallinarum containing these genes and subsequently expressing a high level of vancomycin resistance (12–16). In this study, we characterized a clinical isolate of E. gallinarum containing both vanA and vanB operons conferring vancomycin resistance.

MATERIALS AND METHODS

Case description.

In November 2013, a methyl-alpha-d-glucopyranoside-positive Enterococcus spp. was isolated from a left ischium wound of a 52-year-old man who was a resident in Ottawa, Canada. The isolate was identified biochemically as E. gallinarum and was found to be resistant to ampicillin and vancomycin (MICs of >32 μg/ml and >256 μg/ml, respectively) and susceptible to daptomycin. The susceptibility testing was performed by Etest (AB Biodisk), and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The presence of vanA/vanB genes was tested by PCR using the LC VRE detection kit (Roche, Laval, QC, Canada). Two melting peaks (55.97 and 68.07) were identified and were consistent with the presence of vanA and vanB genes. Given the unusual nature of the isolate, it was referred to the Public Health Ontario Laboratory (PHOL), the reference microbiology laboratory for the province of Ontario, Canada, for confirmation and further susceptibility testing.

Phenotypic and genetic characterization of glycopeptide resistance.

At PHOL, the isolate (designated A6981) was identified using traditional biochemical testing, and the susceptibility testing was done using the reference agar dilution method, and results were interpreted according to the CLSI guidelines (17).

The mechanisms of glycopeptide resistance were identified by using two in-house multiplex PCRs targeting vanA, vanB, vanC1, vanC2/3 and vanD, vanE, ddl_{E. faecalis}, and ddl_{E. faecium}, using previously described primers (18–20). The enriched plasmid DNA was sequenced using the Illumina MiSeq system to genetically characterize acquired glycopeptide resistance in detail. Briefly, the plasmid DNA was extracted using a QIAprep spin miniprep kit (Qiagen) and quantified using a Qubit 2.0 fluorometer (Invitrogen). A total of 1 ng of DNA was used for library preparation using the Nextera XT kit (Illumina, San Diego, CA, USA) according to the manufacturer's protocol. The prepared library was indexed, and its quality was assessed using an Agilent 2100 Hi-Sens DNA chip and the KAPA Library Quantification protocol. The indexed library was normalized to 12 pM and loaded onto an Illumina MiSeq instrument using the 2 × 150-bp paired-end reads with a 300-cycle V2 cartridge.

The quality of the reads (approximately 2.2 million reads) was checked by FastQC:ReadQC. Low-quality reads, including undefined nucleotides, full-length homopolymer runs, reads with Phred quality values of <30, and Nextera adaptor sequences were removed by the FastQ groomer and filter with the quality tool of the Manipulate FASTQ program in Galaxy (21–23). Assembly of the reads was performed using the de novo SPAdes Genome Assembler v3.0 with a read correction module and k-mer sizes of 21, 33, 45, 55, 77, 99, and 127 (24). New primers were designed to close unassembled gaps by PCR and Sanger sequencing.

The sequence annotation and function assignment were done by the NCBI Prokaryotic Genome Automated Annotation Pipeline (PGAAP) (25). The pairwise alignment between two plasmids was performed using Geneious v7.1.7 (Biomatters Ltd., Auckland, New Zealand) (26). Searches of sequences were performed with the BLAST program, available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/).

Nucleotide sequence accession numbers.

The nucleotide sequences of plasmid pA698, the Tn1549/5238-like transposon from this study, and the E. gallinarum chromosomal DNA located in the right and left extremities of Tn1549/5238-like were deposited at DDBJ/EMBL/GenBank under accession no. JMGP00000000.

RESULTS

Identification of isolates to the species level and antimicrobial susceptibility testing.

Isolate A6981 was confirmed as E. gallinarum using the following biochemical testing assays: Gram stain (Gram-positive cocci in chain), pigment (negative reaction [−]), catalase (−), esculin hydrolysis (positive reaction [+]), growth on 6.5% NaCl (+), pyruvate (+), motility (+), lactose (+), d-xylose (+), α-methyl glucosidase (−), arabinose (+), sorbitol (variable reaction [V]), melezitose (V), arginine (+), pyruvate (−), and dextrose (+) (27). In addition, the susceptibility testing confirmed this isolate to be highly resistant to vancomycin (MIC of >256 μg/ml), teicoplanin (MIC = 48) ciprofloxacin, levofloxacin, and erythromycin (Table 1).

TABLE 1.

