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September 22, 2019

Complete genome sequence and characterization of linezolid-resistant Enterococcus faecalis clinical isolate KUB3006 carrying a cfr(B)-transposon on its chromosome and optrA-plasmid.

Linezolid (LZD) has become one of the most important antimicrobial agents for infections caused by gram-positive bacteria, including those caused by Enterococcus species. LZD-resistant (LR) genetic features include mutations in 23S rRNA/ribosomal proteins, a plasmid-borne 23S rRNA methyltransferase gene cfr, and ribosomal protection genes (optrA and poxtA). Recently, a cfr gene variant, cfr(B), was identified in a Tn6218-like transposon (Tn) in a Clostridioides difficile isolate. Here, we isolated an LR Enterococcus faecalis clinical isolate, KUB3006, from a urine specimen of a patient with urinary tract infection during hospitalization in 2017. Comparative and whole-genome analyses were performed to characterize the genetic features and overall antimicrobial resistance genes in E. faecalis isolate KUB3006. Complete genome sequencing of KUB3006 revealed that it carried cfr(B) on a chromosomal Tn6218-like element. Surprisingly, this Tn6218-like element was almost (99%) identical to that of C. difficile Ox3196, which was isolated from a human in the UK in 2012, and to that of Enterococcus faecium 5_Efcm_HA-NL, which was isolated from a human in the Netherlands in 2012. An additional oxazolidinone and phenicol resistance gene, optrA, was also identified on a plasmid. KUB3006 is sequence type (ST) 729, suggesting that it is a minor ST that has not been reported previously and is unlikely to be a high-risk E. faecalis lineage. In summary, LR E. faecalis KUB3006 possesses a notable Tn6218-like-borne cfr(B) and a plasmid-borne optrA. This finding raises further concerns regarding the potential declining effectiveness of LZD treatment in the future.

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September 22, 2019

Genome sequence and metabolic analysis of a fluoranthene-degrading strain Pseudomonas aeruginosa DN1.

Pseudomonas aeruginosa DN1, isolated from petroleum-contaminated soil, showed excellent degradation ability toward diverse polycyclic aromatic hydrocarbons (PAHs). Many studies have been done to improve its degradation ability. However, the molecular mechanisms of PAHs degradation in DN1 strain are unclear. In this study, the whole genome of DN1 strain was sequenced and analyzed. Its genome contains 6,641,902 bp and encodes 6,684 putative open reading frames (ORFs), which has the largest genome in almost all the comparative Pseudomonas strains. Results of gene annotation showed that this strain harbored over 100 candidate genes involved in PAHs degradation, including those encoding 25 dioxygenases, four ring-hydroxylating dioxygenases, five ring-cleaving dioxygenases, and various catabolic enzymes, transcriptional regulators, and transporters in the degradation pathways. In addition, gene knockout experiments revealed that the disruption of some key PAHs degradation genes in DN1 strain, such as catA, pcaG, pcaH, and rhdA, did not completely inhibit fluoranthene degradation, even though their degradative rate reduced to some extent. Three intermediate metabolites, including 9-hydroxyfluorene, 1-acenaphthenone, and 1, 8-naphthalic anhydride, were identified as the dominating intermediates in presence of 50 µg/mL fluoranthene as the sole carbon source according to gas chromatography mass spectrometry analysis. Taken together, the genomic and metabolic analysis indicated that the fluoranthene degradation by DN1 strain was initiated by dioxygenation at the C-1, 2-, C-2, 3-, and C-7, 8- positions. These results provide new insights into the genomic plasticity and environmental adaptation of DN1 strain.

