Salmonella enterica serovar Typhimurium ST34 co-expressing blaNDM-5 and blaCTX-M-55 isolated in China

Emerging Microbes & Infections (2017) 6, e61; doi:10.1038/emi.2017.48; published online 12 July 2017

the transconjugants acquired resistance to imipenem, ampicillin and amoxicillin/clavulanic acid (Supplementary Table S1). Unexpectedly, resistance to cephalosporin antibiotics was also observed. Subsequent sequencing revealed that the cephalosporin resistance gene bla CTX-M-55 was located on the large plasmid, which was simultaneously transferred to the transconjugants. The presence of the bla NDM-5 and bla CTX-M-55 genes in the transconjugants was demonstrated by PCR amplification.
The whole genome of S. Typhimurium SSH006 was sequenced by the Novogene Company (Beijing, China) on the HiSeq 2500 platform. Paired-end reads of 350 bp were assembled using SOAPdenovo (v2.04) with 109-fold coverage. Multi-locus sequence typing analysis showed that SSH006 belongs to sequence-type 34 (ST34). In addition to bla NDM-5 , multiple resistance genes were identified, including bla TEM-1 , bla CTX-M-55 , aadA1, sul1, sul2, sul3, aac(6')-Iaa, tet(A), floR and dfrA12. Interestingly, there were two bla TEM-1 genes: one located on the chromosome and the other truncated and located adjacent to the bla CTX-M-55 gene. A BLAST search indicated that the bla CTX-M-55carrying contig covered 5 kb and consisted of a novel combination of K. pneumoniae plasmid pKP09085 3 and Shewanella sp. ANA-3 plasmid 1 (CP000470, unpublished).
The complete sequence of the bla NDM-5 -carrying plasmid pNDM5-SSH006 is 46 253 bp in length and shares 499% identity with the IncX3 plasmid pNDM_MGR194 that was isolated in India, 4 with 17 nucleotide changes. Twelve of the 17 nucleotide changes are located within the truncated ctuA1 gene, and one is located in the insertion sequence IS26 downstream of the bla NDM-5 gene. In addition to these 13 nucleotide variations, the genetic context of bla NDM-5 in the two plasmids is identical (ISSwil-IS3000-ΔISAba125-IS5-bla NDM-5 -ble-trpF-tat-ΔctuA1-IS26-ΔumuD). The shotgun whole-genome sequence and complete sequence of plasmid pNDM5-SSH006 have been deposited in GenBank under accession number MTKV00000000.
A variety of bla NDM-5 -harboring plasmids have been identified and found to share similar sequences with plasmid pNDM_MGR194, such as pEc1929, 5 pNDM5_0215, 6 pECNDM101, 7 and pNDM5-IncX3, 8 pNDM-QD28 and pNDM-QD29. 9 Most of these have been isolated in China (Supplementary Table S2). All of the above plasmids had the same genetic context of NDM except pNDM5_0215, which had an insertion of IS5 within the truncated insertion sequence IS3000 (Figure 1). Notably, the bla NDM-5 -carrying IncN plasmid pTK1044 from Japan also shared a similar NDM genetic environment with a deletion of the truncated ISAba125 but was~110 kb in length, 10 thus suggesting a potential dissemination of NDM-5 via mobile genetic elements. Plasmids pJEG027 harboring bla NDM-4 11 and pKpN01-NDM7 harboring bla NDM- 7 12 were also found to be almost identical to pNDM5-SSH006 except for the variation of the NDM gene. The bla NDM-7 -harboring IncX3 plasmid pOM26-1 (KP776609, unpublished) is similar to pNDM5-SSH006, except that it has a deletion of ISAba125.
Given that bla NDM-5 and bla NDM-7 differ from bla NDM-4 by a single nucleotide change (G388A and G262T, respectively), the documentation of travel to India indicated that the bla NDM-5 -harboring pNDM_MGR194 and the bla NDM-7 pKpN01-NDM7 plasmid may have evolved from the bla NDM-4 -harboring pJEG027 plasmid, 12 thus raising particular concern regarding their epidemic potential to mediate rapid spread of NDM. However, the pNDM5-SSH006-like plasmids were observed in diverse species at different geographical locations across China, in the absence of documented travel history or epidemiological linkage. The increased occurrence of this highly similar IncX3 plasmid indicated the existence of a natural reservoir and rapid transmission of the plasmid in China.
To the best of our knowledge, this is the first report of S. Typhimurium carrying the bla NDM-5 gene. The bla NDM-5 -carrying plasmids have been reported in a narrow host range in E. coli, K. pneumoniae and recently P. mirabilis. 13 Our work further expands the host range and provides additional evidence of the rapid dissemination of bla NDM-5 among different species of Enterobacteriaceae in China. Given that NDM-5 differs from NDM-1 by two amino-acid substitutions and confers increased resistance to expanded-spectrum cephalosporins and carbapenems, 1 the ability of this bla NDM-5 -harboring plasmid to transfer across species boundaries may pose a great threat to humans and is quite worrisome. S. Typhimurium ST34 clones have raised international concern regarding its rapid spread and multi-drug resistance. A previous study has revealed that S. Typhimurium ST34 clones experienced a rapid expansion in China and exhibit a low percentage susceptibility to cephalosporin antibiotics. 14 However, S. Typhimurium ST34 SSH006 exhibited higher resistance to all tested cephalosporin antibiotics.
Here we report the first case of S. Typhimurium ST34 SSH006 harboring the bla NDM-5 gene. The co-existence of two transferable plasmids carrying bla CTX-M-55 and bla NDM-5 in S. Typhimurium highlights the urgent need for more extensive surveillance and effective action to control its further dissemination.