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July 7, 2019

Identification and characterization of the novel colonization factor CS30 based on whole genome sequencing in enterotoxigenic Escherichia coli (ETEC).

The ability to colonize the small intestine is essential for enterotoxigenic Escherichia coli (ETEC) to cause diarrhea. Although 22 antigenically different colonization factors (CFs) have been identified and characterized in ETEC at least 30% of clinical ETEC isolates lack known CFs. Ninety-four whole genome sequenced “CF negative” isolates were searched for novel CFs using a reverse genetics approach followed by phenotypic analyses. We identified a novel CF, CS30, encoded by a set of seven genes, csmA-G, related to the human CF operon CS18 and the porcine CF operon 987P (F6). CS30 was shown to be thermo-regulated, expressed at 37?°C, but not at 20?°C, by SDS-page and mass spectrometry analyses as well as electron microscopy imaging. Bacteria expressing CS30 were also shown to bind to differentiated human intestinal Caco-2 cells. The genes encoding CS30 were located on a plasmid (E873p3) together with the genes encoding LT and STp. PCR screening of ETEC isolates revealed that 8.6% (n?=?13) of “CF negative” (n?=?152) and 19.4% (n?=?13) of “CF negative” LT?+?STp (n?=?67) expressing isolates analyzed harbored CS30. Hence, we conclude that CS30 is common among “CF negative” LT?+?STp isolates and is associated with ETEC that cause diarrhea.

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July 7, 2019

Identification of novel conjugative plasmids with multiple copies of fosB that confer high-level fosfomycin resistance to vancomycin-resistant Enterococci.

To further characterize the fosB-carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the fosB- and vanA-containing plasmids of Enterococcus faecium (pEMA120) and E. avium (pEA19081) were obtained by single-molecule, real-time sequencing. We found that these two plasmids are essentially identical (99.99% nucleotide sequence identity), which proved the possibility of interspecies transmission. Comparative analysis of the plasmids revealed that the backbone of pEMA120 is 99% similar to a conjugative fosB-negative E. faecium plasmid, pZB18. There is a traE disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact traE. The difference of their transfer frequencies between pEMA120 and pZB18 suggests this interruption of traE might affect conjugative transfer. Two copies of the fosB gene linked to a tnpA gene, forming an ISL3-like transposon, were found at separate locations within pEMA120, which had not been reported previously. These two fosB-carrying transposons were confirmed to form circular intermediates by inverse PCR. The hybridization of plasmid DNA digested by BsaI, having restriction site within the fosB sequence, demonstrated that the presence of multiple copies of fosB per plasmid is common. The total copy number of the fosB gene as revealed by qRT-PCR did not correlate with fosfomycin MICs or growth rates at sub-MICs of fosfomycin in different transconjugants. From susceptibility tests, the fosB gene, regardless of the copy number, conferred high fosfomycin MICs that ranged from 16384 to 65536 µg/ml. This first complete nucleotide sequence of a plasmid carrying two copies of fosB in VRE suggests that the fosB gene can transfer to multiple loci of plasmids by the ISL3 family transposase TnpA, possibly in the form of circular intermediates, leading to the dissemination of high fosfomycin resistance in VRE.

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July 7, 2019

Genome sequencing and comparative genomics of enterohemorrhagic Escherichia coli O145:H25 and O145:H28 reveal distinct evolutionary paths and marked variations in traits associated with virulence & colonization.

