First Isolation of Vancomycin-Resistant Enterococcus faecium Carrying Plasmid-Borne vanD1

ABSTRACT

Vancomycin-resistant Enterococcus faecium carrying the vanD1 gene on plasmid pEF-D was isolated from a fecal sample of a hospitalized patient in Japan. The strain JH5687 showed moderate resistance to vancomycin (MIC, 16 μg/mL) but remained susceptible to teicoplanin (MIC, 1 μg/mL). The backbone gene organization of pEF-D was highly homologous to that of conjugative plasmid pMG1 or pHTβ. The calculated conjugation frequency of JH5687 was 10⁻⁴ to 10⁻⁵ per donor cell.

INTRODUCTION

Vancomycin-resistant Enterococcus faecium (VRE) is an increasingly recognized cause of infection worldwide. Vancomycin resistance is categorized according to the pentapeptide precursors produced by the vancomycin resistance genes, as follows: those encoding d-alanine-d-lactate ligase (VanA, VanB, VanD, VanF, and VanM types) and those encoding d-alanine-d-serine ligase (VanC, VanE, VanG, VanL, and VanN types). VanD is classified into five distinct alleles (VanD1 to VanD5) in enterococci (1). Most vanD genes are harbored on a mobile element that is exclusively chromosomally encoded, and the strains are constitutively resistant to moderate levels of vancomycin (MIC, 64 to 128 μg/mL) and teicoplanin (MIC, 4 to 64 μg/mL) (2–4) with only one exception: VanD-type Enterococcus faecium A902 strain has been reported to be inducibly resistant (5). Chromosomally encoded vanD-type Enterococcus faecium has been sporadically reported in Japan, the United States, Canada, Brazil, and Europe (6–11). In Japan, vanD4-type vancomycin-resistant Enterococcus raffinosus (12) was first isolated in 2006. Here, we report the first case of the isolation of VRE harboring a highly conjugative plasmid, pEF-D, carrying vanD1.

A VRE strain was detected in the feces of an inpatient and was designated JH5687. The antimicrobial susceptibility of JH5687 was determined with a MicroScan WalkAway-96 system (Beckman Coulter, CA, USA) using the Pos/PM2J panel. JH5687 was moderately resistant to vancomycin (MIC, >16 μg/mL) but remained susceptible to teicoplanin (MIC, ≤1 μg/mL). The MICs of other antibiotics were as follows: benzylpenicillin, >8 μg/mL; ampicillin, >8 μg/mL; oxacillin, 4 μg/mL; cefazolin, >16 μg/mL; cefmetazole, >32 μg/mL; imipenem/cilastatin, >8 μg/mL; ampicillin/sulbactam, >16/8 μg/mL; gentamicin, >8 μg/mL; arbekacin, >8 μg/mL; erythromycin, ≤0.25 μg/mL; clindamycin, ≤0.25 μg/mL; minocycline, 8 μg/mL; levofloxacin, >4 μg/mL; daptomycin, 4 μg/mL; sulfamethoxazole/trimethoprim, >2/38 μg/mL; fosfomycin, >16 μg/mL; rifampin, >2 μg/mL; linezolid, >1 μg/mL.

The vanD gene was detected using a multiplex PCR assay for identification of van gene type (13). To investigate the genetic structure, we conducted whole-genome sequencing (WGS) of JH5687. The genomic DNA of JH5687 was extracted using lysozyme and a QIAamp DNA minikit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. An Enzymatics 5× WGS fragmentation mix and WGS ligase reagents (Qiagen, Hilden, Germany) were used to prepare the DNA libraries for sequencing. The 300-bp paired-end sequencing of JH5687 was conducted using the short-read sequencer MiSeq (Illumina, San Diego CA, USA). The reads were assembled using A5-miseq version 20160825 and CLC Genomics Workbench version 9 (Qiagen, Hilden, Germany). Assembly quality of JH5687 was 1,362,927 reads, 212 contigs, de novo-assembled genome sequence with 102-fold mean coverage, and N₅₀ value of 39,053 bp. As a result of blastn analysis, the sequence of the vanD gene cluster was included in the contig of the pMG1-like sequence (accession number AB206333). Next, we sorted and aligned the contigs of pMG1-like sequence using the OSLay software (14). Three gaps of pMG1-like contigs were closed using direct sequencing of PCR products amplified with oligonucleotide primers designed to anneal each end to the neighboring contigs, using an ABI Sanger sequencer (see Fig. S1 in the supplemental material). The gap-filled pMG1-like plasmid was checked with PCR scanning analysis and designated pEF-D (Fig. S1). The pEF-D was examined with Prokka v1.14 and DFAST v1.2.6 used for annotation (15, 16). The fully assembled circular pEF-D was a 61,629-bp plasmid with average GC content of 34.2% and a predicted 75 coding DNA sequences (CDSs). We confirmed the plasmid size of pEF-D using PCR. The annotation of pEF-D identified the vanD gene cluster in the plasmid.

