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Objectives This study aimed to characterise all carbapenemase-producing enterobacteria (CPE) isolates obtained from an outbreak-free setting in Uruguay. Methods We studied 12 CPE isolated from Hospital de Clinicas between 2012–2016. Bacterial identification and antibiotic susceptibility testing were performed using VITEK®2 and Sensititre or agar dilution, respectively. Antimicrobial resistance genes and mobile genetic elements were identified by PCR and sequencing. Multilocus sequence typing was performed for Klebsiella pneumoniae. Plasmid conjugation was assessed, plasmid size was estimated by S1-PFGE and plasmid incompatibility groups were sought by PCR. Results Among 8364 enterobacteria, 12 CPE were isolated from urine, blood culture, wound, peritoneal fluid and punch samples. NDM-1 was the most prevalent carbapenemase, followed by VIM-2 and KPC-2. All isolates were resistant to gentamicin, cefotaxime, ceftazidime, trimethoprim/sulfamethoxazole, ciprofloxacin and imipenem and were susceptible to fosfomycin. We characterised six class 1 integrons: dfrA12–orfF–aadA2; aacA4–blaOXA-2–orfD; aadB–aadA2; dfrA1; aadB–blaOXA-10–aadA1; and blaVIM-2-dfrA7. An association between various aminoglycoside, β-lactam and fluoroquinolone resistance genes were observed, some of them located in transferable plasmids belonging to incompatibility groups IncC, IncHI1 and IncM1. We described a new composite transposon (assigned Tn6935) including blaNDM-1 flanked by two directly-oriented copies of a Tn3-like element ISKox2-like family transposase. The sequence types of K. pneumoniae isolates were ST11, ST14 and ST661. Conclusions The presence of CPE is sporadic and could be due to measures taken by the Public Health Committee. Nevertheless, the coexistence of several resistance mechanisms and their presence in conjugative plasmids and high-risk clones is worrisome.

Objectives The aim of this study was to explore the genomic content of a blaKPC-2- and mcr-1-harbouring Escherichia coli strain and to clarify the molecular mechanism for the transmission of multidrug resistance. Methods Antimicrobial susceptibility testing was conducted by the broth microdilution method to determine the resistance profile. Filter-mating conjugation assays were performed to confirm the plasmid transfer ability. Whole-genome sequence data were acquired by a combination of Illumina paired-end reads and Nanopore long-read sequencing. Results Escherichia coli strain QE11−421 was an mcr-1-positive colistin-resistant isolate that co-harboured the blaKPC-2 gene conferring carbapenem resistance. Genome sequence data confirmed QE11−421 as a sequence type 48 (ST48) E. coli that harboured five large conjugative plasmids encoding several multidrug resistance genes. The blaKPC-2 gene was located on a Tn3–Tn4401 composite transposon, which is part of a 65-kb multidrug-resistant IncN plasmid. The mcr-1 gene was harboured on another 33-kb IncX4 plasmid that was more conserved and presented few antimicrobial resistance determinants. No copies of insertion sequence ISApl1 were found flanking the mcr-1 gene, decreasing the mobility of mcr-1 based on its original Tn6330 transposon. Conclusions Horizontal transfer of multidrug resistance plasmids or resistance-related transposon units was responsible for the emergence of this notorious superbug. The coexistence of blaKPC-2–IncN and mcr-1–IncX4 plasmids in a ST48 E. coli strain in humans poses a great threat.

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated. Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment. Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli . Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials. Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii .

ObjectivesTo characterize the genetic environment of the carbapenem resistance determinant in Proteus vulgaris of swine origin.MethodsThe carbapenem-resistant P. vulgaris strain BC22 was isolated from a faecal swab from a diseased pig with diarrhoea in Sichuan Province of China in 2018. The presence of carbapenemase genes was screened by PCR. WGS and bioinformatics analysis were performed to analyse the genetic environment of the carbapenem resistance determinant.ResultsP. vulgaris strain BC22 was found to harbour the carbapenemase gene blaNDM-1. WGS data revealed that blaNDM-1 was located in a truncated ISAba125 composite transposon. The carbapenem resistance gene blaNDM-1 and 20 other resistance genes, including the multiresistance gene cfr and the bifunctional aminoglycoside/quinolone resistance gene aac(6′)-lb-cr, were located in a novel SXT/R391 integrative and conjugative element (ICE). This new SXT/R391 ICE of 148.7 kb was chromosomally located, and could be transferred to Escherichia coli.ConclusionsHere, we report a carbapenemase gene, blaNDM-1, integrated into an SXT/R391 ICE. Our study highlights that this SXT/R391 ICE may facilitate the dissemination of clinically important resistance genes such as blaNDM-1, cfr and aac(6′)-lb-cr.

