Clonal spread of blaNDM-1-carrying Salmonella enterica serovar Typhimurium clone ST34 and wide spread of IncHI2/ST3-blaNDM-5 plasmid in China.

OBJECTIVES: To characterize blaNDM-carrying Salmonella recovered from a pig slaughterhouse. METHODS: In this study, 46 environment samples were collected from a slaughterhouse in China, and screened for carbapenem-resistant Enterobacterales. WGS, antimicrobial susceptibility testing and conjugation experiments were carried out to identify the isolates' resistance phenotypes and genetic characteristics. The phylogenetic relatedness of the Salmonella isolates obtained in this study and Salmonella (ST34 and ST29) in GenBank was determined. RESULTS: Two ST34 Salmonella Typhimurium and one ST29 Salmonella Stanley, recovered from three environmental samples (6.52%), were positive for blaNDM-1 and blaNDM-5, respectively. The two ST34 S. Typhimurium strains exhibited a close relationship (10-36 SNPs) with two human-derived blaNDM-1-bearing isolates from China (Hong Kong and Guangxi Province) and two blaNDM-negative ST34 Salmonella strains from the UK. The blaNDM-1 genes were located on IncHI2/ST3 plasmids. The capture of blaNDM-1 by the IncHI2/ST3 plasmid seems to be due to homologous recombination mediated by circular structures, as the genetic arrangements of the blaNDM-1 gene contain two IS26 elements of the same orientation. The blaNDM-5 gene was also carried by the IncHI2/ST3 plasmid, which shares highly similar structures with other blaNDM-5-bearing IncHI2/ST3 plasmids from other sources (fish, chicken, duck, human). CONCLUSIONS: This is the first report of a blaNDM-5-carrying IncHI2/ST3 plasmid in Salmonella. The clonal spread of NDM-1-producing ST34 S. Typhimurium across human and animal-associated environments, and the widespread dissemination of epidemic blaNDM-5-carrying IncHI2/ST3 plasmids among Enterobacteriaceae in China indicate the potential of further dissemination of blaNDM among Salmonella, which poses a threat to public health.

Flavomycin restores colistin susceptibility in multidrug-resistant Gram-negative bacteria.

Polymyxin is used as a last resort antibiotics for infections caused by multi-drug resistant (MDR) Gram-negative bacteria and is often combined with other antibiotics to improve clinical effectiveness. However, the synergism of colistin and other antibiotics remains obscure. Here, we revealed a notable synergy between colistin and flavomycin, which was traditionally used as an animal growth promoter and has limited activity against Gram-negative bacteria, using checkerboard assay and time-kill curve analyses. The importance of membrane penetration induced by colistin was assessed by examining the intracellular accumulation of flavomycin and its antimicrobial impact on Escherichia coli (E. coli) strains with truncated lipopolysaccharides. Besides, a mutation in the flavomycin binding site was created to confirm its role in the observed synergy. This synergy is manifested as an augmented penetration of the E. coli outer membrane by colistin, leading to increased intracellular accumulation of flavomycin and enhanced cell killing thereafter. The observed synergy was dependent on the antimicrobial activity of flavomycin, as mutation of its binding site abolished the synergy. In vivo studies confirmed the efficacy of colistin combined with flavomycin against MDR E. coli infections. This study is the first to demonstrate the synergistic effect between colistin and flavomycin, shedding light on their respective roles in this synergism. Therefore, we propose flavomycin as an adjuvant to enhance the potency of colistin against MDR Gram-negative bacteria. IMPORTANCE: Colistin is a critical antibiotic in combating multi-drug resistant Gram-negative bacteria, but the emergence of mobilized colistin resistance (mcr) undermines its effectiveness. Previous studies have found that colistin can synergy with various drugs; however, its exact mechanisms with hydrophobic drugs are still unrevealed. Generally, the membrane destruction of colistin is thought to be the essential trigger for its interactions with its partner drugs. Here, we use clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) for specifically mutating the binding site of one hydrophobic drug (flavomycin) and show that antimicrobial activity of flavomycin is critical for the synergy. Our results first give the evidence that the synergy is set off by colistin's membrane destruction and operated the final antimicrobial function by its partner drugs.