Enterohemorrhagic Escherichia coli (EHEC) O145 are among the top non-O157 serogroups associated with severe human disease worldwide. Two serotypes, O145:H25 and O145:H28 have been isolated from human patients but little information is available regarding the virulence repertoire, origin and evolutionary relatedness of O145:H25. Hence, we sequenced the complete genome of two O145:H25 strains associated with hemolytic uremic syndrome (HUS) and compared the genomes with those of previously sequenced O145:H28 and other EHEC strains.The genomes of the two O145:H25 strains were 5.3 Mbp in size; slightly smaller than those of O145:H28 and other EHEC strains. Both strains contained three nearly identical plasmids and several prophages and integrative elements, many of which differed significantly in size, gene content and organization as compared to those present in O145:H28 and other EHECs. Furthermore, notable variations were observed in several fimbrial gene cluster and intimin types possessed by O145:H25 and O145:H28 indicating potential adaptation to distinct areas of host colonization. Comparative genomics further revealed that O145:H25 are genetically more similar to other non-O157 EHEC strains than to O145:H28.Phylogenetic analysis accompanied by comparative genomics revealed that O145:H25 and O145:H28 evolved from two separate clonal lineages and that horizontal gene transfer and gene loss played a major role in the divergence of these EHEC serotypes. The data provide further evidence that ruminants might be a possible reservoir for O145:H25 but that they might be impaired in their ability to establish a persistent colonization as compared to other EHEC strains.

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July 7, 2019

Complete genome sequences of the plant pathogens Ralstonia solanacearum type strain K60 and R. solanacearum race 3 biovar 2 strain UW551.

Ralstonia solanacearum is a globally distributed plant pathogen that causes bacterial wilt diseases of many crop hosts, threatening both sustenance farming and industrial agriculture. Here, we present closed genome sequences for the R. solanacearum type strain, K60, and the cool-tolerant potato brown rot strain R. solanacearum UW551, a highly regulated U.S. select agent pathogen. Copyright © 2017 Hayes et al.

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July 7, 2019

Parallel evolution of group B Streptococcus hypervirulent clonal complex 17 unveils new pathoadaptive mutations.

Group B Streptococcus (GBS) is a commensal of the gastrointestinal and genitourinary tracts, while a prevailing cause of neonatal disease worldwide. Of the various clonal complexes (CCs), CC17 is overrepresented in GBS-infected newborns for reasons that are still largely unknown. Here, we report a comprehensive genomic analysis of 626 CC17 isolates collected worldwide, identifying the genetic traits behind their successful adaptation to humans and the underlying differences between carriage and clinical strains. Comparative analysis with 923 GBS genomes belonging to CC1, CC19, and CC23 revealed that the evolution of CC17 is distinct from that of other human-adapted lineages and recurrently targets functions related to nucleotide and amino acid metabolism, cell adhesion, regulation, and immune evasion. We show that the most distinctive features of disease-specific CC17 isolates were frequent mutations in the virulence-associated CovS and Stk1 kinases, underscoring the crucial role of the entire CovRS regulatory pathway in modulating the pathogenicity of GBS. Importantly, parallel and convergent evolution of major components of the bacterial cell envelope, such as the capsule biosynthesis operon, the pilus, and Rib, reflects adaptation to host immune pressures and should be taken into account in the ongoing development of a GBS vaccine. The presence of recurrent targets of evolution not previously implicated in virulence also opens the way for uncovering new functions involved in host colonization and GBS pathogenesis. IMPORTANCE The incidence of group B Streptococcus (GBS) neonatal disease continues to be a significant cause of concern worldwide. Strains belonging to clonal complex 17 (CC17) are the most frequently responsible for GBS infections in neonates, especially among late-onset disease cases. Therefore, we undertook the largest genomic study of GBS CC17 strains to date to decipher the genetic bases of their remarkable colonization and infection ability. We show that crucial functions involved in different steps of the colonization or infection process of GBS are distinctly mutated during the adaptation of CC17 to the human host. In particular, our results implicate the CovRS two-component regulator of virulence in the differentiation between carriage- and disease-associated isolates. Not only does this work raise important implications for the ongoing development of a vaccine against GBS but might also drive the discovery of key functions for GBS adaptation and pathogenesis that have been overlooked until now. Author Video: An author video summary of this article is available.

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July 7, 2019

Population structure and local adaptation of MAC lung disease agent Mycobacterium avium subsp. hominissuis.

