Tn6674 Is a Novel Enterococcal optrA-Carrying Multiresistance Transposon of the Tn554 Family

The novel 12,932-bp nonconjugative multiresistance transposon Tn6674 was identified in the chromosomal DNA of a porcine Enterococcus faecalis strain. Tn6674 belongs to the Tn554 family of transposons. It shares the same arrangement of the transposase genes tnpA, tnpB, and tnpC with Tn554. However, in addition to the Tn554-associated resistance genes spc and erm(A), Tn6674 harbored the resistance genes fexA and optrA.

ABSTRACT

The novel 12,932-bp nonconjugative multiresistance transposon Tn6674 was identified in the chromosomal DNA of a porcine Enterococcus faecalis strain. Tn6674 belongs to the Tn554 family of transposons. It shares the same arrangement of the transposase genes tnpA, tnpB, and tnpC with Tn554. However, in addition to the Tn554-associated resistance genes spc and erm(A), Tn6674 harbored the resistance genes fexA and optrA. Circular forms of Tn6674 were detected and suggest the functional activity of this transposon.

INTRODUCTION

Oxazolidinones, such as linezolid and tedizolid, are often used for the treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) in humans (1). Phenicols, such as florfenicol, are approved for the control of respiratory tract infections in food-producing animals (2). The gene optrA, which confers combined resistance to oxazolidinones and phenicols, has emerged during recent years (3) and poses a serious therapeutic challenge to both human and veterinary medicine. Although the optrA gene has been originally described to encode an ABC transporter, a recent study confirmed that OptrA does not mediate the efflux of oxazolidinones and phenicols (4). Meanwhile, OptrA is considered to be an ABC-F protein which acts by protecting the bacterial ribosome (5).

Since the first description of the optrA gene in Enterococcus faecalis and Enterococcus faecium (3), this gene has been identified on various plasmids of different sizes or in the chromosomal DNA of enterococci from humans and food-producing animals (1). In some cases, the optrA gene was also present in combination with cfr, cfr(B), or poxtA on the same strain or plasmid (6–8). In addition, it was also found in Staphylococcus sciuri, together with cfr and other resistance genes on the same plasmid, and in Streptococcus suis and Streptococcus gallolyticus. In S. suis, it was shown to be part of integrative and conjugative elements (ICEs) (9–11). In the present study, we identified a novel chromosome-borne transposon of the Tn554 family, designated Tn6674, which carried the optrA gene together with the resistance genes spc, erm(A), and fexA in an E. faecalis strain.

During a routine survey on antimicrobial resistance in bacteria from pigs, the florfenicol- and linezolid-resistant E. faecalis strain E1731 was isolated from a fecal sample of a pig in Henan Province, China, in 2018. Antimicrobial susceptibility testing (AST) was performed by broth microdilution according to the recommendations given in document M100 (28th ed) of the Clinical and Laboratory Standards Institute (CLSI) (12). S. aureus ATCC 29213 served as the quality control strain in AST. The AST results are shown in Table 1. Multilocus sequence typing (MLST) revealed that it belonged to sequence type 691 (ST691). The presence of the optrA gene was detected by PCR using the primers optrA-fw (5′-GCACCAGACCAATACGATACAA-3′) and optrA-rev (5′-TCCTTCTTAACCTTCTCCTTCTCA-3′), with an annealing temperature of 59°C and amplicon size of 794 bp.

TABLE 1.

Resistance phenotype and genotype of E. faecalis E1731

Antimicrobial agent	MIC (mg/liter)	Associated resistance gene(s) or mutation(s)
Florfenicol	128	optrA, fexA
Linezolid	8	optrA
Erythromycin	512	erm(A), erm(B)
Gentamicin	>512	aac(A)-aph(D)
Lincomycin	512	erm(B)
Ciprofloxacin	64	GyrA S83R, ParC S80I
Tetracycline	64	tet(M), tet(L)
Spectinomycin	64	spc
Vancomycin	2	None

To investigate the transferability of the optrA gene, conjugation and transformation experiments were performed as previously described using E. faecalis E1731 as the donor and E. faecalis JH2-2 (rifampin resistant [Rif^r]) as the recipient (3, 8). For the screening of the transconjugants and transformants, brain heart infusion (BHI) agar was supplemented with 10 mg/liter florfenicol and 50 mg/liter rifampin. However, the conjugation experiments repeatedly failed. The plasmids were then extracted from the donor E. faecalis E1731, and transfer into E. faecalis JH2-2 by electrotransformation was attempted. The transformation experiments also repeatedly failed.

To gain information about the genetic environment of the optrA gene, the whole-genomic DNA of E. faecalis E1731 was sequenced by the PacBio RS II and Illumina MiSeq platforms (Shanghai Personal Biotechnology Co., Ltd., China). The sequences from the Illumina MiSeq platform were assembled using Newbler version 2.8 (454 Life Sciences, Branford, CT, USA), and PacBio sequencing reads were de novo assembled. The prediction of open reading frames (ORFs) and their annotations were performed using Glimmer 3.0. The analysis of regions flanking the optrA gene revealed the presence of a Tn554 family transposon-like element of 12,932 bp, designated Tn6674 according to the nomenclature of transposons (https://transposon.lstmed.ac.uk/). Tn6674 consisted of seven ORFs of more than 150 amino acids (aa). The first three ORFs were indistinguishable from the genes tnpA, tnpB, and tnpC, whose products are involved in the transposition of Staphylococcus aureus transposon Tn554 (13). However, the remaining four ORFs represented the resistance genes spc (resistance to spectinomycin), erm(A) (resistance to macrolides, lincosamides, and streptogramin B antibiotics), fexA (resistance to phenicols), and optrA and replaced its counterpart in Tn554, consisting of only spc and erm(A) (Fig. 1). Sequence analysis also identified several antimicrobial resistance genes or mutations related to the respective phenotypes observed in E. faecalis E1731 (Table 1).

