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March 1, 2019

Identification of Diverse Integron and Plasmid Structures Carrying a Novel Carbapenemase Among Pseudomonas Species.

A novel carbapenem-hydrolyzing beta-lactamase, called IMP-63, was identified in three clonally distinct strains of Pseudomonas aeruginosa and two strains of Pseudomonas putida isolated within a 4 year timeframe in three French hospitals. The blaIMP-63 gene that encodes this carbapenemase turned out to be located in the variable region of four integrons (In1297, In1574, In1573, and In1572) and to coexist with novel or rare gene cassettes (fosM, gcu170, gcuF1) and insertion elements (ISPsp7v, ISPa16v). All these integrons except one (In1574) were flanked by a copy of insertion sequence ISPa17 next to the orf6 putative gene, and were carried by non-conjugative plasmids…

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January 1, 2019

The bile salt glycocholate induces global changes in gene and protein expression and activates virulence in enterotoxigenic Escherichia coli.

Pathogenic bacteria use specific host factors to modulate virulence and stress responses during infection. We found previously that the host factor bile and the bile component glyco-conjugated cholate (NaGCH, sodium glycocholate) upregulate the colonization factor CS5 in enterotoxigenic Escherichia coli (ETEC). To further understand the global regulatory effects of bile and NaGCH, we performed Illumina RNA-Seq and found that crude bile and NaGCH altered the expression of 61 genes in CS5?+?CS6 ETEC isolates. The most striking finding was high induction of the CS5 operon (csfA-F), its putative transcription factor csvR, and the putative ETEC virulence factor cexE. iTRAQ-coupled LC-MS/MS proteomic…

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November 1, 2018

A coupled role for CsMYB75 and CsGSTF1 in anthocyanin hyperaccumulation in purple tea.

Cultivars of purple tea (Camellia sinensis) that accumulate anthocyanins in place of catechins are currently attracting global interest in their use as functional health beverages. RNA-seq of normal (LJ43) and purple Zijuan (ZJ) cultivars identified the transcription factor CsMYB75 and phi (F) class glutathione transferase CsGSTF1 as being associated with anthocyanin hyperaccumulation. Both genes mapped as a quantitative trait locus (QTL) to the purple bud leaf color (BLC) trait in F1 populations, with CsMYB75 promoting the expression of CsGSTF1 in transgenic tobacco (Nicotiana tabacum). Although CsMYB75 elevates the biosynthesis of both catechins and anthocyanins, only anthocyanins accumulate in purple tea,…

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October 1, 2018

Identification of the KPC plasmid pCT-KPC334: New insights on the evolutionary pathway of epidemic plasmids harboring fosA3-blaKPC-2 genes.

A novel, non-conjugative plasmid pKP1034 isolated from a fosfomycin-resistant, carbapenemase-producing Klebsiella pneumonia strain KP1034 was recently reported to carry fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12 and rmtB genes, and was hypothesized to evolve from several recombination events of two closely related plasmids, pHN7A8 and pKPC-LK30 [1]. In this study, a plasmid pCT-KPC334 carrying fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, blaTEM-1, and rmtB genes was identified, providing evidence on the evolutionary pathway of plasmids harboring fosA3-blaKPC-2 genes.

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August 1, 2018

Emergence of XDR Escherichia coli carrying both blaNDM and mcr-1 genes in chickens at slaughter and the characterization of two novel blaNDM-bearing plasmids.

The emergence and spread of carbapenem-resistant isolates, especially New Delhi MBL (NDM)-producing Enterobacteriaceae, has become a global concern. Although NDM-producing Enterobacteriaceae have been mostly observed in clinical cases, they have also been identified in food-producing animals and wildlife. Recently, XDR bacteria harbouring both blaNDMand mcr-1 genes were observed in isolates from animals, posing a potential threat to public health. However, reports on the coexistence of blaNDMand mcr-1 in bacteria isolated from animals at slaughter remains sporadic. Here, we report two Escherichia coli strains, SD133 and SD138, co-producing NDM and MCR-1, isolated from chickens at slaughter in July 2015 in China.

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July 1, 2018

Evolution and comparative genomics of F33:A-:B- plasmids carrying blaCTX-M-55 or blaCTX-M-65 in Escherichia coli and Klebsiella pneumoniae isolated from animals.

To understand the underlying evolution process of F33:A-:B- plasmids among Enterobacteriaceae isolates of various origins in China, the complete sequences of 17 blaCTX-M-harboring F33:A-:B- plasmids obtained from Escherichia coli and Klebsiella pneumoniae isolates from different sources (animals, animal-derived food, and human clinics) in China were determined. F33:A-:B- plasmids shared similar plasmid backbones comprising replication, leading, and conjugative transfer regions and differed by the numbers of repeats in yddA and traD and by the presence of group II intron, except that pHNAH9 lacked a large segment of the leading and transfer regions. The variable regions of F33:A-B- plasmids were distinct and…

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July 1, 2018

Pol V-mediated translesion synthesis elicits localized untargeted mutagenesis during post-replicative gap repair.