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September 22, 2019

SKA: Split Kmer Analysis Toolkit for Bacterial Genomic Epidemiology

Genome sequencing is revolutionising infectious disease epidemiology, providing a huge step forward in sensitivity and specificity over more traditional molecular typing techniques. However, the complexity of genome data often means that its analysis and interpretation requires high-performance compute infrastructure and dedicated bioinformatics support. Furthermore, current methods have limitations that can differ between analyses and are often opaque to the user, and their reliance on multiple external dependencies makes reproducibility difficult. Here I introduce SKA, a toolkit for analysis of genome sequence data from closely-related, small, haploid genomes. SKA uses split kmers to rapidly identify variation between genome sequences, making it possible to analyse hundreds of genomes on a standard home computer. Tests on publicly available simulated and real-life data show that SKA is both faster and more efficient than the gold standard methods used today while retaining similar levels of accuracy for epidemiological purposes. SKA can take raw read data or genome assemblies as input and calculate pairwise distances, create single linkage clusters and align genomes to a reference genome or using a reference-free approach. SKA requires few decisions to be made by the user, which, along with its computational efficiency, allows genome analysis to become accessible to those with only basic bioinformatics training. The limitations of SKA are also far more transparent than for current approaches, and future improvements to mitigate these limitations are possible. Overall, SKA is a powerful addition to the armoury of the genomic epidemiologist. SKA source code is available from Github (https://github.com/simonrharris/SKA).

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September 22, 2019

Update on Tetracycline Susceptibility of Pediococcus acidilactici Based on Strains Isolated from Swiss Cheese and Whey.

Bacterial strains used as starter cultures in the production of fermented foods may act as reservoirs for antibiotic resistance (AbR) genes. To avoid the introduction of such genes into the food chain, the presence of acquired AbR in bacterial strains added to food must be tested. Standard protocols and microbiological cut-off values have been defined to provide practitioners with a basis for evaluating whether their bacterial isolates harbor an acquired resistance to a given antibiotic. Here, we tested the AbR of 24 strains of Pediococcus acidilactici by using the standard protocol and microbiological cut-off values recommended by the European Food Safety Authority. Phenotypic data were complemented by searching for known AbR genes using an in silico analysis of whole genomes. The majority (54.2%) of the strains were able to grow at a tetracycline concentration above the defined cut-off, even though only one strain carried a known tetracycline resistance gene, tetM. The same strain also carried the AbR gene of an erythromycin resistance methylase, ermA, and displayed resistance toward clindamycin and erythromycin. Our results bolster the scarce data on the sensitivity of P. acidilactici to tetracycline and suggest that the microbiological cut-off recommended by the European Food Safety Authority for this antibiotic should be amended.

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September 22, 2019

Antimicrobial resistance profile of mcr-1 positive clinical isolates of Escherichia coli in China From 2013 to 2016.

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.

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September 22, 2019

pYR4 from a Norwegian isolate of Yersinia ruckeri is a putative virulence plasmid encoding both a type IV pilus and a type IV secretion system

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.

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September 22, 2019

The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools.

The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, is selected from among a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars. Chosen on the basis of its favorable properties, the complete genome sequence of NCIMB 14988 is determined and a high-efficiency plasmid transformation protocol devised. The developed DNA transfer procedure allowed demonstration in NCIMB 14988 of the forward and reverse genetic techniques of transposon mutagenesis and gene knockout, respectively. The latter is accomplished through the successful deployment of both group II intron retargeting (ClosTron) and allelic exchange. In addition to gene inactivation, the developed allelic exchange procedure is used to create point mutations in the chromosome, allowing for the effect of amino acid changes in enzymes involved in primary metabolism to be characterized. ClosTron mediated disruption of the currently unannotated non-coding region between genes LF65_05915 and LF65_05920 is found to result in a non-sporulating phenotype.© 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

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September 22, 2019

Comparative genomics of Staphylococcus reveals determinants of speciation and diversification of antimicrobial defense.

