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July 7, 2019

Next-generation sequencing and comparative analysis of sequential outbreaks caused by multidrug-resistant Acinetobacter baumannii at a large academic burn center.

Next-generation sequencing (NGS) analysis has emerged as a promising molecular epidemiological method for investigating health care-associated outbreaks. Here, we used NGS to investigate a 3-year outbreak of multidrug-resistant Acinetobacter baumannii (MDRAB) at a large academic burn center. A reference genome from the index case was generated using de novo assembly of PacBio reads. Forty-six MDRAB isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and sequenced using an Illumina platform. After mapping to the index case reference genome, four samples were excluded due to low coverage, leaving 42 samples for further analysis. Multilocus sequence types (MLST) and the presence of acquired resistance genes were also determined from the sequencing data. A transmission network was inferred from genomic and epidemiological data using a Bayesian framework. Based on single-nucleotide variant (SNV) differences, this MDRAB outbreak represented three sequential outbreaks caused by distinct clones. The first and second outbreaks were caused by sequence type 2 (ST2), while the third outbreak was caused by ST79. For the second outbreak, the MLST and PFGE results were discordant. However, NGS-based SNV typing detected a recombination event and consequently enabled a more accurate phylogenetic analysis. The distribution of resistance genes varied among the three outbreaks. The first- and second-outbreak strains possessed a blaOXA-23-like group, while the third-outbreak strains harbored a blaOXA-40-like group. NGS-based analysis demonstrated the superior resolution of outbreak transmission networks for MDRAB and provided insight into the mechanisms of strain diversification between sequential outbreaks through recombination. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

SMRT sequencing of the Campylobacter coli BfR-CA-9557 genome sequence reveals unique methylation motifs.

Campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. In contrast to Campylobacter jejuni, about 5 % of Campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by DpnI cutting specifically 5′-G(m)ATC-3′ motifs. This indicates significant differences in DNA methylation between both microbial species. The goal of the study was to analyze the methylome of a C. coli strain susceptible to DpnI digestion, to identify its methylation motifs and restriction modification systems (RM-systems), and compare them to related organisms like C. jejuni and Helicobacter pylori. Using one SMRT cell and the PacBio RS sequencing technology followed by PacBio Modification and Motif Analysis the complete genome of the DpnI susceptible strain C. coli BfR-CA-9557 was sequenced to 500-fold coverage and assembled into a single contig of 1.7 Mbp. The genome contains a CJIE1-like element prophage and is phylogenetically closer to C. coli clade 1 isolates than clade 3. 45,881 6-methylated adenines (ca. 2.7 % of genome positions) that are predominantly arranged in eight different methylation motifs and 1,788 4-methylated cytosines (ca. 0.1 %) have been detected. Only two of these motifs correspond to known restriction modification motifs. Characteristic for this methylome was the very high fraction of methylation of motifs with mostly above 99 %.Only five dominant methylation motifs have been identified in C. jejuni, which have been associated with known RM-systems. C. coli BFR-CA-9557 shares one (RAATTY) of these, but four ORFs could be assigned to putative Type I RM-systems, seven ORFs to Type II RM-systems and three ORFs to Type IV RM-systems. In accordance with DpnI prescreening RM-system IIP, methylation of GATC motifs was detected in C. coli BfR-CA-9557. A homologous IIP RM-system has been described for H. pylori. The remaining methylation motifs are specific for C. coli BfR-CA-9557 and have been neither detected in C. jejuni nor in H. pylori. The results of this study give us new insights into epigenetics of Campylobacteraceae and provide the groundwork to resolve the function of RM-systems in C. coli.

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July 7, 2019

Circlator: automated circularization of genome assemblies using long sequencing reads.

The assembly of DNA sequence data is undergoing a renaissance thanks to emerging technologies capable of producing reads tens of kilobases long. Assembling complete bacterial and small eukaryotic genomes is now possible, but the final step of circularizing sequences remains unsolved. Here we present Circlator, the first tool to automate assembly circularization and produce accurate linear representations of circular sequences. Using Pacific Biosciences and Oxford Nanopore data, Circlator correctly circularized 26 of 27 circularizable sequences, comprising 11 chromosomes and 12 plasmids from bacteria, the apicoplast and mitochondrion of Plasmodium falciparum and a human mitochondrion. Circlator is available at http://sanger-pathogens.github.io/circlator/ .

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July 7, 2019

Genome analysis of Kingella kingae strain KWG1 reveals how a ß-Lactamase gene inserted in the chromosome of this species.

