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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 sequence of a commensal bacterium, Hafnia alvei CBA7124, isolated from human feces.

Members of the genus Hafnia have been isolated from the feces of mammals, birds, reptiles, and fish, as well as from soil, water, sewage, and foods. Hafnia alvei is an opportunistic pathogen that has been implicated in intestinal and extraintestinal infections in humans. However, its pathogenicity is still unclear. In this study, we isolated H. alvei from human feces and performed sequencing as well as comparative genomic analysis to better understand its pathogenicity.The genome of H. alvei CBA7124 comprised a single circular chromosome with 4,585,298 bp and a GC content of 48.8%. The genome contained 25 rRNA genes (9 5S rRNA genes, 8 16S rRNA genes, and 8 23S rRNA genes), 88 tRNA genes, and 4043 protein-coding genes. Using comparative genomic analysis, the genome of this strain was found to have 72 strain-specific singletons. The genome also contained genes for antibiotic and antimicrobial resistance, as well as toxin-antitoxin systems.We revealed the complete genome sequence of the opportunistic gut pathogen, H. alvei CBA7124. We also performed comparative genomic analysis of the sequences in the genome of H. alvei CBA7124, and found that it contained strain-specific singletons, antibiotic resistance genes, and toxin-antitoxin systems. These results could improve our understanding of the pathogenicity and the mechanism behind the antibiotic resistance of H. alvei strains.

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July 7, 2019

Complete genome sequence of Clostridioides difficile epidemic strain DH/NAP11/106/ST-42, isolated from stool from a pediatric patient with diarrhea.

We report here the complete genome sequence of Clostridioides difficile strain DH/NAP11/106/ST-42, which is now the most common strain causing C. difficile infection among U.S. adults. This strain was isolated from the stool from a hospitalized pediatric patient with frequent relapses of C. difficile infection. Copyright © 2017 Ozer et al.

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July 7, 2019

Complete genome sequence of Staphylococcus epidermidis ATCC 12228 chromosome and plasmids, generated by long-read sequencing.

Staphylococcus epidermidis ATCC 12228 was sequenced using a long-read method to generate a complete genome sequence, including some plasmid sequences. Some differences from the previously generated short-read sequence of this nonpathogenic and non-biofilm-forming strain were noted. The assembly size was 2,570,371 bp with a total G+C% content of 32.08%. Copyright © 2017 MacLea and Trachtenberg.

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July 7, 2019

Complete genome sequences of four extensively drug-resistant Pseudomonas aeruginosa strains, isolated from adults with ventilator-associated pneumonia at a tertiary referral hospital in Mexico City.

Four extensively drug-resistant Pseudomonas aeruginosa strains, isolated from patients with pneumonia, were sequenced using PacBio RS-II single-molecule real-time (SMRT) technology. Genome sequence analysis identified great variability among mobile genetic elements, as well as some previously undescribed genomic islands and new variants of class 1 integrons (In1402, In1403, In1404, and In1408). Copyright © 2017 Espinosa-Camacho et al.

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July 7, 2019

Pseudomonas aeruginosa clinical isolates in Nepal coproducing metallo-ß-lactamases and 16S rRNA methyltransferases.

A total of 11 multidrug-resistant Pseudomonas aeruginosa clinical isolates were obtained in Nepal. Four of these isolates harbored genes encoding one or more carbapenemases (DIM-1, NDM-1, and/or VIM-2), and five harbored genes encoding a 16S rRNA methyltransferase (RmtB4 or RmtF2). A novel RmtF variant, RmtF2, had a substitution (K65E) compared with the same gene in RmtF. To our knowledge, this is the first report describing carbapenemase- and 16S rRNA methyltransferase-coproducing P. aeruginosa clinical isolates in Nepal. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Complete genome sequences of Lactobacillus curvatus KG6, L. curvatus MRS6, and Lactobacillus sakei FAM18311, isolated from fermented meat products.

The genomes of Lactobacillus curvatus KG6, L. curvatus MRS6, and Lactobacillus sakei FAM18311 were sequenced and assembled using PacBio single-molecule real-time (SMRT) technology. The strains were isolated from Swiss fermented meat products. Circular chromosomes were of 1.98 Mbp (KG6), 2.11 Mbp (MRS6), and 1.95 Mbp (FAM18311), with a G+C content of 41.3 to 42.0%. Copyright © 2017 Jans 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

A novel aerobic degradation pathway of thiobencarb is initiated by a two-component FMN-dependent monooxygenase system TmoAB in Acidovorax sp. T1.

Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. T1. Thiobencarb was oxidized and cleaved at the C-S bond, generating diethylcarbamothioic S-acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in this strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28-32% identities with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum, alkanesulfonate monooxygenase from Pseudomonas savastanoi and dibenzothiophene monooxygenase from Rhodococcus sp.. TmoB shared 25-37% identities with reported flavin reductases and oxidized NADH but not NADPH. TmoAB was an FMN-dependent monooxygenase and catalyzed the C-S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC, was located 7129 bp downstream of tmoAB, and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD(+) as a cofactor. A gene cluster responsible for complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis.IMPORTANCE Microbial degradation is the main factor of thiobencarb dissipation in soil. In previous reports, thiobencarb was degraded initially via N-deethylation, sulfoxidation, hydroxylation and dechlorination. However, enzymes and genes involved in microbial degradation of thiobencarb have not been studied. This study revealed a new thiobencarb degradation pathway in strain Acidovorax sp. T1 and identified a novel two-component FMN-dependent monooxygenase system TmoAB. Under TmoAB-mediated catalysis, thiobencarb was cleaved at the C-S bond, producing diethylcarbamothioic S-acid and 4CDA. Furthermore, the downstream degradation pathway of thiobencarb was proposed. Our study provides the physiological, biochemical and genetic foundation of thiobencarb degradation in this microorganism. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Genome and plasmid sequences of Escherichia coli KV7, an extended-spectrum ß-lactamase isolate derived from feces of a healthy pig.

We present single-contig assemblies for Escherichia coli strain KV7 (serotype O27, phylogenetic group D) and its six plasmids, isolated from a healthy pig, as determined by PacBio RS II and Illumina MiSeq sequencing. The chromosome of 4,997,475 bp and G+C content of 50.75% harbored 4,540 protein-encoding genes. Copyright © 2017 Bateman et al.

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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

Resistance to ceftazidime-avibactam is due to tranposition of KPC in a porin-deficient strain of Klebsiella pneumoniae with increased efflux activity.

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 µg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype. Copyright © 2017 American Society for Microbiology.

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