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July 7, 2019

Genomic analysis of the multi-drug-resistant clinical isolate Myroides odoratimimus PR63039.

Myroides odoratimimus (M. odoratimimus) has been gradually implicated as an important nosocomial pathogen that poses a serious health threat to immunocompromised patients owing to its multi-drug resistance. However, the resistance mechanism is currently unclear. To clarify the antibiotic resistance and infectivity mechanisms of M. odoratimimus, whole genome sequencing was performed on the multi-drug-resistant M. odoratimimus strain PR63039. The genome sequence was completed with single molecule real-time (SMRT) technologies. Then, annotation was performed using RAST and IMG-ER. A number of databases and software programs were used to analyze the genomic characteristics, including GC-Profile, ISfinder, CG viewer, ARDB, CARD, ResFinder, the VFDB database, PHAST and Progressive Mauve. The M. odoratimimus PR63039 genome consisted of a chromosome and a plasmid. The genome contained a large number of resistance genes and virulence factors. The distribution of the resistance genes was distinctive, and a resistance region named MY63039-RR was found. The subsystem features generated by RAST indicated that the annotated genome had 108 genes that were potentially involved in virulence, disease and defense, all of which had strong associations with resistance and pathogenicity. The prophage analysis showed two incomplete prophages in the genome. The genomic analysis of M. odoratimimus PR63039 partially clarified its antibiotic resistance mechanisms and virulence factors. Obtaining a clear understanding of its genomic characteristics will be conducive to the management of multidrug-resistant M. odoratimimus.

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July 7, 2019

The value of new genome references.

Genomic information has become a ubiquitous and almost essential aspect of biological research. Over the last 10-15 years, the cost of generating sequence data from DNA or RNA samples has dramatically declined and our ability to interpret those data increased just as remarkably. Although it is still possible for biologists to conduct interesting and valuable research on species for which genomic data are not available, the impact of having access to a high quality whole genome reference assembly for a given species is nothing short of transformational. Research on a species for which we have no DNA or RNA sequence data is restricted in fundamental ways. In contrast, even access to an initial draft quality genome (see below for definitions) opens a wide range of opportunities that are simply not available without that reference genome assembly. Although a complete discussion of the impact of genome sequencing and assembly is beyond the scope of this short paper, the goal of this review is to summarize the most common and highest impact contributions that whole genome sequencing and assembly has had on comparative and evolutionary biology. Copyright © 2016. Published by Elsevier Inc.

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July 7, 2019

A Clostridioides difficile bacteriophage genome encodes functional binary toxin-associated genes.

Pathogenic clostridia typically produce toxins as virulence factors which cause severe diseases in both humans and animals. Whereas many clostridia like e.g., Clostridium perfringens, Clostridium botulinum or Clostridium tetani were shown to contain toxin-encoding plasmids, only toxin genes located on the chromosome were detected in Clostridioides difficile so far. In this study, we determined, annotated, and analyzed the complete genome of the bacteriophage phiSemix9P1 using single-molecule real-time sequencing technology (SMRT). To our knowledge, this represents the first C. difficile-associated bacteriophage genome that carries a complete functional binary toxin locus in its genome. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Analysis of serial isolates of mcr-1-positive Escherichia coli reveals a highly active ISApl1 transposon.

The emergence of a transferable colistin resistance gene (mcr-1) is of global concern. The insertion sequence ISApl1 is a key component in the mobilization of this gene, but its role remains poorly understood. Six Escherichia coli isolates were cultured from the same patient over the course of 1 month in Germany and the United States after a brief hospitalization in Bahrain for an unconnected illness. Four carried mcr-1 as determined by real-time PCR, but two were negative. Two additional mcr-1-negative E. coli isolates were collected during follow-up surveillance 9 months later. All isolates were analyzed by whole-genome sequencing (WGS). WGS revealed that the six initial isolates were composed of two distinct strains: an initial ST-617 E. coli strain harboring mcr-1 and a second, unrelated, mcr-1-negative ST-32 E. coli strain that emerged 2 weeks after hospitalization. Follow-up swabs taken 9 months later were negative for the ST-617 strain, but the mcr-1-negative ST-32 strain was still present. mcr-1 was associated with a single copy of ISApl1, located on a 64.5-kb IncI2 plasmid that shared >95% homology with other mcr-1 IncI2 plasmids. ISApl1 copy numbers ranged from 2 for the first isolate to 6 for the final isolate, but ISApl1 movement was independent of mcr-1 Some movement was accompanied by gene disruption, including the loss of genes encoding proteins involved in stress responses, arginine catabolism, and l-arabinose utilization. These data represent the first comprehensive analysis of ISApl1 movement in serial clinical isolates and reveal that, under certain conditions, ISApl1 is a highly active IS element whose movement may be detrimental to the host cell. Copyright © 2017 Snesrud et al.

