Retrospective analysis of genome sequences revealed the wide dissemination of optrA in Gram-positive bacteria Purchased

Sir,

Oxazolidinones, including linezolid and tedizolid, are highly effective against infections of enterococci, staphylococci and streptococci, which are clinically important Gram-positive pathogens.^1,cfr is the first gene that is transferable and causes resistance to oxazolidinones; cfr also mediates resistance to phenicols, lincosamides, pleuromutilins and streptogramin A.² Recently, Wang et al.³ first reported a novel transferable resistance gene, optrA, which confers resistance to oxazolidinones and phenicols in enterococcal isolates in China. Shortly after, the optrA gene was detected in clinical enterococcal isolates in Italy⁴ and in Staphylococcus sciuri in China.⁵

WGS is now routine and thousands of bacterial genomes are available in public databases. To investigate the prevalence of optrA in the sequenced genomes, we retrieved the optrA sequence and traced this gene in the GenBank database (Table S1, available as Supplementary data at JAC Online). We found that optrA presented in two enterococcal isolates from human blood in the USA, two environmental samples from swine manure metagenome in China and six Streptococcus suis isolates from healthy pigs in China. These results are the first reported presence of optrA in clinical enterococci in the USA, and in environmental microorganisms and streptococci in China.

optrA genes in those isolates showed 99% nucleotide identity with the original optrA from Enterococcusfaecalis pE349. Comparison of the OptrA amino acid sequences revealed seven variants with alterations at nine positions (Table S1). Further analysis showed that the genetic context of optrA in two US isolates, especially that from E. faecalis 599, was similar to that in the chromosome of E. faecalis E016 though seemingly lacking Tn558 and the Δerm(A)-like segment,⁶ and distinct from that in the plasmid of E. faecalis pE349 and S. sciuri pWo28-3 (Figure 1). The genetic contexts of optrA in two swine manure samples, IN-7 and IN-8 (Figure 1), were similar to those in plasmid pFX13,⁶ i.e. flanked by IS1216E, with the Δerm(A)-like gene located upstream instead of downstream of optrA. We also found that optrA coexisted with tet(O/W/32/O) and tet(L) in sample IN-7 and with lsa(A) and tet(O/W/32/O) in sample IN-8 (Figure 1). The genetic composition of the mosaic segments is unique and no similar structure was found in known mobile genetic elements.

Figure 1

Comparison of genomic sequences harbouring the optrA gene. The Basic Local Alignment Search Tool (BLAST) was used to compare the sequence similarity of optrA using NCBI GenBank nr/nt and WGS databases. Any isolates previously reported were excluded. Isolate information can be seen in Table S1, available as Supplementary data at JAC Online. E. faecalis E016 and pE349 served as chromosome- and plasmid-borne optrA references, respectively. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

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We found that six S.suis isolates from healthy pigs in 2011 also harbour optrA (Table S1). This is surprising as oxazolidinones, often last-resort drugs in clinical treatment, have not been proved to be used in livestock and optrA has not been reported in streptococci. All optrA-carrying segments except strain YS39 were flanked downstream by IS1216E (Figure 1). As the genetic contexts of YS35 and YS39 were not intact due to the over-truncated contigs, we only discuss the other four isolates. In isolate YS57, the ∼8.1 kb optrA contig was comprised of optrA, an upstream araC and downstream an Δerm(A)-like gene, an S-adenosylmethionine (SAM)-dependent methyltransferase (met) gene and two hypothetical genes. The optrA gene block was flanked by IS1216E at both ends in the same orientation, which was found only in plasmids before.⁶ The optrA-carrying IS1216E fragment was inserted between SNF2 and agg, two conserved genes of the 89K pathogenicity island (PAI), which presented in a strain of human S. suis outbreaks in China in 2005.⁷ However, the other conserved genes of the 89K PAI were split into other contigs due to incomplete genome assembly. Significantly, the 89K PAI belongs to the ICESa2603 family of integrative conjugative elements, which is widely distributed in and demonstrates horizontal transfer between streptococci and enterococci.⁸ Figure S1 shows the schematic genetic diagram of the optrA-carrying contig of YS57 with Streptococcus agalactiae ICESa2603 and S. suis 89K. On contigs of YS21, YS49 and YS50, the ∼7.4 kb optrA fragment contains optrA, an upstream truncated araC, a nickase gene and three hypothetical genes. Upstream of optrA, the fragment was flanked by a truncated IS1272-like element not fully sequenced (Figure 1). Downstream of optrA, the fragment was flanked by an IS3L-like element and IS1216E. The optrA fragment was integrated into a larger prophage by replacing the Mega-like element of Streptococcus pyogenes Φm46.1 and the cadA/C-tet(W) fragment of S. suis ΦSsUD.1 (Figure S1).⁹ The Φm46.1 prophage family, which was originally found integrated in the 3′-terminal part of rum loci in S. pyogenes and thereafter in S. agalactiae and S. suis, is transferable to other streptococci.¹⁰ These results suggest the important role of IS1216E in chromosomal integration of the optrA gene and highlight a potential dissemination between streptococci by mobile genetic elements of ICESa2603 and the Φm46.1 family.

The analysis suggests that the optrA gene might have been spreading in Gram-positive bacteria especially in S. suis. We screened the optrA gene in 154 S. suis isolates from routine surveillance for swine streptococcosis in south-east China. optrA was detected in three S. suis isolates (AH0906, SH0918 and NJ1112). Compared with optrA-negative strains, the strains exhibited 4- to 8-fold elevated MICs (2–4 mg/L) of linezolid. Our screen of S. suis and other streptococcal isolates is still in progress and the overall results of the survey will be described later.

Our results showed that optrA-carrying segments can be inserted into the chromosome via IS1216E elements and integrated into ICESa2603 and Φm46.1—a process that plays a role in the chromosomal dissemination of optrA among Gram-positive bacteria. Based on the occurrence of optrA sampled from humans, animals and the environment in China, Italy and the USA, the worldwide distribution of optrA might be underestimated. Therefore, routine surveillance for the presence of optrA in Gram-positive bacteria is warranted.

Funding

This study was supported by the National Natural Science Foundation of China (No. 31572567) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Transparency declarations

None to declare.

Supplementary data

Table S1 and Figure S1 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).

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