The origin and evolution of IncF33 plasmids based on large-scale data sets.

IMPORTANCE: Plasmids that capture multiple antibiotic resistance genes are spreading widely, leading to the emergence and prevalence of multidrug-resistant bacteria. IncF33 plasmids are a newly emerged plasmid type highly prevalent in animal-source Enterobacterales in China, and they are important vectors for transmitting several clinically important antibiotic resistance genes. The study revealed that the IncF33 plasmid is mainly prevalent in China animal-derived Escherichia coli and has the potential for cointegration and intercontinental dissemination. Therefore, it is crucial to enhance surveillance and control measures to limit the spread of IncF33 plasmids and their associated antibiotic resistance genes.

Persistence and molecular epidemiology of blaNDM-positive Gram-negative bacteria in three broiler farms: A longitudinal study (2015-2021).

Although carbapenems have not been approved for animal use, blaNDM-positive bacteria (NPB) are increasingly being detected in farm animals. It is important to investigate the routes and underlying mechanisms of evolution and transmission of animal-borne NPB. In this study, NPB recovered from chicken feces and environmental samples in three adjacent broiler farms were investigated. We found that 13.0% of Escherichia coli strains recovered from chicken feces during the period 2015-2016 carried the blaNDM gene. In 2017-2021, however, as many as 55.8% chicken and environmental samples collected during the breeding period were found to harbor NPB. Importantly, such strains were detectable in samples from farmland (10.3%, 8/78), vegetable fields (7.3%, 3/41), and environment of chicken farms (25.6%, 41/160) which had been left vacant for a long period of time. Intriguingly, different sequence types of NPB became dominant in different years. Both clonal dissemination of NPB and horizontal transmission of blaNDM-bearing plasmids were observed among different farms and among the environment niches inside and outside the farm houses. Worryingly, transmission of NPB and blaNDM-bearing plasmids between these farms and other places was also observed. All in all, our results suggested the persistence of NPB in chickens and farm environments, presumably due to extensive contamination by exogenous materials and transmission of NPB within the farm system. These events were aggravated by the increase in antibiotic usage and poor sanitary conditions in the farm houses. Stringent control measures should be implemented to arrest transmission of animal-borne NPB to the environment and the community.

IS26-Mediated Formation of a Hybrid Plasmid Carrying mcr-1.1.

Purpose: The objective of this study was to elucidate the characteristics and mechanism of formation of the fusion plasmid pHNSHP24 carrying mcr-1.1. Materials and Methods: mcr-1.1-bearing Escherichia coli SHP24 and the corresponding transconjugant were subjected to whole-genome sequencing (WGS) combining the Illumina and MinION platforms to obtain the complete sequences of the fusion plasmid and its original plasmids. Results: Complete sequence analysis and S1 nuclease-pulsed field gel electrophoresis (S1-PFGE) results indicated that E. coli SHP24 carried four plasmids: mcr-1.1-harboring phage-like plasmid pHNSHP24-3, F53:A-:B- plasmid pHNSHP24-4, pHNSHP24-1, and pHNSHP24-2. However, the plasmid pHNSHP24 carrying mcr-1.1 presents in the transconjugant differed from the four plasmids in the donor strain SHP24. Further analysis showed that pHNSHP24 may be the fusion product of pHNSHP24-3 and pHNSHP24-4 and is formed through a replicative transposition mechanism mediated by IS26 in E. coli SHP24. Conclusion: This study is the first to report the fusion of an mcr-1.1-harboring phage-like pO111 plasmid and an F53:A-:B- plasmid mediated by IS26. Our findings revealed the role of phage-like and fusion plasmids in the dissemination of mcr-1.1.

Emergence of a Novel Plasmid-Mediated Tigecycline Resistance Gene Cluster, tmexCD4-toprJ4, in Klebsiella quasipneumoniae and Enterobacter roggenkampii.

The occurrence of transferable tigecycline resistance determinants, tmexCD1-toprJ1, tmexCD2-toprJ2, tmexCD3-toprJ1b, and multiple tet(A) and tet(X) variants, presents an unprecedented challenge to clinical therapeutic options. tmexCD-toprJ-like gene clusters can mediate multidrug resistance and have been detected in a variety of bacteria. Here, we characterized the fourth tmexCD-toprJ-like gene cluster, tmexCD4-toprJ4, identified on untypeable plasmids of Klebsiella quasipneumoniae and Enterobacter roggenkampii isolated from chicken meat and environmental samples from farm markets, respectively. TMexCD4-TOprJ4 was closely related (92 to 99% amino acid identity) to TMexCD1-TOprJ1, TMexCD2-TOprJ2, and TMexCD3-TOprJ1. Phylogenetic analysis revealed that tmexCD4-toprJ4 was not in the same branch as the other three variants. Expression of tmexCD4-toprJ4 increased tigecycline efflux in Escherichia coli and resulted in a 4- to 8-fold increase in MICs of tigecycline in E. coli and Klebsiella pneumoniae. Moreover, tmexCD4-toprJ4 can act synergistically with its upstream gene tet(A) to reduce the susceptibility of E. coli and K. pneumoniae strains to tigecycline. The tmexCD4-toprJ4-containing plasmid is a novel plasmid type and can be transferred to E. coli and K. pneumoniae only via electrotransformation. The increasing emergence of plasmid-mediated tigecycline resistance gene clusters suggests that the spread of tmexCD-toprJ-like gene clusters requires widespread attention. IMPORTANCE The plasmid-mediated tigecycline resistance gene cluster tmexCD1-toprJ1 and other variants have been detected in a variety of strains from multiple sources, including human-derived strains. In addition to tigecycline, these tmexCD-toprJ-like gene clusters reduce susceptibility of the host strain to many other antimicrobials. Here, we identified tmexCD4-toprJ4 in K. quasipneumoniae and E. roggenkampii, suggesting that this gene cluster is already present in the human-associated environment and the risk of transmission to humans is increased. Monitoring tigecycline-resistant Gram-negative bacteria is essential for understanding and addressing the spread of this gene cluster in agriculture and health care.

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.