The multilocus sequence typing of JH5687 revealed sequence type (ST) 203. According to the phylogenic analysis of the van gene amino acid sequence, the vanD of JH5687 belongs to the vanD1 group (see Fig. S2). As shown in Fig. 1, a comparative analysis of pEF-D plasmid demonstrated that the backbone gene organization of pEF-D was highly homologous to that of the conjugative pheromone-independent plasmid pMG1 (accession number AB206333) and pHTβ (accession number AB183714) (17–19).

FIG 1.

Structural comparison of pEF-D and highly conjugative plasmids pMG1 and pHTβ. Sequence reads were processed using the DFAST autoannotation pipeline (16), InSilico molecular cloning software package, genomics edition (InSilico Biology Inc., Yokohama, Japan), and GenomeMatcher software (26). The pEF-D of JH5687 was compared to pMG1 (65,029 bp; GenBank accession number AB206333.1) (15), pHTβ (63,741 bp; GenBank accession number AB183714.1) (18), and vanD gene cluster region on transposon Tn6711 of E. faecium KresVRE0001 (125,858 bp; GenBank accession number MT951615.1). Color shading between plasmids indicates homologous regions. Coding sequences (CDSs) are represented by arrows, and annotated genes are colored as follows: green, van gene cluster; purple, transposase; gray, other genes.

The plasmid sequences indicated that the vanD1 gene cluster of pEF-D was inserted into the topA-encoded DNA topoisomerase of pMG-1 by transposase, thereby generating the 9-bp direct repeat (5′-ATATGACTG-3′). Transposases at both ends of the vanD1-harboring region of pEF-D were classified in the IS256 family by using ISfinder (http://www-is.biotoul.fr//) (20). In addition, although the vanA gene cluster (10,851 bp) of pHTβ was also inserted into the same gene, the exact insertion site (the 8-bp direct repeat, 5′-GATTATGG-3′) was different. The vanD1 gene cluster of pEF-D had a truncated vanY_D gene (encoding the D,d-carboxypeptidase VanY_D) and lacked the two-component system that includes the vanS_D gene (encoding the sensor kinase VanS_D) and vanR_D gene (encoding the transcriptional regulator VanR_D) (Fig. 1; see also Fig. S2). We examined the vanD1-harboring region of pEF-D with blastn search against the NCBI database. The vanD1-harboring region of pEF-D shared highly similarity with the vanD gene cluster region of E. faecium KresVRE0001 transposon Tn6711 (21) (Fig. 1). We suggest that the vanD1-harboring region in pEF-D might represent a putative donor element for this sequence.

The vanD gene cluster is present on a mobile element that is generally located on the chromosome and is not transferable to intraspecies by in vitro conjugation (6). As the vanD1 gene of JH5687 was harbored on a conjugative plasmid, we conducted conjugation experiments using filter mating to confirm the potential horizontal plasmid transfer in vitro using E. faecium BM4105RF and BM4105SS (18) as recipients. The conjugation experiment was performed as described previously (18). The frequency from JH5687 to donors was 10⁻⁴ to 10⁻⁵ per donor cell, which was comparable to intraspecies conjugation by the highly conjugative plasmid pMG1 (18). A similarity search of the IS256 family transposase using a blastp algorithm against the NCBI database indicated the top-hit transposase of Clostridioides difficile DSM 29627 (accession number CP016102; locus_tag CDIF29627_03458) to be the most closely related. This feature was highly similar to the transposase of a bacterial species refractory to in vitro culture of the human gut microbiome, such as Clostridiales (e.g., Flavonifractor plautii strain JCM 32125, accession number CP048436) (22). In support of this, recent studies have identified the vanD gene in anaerobic commensals of human gut microbiomes (14, 23, 24). To date, all characterized VanD-type VRE strains possess mutations in the chromosomal housekeeping gene encoding d-alanyl-d-alanine ligase (Ddl) (2). Similarly, the JH5687 Ddl gene contained a frameshift mutation of a 5-bp (ATATG) insertion at the 22nd base from the 5′-terminal. Therefore, the Ddl of JH5687 is predicted to be inactive based on the internal frameshift. This strain can grow even in the absence of vancomycin, probably because the vanD gene cluster is constitutively expressed as a result of deletions of the vancomycin sensor and regulator genes vanS_D and vanR_D (Fig. S2).

The highly conjugative pMG1-like plasmid containing vanA gene cluster-harboring transposon Tn1546 is widely disseminated in hospitals in the United States and Japan (18, 25). In conclusion, this report describes the first isolation of VRE carrying a highly conjugative plasmid-borne vanD1.

Data availability.

The complete nucleotide sequence of pEF-D was deposited in DDBJ under accession number LC569722. The whole-genome sequence of JH5687 has been deposited in the DDBJ Sequence Read Archive (DRA) under accession number DRA012751.