Microbiological features of strain ZT01-0079
Strain ZT01-0079 was identified as C. freundii. Susceptibility testing showed that C. freundii ZT01-0079 exhibited resistance to aztreonam, nitrofurantoin, ciprofloxacin, piperacillin, cefepime, ceftriaxone, ceftazidime, tobramycin, levofloxacin, and imipenem. S1-PFGE revealed that C. freundii ZT01-0079 contained three plasmids (~143kb, ~89kb and ~53kb) (Figure 1). Electrophoresis and Southern blotting showed that the blaNDM-1 gene was located on the ~53kb plasmid. The NDM-1-carrying plasmid was successfully transferred at a frequency of 1.68×10−3. Transconjugants acquired resistance to ciprofloxacin, piperacillin, cefepime, ceftriaxone, ceftazidime, cefazolin, and imipenem (Table 1). In silico MLST found that C. freundii ZT01-0079 belongs to sequence type (ST)19. In addition to blaNDM-1, C. freundii ZT01-0079 carried multiple resistance genes including blaSHV-12, blaCTX-M-55, blaCMY-152, fosA3, aph (3 '')-Ib, aph (6) -Id, aph (3 '')-Ib, aph (6)–Id and sul2. The blaCMY-152 gene was located on the chromosome, while the other genes were located on plasmids. The 89kb plasmid carried fosA3, blaCTX-M-55, sul2, aph(3``)-Ib and aph(6)-Id genes, whereas the 143kb plasmid carried faph(3'')-Ib, aph(6)-Id and sul2 genes. Plasmid pZY-NDM1 had a length of 53kb, and carried both the blaNDM-1 and blaSHV-12 genes. All three plasmids belonged to type IncX3.
Genetic analysis of pZY-NDM1
Plasmid pZY-NDM1 is a 53573 bp circular plasmid with an average GC content of 49.24% and has 76 open reading frames. pZY-NDM1 belongs to type IncX3. BLAST comparison disclosed that the genome sequence of pZY-NDM1 shared >99% similarity with plasmids pNDM-HK3694 (Genbank accession JX104760.1) and p309074-NDM (Genbank accession MH909346.1). These three plasmids share a typical IncX plasmid backbone composed of replication (repB); plasmid stability (parA, parB); plasmid maintenance (hns, topB); and type IV secretion system (taxA, taxB, taxC and pilx1-pilx11) encoding regions. The 19.66 kb genetic load region of pZY-NDM1 located between hns and resolvease genes contained Tn3, IS3000, the transposon Tn125 encoding blaNDM-1, and a mobile element containing blaSHV-12 and ISL3. The transposon Tn125 served as a mobile element for the transfer of blaNDM-1 and comprised insE (locus tag CS291_RS00115), groEL (locus tag CS291_RS00115), tat (locus tag CS291_RS00130), ΔtrpF (locus tag CS291_RS00135), the bleomycin resistance gene ble (locus tag CS291_RS00140), ΔISAba125, blaNDM-1 (locus tag CS291_RS00145), dct (locus tag CS291_RS00125), and groES (locus tag CS291_RS00120) as previously described [25]. Compared with the prototype Tn125, ΔISAba125 was located upstream of IS5 in the p309074-NDM plasmid, and had a length of 891bp, while ΔISAba125 was deleted in plasmids pZY-NDM1 and pNDM-HK3694.
The blaSHV-12-carrying transposon was located in two opposite IS26 elements and was composed of ΔygbJ (locus tag, CS291_RS00085), ygbI (locus tag, CS291_RS00080) and blaSHV-12 (locus tag, CS291_RS00075). Compared with pNDM-HK3694, the blaSHV-12-carrying transposon of pZY-NDM1 was reversed. The umuD gene was split into two genes, ΔumuD1 and ΔumuD2, which may serve as an insertion sites of regions containing the blaSHV-12-carrying transposons Tn125-like transposon, IS3000 and tnpA.