Antimicrobial resistance is a critical issue that is no longer restricted to hospital settings, but also represents a growing problem involving intensive animal production systems. In this study, we have performed a microbiological and molecular investigation of priority pathogens carrying transferable resistance genes to critical antimicrobials in one-day-old chickens imported from Brazil to Uruguay. Bacterial identification was performed by MALDI-TOF mass spectrometry and antibiotic susceptibility was determined by Sensititre. Antimicrobial resistance genes were sought by polymerase chain reaction and clonality was assessed by PFGE. Four multidrug-resistant (MDR) representative strains were sequenced by Illumina and/or Oxford Nanopore Technologies. Twenty-eight MDR isolates identified as Escherichia coli (n= 14), Enterobacter cloacae (n= 11) and Klebsiella pneumoniae (n= 3). While resistance to oxyiminocephalosporins was due to blaCTX-M-2, blaCTX-M-8, blaCTX-M-15, blaCTX-M-55 and blaCMY-2, plasmid-mediated quinolone resistance was associated with qnrB19, qnrE1, and qnrB2 genes. Finally, resistance to aminoglycosides and fosfomycin was due to the presence of 16S rRNA methyltransferase rmtG and fosA-type genes, respectively. Short and long-read genome sequencing of E. cloacae ODC-Eclo3 strain revealed the presence of IncQ/rmtG (pUR-EC3.1, 7400-pb), IncHI2A/mcr-9.1/blaCTX-M-2 [pUR-EC3.2, ST16 (pMLST), 408,436-bp] and IncN2/qnrB19/aacC3/aph(3'')-Ib (pUR-EC3.3) resistance plasmids. Strikingly, the blaCTX-M-2 gene was carried by a novel Tn1696-like composite transposon designated Tn7337. In summary, we report that imported one-day-old chicks can act as Trojan horses for the hidden spread of WHO critical priority MDR pathogens harboring mcr-9, rmtG and extended-spectrum β-lactamase genes in poultry farms, which is a critical issue within a One Health perspective. Importance section Antimicrobial resistance is considered a significant problem for global health, including within the concept of "One Health", therefore, the food chain is a link that connects human and animal health directly. In this work, we searched for microorganisms resistant to antibiotics considered critical for human health in intestinal microbiota of one-day-old baby chicks imported to Uruguay from Brazil. We described antibiotic-resistant genes to antibiotics named as to watch or reserve for the WHO, such as rmtG or mcr9.1, which confers resistance to all the aminoglycosides and colistin, respectively, among others genes, and their presence in new mobile genetic elements that favor its dissemination. The sustained entry of these microorganisms evades the sanitary measures implemented by the countries and production establishments to reduce the selection of resistant microorganisms. These silently imported resistant microorganisms could explain a considerable part of the antimicrobial resistance problems found in the production stages of the system.

Resistance of bacteria to 3rd generation cephalosporins mediated by beta-lactamases (ESBL, pAmpC) is a public health concern. In this study, 1517 phenotypically cephalosporin-resistant E. coli were screened for the presence of blaSHV genes. Respective genes were detected in 161 isolates. Majority (91%) were obtained from poultry production and meat. The SHV-12 beta-lactamase was the predominant variant (n = 155), while the remaining isolates exhibited SHV-2 (n = 4) or SHV-2a (n = 2). A subset of the isolates (n = 51) was further characterized by PCR, PFGE, or whole-genome sequencing and bioinformatics analysis. The SHV-12-producing isolates showed low phylogenetic relationships, and dissemination of the blaSHV-12 genes seemed to be mainly driven by horizontal gene transfer. In most of the isolates, blaSHV-12 was located on transferable IncX3 (~43 kb) or IncI1 (~100 kb) plasmids. On IncX3, blaSHV-12 was part of a Tn6 composite transposon located next to a Tn3 transposon, which harbored the fluoroquinolone resistance gene qnrS1. On IncI1 plasmids, blaSHV-12 was located on an incomplete class 1 integron as part of a Tn21 transposon. In conclusion, SHV-12 is widely distributed in German poultry production and spreads via horizontal gene transfer. Consumers are at risk by handling raw poultry meat and should take care in appropriate kitchen hygiene.

Escherichia coli sequence type 131 (ST131) is known for its contribution to multidrug resistance and the worldwide spread of this clone has become a global problem. Understanding the trends among ST131 clades will help design strategies to prevent its rapid dissemination. In this study, 72 ST131 strains were subjected to comparative genomic analysis and 64 clade C strains were compared with clade C strains reported from other regions using publicly available whole-genome sequencing data. C1 (n = 31 [48.4%]) and C2 (n = 33 [%51.5]) strains had the same prevalence in our collection, and C1-M27 (n = 22) strains were closely related, carried a unique plasmid type (F1:A2:B20), and exhibited virotype C. Removal of 11 C2 strains with varied virotype patterns and the heterogeneous IncF type identified 22 closely related virotype E/F strains with replicon type F31/F36:A4:B1, forming what we denote as the “C2-subset.” In a global context, the C2-subset constituted a distinct cluster with international virotype E strains and harbored a genomic island, GI-pheU. Association of cnf1/hlyCABD genes with 1 to 7 mobile genetic elements, mostly IS682/ISKpn37 combination within GI-pheU was identified. The C2-subset accounted for excess resistance/virulence of subclade C2 relative to C1 strains. In addition, a conserved chromosomal IS26-mediated composite transposon (IS15DIV-ISEcp1-blaCTX-M-15-WbuC cupin fold metalloprotein-Tn2-IS15DIV) was observed in the C2-subset. The local spread of the C2-subset in the hospital studied, with the carriage of higher virulence/resistance markers and a peculiar F-type plasmid, demonstrates the potential for diversification of the ST131 lineage and the emergence of subpopulations with higher survival potential to cause health care-associated outbreaks.IMPORTANCE Escherichia coli sequence type 131 (ST131) is a globally dominant multidrug-resistant clone that is commonly associated with extraintestinal infections. Specific sublineages have been shown to have emerged and spread within ST131, highlighting the complex nature of ST131 epidemiology. This study systematically compared the Iranian ST131 population to those reported from other countries and found a subpopulation harboring virotype E, a homogeneous profile of plasmid Inc-F type F31/F36:A4:B1 harboring cnf1/hemolysin genes on the genomic island GI-pheU, and up to seven mobile genetic elements (MGEs) flanking cnf1/hemolysin virulence markers. The results of this study highlight the importance of MGEs for virulence gene acquisition and the formation of new subpopulations among pandemic clones such as E. coli ST131.