Mycobacterium avium subsp. hominissuis (MAH) is one of the most common nontuberculous mycobacterial species responsible for chronic lung disease in humans. Despite increasing worldwide incidence, little is known about the genetic mechanisms behind the population evolution of MAH. To elucidate the local adaptation mechanisms of MAH, we assessed genetic population structure, the mutual homologous recombination, and gene content for 36 global MAH isolates, including 12 Japanese isolates sequenced in the present study. We identified five major MAH lineages and found that extensive mutual homologous recombination occurs among them. Two lineages (MahEastAsia1 and MahEastAsia2) were predominant in the Japanese isolates. We identified alleles unique to these two East Asian lineages in the loci responsible for trehalose biosynthesis (treS and mak) and in one mammalian cell entry operon, which presumably originated from as yet undiscovered mycobacterial lineages. Several genes and alleles unique to East Asian strains were located in the fragments introduced via recombination between East Asian lineages, suggesting implication of recombination in local adaptation. These patterns of MAH genomes are consistent with the signature of distribution conjugative transfer, a mode of sexual reproduction reported for other mycobacterial species.© 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

First complete genome sequences of Xanthomonas citri pv. vignicola strains CFBP7111, CFBP7112, and CFBP7113 obtained using long-read technology

Xanthomonas citri pv. vignicola strains cause bacterial blight of the legume crop cowpea. We report whole-genome sequences of three X. citri pv. vignicola strains obtained using PacBio single-molecule real-time sequencing. Such genomic data provide new information on pathogenicity factors, such as transcription activator-like effectors. Copyright © 2017 Ruh et al.

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July 7, 2019

Key features of mcr-1-bearing plasmids from Escherichia coli isolated from humans and food.

Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins.In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5a and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared.MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a single mcr-1 harboring plasmid. Transferable IncI2 (size ca. 60-61 kb) and IncX4 (size ca. 33-35 kb) type plasmids each bearing mcr-1 were found associated with human and food isolates. None of the mcr-1-positive IncI2 and IncX4 plasmids possessed any additional resistance determinants. Surprisingly, all but one of the sequenced mcr-1-positive plasmids lacked the ISApl1 element, which is a key element mediating acquisition of mcr-1 into various plasmid backbones.There is strong evidence that the food chain may be an important transmission route for mcr-1-bearing plasmids. Our data suggest that some “epidemic” plasmids rather than specific E. coli clones might be responsible for the spread of the mcr-1 gene along the food chain.

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July 7, 2019

Convergent evolution of Y chromosome gene content in flies.

Sex-chromosomes have formed repeatedly across Diptera from ordinary autosomes, and X-chromosomes mostly conserve their ancestral genes. Y-chromosomes are characterized by abundant gene-loss and an accumulation of repetitive DNA, yet the nature of the gene repertoire of fly Y-chromosomes is largely unknown. Here we trace gene-content evolution of Y-chromosomes across 22 Diptera species, using a subtraction pipeline that infers Y genes from male and female genome, and transcriptome data. Few genes remain on old Y-chromosomes, but the number of inferred Y-genes varies substantially between species. Young Y-chromosomes still show clear evidence of their autosomal origins, but most genes on old Y-chromosomes are not simply remnants of genes originally present on the proto-sex-chromosome that escaped degeneration, but instead were recruited secondarily from autosomes. Despite almost no overlap in Y-linked gene content in different species with independently formed sex-chromosomes, we find that Y-linked genes have evolved convergent gene functions associated with testis expression. Thus, male-specific selection appears as a dominant force shaping gene-content evolution of Y-chromosomes across fly species.While X-chromosome gene content tends to be conserved, Y-chromosome evolution is dynamic and difficult to reconstruct. Here, Mahajan and Bachtrog use a subtraction pipeline to identify Y-linked genes in 22 Diptera species, revealing patterns of Y-chromosome gene-content evolution.