The bacterial genus Staphylococcus comprises diverse species with most being described as colonizers of human and animal skin. A relational analysis of features that discriminate its species and contribute to niche adaptation and survival remains to be fully described. In this study, an interspecies, whole-genome comparative analysis of 21 Staphylococcus species was performed based on their orthologues. Three well-defined multi-species groups were identified: group A (including aureus/epidermidis); group B (including saprophyticus/xylosus) and group C (including pseudintermedius/delphini). The machine learning algorithm Random Forest was applied to prioritize orthologs that drive formation of the Staphylococcus species groups A-C. Orthologues driving staphylococcal intrageneric diversity comprised regulatory, metabolic and antimicrobial resistance proteins. Notably, the BraSR (NsaRS) two-component system (TCS) and its associated BraDE transporters that regulate antimicrobial resistance showed limited distribution in the genus and their presence was most closely associated with a subset of Staphylococcus species dominated by those that colonize human skin. Divergence of BraSR and GraSR antimicrobial peptide survival TCS and their associated transporters was observed across the staphylococci, likely reflecting niche specific evolution of these TCS/transporters and their specificities for AMPs. Experimental evolution, with selection for resistance to the lantibiotic nisin, revealed multiple routes to resistance and differences in the selection outcomes of the BraSR-positive species S. hominis and S. aureus. Selection supported a role for GraSR in nisin survival responses of the BraSR-negative species S. saprophyticus. Our study reveals diversification of antimicrobial-sensing TCS across the staphylococci and hints at differential relationships between GraSR and BraSR in those species positive for both TCS.

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September 22, 2019

Whole-Genome Analysis of an Extensively Drug-Resistant Acinetobacter baumannii Strain XDR-BJ83: Insights into the Mechanisms of Resistance of an ST368 Strain from a Tertiary Care Hospital in China.

Acinetobacter baumannii is an important pathogen of nosocomial infections. Nosocomial outbreaks caused by antibiotic-resistant A. baumannii remain a significant challenge. Understanding the antibiotic resistance mechanism of A. baumannii is critical for clinical treatment. The purpose of this study was to determine the whole-genome sequence (WGS) of an extensively drug-resistant (XDR) A. baumannii strain, XDR-BJ83, which was associated with a nosocomial outbreak in a tertiary care hospital of China, and to investigate the antibiotic resistance mechanism of this strain. The WGS of XDR-BJ83 was performed using single-molecule real-time sequencing. The complete genome of XDR-BJ83 consisted of a 4,011,552-bp chromosome and a 69,069-bp plasmid. The sequence type of XDR-BJ83 was ST368, which belongs to clonal complex 92 (CC92). The chromosome of XDR-BJ83 carried multiple antibiotic resistance genes, antibiotic efflux pump genes, and mobile genetic elements, including insertion sequences, transposons, integrons, and resistance islands. The plasmid of XDR-BJ83 (pBJ83) was a conjugative plasmid carrying type IV secretion system. These results indicate that the presence of multiple antibiotic resistance genes, efflux pumps, and mobile genetic elements is likely associated with resistance to various antibiotics in XDR-BJ83.

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September 22, 2019

Detection of mcr-1 plasmids in Enterobacteriaceae isolates from human specimens: Comparison with those in Escherichia coli isolates from livestock in Korea.

The emerging mobile colistin resistance gene, mcr-1, is an ongoing worldwide concern and an evaluation of clinical isolates harboring this gene is required in Korea. We investigated mcr-1-possessing Enterobacteriaceae among Enterobacteriaceae strains isolated in Korea, and compared the genetic details of the plasmids with those in Escherichia coli isolates from livestock.Among 9,396 Enterobacteriaceae clinical isolates collected between 2010 and 2015, 1,347 (14.3%) strains were resistant to colistin and those were screened for mcr-1 by PCR. Colistin minimum inhibitory concentrations (MICs) were determined by microdilution, and conjugal transfer of the mcr-1-harboring plasmids was assessed by direct mating. Whole genomes of three mcr-1-positive Enterobacteriaceae clinical isolates and 11 livestock-origin mcr-1-positive E. coli isolates were sequenced.Two E. coli and one Enterobacter aerogenes clinical isolates carried carried IncI2 plasmids harboring mcr-1, which conferred colistin resistance (E. coli MIC, 4 mg/L; E. aerogenes MIC, 32 mg/L). The strains possessed the complete conjugal machinery except for E. aerogenes harboring a truncated prepilin peptidase. The E. coli plasmid transferred more efficiently to E. coli than to Klebsiella pneumoniae or Enterobacter cloacae recipients. Among the three bacterial hosts, the colistin MIC was the highest for E. coli owing to the higher mcr-1-plasmid copy number and mcr-1 expression levels. Ten mcr-1-positive chicken-origin E. coli strains also possessed mcr-1-harboring IncI2 plasmids closely related to that in the clinical E. aerogenes isolate, and the remaining one porcine-origin E. coli possessed an mcr-1-harboring IncX4 plasmid.mcr-1-harboring IncI2 plasmids were identified in clinical Enterobacteriaceae isolates. These plasmids were closely associated with those in chicken-origin E. coli strains in Korea, supporting the concept of mcr-1 dissemination between humans and livestock.© The Korean Society for Laboratory Medicine.

