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July 7, 2019

Genomic epidemiology of NDM-1-encoding plasmids in Latin American clinical isolates reveals insights into the evolution of multidrug resistance

Bacteria that produce the broad-spectrum Carbapenem antibiotic New Delhi Metallo-ß-lactamase (NDM) place a burden on health care systems worldwide, due to the limited treatment options for infections caused by them and the rapid global spread of this antibiotic resistance mechanism. Although it is believed that the associated resistance gene blaNDM-1 originated in Acinetobacter spp., the role of Enterobacteriaceae in its dissemination remains unclear. In this study, we used whole genome sequencing to investigate the dissemination dynamics of blaNDM-1-positive plasmids in a set of 21 clinical NDM-1-positive isolates from Colombia and Mexico (Providencia rettgeri, Klebsiella pneumoniae, and Acinetobacter baumannii) as well as six representative NDM-1-positive Escherichia coli transconjugants. Additionally, the plasmids from three representative P. rettgeri isolates were sequenced by PacBio sequencing and finished. Our results demonstrate the presence of previously reported plasmids from K. pneumoniae and A. baumannii in different genetic backgrounds and geographically distant locations in Colombia. Three new previously unclassified plasmids were also identified in P. rettgeri from Colombia and Mexico, plus an interesting genetic link between NDM-1-positive P. rettgeri from distant geographic locations (Canada, Mexico, Colombia, and Israel) without any reported epidemiological links was discovered. Finally, we detected a relationship between plasmids present in P. rettgeri and plasmids from A. baumannii and K. pneumoniae. Overall, our findings suggest a Russian doll model for the dissemination of blaNDM-1 in Latin America, with P. rettgeri playing a central role in this process, and reveal new insights into the evolution and dissemination of plasmids carrying such antibiotic resistance genes.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Genomic characterization of a large plasmid containing a bla NDM-1 gene carried on Salmonella enterica serovar Indiana C629 isolate from China.

The bla NDM-1 gene in Salmonella species is mostly reported in clinical cases, but is rarely isolated from red and white meat in China.A Salmonella Indiana (S. Indiana) isolate was cultured from a chicken carcass procured from a slaughterhouse in China. Antimicrobial susceptibility was tested against a panel of agents. Whole-genome sequencing of the isolate was carried out and data was analyzed.A large plasmid, denoted as plasmid pC629 (210,106 bp), containing a composite cassette, consisting of IS26-bla NDM-1-ble MBL -?trpF-tat-cutA-ISCR1-sul1-qacE?1-aadA2-dfrA12-intI1-IS26 was identified. The latter locus was physically linked with bla OXA-1, bla CTX-M-65, bla TEM-1-encoding genes. A mercury resistance operon merACDEPTR was also identified; it was flanked on the proximal side, among IS26 element and the distally located on the bla NDM-1 gene. Plasmid pC629 also contained 21 other antimicrobial resistance-encoding genes, such as aac(6′)-Ib-cr, aac(3)-VI, aadA5, aph(4)-Ia, arr-3, blmS, brp, catB3, dfrA17, floR, fosA, mph(A), mphR, mrx, nimC/nimA, oqxA, oqxB, oqxR, rmtB, sul1, sul2. Two virulence genes were also identified on plasmid pC629.To the best of our knowledge, this is the first report of bla NDM-1 gene being identified from a plasmid in a S. Indiana isolate cultured from chicken carcass in China.

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July 7, 2019

Detection of an Escherichia coli sequence type 167 strain with two tandem copies of blaNDM-1 in the chromosome.