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July 7, 2019

The rapid in vivo evolution of Pseudomonas aeruginosa in ventilator-associated pneumonia patients leads to attenuated virulence.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe airway infections in humans. These infections are usually difficult to treat and associated with high mortality rates. While colonizing the human airways, P. aeruginosa could accumulate genetic mutations that often lead to its better adaptability to the host environment. Understanding these evolutionary traits may provide important clues for the development of effective therapies to treat P. aeruginosa infections. In this study, 25 P. aeruginosa isolates were longitudinally sampled from the airways of four ventilator-associated pneumonia (VAP) patients. Pacbio and Illumina sequencing were used to analyse the in vivo evolutionary trajectories of these isolates. Our analysis showed that positive selection dominantly shaped P. aeruginosa genomes during VAP infections and led to three convergent evolution events, including loss-of-function mutations of lasR and mpl, and a pyoverdine-deficient phenotype. Specifically, lasR encodes one of the major transcriptional regulators in quorum sensing, whereas mpl encodes an enzyme responsible for recycling cell wall peptidoglycan. We also found that P. aeruginosa isolated at late stages of VAP infections produce less elastase and are less virulent in vivo than their earlier isolated counterparts, suggesting the short-term in vivo evolution of P. aeruginosa leads to attenuated virulence.© 2017 The Authors.

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July 7, 2019

Complete genome of Arthrobacter alpinus strain R3.8, bioremediation potential unraveled with genomic analysis.

Arthrobacter alpinus R3.8 is a psychrotolerant bacterial strain isolated from a soil sample obtained at Rothera Point, Adelaide Island, close to the Antarctic Peninsula. Strain R3.8 was sequenced in order to help discover potential cold active enzymes with biotechnological applications. Genome analysis identified various cold adaptation genes including some coding for anti-freeze proteins and cold-shock proteins, genes involved in bioremediation of xenobiotic compounds including naphthalene, and genes with chitinolytic and N-acetylglucosamine utilization properties and also plant-growth-influencing properties. In this genome report, we present a complete genome sequence of A. alpinus strain R3.8 and its annotation data, which will facilitate exploitation of potential novel cold-active enzymes.

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July 7, 2019

Genetic plasticity of the Shigella virulence plasmid is mediated by intra- and inter-molecular events between insertion sequences.

Acquisition of a single copy, large virulence plasmid, pINV, led to the emergence of Shigella spp. from Escherichia coli. The plasmid encodes a Type III secretion system (T3SS) on a 30 kb pathogenicity island (PAI), and is maintained in a bacterial population through a series of toxin:antitoxin (TA) systems which mediate post-segregational killing (PSK). The T3SS imposes a significant cost on the bacterium, and strains which have lost the plasmid and/or genes encoding the T3SS grow faster than wild-type strains in the laboratory, and fail to bind the indicator dye Congo Red (CR). Our aim was to define the molecular events in Shigella flexneri that cause loss of Type III secretion (T3S), and to examine whether TA systems exert positional effects on pINV. During growth at 37°C, we found that deletions of regions of the plasmid including the PAI lead to the emergence of CR-negative colonies; deletions occur through intra-molecular recombination events between insertion sequences (ISs) flanking the PAI. Furthermore, by repositioning MvpAT (which belongs to the VapBC family of TA systems) near the PAI, we demonstrate that the location of this TA system alters the rearrangements that lead to loss of T3S, indicating that MvpAT acts both globally (by reducing loss of pINV through PSK) as well as locally (by preventing loss of adjacent sequences). During growth at environmental temperatures, we show for the first time that pINV spontaneously integrates into different sites in the chromosome, and this is mediated by inter-molecular events involving IS1294. Integration leads to reduced PAI gene expression and impaired secretion through the T3SS, while excision of pINV from the chromosome restores T3SS function. Therefore, pINV integration provides a reversible mechanism for Shigella to circumvent the metabolic burden imposed by pINV. Intra- and inter-molecular events between ISs, which are abundant in Shigella spp., mediate plasticity of S. flexneri pINV.