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July 7, 2019

Analysis of the complete genome sequence of Nocardia seriolae UTF1, the causative agent of fish nocardiosis: The first reference genome sequence of the fish pathogenic Nocardia species.

Nocardiosis caused by Nocardia seriolae is one of the major threats in the aquaculture of Seriola species (yellowtail; S. quinqueradiata, amberjack; S. dumerili and kingfish; S. lalandi) in Japan. Here, we report the complete nucleotide genome sequence of N. seriolae UTF1, isolated from a cultured yellowtail. The genome is a circular chromosome of 8,121,733 bp with a G+C content of 68.1% that encodes 7,697 predicted proteins. In the N. seriolae UTF1 predicted genes, we found orthologs of virulence factors of pathogenic mycobacteria and human clinical Nocardia isolates involved in host cell invasion, modulation of phagocyte function and survival inside the macrophages. The virulence factor candidates provide an essential basis for understanding their pathogenic mechanisms at the molecular level by the fish nocardiosis research community in future studies. We also found many potential antibiotic resistance genes on the N. seriolae UTF1 chromosome. Comparative analysis with the four existing complete genomes, N. farcinica IFM 10152, N. brasiliensis HUJEG-1 and N. cyriacigeorgica GUH-2 and N. nova SH22a, revealed that 2,745 orthologous genes were present in all five Nocardia genomes (core genes) and 1,982 genes were unique to N. seriolae UTF1. In particular, the N. seriolae UTF1 genome contains a greater number of mobile elements and genes of unknown function that comprise the differences in structure and gene content from the other Nocardia genomes. In addition, a lot of the N. seriolae UTF1-specific genes were assigned to the ABC transport system. Because of limited resources in ocean environments, these N. seriolae UTF1 specific ABC transporters might facilitate adaptation strategies essential for marine environment survival. Thus, the availability of the complete N. seriolae UTF1 genome sequence will provide a valuable resource for comparative genomic studies of N. seriolae isolates, as well as provide new insights into the ecological and functional diversity of the genus Nocardia.

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July 7, 2019

Prevalence and molecular characterization of mcr-1-positive Salmonella strains recovered from clinical specimens in China.

The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ~30 to ~250 kb, among which there were conjugative plasmids of ~30 kb, ~60 kb, and ~250 kb and nonconjugative plasmids of ~140 kb, ~180 kb, and ~240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Identification of IncA/C plasmid replication and maintenance genes and development of a plasmid multilocus sequence typing scheme.

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of loci encoding increased protein quality control mechanisms.

Salmonella enterica subsp. enterica bacteria are important foodborne pathogens with major economic impact. Some isolates exhibit increased heat tolerance, a concern for food safety. Analysis of a finished-quality genome sequence of an isolate commonly used in heat resistance studies, S. enterica subsp. enterica serovar Senftenberg 775W (ATCC 43845), demonstrated an interesting observation that this strain contains not just one, but two horizontally acquired thermotolerance locus homologs. These two loci reside on a large 341.3-kbp plasmid that is similar to the well-studied IncHI2 R478 plasmid but lacks any antibiotic resistance genes found on R478 or other IncHI2 plasmids. As this historical Salmonella isolate has been in use since 1941, comparative analysis of the plasmid and of the thermotolerance loci contained on the plasmid will provide insight into the evolution of heat resistance loci as well as acquisition of resistance determinants in IncHI2 plasmids. IMPORTANCE Thermal interventions are commonly used in the food industry as a means of mitigating pathogen contamination in food products. Concern over heat-resistant food contaminants has recently increased, with the identification of a conserved locus shown to confer heat resistance in disparate lineages of Gram-negative bacteria. Complete sequence analysis of a historical isolate of Salmonella enterica serovar Senftenberg, used in numerous studies because of its novel heat resistance, revealed that this important strain possesses two distinct copies of this conserved thermotolerance locus, residing on a multireplicon IncHI2/IncHI2A plasmid. Phylogenetic analysis of these loci in comparison with homologs identified in various bacterial genera provides an opportunity to examine the evolution and distribution of loci conferring resistance to environmental stressors, such as heat and desiccation.