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September 22, 2019

Prevalence, antimicrobial resistance and phylogenetic characterization of Yersinia enterocolitica in retail poultry meat and swine feces in parts of China

Yersinia enterocolitica is an enteropathogen transmitted by contaminated food. In this study, a total of 500 retail poultry meat samples from 4 provinces and 145 swine feces samples from 12 provinces in China was tested for Y. enterocolitica and 26 isolates were obtained for further bio-serotyping, testing with antimicrobial susceptibility testing to a panel of antimicrobial compounds, and genetically characterization based on the whole genome sequencing. Higher prevalence (4.8%) of Y. enterocolitica contamination in retail poultry meat than that in swine feces (2.76%) was observed. No difference in bio-serotypes, multilocus sequence typing (MLST) and virulence genes distribution between swine and poultry origin were found. All isolates were resistant to ampicillin, amoxicillin/clavulanic acid, and cefazolin and were multi-drug resistant (MDR). The most predominant drug-resistance profile was AMP-CFZ-AMC-FOX (42.31%). A pathogenic isolate with bio-serotype 3/O:3 and ST135 was cultured from retail fresh chicken meat for the first time in China. Based on the whole-genome single nucleotide polymorphisms (SNPs) tree analysis, pathogenic isolates clustered closely, while nonpathogenic isolates exhibited high genetic heterogeneity. These indicated that pathogenic isolates were conserved on genetic level. The whole-genome SNP tree also revealed that Y. enterocolitica of swine, chicken and duck origin may share a common ancestor. The findings highlight the emergence of drug-resistant pathogenic Y. entrocoliticas in retailed poultry meats in China.

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September 22, 2019

Genomic analysis of multi-resistant Staphylococcus capitis associated with neonatal sepsis.

Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis, are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally. Here, 122 S. capitis isolates from New Zealand (NZ) underwent whole-genome sequencing (WGS), and these data were supplemented with publicly available S. capitis sequence reads. Phylogenetic and comparative genomic analyses were performed, as were phenotypic assessments of antimicrobial resistance, biofilm formation, and plasmid segregational stability on representative isolates. A distinct lineage of S. capitis was identified in NZ associated with neonates and the NICU environment. Isolates from this lineage produced increased levels of biofilm, displayed higher levels of tolerance to chlorhexidine, and were multidrug resistant. Although similar to globally circulating NICU-associated S. capitis strains at a core-genome level, NZ NICU S. capitis isolates carried a novel stably maintained multidrug-resistant plasmid that was not present in non-NICU isolates. Neonatal blood culture isolates were indistinguishable from environmental S. capitis isolates found on fomites, such as stethoscopes and neonatal incubators, but were generally distinct from those isolates carried by NICU staff. This work implicates the NICU environment as a potential reservoir for neonatal sepsis caused by S. capitis and highlights the capacity of genomics-based tracking and surveillance to inform future hospital infection control practices aimed at containing the spread of this important neonatal pathogen. Copyright © 2018 Carter et al.

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September 22, 2019

FRI-4 carbapenemase-producing Enterobacter cloacae complex isolated in Tokyo, Japan.