Objectives This study describes the first draft genome sequence of a multidrug-resistant (MDR) Escherichia coli D-ST69 clinical isolate from Egypt carrying blaNDM-1 and blaOXA-244. Methods The strain was isolated in December 2014 from a wound pus swab of a male patient in the city of Kafr El-Sheikh using MacConkey agar containing 2 μg/mL meropenem. The strain was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole-genome sequencing using an Illumina MiSeq platform. Results The draft genome of the strain (HR14_AS) was 5.08 Mbp in size containing a total of 90 contigs encoding 4677 predicted genes with an average G+C content of 50.7%. Strain HR14_AS belongs to sequence type 69 (ST69), phylogroup D and exhibits an MDR phenotype, with minimum inhibitory concentrations (MICs) of 64 μg/mL and 32 μg/mL for meropenem and doripenem, respectively. Multiple acquired antimicrobial resistance genes conferring resistance to macrolides [mdf(A)], fluoroquinolones [aac(6')-Ib-cr], quinolones (qnrS1), trimethoprim (dfrA14), β-lactams (blaNDM-1, blaOXA-244, blaCTX-M-15, blaOXA-9 and blaTEM-1B) and aminoglycosides [aac(3)-IId, aac(6')-Ib, aadA1 and aph(3')-VI] were detected. The blaOXA-244 and blaNDM-1 genes were located on the chromosome (Tn6237) and on an IncI1-type self-conjugative plasmid of >93 kb in size, respectively. Conclusions Here we report the first draft genome sequence of a MDR E. coli D-ST69 isolate carrying blaNDM-1 and blaOXA-244. Besides clonal expansion of the E. coli ST38 pandemic clone, this study further identified that the spread of OXA-244-producing E. coli could be related to mobilisation of the IS1R-made composite transposon (Tn6237) carrying blaOXA-244.

Objectives: The aim of the study was to determine the presence of antimicrobial-resistance (AMR) genes, virulence genes, and mobile genetic elements (MGEs) in 14 biofilm-producing clinical isolates of Acinetobacter baumannii. Materials and Methods: PCR amplification was performed to analyse the prevalence of genes associated with antibiotic resistance (extended-spectrum β-lactamases [ESBLs] and metallo-β-lactamases [MBLs]), virulence factors, MGEs (class 1 integron, Tn1213, and A. baumannii antibiotic resistance [AbaR]), and comM among the study isolates. Random amplified polymorphic DNA (RAPD) PCR was then deployed to understand their phylogenetic relationship. All the isolates were investigated for biofilm production. Results: Two isolates were antibiotic-sensitive (AS), 3 were multi-drug-resistant (MDR), and the remaining 9 were extensively drug-resistant (XDR). The majority of the isolates were found to be positive for biofilm production and were sensitive against tetracycline and colistin only. Ab14 and Ab11 were found to be resistant to minocycline and colistin, respectively. blaTEM, blaOXA, blaNDM, blaVIM, blaSIM, and blaPER-1; class 1 integron; composite transposon Tn1213; AbaR island, and virulence factor genes were detected among the isolates. These pathogens were found to have originated from multiple clonal lineages. Conclusion: Biofilm-producing A. baumannii with multiple virulence and AMR genes pose serious clinical challenges. The presence of MGEs further compounds the situation as these isolates serve as potential reservoirs of AMR and virulence genes. Together with their capacity for natural competence, A. baumannii, if left unchecked, will lead to the spread of resistance determinants to previously sensitive bacteria and may aid in the emergence of untreatable pan-drug-resistant phenotypes.

Transposable elements in prokaryotes are found in many forms and therefore a robust nomenclature system is needed in order to allow researchers to describe and search for them in publications and databases. Here we provide an update on The Transposon Registry which allocates numbers to any prokaryotic transposable element. Additionally, we present the completion of registry records for all transposons assigned Tn numbers from Tn1 onwards where sequence data or publications exist.

There is emerging evidence that food of animal origin may be responsible for the spread of multidrug resistant extraintestinal pathogenic Escherichia coli in the community. Here, we describe the emergence of colistin resistance gene, mcr-1, in a strain belonging to the dominant uropathogenic E. coli ST69 lineage. E. coli strain CFSAN061770 was isolated during monitoring of the popular Egyptian raw milk cheese, karish cheese, for the presence of colistin resistance. The complete genome of E. coli strain CFSAN061770 comprises a chromosome of 5,292,297 bp with a G + C content of 50.6%. Further, three plasmids named pEGY1-MCR-1, pEGY2 and pEGY3 of 228,947 bp, 103,234 bp and 87,012 bp were detected, respectively. Plasmid pEGY1-MCR-1 belongs to the IncHI2 incompatibility group and carries the colistin resistance mcr-1 gene flanked by two ISApl1 elements and forms a composite transposon. It mediates resistance to aminoglycosides (aadA1 and aadA2), phenicol (cmlA1 and floR), sulfonamides (sul3), and tetracycline (tet(A)), and these loci were found clustered in a multidrug resistant region. Plasmid pEGY3 carries a complex multiple resistance locus (CMR) (aph(3′)-Ia, strA, strB, sul2, and blaTEM-1) encoding resistance to different classes of antibiotics. Interestingly, the closest plasmids to plasmid pEGY1-MCR-1 detected from the NCBI Blast search belonged to the incompatibility group IncHI2 and were from the Kingdom of Saudi Arabia and Qatar which suggests a dissemination of pEGY1-MCR-1-like plasmids in the Middle East. Most striking, and of great public health concern is that strain CFSAN061770 carries five virulence genes (iss, fimH, iutA, kpsMIII and kpsTIII) which were identified in clinical extraintestinal pathogenic E. coli. Besides that, it carries the astA gene, which codes for the enteroaggregative E. coli heat-stable toxin 1 (EAST1).