Promoter regulatory mode evolution enhances the high multidrug resistance of tmexCD1-toprJ1.

Antibiotic resistance could rapidly emerge from acquiring the mobile antibiotic resistance genes, which are commonly evolved from an intrinsic gene. The emergence of the plasmid-borne mobilized efflux pump gene cluster tmexCD1-toprJ1 renders the last-resort antibiotic tigecycline ineffective, although its evolutionary mechanism remains unclear. In this study, we investigate the regulatory mechanisms of the progenitor NfxB-MexCD-OprJ, a chromosomally encoded operon that does not mediate antibiotic resistance in the wild-type version, and its homologs, TNfxB1-TMexCD1-TOprJ1 mediating high-level tigecycline resistance, and TNfxB3-TMexCD3-TOprJ1. Mechanistic studies demonstrated that in nfxB-mexCD-oprJ, MexCD expression was under a weaker promoter, PmexC and inhibited by a strong repressor NfxB. For tmexCD1-toprJ1, TMexCD1 was highly expressed owing to the presence of a strong promoter, PtmexC1, and an inactive suppressor, TNfxB1, with a T39R mutation that rendered it unable to bind to promoter DNA. In tnfxB3-tmexCD3-toprJ1b, TMexCD3 expression was intermediate because of the local regulator TNfxB3, which binds to two inverted repeat sequences of PtmexC. Additionally, TNfxB3 exhibited lower protein expression and weaker DNA binding affinity than its ancestor NfxB, together with their promoter activities difference explaining the different expression levels of tmexCD-toprJ homologs. Distinct fitness burdens on these homologs-carrying bacteria were observed due to the corresponding expression level, which might be associated with their global prevalence. In summary, our data depict the mechanisms underlying the evolution and dissemination of an important mobile antibiotic resistance gene from an intrinsic chromosomal gene.IMPORTANCEAs antibiotic resistance seriously challenges global health, tigecycline is one of the few effective drugs in the pipeline against infections caused by multidrug-resistant pathogens. Our previous work identified a novel tigecycline resistance efflux pump gene cluster tmexCD1-toprJ1 in animals and humans, together with its various variants, a rising clinical concern. Herein, this study focused on how the local regulation modes of tmexCD1-toprJ1 evolved to a highly expressed efflux pump. Through comparative analysis between three tnfxB-tmexCD-toprJ homologs and their progenitor nfxB-mexCD-oprJ, modes, we demonstrated the evolutionary dynamics from a chromosomal silent gene to an active state. We found the de-repression of the local regulator and an increase of the promoter activity work together to promote a high production of drug efflux machines and enhance multidrug resistance. Our findings revealed that TMexCD1-TOprJ1 adopts a distinct evolutionary path to achieve higher multidrug resistance, urgently needing tight surveillance.

Emergence of tet(X4)-positive Enterobacterales in retail eggs and the widespread of IncFIA(HI1)-HI1A-HI1B(R27) plasmids carrying tet(X4).