FIG 1.

Structural comparison of E. faecalis transposon Tn6674 with other related Tn554 family transposons. Accession numbers are as follows: Tn554, X03216; Tn5406, AF186237; Tn558, AJ715531; Tn559, FN677369; Tn6133, FR772051; Tn6188, HF565366; Tn6260, KX470419; and Tn6674, MK737778. A distance scale in kilobases is given. The transposase genes (tnpA, tnpB, and tnpC), antimicrobial and disinfectant resistance genes, and genes that code for other functions are shown in blue, red, and black, respectively. The 6-bp nucleotide sequences at the transposon junctions are shown in boxes. The disrupted radC genes are indicated by asterisks. The positions of primers circ-fw/circ-rev used to detect the presence of the circular intermediates are shown by black arrows.

An NCBI BLASTN search with the Tn6674 sequence as a query sequence showed that it displayed 99% nucleotide sequence identity (query cover, 100%) with sequences deposited in GenBank (E. faecalis A101, accession no. MH018572.1; E. faecalis TZ2, accession no. MH225421.1) (14), 99% nucleotide sequence identity (query cover, 93%) with that deposited in GenBank (E. faecalis 743142, accession no. MF443377.1) (15), and 99% nucleotide sequence identity (query cover, 100%) with those of whole-genome shotgun contigs (E. faecalis EF294, accession no. QDDM01000007.1; E. faecalis 33710, accession no. QNHF01000012.1) (16, 17). In addition, a similar structure was also described in an optrA-carrying ST16 E. faecalis strains (18).

The analysis of the regions flanking Tn6674 revealed that Tn6674 inserted into the gene radC gene, which encodes a DNA repair protein (Fig. 1). The radC gene had been described to be a common integration site for transposons of the Tn554 family, such as Tn554, Tn5406, Tn558, Tn559, Tn6133, Tn6188, and Tn6260 in various Gram-positive bacteria (19–25). In E. faecalis E1731, Tn6674 displayed the hexanucleotide sequence 5′-AATCCG-3′ at the left-end junction and the sequence 5′-GATCCT-3′ at the right-end junction, which marked the boundaries of the integrated segment. This is similar to that in the Staphylococcus lentus transposon Tn558 (21). Transposons of the Tn554 family, including Tn6674, do not contain inverted repeats at their termini and also do not generate a duplication of the target sequence at the integration site. Instead, studies on serial transposition of Tn554 into primary and secondary target sites revealed that the sequences at the junctions of Tn554 varied with respect to the target sites, as follows: with each new transposition event, the sequence originally present in the target site is found at the left terminus of Tn554, whereas the former left-end junction is now found at the right terminus, and the former right-end junction is lost (21, 26).

Tn6674 has an overall structure similar to that of other members of the Tn554 family of transposons. However, all so-far-known Tn554-like transposons differ distinctly in their resistance gene regions. These are composed of spc and erm(A) in Tn554 from S. aureus (13), a vga(A) variant in Tn5406 from S. aureus (20), fexA in Tn558 from S. lentus (21), dfrK in Tn559 from S. aureus (22), spc, erm(A), and vga(E) in Tn6133 from S. aureus (23), lnu(G) in Tn6260 from E. faecalis (25), and spc, erm(A), fexA, and optrA in Tn6674 from E. faecalis.

Previous studies have shown that the active transposons, such as Tn5406, Tn558, and Tn559, can excise from their host DNA and form circular intermediates which precede their integration into a new target site (20–22). Thus, an inverse PCR assay was set up to detect whether these circular intermediates were present in E. faecalis E1731. For this, the pair of primers circ-fw (5′-TAGATGAACCGACAAACC-3′) and circ-rev (5′-TCAACCAACCTACGAAGT-3′), with an annealing temperature of 47°C and amplicon size of 1,445 bp, was used. Sequence analysis of this amplicon consisted of 380 bp, which included part of tnpA and its upstream region at the left end of Tn6674, whereas the remaining 1,065 bp of the amplicon represented the right end of Tn6674. The presence of circular Tn6674 forms suggested that this transposon was active in E. faecalis E1731.

On the one hand, the location of optrA as part of transposon Tn6674 suggests a novel mechanism for the dissemination of optrA in enterococci, which is different from IS1216-mediated recombination that was previously described in other studies (27, 28). On the other hand, its location as an integral part of a functionally active transposon will likely accelerate the dissemination of the optrA gene. Since the optrA gene confers combined resistance to oxazolidinones used in human medicine (1) and phenicols used in food-producing animals (3, 8, 9), the prudent use of both classes of antimicrobial agents in their respective fields is urgently needed.

Data availability.

The complete sequence of Tn6674 determined in this study has been deposited in GenBank under accession number MK737778.