New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Enterobacteriaceae has disseminated rapidly throughout the world and poses an urgent threat to public health. Previous studies confirmed that the blaNDM-1 gene is typically carried in plasmids but rarely in chromosome. We discovered a multidrug-resistant Escherichia coli strain Y5, originating from a urine sample and containing the blaNDM-1 gene, which did not transfer by either conjugation or electrotransformation. We confirmed the possibility of its chromosome location by S1-pulsed-field gel electrophoresis (PFGE) and XbaI-PFGE, followed by Southern blotting. To determine the genomic background of blaNDM-1, the genome of Y5 was completely sequenced and compared to other reference genomes. The results of our study revealed that this isolate consists of a 4.8-Mbp chromosome and three plasmids, it is an epidemic clone of sequence type (ST) 167, and it shows 99% identity with Escherichia coli 6409 (GenBank accession no. CP010371), which lacks the same blaNDM-1 gene-surrounding structure as Y5. The blaNDM-1 gene is embedded in the chromosome along with two tandem copies of an insertion sequence common region 1 (ISCR1) element (sul1-ARR-3-cat-blaNDM-1-bleo-ISCR1), which appears intact in the plasmid from Proteus mirabilis (GenBank accession no. KP662515). The genomic context indicates that the ISCR1 element mediated the blaNDM-1 transposition from a single source plasmid to the chromosome. Our study is the first report of an Enterobacteriaceae strain harboring a chromosomally integrated blaNDM-1, which directly reveals the vertical spreading pattern of the gene. Close surveillance is urgently needed to monitor the emergence and potential spread of ST167 strains that harbor blaNDM-1. Copyright © 2016 American Society for Microbiology.

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July 7, 2019

Emergence of Klebsiella variicola positive for NDM-9, a variant of New Delhi metallo-ß-lactamase, in an urban river in South Korea.

To examine the presence of pathogenic bacteria carrying New Delhi metallo-ß-lactamase in the environment and to characterize the genome structures of these strains.Phenotypic screening of antimicrobial susceptibility and WGS were conducted on three Klebsiella variicola strains possessing NDM-9 isolated from an urban river.Three carbapenem-resistant K. variicola isolated from Gwangju tributary were found to possess bla NDM-9 genes. Antimicrobial susceptibility testing indicated resistance of these strains to aminoglycosides, carbapenems, cephems, folate pathway inhibitors, fosfomycin and penicillins, but susceptibility to fluoroquinolones, phenicols, tetracyclines and miscellaneous agents. WGS revealed that the 108 kb IncFII(Y)-like plasmids carry bla NDM-9 sandwiched between IS 15 for the GJ1 strain, IS 26 for the GJ2 strain, IS 15D1 for the GJ3 strain and IS Vsa3 , and further bracketed by IS 26 and Tn AS3 along with the mercury resistance operon upstream and the class 1 integron composed of gene cassettes of aadA2 , dfrA12 and sul1 downstream. An aph(3′)-Ia gene conferring resistance to aminoglycosides is located after the integrons. Chromosomally encoded bla LEN-13 , fosA , aqxA and oqxB genes, as well as plasmid-mediated bla TEM-1B and bla CTX-M-65 encoding ESBL, ant(3′)-Ia and mph (A) genes, were also identified.The findings of the present study provide us with the information that NDM-9 has been spreading into the environment. Dissemination of NDM-9 in the environment has raised a health risk alarm as this variant of NDM carries MDR genes with highly transferable mobile genetic elements, increasing the possibility of resistance gene transfer among microorganisms in the environment.

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July 7, 2019

Expanding landscapes of the diversified mcr-1-bearing plasmid reservoirs.

Polymyxin is a cationic polypeptide antibiotic that can disrupt bacterial cell membrane by interacting with its lipopolysaccharide molecules and is used as a last resort drug against lethal infections by the carbapenem-resistant superbugs (like NDM-1). However, global discovery of the MCR-1 colistin resistance dramatically challenges the newly renewed interest in colistin for clinical use.The mcr-1-harboring plasmids were acquired from swine and human Escherichia coli isolated in China, from 2015 to 2016, and subjected to Illumina PacBio RSII and Hi-Seq2000 for full genome sequencing. PCR was applied to close the gap of the assembled contigs. Ori-Finder was employed to predict the replication origin (oriC) in plasmids. The phenotype of MCR-1-producing isolates was evaluated on the LBA plates with various level of colistin. Genetic deletion was used to test the requirement of the initial “ATG” codon for the MCR-1 function.Here, we report full genomes of over 10 mcr-1-harboring plasmids with diversified replication incompatibilities. A novel hybrid IncI2/IncFIB plasmid pGD17-2 was discovered and characterized from a swine isolate with colistin resistance. Intriguingly, co-occurrence of two unique mcr-1-bearing plasmids (pGD65-3, IncI2, and pGD65-5, IncX4) was detected in a single isolate GD65, which might accelerate dissemination of the mcr-1 under environmental selection pressure. Genetic analyses of these plasmids mapped mobile elements in the context of antibiotic resistance and determined two insertion sequences (ISEcp1 and ISApl1) that are responsible for the mobilization of mcr-1. Gene deletion also proved that the first ATG codon is redundant in the mcr-1 gene.Collectively, our results extend landscapes of the diversified mcr-1-bearing plasmid reservoirs.