The proliferation of antimicrobial-resistant microbes and resistance genes in various foods poses a serious hazard to public health. The plasmid-mediated tigecycline resistance gene tet(X4) has been detected in Enterobacterales from various niches but has not yet been reported in eggs. This study aimed to investigate the occurrence and characteristics of tigecycline-resistant strains from retail eggs. A total of 144 eggs were purchased from farmers' markets in Guangdong province, China, and eggshell (n = 144) and egg content (n = 96) samples were used to screen for tigecycline-resistant strains. Eight Escherichia coli strains (two ST195, one ST48, ST8165, ST752, ST93, ST189, and ST224) and one Klebsiella pneumoniae strain (ST252) recovered from eight (5.56 %, 8/144) egg samples (eggshells, n = 6; egg content, n = 2) were positive for tet(X4). Notably, the two E. coli ST195 strains were closely (15-54 SNPs) related to all the tet(X4)-positive E. coli ST195 from various origins (food animals, foods, migratory birds, human, and environment) deposited in GenBank. The E. coli ST224 showed a close phylogenetic relationship (9-12 SNPs) with two tet(X4)-positive E. coli strains from chicken feces and retail chicken in Guangdong province. The hybrid plasmid IncFIA(HI1)-HI1A-HI1B(R27) constitutes the predominant tet(X4) vector both herein (7/9, 77.78 %) and in the GenBank database (32/160, 20 %). The tet(X4)-positive IncFIA(HI1)-HI1A-HI1B(R27) plasmids, sharing highly similar structures, have been widely disseminated across China. However, the IncFIA(HI1)-HI1A-HI1B(R27) plasmids exhibit poor stability and low conjugation frequency. The contamination of tet(X4)-positive bacteria internally and externally in retail eggs poses a prospective food safety threat. More attention should be paid to the spread of the tet(X4) gene via epidemic clone E. coli ST195 and the plasmid IncFIA(HI1)-HI1A-HI1B(R27).

Successful spread of mcr-1-bearing IncX4 plasmids is associated with variant in replication protein of IncX4 plasmids.

IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2, respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1. The copy number of pir-2 encoding plasmids (3.15 ± 0.9 copies) are higher than that of pir-1 encoding plasmids (0.85 ± 0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2, the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared with mcr-1-positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2. Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.

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.

The evolution of infectious transmission promotes the persistence of mcr-1 plasmids.

Conjugative plasmids play a vital role in bacterial evolution and promote the spread of antibiotic resistance. They usually cause fitness costs that diminish the growth rates of the host bacteria. Compensatory mutations are known as an effective evolutionary solution to reduce the fitness cost and improve plasmid persistence. However, whether the plasmid transmission by conjugation is sufficient to improve plasmid persistence is debated since it is an inherently costly process. Here, we experimentally evolved an unstable and costly mcr-1 plasmid pHNSHP24 under laboratory conditions and assessed the effects of plasmid cost and transmission on the plasmid maintenance by the plasmid population dynamics model and a plasmid invasion experiment designed to measure the plasmid's ability to invade a plasmid-free bacterial population. The persistence of pHNSHP24 improved after 36 days evolution due to the plasmid-borne mutation A51G in the 5'UTR of gene traJ. This mutation largely increased the infectious transmission of the evolved plasmid, presumably by impairing the inhibitory effect of FinP on the expression of traJ. We showed that increased conjugation rate of the evolved plasmid could compensate for the plasmid loss. Furthermore, we determined that the evolved high transmissibility had little effect on the mcr-1-deficient ancestral plasmid, implying that high conjugation transfer is vital for maintaining the mcr-1-bearing plasmid. Altogether, our findings emphasized that, besides compensatory evolution that reduces fitness costs, the evolution of infectious transmission can improve the persistence of antibiotic-resistant plasmids, indicating that inhibition of the conjugation process could be useful to combat the spread of antibiotic-resistant plasmids. IMPORTANCE Conjugative plasmids play a key role in the spread of antibiotic resistance, and they are well-adapted to the host bacteria. However, the evolutionary adaptation of plasmid-bacteria associations is not well understood. In this study, we experimentally evolved an unstable colistin resistance (mcr-1) plasmid under laboratory conditions and found that increased conjugation rate was crucial for the persistence of this plasmid. Interestingly, the evolved conjugation was caused by a single-base mutation, which could rescue the unstable plasmid from extinction in bacterial populations. Our findings imply that inhibition of the conjugation process could be necessary for combating the persistence of antibiotic-resistance plasmids.

Characterization of an International High-Risk Escherichia coli ST410 Clone Coproducing NDM-5 and OXA-181 in a Food Market in China.