Background Clindamycin is a lincosamide antibiotic used to treat staphylococcal and streptococcal infections. Reports of clinical Streptococcus agalactiae isolates with the rare lincosamide resistance/macrolide susceptibility (LR/MS) phenotype are increasing worldwide. In this study, we characterised three clinical S. agalactiae strains with the unusual L phenotype from China. Methods Three clinical S. agalactiae strains, Sag3, Sag27 and Sag4104, with the L phenotype were identified from 186 isolates collected from 2016 to 2018 in Shanghai, China. The MICs of clindamycin, erythromycin, tetracycline, levofloxacin, and penicillin were determined using Etest. PCR for the lnu(B) gene was conducted. Whole genome sequencing and sequence analysis were carried out to investigate the genetic context of lnu(B). Efforts to transfer lincomycin resistance by conjugation and to identify the circular form by inverse PCR were made. Results Sag3, Sag27, and Sag4104 were susceptible to erythromycin (MIC ≤0.25 mg/L) but resistant to clindamycin (MIC ≥1 mg/L). lnu(B) was found to be responsible for the L phenotype. lnu(B) in Sag3 and Sag27 were chromosomally located in an aadE-spw-lsa(E)-lnu(B) resistance gene cluster adjacent to an upstream 7-kb tet(L)-cat resistance gene cluster. Two resistance gene clusters were flanked by the IS6-like element, IS1216. Sag4104 only contained partial genes of aadE-spw-lsa(E)-lnu(B) resistance gene cluster and was also flanked by IS1216. Conclusion These results established the presence of the L phenotype associated with lnu(B) in clinical S. agalactiae isolates in China. The lnu(B)-containing multi-resistance gene cluster possibly acts as a composite transposon flanked by IS1216 and as a vehicle for the dissemination of multidrug resistance among S. agalactiae.

Sulfonamide-degrading bacteria have been discovered in various environments, suggesting the presence of novel resistance mechanisms via drug inactivation. In this study, Microbacterium sp. CJ77 capable of utilizing various sulfonamides as a sole carbon source was isolated from a composting facility. Genome and proteome analyses revealed that a gene cluster containing a flavin-dependent monooxygenase and a flavin reductase was highly up-regulated in response to sulfonamides. Biochemical analysis showed that the two-component monooxygenase system was key enzymes for the initial cleavage of sulfonamides. Co-expression of the two-component system in Escherichia coli conferred decreased susceptibility to sulfamethoxazole, indicating that the genes encoding drug-inactivating enzymes are potential resistance determinants. Comparative genomic analysis revealed that the gene cluster containing sulfonamide monooxygenase (renamed as sulX) and flavin reductase (sulR) was highly conserved in a genomic island shared among sulfonamide-degrading actinobacteria, all of which also contained sul1-carrying class 1 integrons. These results suggest that the sulfonamide metabolism may have evolved in sulfonamide-resistant bacteria which had already acquired the class 1 integron under sulfonamide selection pressures. Furthermore, the presence of multiple insertion sequence elements and putative composite transposon structures containing the sulX gene cluster indicated potential mobilization. This is the first study to report that sulX responsible for both sulfonamide degradation and resistance is prevalent in sulfonamide-degrading actinobacteria and its genetic signatures indicate horizontal gene transfer of the novel resistance gene. Keywords: Sulfonamide resistance, Sulfonamide degradation, Genomic island, Composite transposon, Horizontal gene transfer

Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in dissemination and modification of antibiotic resistance phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over four days from an inpatient with prolonged bacteremia. Isolates from both species harbored similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events, and IS-medicated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the IS in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype-phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium offering a new paradigm for the study of daptomycin-resistance and novel insights into the route of daptomycin resistance dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.

Here, we characterized a carbapenem-resistant Vibrio diabolicus strain of shrimp origin with various experiments and bioinformatics analysis. A novel metallo-β-lactamase (MBL) gene, bla VMB-2 , that confers resistance to β-lactams, including meropenem and cephalosporins, was identified on a plasmid-borne composite transposon, IS Shfr9 -IS CR1 - bla VMB-2 - bla CARB-12 - aadA1- IS Shfr9 , which is capable of generating a bla VMB-2 -bearing circular intermediate. IS Shfr9 was found to be disseminated in multidrug-resistant (MDR) pathogens, arousing the concern of further transmission of a bla VMB-2 -bearing circular intermediate to clinical Enterobacterales strains via such insertion sequences, which warrants further investigations.

Extended-spectrum β-lactamases (ESBLs) production and (fluoro)quinolone (FQ) resistance among Salmonella pose a public health threat. The objective of this study was the phenotypic and genotypic characterization of an ESBL-producing and nalidixic acid-resistant Salmonella enterica serovar Gloucester isolate (serotype 4:i:l,w) of sequence type 34 (ST34) from ready-to-eat (RTE) meat products in China. Whole-genome short and long read sequencing (HiSeq and MinION) results showed that it contained blaCTX–M–55, qnrS1, and tetB genes, with blaCTX–M–55 and qnrS1 located in chromosomal IS26-mediated composite transposon (IS26–qnrS1–IS3–Tn3–orf–blaCTX–M–55–ISEcp1–IS26). The same genetic structure was found in the chromosome of S. enterica subsp. enterica serovar Typhimurium strain and in several plasmids of Escherichia coli, indicating that the IS26-mediated composite transposon in the chromosome of S. Gloucester may originate from plasmids of E. coli and possess the ability to disseminate to Salmonella and other bacterial species. Besides, the structural unit qnrS1–IS3–Tn3–orf–blaCTX–M–55 was also observed to be linked with ISKpn19 in both the chromosomes and plasmids of various bacteria species, highlighting the contribution of the insertion sequences (IS26 and ISKpn19) to the co-dissemination of blaCTX–M–55 and qnrS1. To our knowledge, this is the first description of chromosomal blaCTX–M–55 and qnrS in S. Gloucester from RTE meat products. Our work expands the host range and provides additional evidence of the co-transfer of blaCTX–M–55 and qnrS1 among different species of Salmonella through the food chain.

Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides., Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module. IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.

ObjectivesThe emergence of mobile colistin resistance genes has compromised the efficacy of the last resort antibiotic, colistin, in clinical treatment. The mcr-2 gene was first identified in Belgium in association with the insertion sequence ISEc69. However, the molecular mechanisms of mcr-2 mobilization are not well understood.MethodsTo further explore the mobilization of mcr-2 gene via ISEc69, we constructed a conjugative plasmid that carries an intact composite transposon Tn7052. Transposition assays were performed by conjugation, the transposition sites were characterized by arbitrary primed PCR and DNA sequencing.ResultsIn this study, we experimentally demonstrated that mcr-2 could be mobilized as a composite transposon Tn7052 and its transposition generated 8-bp AT-rich duplications in the host genome.ConclusionThese results indicate that mcr-2 gene could be mobilized by ISEc69, the current investigations provide mechanistic insights in the transposition of mcr-2.

Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125 whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3 and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids whereas pAC1633-2 is a 12,651 bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.

The mobile colistin resistance gene mcr-3 has globally disseminated since it was first reported in 2017 in Escherichia coli . In vitro mobilization assays in this study demonstrate the functionality of the composite transposon structure IS Kpn40-mcr-3.11-dgkA -IS Kpn40 in wild-type and recA − E. coli strains. These transpositions generated 4-bp duplications at the target sites.

We described the genome and resistome characterization of a carbapenem-resistant Klebsiella pneumoniae ST307 strain carrying blaIMP-38 in China. This report highlights that the high-risk ST307 clone continues to acquire different antimicrobial resistance genes, posing significant challenges to clinical practice, and should be closely monitored., An emerging multidrug-resistant Klebsiella pneumoniae high-risk clone of sequence type 307 (ST307) has been increasingly reported worldwide. Here, we described the genomic characteristics of an IMP-38-producing ST307 K. pneumoniae strain and investigated the prevalence of blaIMP-38 among carbapenem-resistant Klebsiella pneumoniae isolates from a tertiary care hospital in central China. A total of 14 IMP-38-producing ST307 K. pneumoniae strains were identified from 2013 to 2016, with 13 strains isolated from patients with neonatal sepsis in the neonatal ward. PacBio and Illumina whole-genome sequencing analysis performed on a representative IMP-38-producing K. pneumoniae strain, WCGKP294, showed that it contained a circular chromosome and two plasmids. Carbapenemase gene blaIMP-38 is colocated with blaCTX-M-3 in transposon Tn6382 on an IncHI5 plasmid (pWCGKP294-2). WCGKP294 harbors another IncFIB plasmid, pWCGKP294-1, carrying three copies of tandem-repeated IS26-blaSHV-2A-deoR-ygbJ-ygbK-fucA-IS26 composite transposon elements. Phylogenetic analysis placed WCGKP294 in the global ST307 cluster, distant from the U.S. (Texas) and South Africa clusters. Nevertheless, WCGKP294 does not contain the chromosomal fluoroquinolone resistance-associated mutations and IncFIIK/IncFIBK plasmid-associated blaCTX-M-15 gene that are frequently found in other global ST307 strains. IMPORTANCE We described the genome and resistome characterization of a carbapenem-resistant Klebsiella pneumoniae ST307 strain carrying blaIMP-38 in China. This report highlights that the high-risk ST307 clone continues to acquire different antimicrobial resistance genes, posing significant challenges to clinical practice, and should be closely monitored.

In Enterobacterales, the most common carbapenemases are Ambler’s class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The blaOXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the blaOXA-23 gene in that isolate. By contrast to the blaOXA-23 genes, the blaOXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the blaOXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.

Knowledge of pneumococcal lineages, their geographic distribution and antibiotic resistance patterns, can give insights into global pneumococcal disease. We provide interactive bioinformatic outputs to explore such topics, aiming to increase dissemi-nation of genomic insights to the wider community, without the need for specialist training. We prepared 12 country-specific phylogenetic snapshots, and international phylogenetic snapshots of 73 common Global Pneumococcal Sequence Clusters (GPSCs) previously defined using PopPUNK, and present them in Microreact. Gene presence and absence defined using Roary, and recombination profiles derived from Gubbins are presented in Phandango for each GPSC. Temporal phylogenetic signal was assessed for each GPSC using BactDating. We provide examples of how such resources can be used. In our example use of a country-specific phylogenetic snapshot we determined that serotype 14 was observed in nine unrelated genetic backgrounds in South Africa. The international phylogenetic snapshot of GPSC9, in which most serotype 14 isolates from South Africa were observed, highlights that there were three independent sub-clusters represented by South African serotype 14 isolates. We estimated from the GPSC9-dated tree that the sub-clusters were each established in South Africa during the 1980s. We show how recombination plots allowed the identification of a 20 kb recombination spanning the capsular polysaccharide locus within GPSC97. This was consistent with a switch from serotype 6A to 19A estimated to have occured in the 1990s from the GPSC97-dated tree. Plots of gene presence/absence of resistance genes (tet, erm, cat) across the GPSC23 phylogeny were consistent with acquisition of a composite transposon. We estimated from the GPSC23-dated tree that the acquisition occurred between 1953 and 1975. Finally, we demonstrate the assignment of GPSC31 to 17 externally generated pneumococcal serotype 1 assemblies from Utah via Pathogenwatch. Most of the Utah isolates clustered within GPSC31 in a USA-specific clade with the most recent common ancestor estimated between 1958 and 1981. The resources we have provided can be used to explore to data, test hypothesis and generate new hypotheses. The accessible assignment of GPSCs allows others to contextualize their own collections beyond the data presented here.