We describe the genome of a penicillinase-producing Kingella kingae strain (KWG1), the first to be isolated in continental Europe, whose blaTEM-1 gene was, for the first time in this species, found to be chromosomally inserted. The blaTEM gene is located in an integrative and conjugative element (ICE) inserted in Met-tRNA and comprising genes that encode resistance to sulfonamides, streptomycin, and tetracycline. This ICE is homologous to resistance-conferring plasmids of K. kingae and other Gram-negative bacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Complete sequence of conjugative IncA/C plasmid encoding CMY-2 ß-lactamase and RmtE 16S rRNA methyltransferase.

RmtE is a rare 16S-RMTase which was first reported in an aminoglycoside-resistant Escherichia coli strain of calf origin (1). Subsequently, we reported the first human case of infection caused by RmtE-producing E. coli (2). The rmtE gene is carried on a self-conjugative plasmid (pYDC637) in the latter strain. The present work aimed to elucidate the genetic context of rmtE. The sequencing approach has been described previously (3). In brief, the plasmid was extracted from an E. coli TOP10 transformant carrying pYDC637 and sequenced on a PacBio RS II sequencing instrument (Pacific Biosciences, Menlo Park, CA). Assembly was also conducted using the HGAP pipeline (Pacific Biosciences) as previously described (3).

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July 7, 2019

Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria.

In recent years, the number of human infections caused by opportunistic pathogens has increased dramatically. Plant rhizospheres are one of the most typical natural reservoirs for these pathogens but they also represent a great source for beneficial microbes with potential for biotechnological applications. However, understanding the natural variation and possible differences between pathogens and beneficials is the main challenge in furthering these possibilities. The genus Stenotrophomonas contains representatives found to be associated with human and plant host.We used comparative genomics as well as transcriptomic and physiological approaches to detect significant borders between the Stenotrophomonas strains: the multi-drug resistant pathogenic S. maltophilia and the plant-associated strains S. maltophilia R551-3 and S. rhizophila DSM14405T (both are biocontrol agents). We found an overall high degree of sequence similarity between the genomes of all three strains. Despite the notable similarity in potential factors responsible for host invasion and antibiotic resistance, other factors including several crucial virulence factors and heat shock proteins were absent in the plant-associated DSM14405T. Instead, S. rhizophila DSM14405T possessed unique genes for the synthesis and transport of the plant-protective spermidine, plant cell-wall degrading enzymes, and high salinity tolerance. Moreover, the presence or absence of bacterial growth at 37°C was identified as a very simple method in differentiating between pathogenic and non-pathogenic isolates. DSM14405T is not able to grow at this human-relevant temperature, most likely in great part due to the absence of heat shock genes and perhaps also because of the up-regulation at increased temperatures of several genes involved in a suicide mechanism.While this study is important for understanding the mechanisms behind the emerging pattern of infectious diseases, it is, to our knowledge, the first of its kind to assess the risk of beneficial strains for biotechnological applications. We identified certain traits typical of pathogens such as growth at the human body temperature together with the production of heat shock proteins as opposed to a temperature-regulated suicide system that is harnessed by beneficials.

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July 7, 2019

Complete genome sequences of nitrofurantoin-sensitive and -resistant Escherichia coli ST540 and ST2747 strains.

Widespread multidrug resistance in Escherichia coli has necessitated the reintroduction of older antibiotics, such as nitrofurantoin. However, mechanisms by which resistance to nitrofurantoin emerges in E. coli are not well elucidated. Toward this aim, we sequenced two nitrofurantoin-sensitive E. coli sequence types (ST540 and ST2747) and their four nitrofurantoin-resistant derivatives generated in vitro under aerobic and anaerobic growth conditions.

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July 7, 2019

vanG element insertions within a conserved chromosomal site conferring vancomycin resistance to Streptococcus agalactiae and Streptococcus anginosus.