During a 2020 routine epidemiological investigation of carbapenem-resistant Enterobacterales at a local food market in Guangzhou, China, two Escherichia coli ST410 isolates coproducing NDM-5 and OXA-181 were obtained from environmental samples. Antimicrobial susceptibility testing, whole-genome sequencing, and conjugation assays were applied to identify their resistance phenotypes, phylogenetic relatedness, and genetic characteristics. Phylogenetic analysis showed that the two isolates were clonally related with only one core-genome single-nucleotide polymorphism (SNP) difference and clustered into a branch with 87 E. coli ST410 isolates deposited in GenBank. These 89 ST410 isolates were closely related (≤51 SNPs), and most were from humans in Southeast Asian countries (n = 47). A Vietnamese clinical isolate collected in 2017 showed the strongest epidemiological link (seven SNPs) to the two ST410 isolates detected in this study. Complete-genome analysis revealed that the carbapenem resistance determinants blaNDM-5 and blaOXA-181 were located on an IncF1:A1:B49-IncQ1 plasmid and IncX3 plasmid, respectively. Conjugation experiments confirmed that the IncX3 plasmid was self-transmissible while the IncF1:A1:B49-IncQ1 plasmid was nonconjugative. BLASTn analysis indicated that the two plasmids showed high similarity to other blaNDM-5-bearing IncF1:A1:B49-IncQ1 and blaOXA-181-bearing IncX3 plasmids from other countries. Altogether, the high similarity of the core genomes and plasmids between the ST410 isolates found in this study and those human source isolates from foreign countries suggested the clonal spread of E. coli ST410 strains and horizontal transmission of blaOXA-181-bearing IncX3 plasmids across Southeast Asian countries. Stringent sanitary management of food markets is important to prevent the dissemination of high-risk clones to the public. IMPORTANCE This is the first report of an Escherichia coli ST410 clone that coproduces NDM-5 and OXA-181 in China. The high similarity of the core genomes and plasmids between the ST410 isolates characterized in this study and human source isolates from foreign countries strongly suggests that this ST410 lineage is an international high-risk clone, highlighting the need for continuous global surveillance of ST410 clones.

Pseudomonas Acts as a Reservoir of Novel Tigecycline Resistance Efflux Pump tmexC6D6-toprJ1b and tmexCD-toprJ Variants.

Several variants of the plasmid-carried tigecycline resistance gene cluster, tmexCD-toprJ, have been identified. This study characterized another novel variant, tmexC6D6-toprJ1b, located on the chromosome of environmental-origin Pseudomonas mendocina. TMexC6D6-TOprJ1 mediates resistance to multiple drugs, including tigecycline. The promoter activity of tmexC6D6-toprJ1b and negative transcriptional repression by the upstream regulator tnfxB6 are crucial for the expression of tmexC6D6-toprJ1b. tmexC6D6-toprJ1b was found in the plasmids or chromosomes of different Pseudomonas species from six countries. Two genetic backgrounds, class 1 integrons and int-carrying integrase units, were found adjacent to the tmexC6D6-toprJ1b gene cluster and might mediate the transfer of this novel efflux pump gene cluster in Pseudomonas. Further phylogenetic analysis revealed Pseudomonas as the major reservoir of tmexCD-toprJ variants, warranting closer monitoring in the future. IMPORTANCE Tigecycline is one of the treatment options for serious infections caused by multidrug-resistant bacteria, and tigecycline resistance has gained extensive attention. The emergence of a transferable tigecycline resistance efflux pump gene cluster, tmexCD-toprJ, severely challenged the efficiency of tigecycline. In this study, we identified another novel tmexCD-toprJ variant, tmexC6D6-toprJ1b, which could confer resistance to multiple classes of antibiotics, including tigecycline. Although tmexC6D6-toprJ1b was found only in Pseudomonas species, tmexC6D6-toprJ1b might spread to Enterobacteriaceae hosts via mobile genetic elements resembling those of other tmexCD-toprJ variants, compromising the therapeutic strategies. Meanwhile, novel transferable tmexCD-toprJ variants are constantly emerging and mostly exist in Pseudomonas spp., indicating Pseudomonas as the important hidden reservoir and origin of tmexCD-toprJ variants. Continuous monitoring and investigations of tmexCD-toprJ are urgent to control its spread.

Greater Invasion and Persistence of mcr-1-Bearing Plasmids in Escherichia coli than in Klebsiella pneumoniae.

