Novel tigecycline resistance gene cluster tnfxB3-tmexCD3-toprJ1b in Proteus spp. and Pseudomonas aeruginosa, co-existing with tet(X6) on an SXT/R391 integrative and conjugative element.

OBJECTIVES: To characterize a novel MDR efflux pump gene cluster tnfxB3-tmexCD3-toprJ1b carried by Proteus spp. and Pseudomonas aeruginosa strains from chickens. METHODS: Antimicrobial susceptibility testing, conjugation and WGS were performed to characterize tnfxB3-tmexCD3-toprJ1b-positive isolates. Cloning and reverse transcription-quantitative PCR were performed to investigate the function of tnfxB3-tmexCD3-toprJ1b. RESULTS: The WGS data revealed that a novel efflux pump gene cluster, tnfxB3-tmexCD3-toprJ1b, was identified on the chromosome of the Proteus cibarius strain SDQ8C180-2T, where an SXT/R391-family integrative and conjugative element (ICE) was found to co-carry tet(X6) and tnfxB3-tmexCD3-toprJ1b. Further retrospective analysis found two other tnfxB3-tmexCD3-toprJ1b variants in a Proteus mirabilis isolate and a P. aeruginosa isolate, respectively. tmexCD3-toprJ1b and its variants increased the MICs of tigecycline (8-fold) and other antibiotics (2-8-fold) in Escherichia coli host strains. The TNfxB3 protein down-regulated the expression of the tmexCD3-toprJ1b operon. Moreover, genetic-context analyses showed that tnfxB3-tmexCD3-toprJ1b together with adjacent integrase genes appeared to compose a transferable module 'int1-like+int2-like+hp1+hp2+ISCfr1+tnfxB3-tmexCD3-toprJ1b', which was inserted into the umuC-like gene of this ICE. Further analysis of the tnfxB3-tmexCD3-toprJ1b-harbouring sequences deposited in GenBank revealed similar transferable modules inserted into umuC-like genes in plasmids or chromosomes of Klebsiella pneumoniae, Pseudomonas spp. and Aeromonas spp., implying that these modules could be transferred across different bacterial species. CONCLUSIONS: To the best of our knowledge, this is the first identification of a novel tigecycline gene cluster, tmexCD3-toprJ1b, which co-exists with tet(X6) within an ICE. More attention should be paid to the co-transfer of these two tigecycline resistance determinants via an ICE to other Gram-negative bacteria.

Clonal spread of Escherichia coli O101: H9-ST10 and O101: H9-ST167 strains carrying fosA3 and bla CTX-M-14 among diarrheal calves in a Chinese farm, with Australian Chroicocephalus as the possible origin of E. coli O101: H9-ST10.

During a 2018 antimicrobial resistance surveillance of Escherichia coli isolates from diarrheal calves in Xinjiang Province, China, an unexpectedly high prevalence (48.5%) of fosfomycin resistance was observed. This study aimed to reveal the determinants of fosfomycin resistance and the underlying transmission mechanism. Polymerase chain reaction (PCR) screening showed that all fosfomycin-resistant E. coli carried the fosA3 gene. Pulsed-field gel electrophoresis (PFGE) and southern blot hybridization revealed that the 16 fosA3-positive isolates belonged to four different PFGE patterns (i.e., A, B, C, D). The fosA3 genes of 11 clonally related strains (pattern D) were located on the chromosome, while others were carried by plasmids. Whole-genome and long-read sequencing indicated that the pattern D strains were E. coli O101: H9-ST10, and the pattern C, B, and A strains were O101: H9-ST167, O8: H30-ST1431, and O101: H9 with unknown ST, respectively. Among the pattern C strains, the bla CTX-M-14 gene was co-localized with the fosA3 gene on the F18: A-: B1 plasmids. Interestingly, phylogenetic analysis based on core genome single nucleotide polymorphisms (cgSNPs) showed that the O101: H9-ST10 strains were closely related to a Australian-isolated Chroicocephalus-origin E. coli O101: H9-ST10 strain producing CTX-M-14 and FosA3, with a difference of only 11 SNPs. These results indicate possible international dissemination of the high-risk E. coli clone O101: H9-ST10 by migratory birds.

A Novel Transferable Resistance-Nodulation-Division Pump Gene Cluster, tmexCD2-toprJ2, Confers Tigecycline Resistance in Raoultella ornithinolytica.

We recently identified a novel plasmid-mediated resistance-nodulation-division (RND)-type efflux pump gene cluster, tmexCD1-toprJ1, in Klebsiella pneumoniae that conferred resistance to multiple antimicrobials, including tigecycline. While homologs of tmexCD1-toprJ1 were found encoded in many other bacterial species in GenBank, their functions and transfer mechanisms remain unknown. This study identified another mobile gene cluster, tmexCD2-toprJ2, co-occurring on both a plasmid (pHNNC189-2) and the chromosome of a clinical Raoultella ornithinolytica isolate, strain NC189, producing KPC-2, NDM-1, and RmtC. tmexCD2-toprJ2 shares high similarity at the nucleotide level with tmexCD1-toprJ1, with 98.02%, 96.75%, and 99.93% identities to tmexC1, tmexD1, and toprJ1, respectively. Phylogenetic analysis revealed that tmexCD2-toprJ2 may have originated from the chromosome of a Pseudomonas species. The expression of tmexCD2-toprJ2 in an Escherichia coli strain resulted in an 8-fold increase in the tigecycline MIC and decreased susceptibility to other antimicrobials. Genetic context analyses demonstrated that tmexCD2-toprJ2, together with the adjacent hypothetical site-specific integrase genes, was possibly captured and mobilized by a XerD-like tyrosine recombinase system, forming a putative transposition unit (xerD-like-int3-like-thf2-ybjD-umuD-ΔumuC1-int1-like-int2-like-hp1-hp2-tnfxB2-ISBvi2-tmexCD2-toprJ2-ΔumuC1), which was inserted into umuC-like genes in both the NC189 plasmid pHNNC189-2 and the chromosome. Since tmexCD1-toprJ1 and tmexCD2-toprJ2 could confer multidrug resistance, the spread of these gene clusters, associated with the new recombinase system, calls for more attention.