In vivo, replication forks proceed beyond replication-blocking lesions by way of downstream repriming, generating daughter strand gaps that are subsequently processed by post-replicative repair pathways such as homologous recombination and translesion synthesis (TLS). The way these gaps are filled during TLS is presently unknown. The structure of gap repair synthesis was assessed by sequencing large collections of single DNA molecules that underwent specific TLS events in vivo. The higher error frequency of specialized relative to replicative polymerases allowed us to visualize gap-filling events at high resolution. Unexpectedly, the data reveal that a specialized polymerase, Pol V, synthesizes stretches of DNA both upstream and…

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June 27, 2018

Clinical Staphylococcus argenteus develops to small colony variants to promote persistent infection.

Staphylococcus argenteus is a novel staphylococcal species (also considered as a part of Staphylococcus aureus complex) that is infrequently reported on, and clinical S. argenteus infections are largely unstudied. Here, we report a persistent and recurrent hip joint infection case in which a S. argenteus strain and its small colony variants (SCVs) strain were successively isolated. We present features of the two S. argenteus strains and case details of their pathogenicity, explore factors that induce S. argenteus SCVs formation in the course of anti-infection therapy, and reveal potential genetic mechanisms for S. argenteus SCVs formation. S. argenteus strains were identified…

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June 1, 2018

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should…

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May 1, 2018

Characterization of the complete sequences and stability of plasmids carrying the genes aac(6′)-Ib-cr or qnrS in Shigella flexneri in the Hangzhou area of China.

The aim of this study was to explore the fluoroquinolone resistance mechanism of aac (6')-Ib-cr and qnrS gene by comparing complete sequences and stability of the aac(6')-Ib-cr- and qnrS-positive plasmids from Shigella isolates in the Hangzhou area of China. The complete sequences of four newly acquired plasmids carrying aac(6')-Ib-cr or qnrS were compared with those of two plasmids obtained previously and two similar reference Escherichia coli plasmids. The results showed that the length, antibiotic resistance genes and genetic environment were different among the plasmids. Moreover, the plasmid stability of three wild-type isolates and five plasmid transformants carrying aac(6')-Ib-cr and/or qnrS…

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May 1, 2018

An improved medium for colistin susceptibility testing.

The plasmid-located colistin resistance gene mcr-1 confers low-level resistance to colistin, a last-line antibiotic against multidrug-resistant Gram-negative bacteria. Current CLSI-EUCAST recommendations require the use of a broth microdilution (BMD) method with cation-adjusted Mueller-Hinton (CA-MH) medium for colistin susceptibility testing, but approximately 15% of all MCR-1 producers are classified as sensitive in that broth. Here we report on an improved calcium-enhanced Mueller-Hinton (CE-MH) medium that permits simple and reliable determination of mcr-1-containing Enterobacteriaceae Colistin susceptibility testing was performed for 50 mcr-1-containing Escherichia coli and Klebsiella pneumoniae isolates, 7 intrinsically polymyxin-resistant species, K. pneumoniae and E. coli isolates with acquired resistance to…

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March 1, 2018

A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering.

Despite the need for inducible promoters in strain development efforts, the majority of engineering in Saccharomyces cerevisiae continues to rely on a few constitutively active or inducible promoters. Building on advances that use the modular nature of both transcription factors and promoter regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein. Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain…

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February 5, 2018

High genetic plasticity in multidrug-resistant sequence type 3-IncHI2 plasmids revealed by sequence comparison and phylogenetic analysis.

We report a novel fusion plasmid, pP2-3T, cointegrating sequence type 3 (ST3)-IncHI2 with an IncFII plasmid backbone mediating multidrug resistance (MDR) and virulence. Phylogenetic analysis and comparative genomics revealed that pP2-3T and other MDR ST3-IncHI2 plasmids clustered together, representing a unique IncHI2 lineage that exhibited high conservation in backbones of plasmids but possessed highly genetic plasticity in various regions by acquiring numerous antibiotic resistance genes and fusing with other plasmids. Surveillance studies should be performed to monitor multiresistance IncHI2 plasmids among Enterobacteriaceae. Copyright © 2018 American Society for Microbiology.

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January 6, 2018

Functional metagenomics reveals a novel carbapenem-hydrolyzing mobile beta-lactamase from Indian river sediments contaminated with antibiotic production waste.

Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to…

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January 1, 2018

Extensively drug-resistant Escherichia coli sequence type 1642 carrying an IncX3 plasmid containing the blaKPC-2 gene associated with transposon Tn4401a.

Extensively drug-resistant (XDR) Enterobacteriaceae carrying the bla(KPC) gene have emerged as a major global therapeutic concern. The purpose of this study was to analyze the complete sequences of plasmids from KPC-2 carbapenemase-producing XDR Escherichia coli sequence type (ST) 1642 isolates.We performed antimicrobial susceptibility testing, PCR, multilocus sequence typing (MLST), and whole-genome sequencing to characterize the plasmid-mediated KPC-2-producing E. coli clinical isolates.The isolates were resistant to most available antibiotics, including meropenem, ampicillin, ceftriaxone, gentamicin, and ciprofloxacin, but susceptible to tigecycline and colistin. The isolates were identified as the rare ST1642 by MLST. The isolates carried four plasmids: the first 69-kb conjugative…

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