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July 7, 2019

Emergence and evolution of multidrug-resistant Klebsiella pneumoniae with both blaKPC and blaCTX-M integrated in the chromosome.

The extended-spectrum-ß-lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae, we identified three clinical isolates, CN1, CR14, and NY9, carrying both blaCTX-M and blaKPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short- and long-read whole-genome sequencing. In CR14 and NY9, blaCTX-M and blaKPC were carried on two different plasmids. In contrast, CN1 had one copy of blaKPC-2 and three copies of blaCTX-M-15 integrated in the chromosome, for which the blaCTX-M-15 genes were linked to an insertion sequence, ISEcp1, whereas the blaKPC-2 gene was in the context of a Tn4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn4401a-blaKPC-2-prophage genomic island, CN1 also carried a clustered regularly interspaced short palindromic repeat (CRISPR)-cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of type I-E CRISPR-cas in K. pneumoniae strains and suggested that the evolving CRISPR-cas, with its acquired novel spacer, induced the mobilization of antimicrobial resistance genes from plasmids into the chromosome. The integration and dissemination of multiple copies of blaCTX-M and blaKPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae Additionally, the implications from this study also raise concerns for the application of a CRISPR-cas strategy against antimicrobial resistance. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Complete genome sequences of three representative Mycobacterium tuberculosis Beijing family strains belonging to distinct genotype clusters in Hanoi, Vietnam, during 2007 to 2009.

We present here three complete genome sequences of Mycobacterium tuberculosis Beijing family strains isolated in Hanoi, Vietnam. These three strains were selected from major genotypic clusters (15-MIRU-VNTR) identified in a previous population-based study. We emphasize their importance and potential as reference strains in this Asian region. Copyright © 2017 Wada et al.

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July 7, 2019

Completed genome sequences of Borrelia burgdorferi sensu stricto B31(NRZ) and closely related patient isolates from Europe.

Borrelia burgdorferi sensu stricto is a causative agent of human Lyme borreliosis in the United States and Europe. We report here the completed genome sequences of strain B31 isolated from a tick in the United States and two closely related strains from Europe, PAli and PAbe, which were isolated from patients with erythema migrans and neuroborreliosis, respectively. Copyright © 2017 Margos et al.

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July 7, 2019

Next-generation sequence analysis reveals transfer of methicillin resistance to a methicillin-susceptible Staphylococcus aureus strain that subsequently caused a methicillin-resistant Staphylococcus aureus outbreak: a descriptive study.

Resistance to methicillin in Staphylococcus aureus is caused primarily by the mecA gene, which is carried on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec). Horizontal transfer of this element is supposed to be an important factor in the emergence of new clones of methicillin-resistant Staphylococcus aureus (MRSA) but has been rarely observed in real time. In 2012, an outbreak occurred involving a health care worker (HCW) and three patients, all carrying a fusidic acid-resistant MRSA strain. The husband of the HCW was screened for MRSA carriage, but only a methicillin-susceptible S. aureus (MSSA) strain, which was also resistant to fusidic acid, was detected. Multiple-locus variable-number tandem-repeat analysis (MLVA) typing showed that both the MSSA and MRSA isolates were MT4053-MC0005. This finding led to the hypothesis that the MSSA strain acquired the SCCmec and subsequently caused an outbreak. To support this hypothesis, next-generation sequencing of the MSSA and MRSA isolates was performed. This study showed that the MSSA isolate clustered closely with the outbreak isolates based on whole-genome multilocus sequence typing and single-nucleotide polymorphism (SNP) analysis, with a genetic distance of 17 genes and 44 SNPs, respectively. Remarkably, there were relatively large differences in the mobile genetic elements in strains within and between individuals. The limited genetic distance between the MSSA and MRSA isolates in combination with a clear epidemiologic link supports the hypothesis that the MSSA isolate acquired a SCCmec and that the resulting MRSA strain caused an outbreak. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

First report of Klebsiella oxytoca strain simultaneously producing NDM-1, IMP-4 and KPC-2 carbapenemases.