MICs of various antimicrobial agents obtained for the clinical isolate E. gallinarum A6981

Antibiotic	MIC (μg/ml)	Interpretation (CLSI)a	Presumed resistant mechanism
Vancomycin	≥256	R	vanA, van B
Teicoplanin	48	R	vanA
Erythromycin	≥256	R	ErmB
Ciprofloxacin	≥32	R
Levofloxacin	≥32	R
Tetracycline	0.12	S
Penicillin	4	S
Ampicillin	2	S
Chloramphenicol	2	S
Linezolid	0.5	S
Cotrimoxazole	≤0.006	−
Streptomycin	≥1024	−	aadE-sat4-aphA-3 gene cluster
Gentamicin	2	S

^aR, resistant; S, susceptible; −, no CLSI breakpoint interpretations exist.

Genetic characterization of vancomycin resistance.

Isolate A6981 was positive for vanC1, vanA, and vanB genes by PCR. Considering that vanA and vanB clusters are commonly acquired through mobile, conjugative elements, the plasmid DNA from the isolate was extracted and sequenced. The assembly resulted in 175 contigs (range, 79 to 458,023 bp). Contigs with low coverage as well as those with <1 kb length or with no match with enterococci in the GenBank database were removed from the analysis. Five contigs with higher coverage (average 632×) with significant homology to plasmid pS177 were identified by BLAST. The vanA operon was detected on one of these contigs (104,308 bp) with average coverage of 582×. In addition, 19 contigs (29,779 to 458,023- bp; N50 = 241,184 bp) matching to E. gallinarum chromosomal DNA were identified. These contigs were the result of residual chromosomal DNA in the plasmid extractions, easily recognized by their low read coverage, resulting in a mean chromosome/plasmid ratio of 1:19 (34× for chromosomal contigs versus 632× for plasmid contigs).The largest contig (458,023 bp) was found to carry the genes normally located on the chromosome of E. gallinarum as well as the vanB cluster.

General properties of plasmid containing vanA operon.

The 5 contigs with higher coverage showed high nucleotide identity with the previously characterized vanA carrying plasmid pS177 (GenBank accession no. HQ115078). After filling the gaps, the complete circular sequence of the plasmid, named pA6981, was found to be 35,608 bp in size (average G+C content of 35.4%) with 99% nucleotide identity to pS177 over 32,759 bp (92% coverage). Based on a similarity search against the BLASTp reference protein database, 45 coding sequences (CDSs) were identified (see Table S1 in the supplemental material).

The vancomycin resistance cluster (vanRSHAXY), present on transposon Tn1546 on plasmid pA6981, showed 99% (8,132/8,133) overall sequence identity to that of pS177 (Fig. 1) (28). Further analysis of Tn1546 revealed the presence of an IS1251-like element inserted into the vanSH intergenic region as well as the elimination of orf1 (transposase of Tn1546), replaced by a 681-bp IS1216 element followed by a 335-bp coding region with 100% homology to a predicted transcriptional regulator in Enterococcus (GenBank accession no. WP_002321977.1) (Fig. 1). Two toxin-antitoxin (TA) type II systems were identified, including the genes for the axe-txe and relE-like toxin/antitoxin components of the TA systems usually found in E. faecium. In addition, this plasmid harbors the aadE-sat4-aphA-3 gene cluster, which confers resistance to streptomycin, streptothricin, and kanamycin (29), and the ermB gene, known to confer resistance to erythromycin by methylation of bacterial 23S rRNA (30). The presence of these mechanisms of resistance in E. gallinarum A6981 explain the high MIC values for streptomycin (≥1024 mg/liter) and erythromycin (≥8 mg/liter) (Table 1).

FIG 1.

Genetic map of pA6981 compared to that of the closest plasmid homolog, pS177 (GenBank accession no. HQ115078) (9). The numbering of the plasmids commences at the first nucleotide of the ATG start codon of orf1, which is predicted to encode replication control protein PrgN. Regions with high homology are represented by light blue shading. Arrows indicate predicted open reading frames (ORFs): stability and accessory genes (gray), antimicrobial resistance genes (orange; the vanA operon is indicated in red), transposon-related genes (green), and hypothetical proteins (black). The numbering above the pA6981 plasmid corresponds to the ORF numbers in Table S1 in the supplemental material.

General properties of transposon-containing vanB operon.

