Emergence of a clinical Salmonella enterica serovar 1,4,[5], 12: i:-isolate, ST3606, in China with susceptibility decrease to ceftazidime-avibactam carrying a novel blaCTX-M-261 variant and a blaNDM-5

Abstract

Purpose: The detection rate of Salmonella enterica serovar 1,4,[5], 12: i: - (S. 1,4,[5], 12: i: -) has increased as the most common serotype globally. A S. 1,4,[5], 12: i: - strain named ST3606 (sequence type 34), isolated from a fecal specimen of a child with acute diarrhea hospitalized in a tertiary hospital in China, was firstly reported to be resistant to carbapenem and ceftazidime-avibactam. The aim of this study was to characterize the whole-genome sequence of S. 1,4,[5], 12: i: - isolate, ST3606, and explore its antibiotic resistance genes and their genetic environments.

Methods: The genomic DNA of S. 1,4,[5], 12: i: - ST3606 was extracted and performed with single-molecule real-time sequencing. Resistance genes, plasmid replicon type, mobile elements, and multilocus sequence types (STs) of ST3606 were identified by ResFinder 3.2, PlasmidFinder, OriTfinder database, ISfinder database, and MLST 2.0, respectively. The conjugation experiment was utilized to evaluate the conjugation frequency of pST3606-2. Protein expression and enzyme kinetics experiments of CTX-M were performed to analyze hydrolytic activity of a novel CTX-M-261 enzyme toward several antibiotics.

Results: Single-molecule real-time sequencing revealed the coexistence of a 109-kb IncI1-Iα plasmid pST3606-1 and a 70.5-kb IncFII plasmid pST3606-2. The isolate carried resistance genes, including bla_NDM-5, sul1, qacE, aadA2, and dfrA12 in pST3606-1, bla_TEM-1B, aac(3)-lld, and bla_CTX-M-261, a novel bla_CTX-M-1 family member, in pST3606-2, and aac(6')-Iaa in chromosome. The bla_CTX-M-261 was derived from bla_CTX-M-55 by a single-nucleotide mutation 751G>A leading to amino acid substitution of Val for Met at position 251 (Val251Met), which conferred CTX-M increasing resistance to ceftazidime verified by antibiotics susceptibility testing of transconjugants carrying pST3606-2 and steady-state kinetic parameters of CTX-M-261. pST3606-1 is an IncI1-α incompatibility type that shares homology with plasmids of pC-F-164_A-OXA140, pE-T654-NDM-5, p_dm760b_NDM-5, and p_dmcr749c_NDM-5. The conjugation experiment demonstrated that pST3606-2 was successfully transferred to the Escherichia coli recipient C600 with four modules of OriTfinder.

Conclusion: Plasmid-mediated horizontal transfer plays an important role in bla_NDM-5 and bla_CTX-M-261 dissemination, which increases the threat to public health due to the resistance to most β-lactam antibiotics. This is the first report of bla_CTX-M-261 and bla_NDM-5 in S. 1,4,[5], 12: i: -. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of CTX-M enzymes and confirms urgency to control resistance of S. 1,4,[5], 12: i: -.

Keywords: Bla CTX-M-261; Bla NDM-5; Salmonella enterica serovar 1,4,[5]; 12: i: -; Ceftazidime-avibactam; IncI1-I(α).

Fig. 1

Differentiation of diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium (S. 1,4,[5], 12: i: -). A 1389-bp product from Salmonella Typhimurium that possesses a phase 2 flagellar antigen and no product from S. 1,4,[5], 12: i:—that lacks a phase 2 flagellar antigen

Fig. 2

A 10.9-kb IncI1-α sequence of the genetic context of the NDM-5-harboring plasmid pST3606-1 is shown. Linear genetic structure comparison of plasmid pST3606-1 (Salmonella Typhimurium (S. 1,4,[5], 12: i: -), GenBank accession number CP094333), pC-F-164_A-OXA140 (Escherichia coli, GenBank accession number CP048368), pE-T654-NDM-5 (Escherichia coli, GenBank accession number CP090291), p_dm760b_NDM-5 (Klebsiella pneumoniae, GenBank accession number CP095648), and p_dmcr749c_NDM-5 (Citrobacter sedlakii, GenBank accession number CP095669). The arrows indicate open reading frames. The red arrows represent antibiotic resistance gene. The blue arrows represent the transposon and IS elements. The yellow arrows represent Integrase gene. Light green shading denotes homology regions. The depth of shadowing is indicative of the BLASTn matching degree. IS, insertion sequences. Color images are available online. Comparisons performed using a nucleotide basic local alignment sequence tool search and visualized using in silico molecular cloning genomics edition software

Fig. 3

Circular representation of pST3606-2. The innermost circle represents GC content; the second inner circle represents GC skew (green, GC + ; purple, GC-). These mcr-8-carrying plasmids pST3606-2 (Salmonella Typhimurium (S. 1,4,[5], 12: i: -), GenBank accession number CP094334), p51015_CTX_M_15 (Klebsiella pneumoniae, GenBank accession number CP050379), pST90-1 (Salmonella enterica, GenBank accession number CP050735), pEC31-1 (Escherichia coli, GenBank accession number CP056037), and plasmid unnamed 2 (Shigella flexneri, GenBank accession number CP058841) were represented by pink, light blue, purple, dark blue, and yellow circles, respectively. The outermost red circle depicts gene distribution of the resistance gene, transposon, IS elements, and integrative and conjugative element. bla_CTX-M-261, CTX-M-261 extended spectrum β-lactamase gene; aac(3)-lld gene, aminoglycoside resistance gene; ATPase, adenosine triphosphate synthase; hypothetical protein

Fig. 4

The protein expression levels of CTX-M-1/55/261 in E. coli BL21 and E. coli TOP10 were compared