Carbapenem resistance in Enterobacterales is a public health threat. Klebsiella pneumoniae carbapenemase (encoded by alleles of the bla KPC family) is one of the most common transmissible carbapenem resistance mechanisms worldwide. The dissemination of bla KPC historically has been associated with distinct K. pneumoniae lineages (clonal group 258 [CG258]), a particular plasmid family (pKpQIL), and a composite transposon (Tn4401). In the United Kingdom, bla KPC has represented a large-scale, persistent management challenge for some hospitals, particularly in North West England. The dissemination of bla KPC has evolved to be polyclonal and polyspecies, but the genetic mechanisms underpinning this evolution have not been elucidated in detail; this study used short-read whole-genome sequencing of 604 bla KPC-positive isolates (Illumina) and long-read assembly (PacBio)/polishing (Illumina) of 21 isolates for characterization. We observed the dissemination of bla KPC (predominantly bla KPC-2; 573/604 [95%] isolates) across eight species and more than 100 known sequence types. Although there was some variation at the transposon level (mostly Tn4401a, 584/604 [97%] isolates; predominantly with ATTGA-ATTGA target site duplications, 465/604 [77%] isolates), bla KPC spread appears to have been supported by highly fluid, modular exchange of larger genetic segments among plasmid populations dominated by IncFIB (580/604 isolates), IncFII (545/604 isolates), and IncR (252/604 isolates) replicons. The subset of reconstructed plasmid sequences (21 isolates, 77 plasmids) also highlighted modular exchange among non-bla KPC and bla KPC plasmids and the common presence of multiple replicons within bla KPC plasmid structures (>60%). The substantial genomic plasticity observed has important implications for our understanding of the epidemiology of transmissible carbapenem resistance in Enterobacterales for the implementation of adequate surveillance approaches and for control.

A novel phenotype ofEscherichia coliand Klebsiella pneumoniae resistant to piperacillin/tazobactam (TZP), but susceptible to carbapenems and 3rdgeneration cephalosporins has recently emerged. The resistance mechanism of this phenotype has been identified as hyperproduction of the β-lactamaseblaTEM, however the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood. We sought to understand this mechanism by focussing on a pair of isolates obtained from an individual patient across two infection episodes and displaying within-patient evolution to TZP resistance. Following confirmation that the two isolates were clonal, we found that the TZP-resistant isolate hyperproduced a β-lactamase but lacked mutations within β-lactamase promoter regions. Hybrid assembly of long and short sequencing reads of the two isolates revealed both harboured a novel IS26-flanked composite transposon containing several antibiotic resistance genes, includingblaTEM-1B, which was designated Tn6762. These resistance genes are also found to be present on a translocatable unit which had excised from Tn6762in the TZP-resistant isolate. By replicating the evolutionary event leading to TZP resistance we were able to observe excision of the translocatable unit from Tn6762following exposure to TZP and capture the TU in a plasmid containing a copy of IS26. Subsequent amplification of the TU, and by extensionblaTEM-1B, leads to β-lactamase hyperproduction and TZP resistance. Despite a significant increase in gene copy number (P value = blaTEM-1Bis an important consideration when using genomic data to predict resistance/susceptibility to TZP.

Regulation of the expression of the gene for chlorite dismutase (cld), located on the chlorate reduction composite transposon of the chlorate reducer Ideonella dechloratans, was studied. A 200 bp upstream sequence of the cld gene, and mutated and truncated versions thereof, was used in a reporter system in Escherichia coli. It was found that a sequence within this upstream region, which is nearly identical to the canonical FNR‐binding sequence of E. coli, is necessary for anaerobic induction of the reporter gene. Anaerobic induction was regained in an FNR‐deficient strain of E. coli when supplemented either with the fnr gene from E. coli or with a candidate fnr gene cloned from I. dechloratans. In vivo transcription of the suggested fnr gene of I. dechloratans was demonstrated by qRT‐PCR. Based on these results, the cld promoter of I. dechloratans is suggested to be a class II‐activated promoter regulated by an FNR‐type protein of I. dechloratans. No fnr‐type genes have been found on the chlorate reduction composite transposon of I. dechloratans, making anaerobic upregulation of the cld gene after a gene transfer event dependent on the presence of an fnr‐type gene in the recipient., Activation of the chlorite dismutase (cld) gene, located in the chlorate composite transposon of the chlorate reducer Ideonella dechloratans, was studied in a transcriptional reporter system in an fnr‐deficient Escherichia coli strain. The results demonstrate that an fnr‐type regulator of I. dechloratans, not present in the chlorate composite transposon, induces this gene under anaerobic conditions. Implications for horizontal gene transfer of the capability of dissimilatory chlorate reduction are discussed.

In Enterobacteriaceae, the blaOXA-48-like genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA-48-like enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the blaOXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the blaOXA-370 gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The blaOXA-370 gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera.

Objectives To determine the location of the antibiotic resistance genes in the MDR Proteus mirabilis PmPHI clinical isolate. Methods WGS and de novo assembly were performed. BLAST searches were used to identify relevant contigs. PCR and Sanger sequencing were used to link the fragments of interest and fill the gaps. Results P. mirabilis PmPHI was resistant to six classes of antibiotics: penicillins, aminoglycosides, phenicols, tetracyclines, folate inhibitors and fluoroquinolones. A novel genomic resistance island (GIPmi1) of 55.8 kb was located at the 3' end of trmE. The backbone shared 93% identity with a genomic sequence of Enterobacter cloacae DSM 16690. The MDR region was composed of two class 1 integrons [one Tn402-type (estX-qacE) and one In5-type (aadB-aadA2)], separated by a region containing many parts of transposons. An external circular form of GIPmi1 was detected; however, mobilization by an A/C plasmid failed. In addition, an SXT/R391 integrative and conjugative element (ICEPmiFra1) was inserted into prfC. It carried floR, the sul2-strA-strB cluster and a composite transposon flanked by two copies of a tISPpu12 element that contains a class 1 integron (dfrA32-ereA1-aadA2), Tn4352 (aphA1a) and tetA(C). A class 2 integron (dfrA1-sat2-aadA1) was also identified on Tn7 as well as point mutations in gyrA and parC accounting for quinolone resistance. Conclusions The finding of the new genomic island GIPmi1 belonging to the same superfamily of genomic islands as SGI1/SGI2/PGI1/AGI1 and of the integrative conjugative element ICEPmiFra1 (SXT/R391 family) suggested that these genetic elements might be key mediators of resistance gene acquisition in P. mirabilis.