The nucleotide sequences of five plasmids from one Klebsiella oxytoca isolate were determined using the PacBio RS II system. Plasmid analysis revealed that blaNDM-1 was carried on an IncX3 plasmid. The blaIMP-4 and blaKPC-2 genes were located on IncN and IncP-6 plasmids, respectively. Comparative sequence analysis highlighted the successful spread of carbapenemase-harboring plasmids among different enterobacterial species. We report for the first time, to our knowledge, coproducing NDM-1, KPC-2, and IMP-4 carbapenemases on a K. oxytoca isolate. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Genome evolution to penicillin resistance in serotype 3 Streptococcus pneumoniae by capsular switching.

Streptococcus pneumoniae isolates of serotype 3 were collected from cases of invasive pneumococcal disease (n= 124) throughout Japan between April 2010 and March 2013. A penicillin-resistantS. pneumoniae(PRSP) isolate from an adult patient, strain KK0981 of serotype 3, was identified among these strains. Whole-genome analysis characterized this PRSP as a recombinant strain derived from PRSP of serotype 23F with thecpslocus (20.3 kb) replaced by that of a penicillin-susceptible strain of serotype 3. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Complete genome sequence of Staphylococcus lutrae ATCC 700373, a potential pathogen isolated from deceased otters.

Despite their relevance to human health, not all staphylococcal species have been characterized. As such, the potential zoonotic threats posed by uninvestigated species and their contribution to the staphylococcal pangenome are unclear. Here, we report the complete genome sequence of Staphylococcus lutrae ATCC 700373, a coagulase-positive species isolated from deceased otters. Copyright © 2017 Veseli et al.

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July 7, 2019

Rapid and consistent evolution of colistin resistance in XDR Pseudomonas aeruginosa during morbidostat culture.

Colistin is a last resort antibiotic commonly used against multidrug-resistant strains of Pseudomonas aeruginosa To investigate the potential for in-situ evolution of resistance against colistin and to map the molecular targets of colistin resistance, we exposed two P. aeruginosa isolates to colistin using a continuous culture device known as morbidostat. As a result, colistin resistance reproducibly increased 10-fold within ten days, and 100-fold within 20 days, along with highly stereotypic, yet strain specific mutation patterns. The majority of mutations hit the pmrAB two component signaling system and genes involved in lipopolysaccharide (LPS) synthesis, including lpxC, pmrE, and migA We tracked the frequencies of all arising mutations by whole genome deep sequencing every 3-4 days to provide a detailed picture of the dynamics of resistance evolution, including competition and displacement among multiple resistant sub-populations. In seven out of 18 cultures, we observed mutations in mutS along with a mutator phenotype that seemed to facilitate resistance evolution. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia.

In this study, we report the comparative genomics and phylogenetic analysis of Corynebacterium diphtheriae strain B-D-16-78 that was isolated from a clinical specimen in 2016. The complete genome of C. diphtheriae strain B-D-16-78 was sequenced using PacBio Single Molecule, Real-Time sequencing technology and consists of a 2,474,151-bp circular chromosome with an average GC content of 53.56%. The core genome of C. diphtheriae was also deduced from a total of 74 strains with complete or draft genome sequences and the core genome-based phylogenetic analysis revealed close genetic relationship among strains that shared the same MLST allelic profile. In the context of CRISPR-Cas system, which confers adaptive immunity against re-invading DNA, 73 out of 86 spacer sequences were found to be unique to Malaysian strains which harboured only type-II-C and/or type-I-E-a systems. A total of 48 tox genes which code for the diphtheria toxin were retrieved from the 74 genomes and with the exception of one truncated gene, only nucleotide substitutions were detected when compared to the tox gene sequence of PW8. More than half were synonymous substitution and only two were nonsynonymous substitutions whereby H24Y was predicted to have a damaging effect on the protein function whilst T262V was predicted to be tolerated. Both toxigenic and non-toxigenic toxin-gene bearing strains have been isolated in Malaysia but the repeated isolation of toxigenic strains with the same MLST profile suggests the possibility of some of these strains may be circulating in the population. Hence, efforts to increase herd immunity should be continued and supported by an effective monitoring and surveillance system to track, manage and control outbreak of cases. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts.

A restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities.Two taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems.Our comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

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