The emergence of the plasmid-borne polymyxin resistance gene mcr-1 threatens the clinical utility of last-line polymyxins. Although mcr-1 has disseminated to various Enterobacterales species, the prevalence of mcr-1 is the highest among Escherichia coli isolates while remaining low in Klebsiella pneumoniae. The reason for such a difference in prevalence has not been investigated. In this study, we examined and compared the biological characteristics of various mcr-1 plasmids in these two bacterial species. Although mcr-1-bearing plasmids were stably maintained in both E. coli and K. pneumoniae, the former presented itself to be superior by demonstrating a fitness advantage while carrying the plasmid. The inter- and intraspecies transferability efficiencies were evaluated for common mcr-1-harboring plasmids (IncX4, IncI2, IncHI2, IncP, and IncF types) with native E. coli and K. pneumoniae strains as donors. Here, we found that the conjugation frequencies of mcr-1 plasmids were significantly higher in E. coli than in K. pneumoniae, regardless of the donor species and Inc types of the mcr-1 plasmids. Plasmid invasion experiments revealed that mcr-1 plasmids displayed greater invasiveness and stability in E. coli than in K. pneumoniae. Moreover, K. pneumoniae carrying mcr-1 plasmids showed a competitive disadvantage when cocultured with E. coli. These findings indicate that mcr-1 plasmids could spread more easily among E. coli than among K. pneumoniae isolates and that mcr-1 plasmid-carrying E. coli has a competitive advantage over K. pneumoniae, leading to E. coli being the main mcr-1 reservoir. IMPORTANCE As infections caused by multidrug-resistant "superbugs" are increasing globally, polymyxins are often the only viable therapeutic option. Alarmingly, the wide spread of the plasmid-mediated polymyxin resistance gene mcr-1 is restricting the clinical utility of this last-line treatment option. With this, there is an urgent need to investigate the factors contributing to the spread and persistence of mcr-1-bearing plasmids in the bacterial community. Our research highlights that the higher prevalence of mcr-1 in E. coli than in K. pneumoniae is attributed to the greater transferability and persistence of mcr-1-bearing plasmid in the former species. By gaining these important insights into the persistence of mcr-1 in different bacterial species, we will be able to formulate effective strategies to curb the spread of mcr-1 and prolong the clinical life span of polymyxins.

First detection of tet(X4)-positive Enterobacterales in retail vegetables and clonal spread of Escherichia coli ST195 producing Tet(X4) in animals, foods, and humans across China and Thailand.

The mobile tigecycline-resistant gene tet(X4), which confers resistance to all tetracyclines, has been identified in bacterial isolates from various sources. However, there are no reports on the occurrence of tet(X4) in bacterial isolates of ready-to-eat fresh vegetables. In this study, 113 vegetable samples from farmers' markets were screened for tigecycline-resistant strains. Ten Escherichia coli (two ST195, two ST48, and one ST10, ST58, ST88, ST394, ST641, and ST101) and one Klebsiella pneumoniae (ST327) recovered from nine vegetable samples (7.96 %) were identified as carrying tet(X4). The core genome sequences of the two E. coli ST195 isolates showed a close relationship (14-41 single-nucleotide polymorphisms) with 31 tet(X4)-bearing E. coli ST195 isolates from humans, pigs, pork, and bird in China and Thailand, and the 33 E. coli ST195 isolates producing Tet(X4) shared similar resistance genes and plasmid replicons. Nanopore sequencing and conjugation experiments confirmed that the tet(X4) genes were located on the hybrid plasmids IncFIA-HI1A-HI1B (n = 6), IncX1 (n = 3), and IncFII2 (n = 1) in E. coli, and IncFII plasmid in K. pneumoniae. IncFIA-HI1A-HI1B and IncX1 plasmids shared highly similar structures with plasmids from various sources in the GenBank database. This is the first study to report the observation of tet(X4)-positive bacteria in retail vegetables. The epidemic clones and plasmids contribute to tet(X4) dissemination in vegetables. The clonal spread of Tet(X4)-producing E. coli ST195 across multiple niches and countries could pose a potential threat to public health.