The increasing incidence of carbapenem-resistant Enterobacteriaceae has become a challenge for clinical therapy. In our study, we analysed the molecular characteristics of imipenem-hydrolyzing β-lactamase (IMI) in Enterobacteriaceae isolates. Two reported clinical isolates, the IMI-3-producing Raoultella ornithinolytica RJ46C and the IMI-2-producing Escherichia coli RJ18 were identified in our retrospective review of isolates collected from June 2010 to June 2013, both isolates were resistant to carbapenem but sensitive to expanded-spectrum cephalosporins. The blaIMI gene was located on different â¼170-kb plasmids in both isolates. The blaIMI-3 gene was carried by the plasmid pRJ46C, which was extracted from the transconjugant and identified to be a 166,620-bp conjugative IncFIIY plasmid that contained 193 open reading frames, including replication-, plasmid conjugal transfer-, partitioning-, and mobilization-associated structures. The blaIMI-3 gene was located on a 15-kb region with a completely inverted sequence relative to that of plasmid pGA45, two ISEcl1-like elements containing two 33-bp complete inverted repeats were in an inverted orientation on both sides of the 15-kb region. We identified this typical structure as a novel composite transposon named Tn6306, indicating the occurrence of transposition. In addition, the blaIMI-2-carrying pRJ18 was an IncFIB plasmid, and a similar ISEcl1-like element was identified in an inverted direction upstream of IMI-2 in pRJ18. The identification of blaIMI in R. ornithinolytica and E. coli highlights the diversity of spreading carbapenemases in Enterobacteriaceae between hospitals and the environment in China. The novel transposon Tn6306, and other insert sequences, may play important roles in blaIMI mobilization. Keywords: blaIMI in Enterobacteriaceae, Genetic environment, Plasmids, Novel transposon structure Tn6306, ISEcl1-like element

Objectives To determine the occurrence of mcr-1 and mcr-2 genes in Gram-negative bacteria isolated from healthy pigs in Great Britain. Methods Gram-negative bacteria (n = 657) isolated from pigs between 2014 and 2015 were examined by WGS. Results Variants of mcr-1 and mcr-2 were identified in Moraxella spp. isolated from pooled caecal contents of healthy pigs at slaughter collected from six farms in Great Britain. Other bacteria, including Escherichia coli from the same farms, were not detected harbouring mcr-1 or mcr-2. A Moraxella porci-like isolate, MSG13-C03, harboured MCR-1.10 with 98.7% identity to MCR-1, and a Moraxella pluranimalium-like isolate, MSG47-C17, harboured an MCR-2.2 variant with 87.9% identity to MCR-2, from E. coli; the isolates had colistin MICs of 1-2 mg/L. No intact insertion elements were identified in either MSG13-C03 or MSG47-C17, although MSG13-C03 harboured the conserved nucleotides abutting the ISApl1 composite transposon found in E. coli plasmids and the intervening ∼2.6 kb fragment showed 97% identity. Six Moraxella osloensis isolates were positive for phosphoethanolamine transferase (EptA). They shared 62%-64.5% identity to MCR-1 and MCR-2, with colistin MICs from 2 to 4 mg/L. Phylogenetic analysis indicated that MCR and EptA have evolved from a common ancestor. In addition to mcr, the β-lactamase gene, blaBRO-1, was found in both isolates, whilst the tetracycline resistance gene, tetL, was found in MSG47-C17. Conclusions Our results add further evidence for the mobilization of the mcr-pap2 unit from Moraxella via composite transposons leading to its global dissemination. The presence of mcr-pap2 from recent Moraxella isolates indicates they may comprise a reservoir for mcr.

Two novel New Delhi metallo-β-lactamase-1 (NDM-1)-positive plasmids containing a complete composite transposon, Tn125, from two respective Acinetobacter towneri isolates were characterized. Plasmid pNDM-GJ01 (30,293 bp) isolated from A. towneri G165 did not show homology to any known plasmid structure, except for the transposon Tn125 containing blaNDM-1. A novel repB gene and two XRE-type transcriptional regulators were found in pNDM-GJ01. Plasmid pNDM-GJ02 (62,011 bp) isolated from A. towneri G295 showed the highest homology to pBJAB0715 (41% coverage, 99% nucleotide identity). In addition to the blaNDM-1-harbouring transposon Tn125, pNDM-GJ02 also had an IS26-composite transposon, which contains ISCR1 and two class 1 integrons carrying different cassette arrays. Both clinical isolates were highly resistant to β-lactams and susceptible to tigecycline and colistin. Ten other resistance genes were detected in G295, and one other resistance gene was detected in G165. No transconjugant was obtained from any of the donors by broth and filter mating. The emergence of these two novel plasmids carrying NDM-1 in Acinetobacter spp., pNDM-GJ01 and pNDM-GJ02, suggests Tn125 mobile integration.

Background The spread of carbapenem-resistant Enterobacteriaceae is an important challenge and an increasing healthcare problem. OXA-48 is a class D carbapenemase that is usually localized on a conjugative plasmid belonging to the IncL incompatibility group. Methods In this study, we used a combination of short- and long-read WGS approaches and molecular typing techniques to characterize the genetic environment of the smallest reported 27 029 bp IncFII plasmid carrying blaOXA-48 (pLAU-OXA48). Results The plasmid recovered from a clinical Escherichia coli isolate was positive for blaOXA-48, which was located within the Tn6237 composite transposon. Primers targeting junctions between the IncF fragment and Tn6237 for the rapid identification of pLAU-OXA48-like plasmids were designed. Conclusions To our knowledge, this is the first report showing the complete sequence of an IncFII plasmid carrying blaOXA-48 within Tn6237 using hybrid assembly of long- and short-read sequencing.

