3.1 Bacterial Strains and Antimicrobial Susceptibility Testing
Seven blaNDM-5-positive isolates were recovered from seven hospitalized patients from three sources in three different cities in China. EBJ001 and EBJ003 were isolated from Beijing, ESY001, ESY002, and ESY003 from Shenyang, EJN001 and EJN003 from Jinan. (Table 1). None of the patients had ever traveled abroad. All E. coli strains were resistant to carbapenems, cephalosporins, quinolones, aztreonam, ampicillin, and sulfamethoxazole/trimethoprim, and were mostly susceptible to amikacin (Table 2).
3.2 Genetic Relatedness of all Strains
MLST analysis revealed that strain EBJ003 belonged to ST405, which fell into phylogenetic group D and was distinguishable from other strains by PFGE. The other six strains belonged to ST167 and phylogenetic group A (Fig. 1). ESY001 and ESY002 were classified into the same pulsotype, whereas EJN001, EJN003, ESY003, and EBJ001 were classified into different pulsotypes (Fig. 2), suggesting both clonal and non-clonal dissemination.
3.3 Virulence and Resistance Genes in All Strains
The seven strains harbored type II and III secretion systems. Certain virulence factors were exclusively observed in EBJ003, such as fim (encoding type I fimbriae regulatory proteins), iuc (encoding aerobactin siderophore biosynthesis proteins), and irp and ybt (encoding yersiniabactin biosynthetic proteins) genes.
In addition to blaNDM-5, other antimicrobial resistance genes were also frequently identified in the seven E. coli strains including blaCTX-M, blaTEM, aac(3)-IIa, aada5, dfrA7 and mphA. The coexistence of a blaCTX-M-14 gene and a blaCTX-M-55 gene was observed in EBJ003 (Table 1). A blaCTX-M-24 gene was identified in EBJ001, and blaCTX-M-55 was identified in EJN003, whereas other strains harbored a single blaCTX-M-14. Also, blaCMY-42 was detected in EJN001 and EBJ001, and blaOXA-1 was identified in EBJ003. Strain ESY001 harbored two blaTEM variants designated as blaTEM-230 and blaTEM-231. The deduced protein sequence of blaTEM-230 had a single amino acid substitution at position 233 (Ser→Thr) relative to its closest homolog TEM-168. The blaTEM-230 gene was located on an IncX6 plasmid and inserted between the TivB5 and TivB6 genes related to type IV secretion systems. The blaTEM-231 gene was located on an IncFIB plasmid and inserted between a Tn3 mobile element and sul2.
3.4 Characteristics of the blaNDM−5-Carrying Plasmids
In this study, all seven strains successfully transferred the blaNDM−5 gene to the recipient with high frequency ranging from 6.56 × 10-3 to 1.33 × 10-2 per donor cell. The transfer of the blaNDM-5 gene and carbapenem resistance to the transconjugants was confirmed by PCR amplification and antimicrobial susceptibility testing. S1-PFGE and subsequent Southern hybridization revealed that blaNDM−5 was carried by plasmids of the same size (∼45 kb) (Fig.3)
Sequence analysis revealed that the blaNDM-5-carrying plasmid was identical in ESY001 (pNDM5-ESY001), ESY002 (pNDM5-ESY002), ESY003 (pNDM5-ESY003), EBJ001 (pNDM5-EBJ001), EBJ003 (pNDM5-EBJ003), and EJN001 (pNDM5-EJN001). pNDM5-ESY001 was 46,161 bp in length and had only two single nucleotide substitutions compared with the blaNDM-5-carrying plasmid pNDM5-EJN003 in EJN003, and the two single nucleotide substitutions were located within VirB4 and the non-coding region, respectively. A BLASTn search against the NCBI database revealed that pNDM5-ESY001 shared 100% coverage and >99% identity with plasmid pNDM5-SSH006 from Shanghai, China[20]. A single nucleotide substitution located within a truncated cutA1 gene downstream of blaNDM-5 was identified. A variety of blaNDM-5-harboring plasmids show high similarity with the plasmid pNDM5-ESY001 including pNDM_MGR194[21], pEc1929[22], pNDM-QD28, pNDM-QD29[15], pECNDM101, and pNDM5-IncX3[23]. The nucleotide differences among these plasmids were mostly located within insertion sequences and genes associated with type IV secretion systems, partition, and DNA distortion (Fig. 4). The plasmids pJEG027[24] carrying blaNDM-4 and pKpN01-NDM7[25] carrying blaNDM-7 were also highly identical with pNDM5-ESY001 except for the variation of blaNDM alleles.
All the above plasmids shared the same genetic context of the blaNDM-5 gene (ISSwil-IS3000-ΔISAba125-IS5-blaNDM-5-ble-trpF-tat-ΔctuA1-IS26-ΔumuD) (Fig. 5). pNDM5_0215[26], which was isolated from Chengdu, China, had an insertion of IS5 into IS3000. Another blaNDM-5-harboring IncX3 plasmid, pP744T-NDM5[27], from China had a similar NDM genetic context except for the deletion of a segment of the truncated ISAba125, whereas pTK1044[28] from Japan had a shorter ISAba125 remnant of 73bp, suggesting a possible dissemination of blaNDM-5 via mobile elements. A recently identified fusion plasmid pBJ114T-190[29] comprising both IncFIB and IncX3 replicons from China lost the ISSWil and IS3000 upstream of the blaNDM-5 gene, suggesting the continuing variation of the blaNDM-5 genetic context.