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July 7, 2019

A nosocomial outbreak of extensively drug resistant (XDR) Acinetobacter baumannii isolates containing blaOXA-237 encoded on a plasmid.

Carbapenem antibiotics are among the mainstay for treating infections caused by Acinetobacter baumannii, especially in the Northwest United States where carbapenem resistant A. baumannii remain relatively rare. However, between June 2012 and October 2014, an outbreak of carbapenem-resistant A. baumannii occurred in 16 patients from 5 healthcare facilities in the state of Oregon. All isolates were defined as extensively-drug resistant (XDR). MLST revealed that the isolates belonged to sequence type 2 (international clone 2, IC2), and were greater than 95% similar by rep-PCR analysis. Multiplex PCR revealed the presence of a blaOXA carbapenemase gene, later identified as blaOXA-237 Whole genome sequencing of all isolates revealed a well-supported separate branch within a global A. baumannii phylogeny. Pacific Biosciences (PacBio) SMRT sequencing was also performed on one isolate to gain insight into the genetic location of the carbapenem resistance gene. We discovered that blaOXA-237, flanked on either side by ISAba1 elements in opposite orientations, was carried by a 15,198 bp plasmid designated pORAB01-3, and was present in all 16 isolates. The plasmid also contained genes encoding for: a TonB-dependent receptor, septicolysin, a type IV secretory system conjugative DNA transfer family protein, an integrase, a RepB family plasmid DNA replication initiator protein, an a/ß hydrolase, and a BrnT/BrnA type II toxin-antitoxin system. This is the first reported outbreak associated with this specific carbapenemase. Particularly worrisome is that blaOXA-237 was plasmid encoded and found in the most prominent worldwide clonal group IC2, potentially giving pORAB01-3 great capacity for future widespread dissemination. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Distinct mechanisms of acquisition of mcr-1 -bearing plasmid by Salmonella strains recovered from animals and food samples.

Since the report of its discovery in E. coli in late 2015, the plasmid-mediated colistin resistance gene, mcr-1, has been detected in various bacterial species in clinical setting and various environmental niches. However, the transmission mechanisms of this gene in Salmonella is less defined. In this study, we conducted a comprehensive study to characterize the genetic features of mcr-1-positive Salmonella strains isolated from animals and foods. Our data revealed that Salmonella recovered from animals and food specimens exhibited highly different PFGE patterns, and acquired mcr-1-encoding plasmids via different mechanism. Plasmids harboring mcr-1 in Salmonella food isolates were all conjugative and similar as plasmids reported in other species of Enterobacteriaceae, whereas mcr-1-bearing plasmids from animal Salmonella isolates were not conjugative, and belonged to the IncHI2 type. The lack of a region carrying the tra genes was found to account for the inability to undergo conjugation for various sizes of IncHI2 plasmids harbored by animal strains. These data suggest that transmission of mcr-1-positive Salmonella from animal to food might not be a common event and food isolates may have acquired mcr-1-bearing plasmids from other mcr-1-positive bacteria such as E. coli, which co-exist in food samples.

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July 7, 2019

Whole-genome sequencing identification of a multidrug-resistant Salmonella enterica serovar Typhimurium strain carrying blaNDM-5 from Guangdong, China.

A carbapenem-resistant Salmonella enterica serovar Typhimurium (sequence type 34 [ST34]) strain was isolated from a fecal specimen from a child with acute diarrhea. Whole-genome sequencing revealed that the 84.5-kb IncFII plasmid pST41-NDM carrying the NDM-5 carbapenemase gene possesses a structure identical to that of the IncFII-type plasmid backbone. However, the blaNDM-5 flanking sequence found in this plasmid is identical to the blaNDM-5-positive IncX3 plasmids carried by 10 strains of Enterobacteriaceae identified in the same hospital. Copyright © 2017 Elsevier B.V. All rights reserved.

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