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July 7, 2019

Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts.

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.

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July 7, 2019

The histidine decarboxylase gene cluster of Lactobacillus parabuchneri was gained by horizontal gene transfer and is mobile within the species.

Histamine in food can cause intolerance reactions in consumers. Lactobacillus parabuchneri (L. parabuchneri) is one of the major causes of elevated histamine levels in cheese. Despite its significant economic impact and negative influence on human health, no genomic study has been published so far. We sequenced and analyzed 18 L. parabuchneri strains of which 12 were histamine positive and 6 were histamine negative. We determined the complete genome of the histamine positive strain FAM21731 with PacBio as well as Illumina and the genomes of the remaining 17 strains using the Illumina technology. We developed the synteny aware ortholog finding algorithm SynOrf to compare the genomes and we show that the histidine decarboxylase (HDC) gene cluster is located in a genomic island. It is very likely that the HDC gene cluster was transferred from other lactobacilli, as it is highly conserved within several lactobacilli species. Furthermore, we have evidence that the HDC gene cluster was transferred within the L. parabuchneri species.

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July 7, 2019

Elucidation of quantitative structural diversity of remarkable rearrangement regions, shufflons, in IncI2 plasmids.

A multiple DNA inversion system, the shufflon, exists in incompatibility (Inc) I1 and I2 plasmids. The shufflon generates variants of the PilV protein, a minor component of the thin pilus. The shufflon is one of the most difficult regions for de novo genome assembly because of its structural diversity even in an isolated bacterial clone. We determined complete genome sequences, including those of IncI2 plasmids carrying mcr-1, of three Escherichia coli strains using single-molecule, real-time (SMRT) sequencing and Illumina sequencing. The sequences assembled using only SMRT sequencing contained misassembled regions in the shufflon. A hybrid analysis using SMRT and Illumina sequencing resolved the misassembled region and revealed that the three IncI2 plasmids, excluding the shufflon region, were highly conserved. Moreover, the abundance ratio of whole-shufflon structures could be determined by quantitative structural variation analysis of the SMRT data, suggesting that a remarkable heterogeneity of whole-shufflon structural variations exists in IncI2 plasmids. These findings indicate that remarkable rearrangement regions should be validated using both long-read and short-read sequencing data and that the structural variation of PilV in the shufflon might be closely related to phenotypic heterogeneity of plasmid-mediated transconjugation involved in horizontal gene transfer even in bacterial clonal populations.

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July 7, 2019

The two-component monooxygenase MeaXY initiates the downstream pathway of chloroacetanilide herbicide catabolism in sphingomonads.

Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through N-dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. Sphingobium baderi DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of S. baderi DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of meaX and meaY are responsible for the initial step of MEA degradation in S. baderi DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the para-position. Transcription of meaX was enhanced remarkably upon induction of MEA or DEA in S. baderi DE-13. Additionally, meaX and meaY were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads. IMPORTANCE Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial degradation is considered an important mechanism in the degradation of these compounds. Bacterial degradation of chloroacetanilide herbicides has been investigated in many recent studies. Pure cultures or consortia able to mineralize these herbicides have been obtained. The catabolic pathway has been proposed, and most key genes involved have been identified. However, the genes responsible for the initiation step (from MEA to hydroxylated MEA or from DEA to hydroxylated DEA) of the downstream pathway have not been reported. The present study demonstrates that a two-component flavin-dependent monooxygenase system, MeaXY, catalyzes the para-hydroxylation of MEA or DEA in sphingomonads. Therefore, this work finds a missing link in the biochemical pathway that carries out the mineralization of chloroacetanilide herbicides in sphingomonads. Additionally, the results expand our understanding of the degradation of a special kind of aniline derivative. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Population and clinical genetics of human transposable elements in the (post) genomic era.

Recent technological developments-in genomics, bioinformatics and high-throughput experimental techniques-are providing opportunities to study ongoing human transposable element (TE) activity at an unprecedented level of detail. It is now possible to characterize genome-wide collections of TE insertion sites for multiple human individuals, within and between populations, and for a variety of tissue types. Comparison of TE insertion site profiles between individuals captures the germline activity of TEs and reveals insertion site variants that segregate as polymorphisms among human populations, whereas comparison among tissue types ascertains somatic TE activity that generates cellular heterogeneity. In this review, we provide an overview of these new technologies and explore their implications for population and clinical genetic studies of human TEs. We cover both recent published results on human TE insertion activity as well as the prospects for future TE studies related to human evolution and health.

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