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.

Multiple Copies of Mobile Tigecycline Resistance Efflux Pump Gene Cluster tmexC2D2.2-toprJ2 Identified in Chromosome of Aeromonas spp.

The appearance and prevalence of novel plasmid-encoded tigecycline resistance efflux pump gene clusters tmexC1D1-toprJ1 and tmexC2D2-toprJ2 in Enterobacteriaceae have raised a threat to public health. Here, another tigecycline resistance gene cluster, tmexC2D2.2-toprJ2, was identified in two Aeromonas isolates recovered from fish meat and vegetables. Cloning confirmed the expression of tmexC2D2.2-toprJ2 mediated the resistance to tigecycline and decreased susceptibility to tetracyclines and cephalosporins in both Escherichia coli and Aeromonas. In an Aeromonas veronii strain, four copies of tmexC2D2.2-toprJ2 were located on the chromosome. Further analysis revealed that tmexC2D2.2-toprJ2 has been detected in the chromosomes of A. veronii, Aeromonas hydrophila, and Aeromonas caviae with one to four copies due to the insertion of a potential integrative transferable unit. The occurrence of multiple copies of chromosomal tmexC2D2.2-toprJ2 may act as a sink for this tigecycline resistance gene cluster, which requires continuous monitoring. IMPORTANCE Tigecycline is regarded as one of the few effective drugs against multidrug-resistant bacterial infection. However, mobile tigecycline resistance efflux pump gene clusters such as tmexC1D1-toprJ1 and its variants have been identified in both animal- and human-origin Enterobacteriaceae. In this study, we first found another efflux pump gene cluster, tmexC2D2.2-toprJ2, in the Aeromonas chromosome. This gene cluster could mediate tigecycline resistance and decrease susceptibility to tetracyclines and cephalosporins in the Aeromonas host strain. Meanwhile, tmexC2D2.2-toprJ2 was detected with multiple copies in Aeromonas spp. This multidrug resistance efflux pump gene cluster with multiple copy numbers might stably exist in Aeromonas and serve as a reservoir for tmexCD2-toprJ2, facilitating its persistent presence and spread.

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.

Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp ( Ctenopharyngodon idella) and evidence of bla NDM-5-bearing IncHI2 plasmid transfer between ducks and fish.

We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou, China. A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes. Characterization of bla NDM-5 positive isolates and plasmids was determined by antimicrobial susceptibility testing, conjugation experiments, Illumina HiSeq, and Nanopore sequencing. One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as bla NDM-5 carriers (3.57%, 7/196). The bla NDM-5 genes were located on the IncX3 ( n=5), IncHI2 ( n=1), or IncHI2-IncF ( n=1) plasmids. All bla NDM-5-bearing plasmids were transferred by conjugation at frequencies of ~10 -4-10 -6. Based on sequence analysis, the IncHI2 plasmid pHNBYF33-1 was similar to other bla NDM-5-carrying IncHI2 plasmids deposited in GenBank from Guangdong ducks. In all IncHI2 plasmids, bla NDM-5 was embedded in a novel transposon, Tn 7051 (IS 3000-ΔIS Aba125-IS 5-ΔIS Aba125- bla NDM-5- ble MBL- trpF- tat-∆ dct-IS 26-∆ umuD-∆IS Kox3-IS 3000), which was identical to the genetic structure surrounding bla NDM-5 found in some IncX3 plasmids. The IncHI2-IncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of the bla NDM-5-carrying IncHI2 plasmid and a heavy-metal-resistant IncF plasmid through ∆Tn 1721. To the best of our knowledge, this is the first report on the characterization of bla NDM-5-bearing plasmids in fish in China. The IncHI2 plasmid pHNBYF33-1 may be transmitted from ducks, considering the common duck-fish freshwater aquaculture system in Guangdong. Tn 7051 is likely responsible for the transfer of bla NDM-5 from IncX3 to IncHI2 plasmids in Enterobacteriaceae, resulting in the expansion of transmission vectors of bla NDM-5.