Three vancomycin-resistant streptococcal strains carrying vanG elements (two invasive Streptococcus agalactiae isolates [GBS-NY and GBS-NM, both serotype II and multilocus sequence type 22] and one Streptococcus anginosus [Sa]) were examined. The 45,585-bp elements found within Sa and GBS-NY were nearly identical (together designated vanG-1) and shared near-identity over an ~15-kb overlap with a previously described vanG element from Enterococcus faecalis. Unexpectedly, vanG-1 shared much less homology with the 49,321-bp vanG-2 element from GBS-NM, with widely different levels (50% to 99%) of sequence identity shared among 44 related open reading frames. Immediately adjacent to both vanG-1 and vanG-2 were 44,670-bp and 44,680-bp integrative conjugative element (ICE)-like sequences, designated ICE-r, that were nearly identical in the two group B streptococcal (GBS) strains. The dual vanG and ICE-r elements from both GBS strains were inserted at the same position, between bases 1328 and 1329, within the identical RNA methyltransferase (rumA) genes. A GenBank search revealed that although most GBS strains contained insertions within this specific site, only sequence type 22 (ST22) GBS strains contained highly related ICE-r derivatives. The vanG-1 element in Sa was also inserted within this position corresponding to its rumA homolog adjacent to an ICE-r derivative. vanG-1 insertions were previously reported within the same relative position in the E. faecalis rumA homolog. An ICE-r sequence perfectly conserved with respect to its counterpart in GBS-NY was apparent within the same site of the rumA homolog of a Streptococcus dysgalactiae subsp. equisimilis strain. Additionally, homologous vanG-like elements within the conserved rumA target site were evident in Roseburia intestinalis. Importance: These three streptococcal strains represent the first known vancomycin-resistant strains of their species. The collective observations made from these strains reveal a specific hot spot for insertional elements that is conserved between streptococci and different Gram-positive species. The two GBS strains potentially represent a GBS lineage that is predisposed to insertion of vanG elements. Copyright © 2014 Srinivasan et al.

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July 7, 2019

Complete genome sequences of Salmonella enterica serovar Heidelberg strains associated with a multistate food-borne illness investigation.

Next-generation sequencing is being evaluated for use with food-borne illness investigations, especially when the outbreak strains produce patterns that cannot be discriminated from non-outbreak strains using conventional procedures. Here we report complete genome assemblies of two Salmonella enterica serovar Heidelberg strains with a common pulsed-field gel electrophoresis pattern isolated during an outbreak investigation.

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July 7, 2019

Complete closed genome sequences of three Bibersteinia trehalosi nasopharyngeal isolates from cattle with shipping fever.

Bibersteinia trehalosi is a respiratory pathogen affecting cattle and related ruminants worldwide. B. trehalosi is closely related to Mannheimia haemolytica and is often associated with bovine respiratory disease complex (BRDC), a polymicrobial multifactorial disease. We present three complete closed genome sequences of this species generated using an automated assembly pipeline.

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July 7, 2019

Draft genome sequence of Kitasatospora cheerisanensis KCTC 2395, which produces plecomacrolide against phytopathogenic fungi.

Kitasatospora cheerisanensis KCTC 2395, which produces antifungal metabolites with bafilomycin derivatives, including bafilomycin C1-amide, was isolated from a soil sample at Mt. Jiri, South Korea. Here, we report its draft genome sequence, which contains 8.04 Mb with 73.6% G+C content and 7,810 protein-coding genes. Copyright © 2014 Hwang et al.

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July 7, 2019

First genome sequences of Achromobacter phages reveal new members of the N4 family.

Multi-resistant Achromobacter xylosoxidans has been recognized as an emerging pathogen causing nosocomially acquired infections during the last years. Phages as natural opponents could be an alternative to fight such infections. Bacteriophages against this opportunistic pathogen were isolated in a recent study. This study shows a molecular analysis of two podoviruses and reveals first insights into the genomic structure of Achromobacter phages so far.Growth curve experiments and adsorption kinetics were performed for both phages. Adsorption and propagation in cells were visualized by electron microscopy. Both phage genomes were sequenced with the PacBio RS II system based on single molecule, real-time (SMRT) technology and annotated with several bioinformatic tools. To further elucidate the evolutionary relationships between the phage genomes, a phylogenomic analysis was conducted using the genome Blast Distance Phylogeny approach (GBDP).In this study, we present the first detailed analysis of genome sequences of two Achromobacter phages so far. Phages JWAlpha and JWDelta were isolated from two different waste water treatment plants in Germany. Both phages belong to the Podoviridae and contain linear, double-stranded DNA with a length of 72329 bp and 73659 bp, respectively. 92 and 89 putative open reading frames were identified for JWAlpha and JWDelta, respectively, by bioinformatic analysis with several tools. The genomes have nearly the same organization and could be divided into different clusters for transcription, replication, host interaction, head and tail structure and lysis. Detailed annotation via protein comparisons with BLASTP revealed strong similarities to N4-like phages.Analysis of the genomes of Achromobacter phages JWAlpha and JWDelta and comparisons of different gene clusters with other phages revealed that they might be strongly related to other N4-like phages, especially of the Escherichia group. Although all these phages show a highly conserved genomic structure and partially strong similarities at the amino acid level, some differences could be identified. Those differences, e.g. the existence of specific genes for replication or host interaction in some N4-like phages, seem to be interesting targets for further examination of function and specific mechanisms, which might enlighten the mechanism of phage establishment in the host cell after infection.

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