A carbapenem-resistant Enterobacter cloacae complex isolated in Tokyo, Japan, produced a carbapenemase that was detected by a Carba NP test and a modified carbapenem inactivation method, but none of the ‘Big Five’ carbapenemase genes was detected by PCR. This study aimed to identify the carbapenemase.Carbapenemase genes were screened by WGS. Next, we generated a recombinant plasmid in which the carbapenemase gene was inserted. We also extracted the carbapenemase gene-carrying plasmid from the E. cloacae complex. The effects of both plasmids on the antibiotic susceptibility of Escherichia coli were then tested. The carbapenemase gene-carrying plasmid in the E. cloacae complex was completely sequenced.A novel carbapenemase gene, blaFRI-4, encoded an amino acid sequence that was 93.2% identical to French imipenemase (FRI-1). E. coli transformed with blaFRI-4 showed reduced carbapenem susceptibility. A complete sequence of the blaFRI-4-carrying 98?508?bp IncFII/IncR plasmid (pTMTA61661) showed that blaFRI-4 and the surrounding region (18.7?kb) were duplicated.The FRI-4-producing E. cloacae complex was isolated in Japan, whereas all other FRI variants have been found in Europe, suggesting that the spread of FRI carbapenemases is global.

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September 22, 2019

Phylogenomics of colistin-susceptible and resistant XDR Acinetobacter baumannii.

Acinetobacter baumannii is a healthcare-associated pathogen with high rates of carbapenem resistance. Colistin is now routinely used for treatment of infections by this pathogen. However, colistin use has been associated with development of resistance to this agent.To elucidate the phylogenomics of colistin-susceptible and -resistant A. baumannii strain pairs from a cohort of hospitalized patients at a tertiary medical centre in the USA.WGS data from 21 pairs of colistin-susceptible and -resistant, XDR clinical strains were obtained and compared using phylogeny of aligned genome sequences, assessment of pairwise SNP differences and gene content.Fourteen patients had colistin-resistant strains that were highly genetically related to their own original susceptible strain with a median pairwise SNP distance of 5.5 (range 1-40 SNPs), while seven other strain pairs were divergent with =84 SNP differences. In addition, several strains from different patients formed distinct clusters on the phylogeny in keeping with closely linked transmission chains. The majority of colistin-resistant strains contained non-synonymous mutations within the pmrAB locus suggesting a central role for pmrAB mutations in colistin resistance. Excellent genotype-phenotype correlation was also observed for carbapenems, aminoglycosides and tetracyclines.The findings suggest that colistin resistance in the clinical setting arises through both in vivo evolution from colistin-susceptible strains and reinfection by unrelated colistin-resistant strains, the latter of which may involve patient-to-patient transmission.

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September 22, 2019

Plasmid and chromosomal integration of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.

To provide detailed genetic characterization of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.P. aeruginosa 60512, K. pneumoniae 447, P. aeruginosa 12939 and Enterobacter sp. A1137 were subjected to genome sequencing. The complete nucleotide sequences of two plasmids (p60512-IMP from the 60512 isolate and p447-IMP from the 447 isolate) and two chromosomes (the 12939 and A1137 isolates) were determined, then a genomic comparison of p60512-IMP, p447-IMP and four novel blaIMP-carrying transposons (Tn6394, Tn6375, Tn6411 and Tn6397) with related sequences was performed. Transferability of the blaIMP gene and bacterial antimicrobial susceptibility were tested.Tn6394 and Tn6375 were located in p60512-IMP and p447-IMP, respectively, while Tn6411 and Tn6397 were integrated into the 12939 and A1137 chromosomes, respectively. Tn6394 was an ISPa17-based transposition unit that harboured the integron In992 (carrying blaIMP-1). In73 (carrying blaIMP-8), In73 and In992, together with the ISEcp1:IS1R-blaCTX-M-14-IS903D unit, the macAB-tolC region and the truncated aacC2-tmrB region, respectively, were integrated into the prototype transposons Tn1722, Tn1696 and Tn7, respectively, generating the Tn3-family unit transposons, Tn6375 and Tn6378, and the Tn7-family unit transposon Tn6411, respectively. Tn6397 was a large integrative and conjugative element carrying Tn6378.Complex events of transposition and homologous recombination have occurred during the original formation and further plasmid and chromosomal integration of these four transposons, promoting accumulation and spread of antimicrobial resistance genes.

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