Linezolid and vancomycin are among the last-resort antimicrobial agents in the treatment of multidrug-resistant Gram-positive bacterial infections. Linezolid- and vancomycin-resistant (LVR) Gram-positive bacteria may pose severe threats to public health. In this study, three optrA- and vanG-positive Streptococcus suis strains were isolated from two farms of different cities. There were only 1 and 343 single-nucleotide polymorphisms in coding region (cSNPs) of HCB4 and YSJ7 to YSJ17, respectively. Mobilome analysis revealed the presence of vanG, erm(B), tet(O/W/32/O), and aadE-apt-sat4-aphA3 cluster on an integrative and conjugative element, ICESsuYSJ17, and erm(B), aphA3, aac(6′)-aph(2″), catpC194, and optrA on a prophage, ΦSsuYSJ17-3. ICESsuYSJ17 exhibited a mosaic structure and belongs to a highly prevalent and transferable ICESa2603 family of Streptococcus species. ΦSsuYSJ17-3 shared conserved backbone to a transferable prophage Φm46.1. A novel composite transposon, IS1216E-araC-optrA-hp-catpC194-IS1216E, which can be circulated as translocatable unit (TU) by IS1216E, was integrated on ΦSsuYSJ17-3. Vancomycin resistance phenotype and vanG transcription assays revealed that the vanG operon was inducible. The LVR strain YSJ17 exhibited moderate virulence in a zebrafish infection model. To our knowledge, this is the first report of LVR isolate, which is mediated by acquired resistance genes optrA and vanG operons in Gram-positive bacteria. Since S. suis has been recognized as an antimicrobial resistance reservoir in the spread of resistance genes to major streptococcal pathogens, the potential risks of disseminating of optrA and vanG from S. suis to other Streptococcus spp. are worrisome and routine surveillance should be strengthened.

A qepA8+ Shigella flexneri was cultured from the stool of a traveler returning from India and East Asia. This chromosomally encoded qepA variant, has a six-base insertion, and may have been mobilized as part of a complex IS1-mediated composite transposon including catA1, aadA1, and blaOXA-1. In laboratory E. coli, qepA8 alone only conferred decreased ciprofloxacin susceptibility; however, it may work in combination with additional mechanisms to confer clinical resistance.

Brevibacterium aurantiacum is an actinobacterium that confers key organoleptic properties to washed-rind cheeses during the ripening process. Although this industrially relevant species has been gaining an increasing attention in the past years, its genome plasticity is still understudied due to the unavailability of complete genomic sequences. To add insights on the mobilome of this group, we sequenced the complete genomes of five dairy Brevibacterium strains and one non-dairy strain using PacBio RSII. We performed phylogenetic and pan-genome analyses, including comparisons with other publicly available Brevibacterium genomic sequences. Our phylogenetic analysis revealed that these five dairy strains, previously identified as Brevibacterium linens, belong instead to the B. aurantiacum species. A high number of transposases and integrases were observed in the Brevibacterium spp. strains. In addition, we identified 14 and 12 new insertion sequences (IS) in B. aurantiacum and B. linens genomes, respectively. Several stretches of homologous DNA sequences were also found between B. aurantiacum and other cheese rind actinobacteria, suggesting horizontal gene transfer (HGT). A HGT region from an iRon Uptake/Siderophore Transport Island (RUSTI) and an iron uptake composite transposon were found in five B. aurantiacum genomes. These findings suggest that low iron availability in milk is a driving force in the adaptation of this bacterial species to this niche. Moreover, the exchange of iron uptake systems suggests cooperative evolution between cheese rind actinobacteria. We also demonstrated that the integrative and conjugative element BreLI (Brevibacterium Lanthipeptide Island) can excise from B. aurantiacum SMQ-1417 chromosome. Our comparative genomic analysis suggests that mobile genetic elements played an important role into the adaptation of B. aurantiacum to cheese ecosystems.

Here, we identified mcr-4.3 in Acinetobacter baumannii, which had not been previously observed to carry an mcr gene. The mcr-4.3-harboring A. baumannii strain AB18PR065 was isolated from pig feces from a slaughterhouse in Guangdong Province of China. The mcr-4.3-carrying pAB18PR065 is 25,602 bp in size and could not be transferred in conjugation, transformation, and electroporation experiments, as we did not find any conjugation-related genes therein. pAB18PR065 harbors two copies of type II toxin-antitoxin systems, which are functional in plasmid stabilization and maintenance. pAB18PR065 shares similarity only with one recently identified plasmid, pAb-MCR4.3 (35,502 bp), from a clinical A. baumannii strain. It is likely that the emergence of pAb-MCR4.3 was due to the insertion of an 11,386-bp, ISAba19-based, composite transposon into pAB18PR065. These data indicate that mcr-4.3 was captured by an A. baumannii-original plasmid via horizontal gene transfer.

Objectives bla OXA-23 is a class D carbapenemase-encoding gene typical of the Acinetobacter genus. However, its occurrence in the Enterobacteriaceae is uncommon. Here we provide the genome characterization of blaOXA-23-positive Proteus mirabilis. Methods In Singapore, a national surveillance of carbapenem non-susceptible clinical Enterobacteriaceae has enabled the collection of OXA-23 bearing isolates. Three clinical P. mirabilis were whole-genome sequenced using Oxford Nanopore MinION and Illumina platforms. The sequence accuracy of MinION long-read contigs was enhanced by polishing with Illumina-derived short-read data. Results In two P. mirabilis genomes, blaOXA-23 was detected as two copies, present on the chromosome and on a 60018 bp plasmid. blaOXA-23 was associated with the classic Acinetobacter composite transposon Tn2006, bounded by two copies of ISAba1 bracketing the carbapenemase gene. The Tn2006 itself was embedded within an Acinetobacter baumannii AbaR4 resistance island. In the chromosome, the AbaR4 was found integrated into the comM gene, which is also the preferred ‘hotspot’ in A. baumannii. In the plasmid, AbaR4 integrated into a putative colicin gene. Conclusions Our description of an A. baumannii AbaR4 encoding blaOXA-23 in P. mirabilis is to our knowledge the first description of an Acinetobacter resistance island in Proteus and suggests that P. mirabilis may be a reservoir for this class D carbapenemase gene.