The genome of E. gallinarum A6981 carries the complete nucleotide sequence of the Tn1549/5382-like transposon consisting of 41,541 bp within the left (IR_L, AAAATTTTAGG) and right (IR_R, CATATAATTTT) inverted repeats (Fig. 2). This transposon was inserted downstream of the membrane-bound lytic murein transglycosylase D precursor protein-encoding gene (autolysin), homologous in GenBank to accession no. WP_005470940.1, and upstream of a putative cell wall-binding protein-encoding gene and a disrupted l-lactate dehydrogenase-encoding gene (see Table S3 in the supplemental material). Compared to the complete sequences of Tn1549 and the Tn5382-like transposon from Clostridium sp. CCRI-9842 (accession no. AY772783) and Clostridium sp. CCRI-9842 (accession no. AY772783), respectively, the mobile element in E. gallinarum A6981 was 7,736 bp larger (10). This difference is the result of 3 large DNA insertions into the Tn1549/5382 of E. gallinarum A6981. The 1,695-bp sequence of the TrsK-like protein (identified as orf16 in Tn1549) is interrupted by a 2,790-bp DNA insertion in E. gallinarum A6981, which encodes a reverse transcriptase (RefSeq WP_016283879.1) and 2 putative conjugal transfer proteins with homology to TraG in E. faecalis. In addition, the TrsE-like protein (orf20 in Tn1549) was truncated by an early stop codon resulting from the insertion of a 2,437-bp DNA sequence encoding the conjugal transfer protein TraE, an excisionase, and an additional hypothetical protein. A stretch of 2,502 bp was found to be inserted in the orf28 (relaxase-like protein) region causing replacement of orf28 with 4 orfs predicted to encode for endonucleases, excisionase, and two hypothetical proteins (Fig. 2).

FIG 2.

Genetic maps of Tn1549-like transposon found on the chromosome of E. gallinarum A6981 (Ega A6981) and the prototype Tn1549 (GenBank accession no. AF192329) (10). Regions with high homology are represented by light blue shading. Arrows indicate predicted ORFs: conjugation and accessory genes (gray), transposition (orange), and hypothetical proteins (black). The vanB operon is indicated in blue. Chromosomal genes (brown) are also indicated. Insertions disrupting ORFs described in Tn1549 are shown in green. The Tn1549 inverted repeats (IR_R and IR_L) are represented as yellow circles. Red arrows indicate ORFs that were not annotated in the prototype Tn1549. The numbering and predicted protein function of E. gallinarum A6981 ORFs represented in this figure are described in Table S3 in the supplemental material.

Analysis of the complete sequences of the E. gallinarum A6981 vanB cluster revealed slightly higher sequence identity to Tn5382 than to Tn1549 at both the nucleotide and amino acid levels. Despite close homology to Tn5382, the vanB operon in E. gallinarum A6981 displayed 1 novel nonsynonymous substitution in each vanS_B, vanW, and vanX_B genes. Two additional novel nonsynonymous substitutions with unknown effects (M308L and V321M) in the N-terminal substrate-binding domain of VanB were identified (see Table S2 in the supplemental material).

DISCUSSION

Vancomycin resistance mediated by vanA and vanB gene clusters is primarily found in E. faecium and to a lesser extent in E. faecalis. Other Enterococcus spp. encountered in human infections, including E. gallinarum and E. casseliflavus/E. flavescens, are intrinsically less susceptible to vancomycin (MICs of 2 to 16 μg/ml) and susceptible to teicoplanin (31). The low-level resistance is mediated by a nontransferable chromosomally mediated vanC gene and therefore does not represent significant infection control challenges. In this report, we identified an isolate of E. gallinarum carrying vanA and vanB genes conferring high-level resistance to vancomycin and teicoplanin. Genetic characterization of these resistant determinants showed that the Tn1546-containing vanA transposon was located on a plasmid highly similar to the previously characterized pS177. The transposon-containing vanB gene cluster was found as part of a Tn5382-like transposon on the chromosome of E. gallinarum A6981.

Although motile enterococci, including E. gallinarum, are not commonly found to be clinically significant and, therefore, are not processed extensively in the laboratory setting, our study shows that their isolation and characterization is important due to its ability to acquire high-level vancomycin resistance genes such as vanA or vanB. In addition, the presence of the vanA or vanB gene in motile enterococci may contribute to the spread of vancomycin resistance among other enterococcal species. The ability of E. faecium to transfer the vanA gene cluster to E. gallinarum has previously been documented (32). E. gallinarum containing the transferable vancomycin resistance determinant is extremely rare, although the current algorithm in clinical microbiology laboratories does not routinely test for such resistant determinants in these species. As a result, this report highlights the need for further investigation of any isolated motile enterococci such as E. gallinarum in clinical settings.