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July 7, 2019  |  

Genomic analyses of multidrug resistant Pseudomonas aeruginosa PA1 resequenced by single-molecule real-time sequencing.

As a third-generation sequencing (TGS) method, single-molecule real-time (SMRT) technology provides long read length, and it is well suited for resequencing projects and de novo assembly. In the present study, Pseudomonas aeruginosa PA1 was characterized and resequenced using SMRT technology. PA1 was also subjected to genomic, comparative and pan-genomic analyses. The multidrug resistant strain PA1 possesses a 6,498,072 bp genome and a sequence type of ST-782. The genome of PA1 was also visualized, and the results revealed the details of general genome annotations, virulence factors, regulatory proteins (RPs), secretion system proteins, type II toxin-antitoxin (T-A) pairs and genomic islands. Whole genome comparison analysis suggested that PA1 exhibits similarity to other P. aeruginosa strains but differs in terms of horizontal gene transfer (HGT) regions, such as prophages and genomic islands. Phylogenetic analyses based on 16S rRNA sequences demonstrated that PA1 is closely related to PAO1, and P. aeruginosa strains can be divided into two main groups. The pan-genome of P. aeruginosa consists of a core genome of approximately 4,000 genes and an accessory genome of at least 6,600 genes. The present study presented a detailed, visualized and comparative analysis of the PA1 genome, to enhance our understanding of this notorious pathogen. © 2016 The Author(s).


July 7, 2019  |  

Whole genome analysis of Yersinia ruckeri isolated over 27 years in Australia and New Zealand reveals geographical endemism over multiple lineages and recent evolution under host selection.

Yersinia ruckeri is a salmonid pathogen with widespread distribution in cool-temperate waters including Australia and New Zealand, two isolated environments with recently developed salmonid farming industries. Phylogenetic comparison of 58 isolates from Australia, New Zealand, USA, Chile, Finland and China based on non-recombinant core genome SNPs revealed multiple deep-branching lineages, with a most recent common ancestor estimated at 18?500 years BP (12?355-24?757 95% HPD) and evidence of Australasian endemism. Evolution within the Tasmanian Atlantic salmon serotype O1b lineage has been slow, with 63 SNPs describing the variance over 27 years. Isolates from the prevailing lineage are poorly/non-motile compared to a lineage pre-vaccination, introduced in 1997, which is highly motile but has not been isolated since from epizootics. A non-motile phenotype has arisen independently in Tasmania compared to Europe and USA through a frameshift in fliI, encoding the ATPase of the flagella cluster. We report for the first time lipopolysaccharide O-antigen serotype O2 isolates in Tasmania. This phenotype results from deletion of the O-antigen cluster and consequent loss of high-molecular-weight O-antigen. This phenomenon has occurred independently on three occasions on three continents (Australasia, North America and Asia) as O2 isolates from the USA, China and Tasmania share the O-antigen deletion but occupy distant lineages. Despite the European and North American origins of the Australasian salmonid stocks, the lineages of Y. ruckeri in Australia and New Zealand are distinct from those of the northern hemisphere, suggesting they are pre-existing ancient strains that have emerged and evolved with the introduction of susceptible hosts following European colonization.


July 7, 2019  |  

Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.

Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under ‘priming’ conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30?kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences – but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event – with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.© 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.


July 7, 2019  |  

Genome sequence of a commensal bacterium, Enterococcus faecalis CBA7120, isolated from a Korean fecal sample.

Enterococcus faecalis, the type strain of the genus Enterococcus, is not only a commensal bacterium in the gastrointestinal tract in vertebrates and invertebrates, but also causes serious disease as an opportunistic pathogen. To date, genome sequences have been published for over four hundred E. faecalis strains; however, pathogenicity of these microbes remains complicated. To increase our knowledge of E. faecalis virulence factors, we isolated strain CBA7120 from the feces of an 81-year-old female from the Republic of Korea and performed a comparative genomic analysis.The genome sequence of E. faecalis CBA7120 is 3,134,087 bp in length, with a G + C content of 37.35 mol%, and is comprised of four contigs with an N50 value of 2,922,046 bp. The genome showed high similarity with other strains of E. faecalis, including OG1RF, T13, 12107 and T20, based on OrthoANI values. Strain CBA7120 contains 374 pan-genome orthologous groups (POGs) as singletons, including “Phages, Prophages, Transposable elements, Plasmids,” “Carbohydrates,” “DNA metabolism,” and “Virulence, Disease and Defense” subsystems. Genes related to multidrug resistance efflux pumps were annotated in the genome.The comparative genomic analysis of E. faecalis strains presented in this study was performed using a variety of analysis methods and will facilitate future identification of hypothetical proteins.


July 7, 2019  |  

Use of single molecule sequencing for comparative genomics of an environmental and a clinical isolate of Clostridium difficile ribotype 078.

How the pathogen Clostridium difficile might survive, evolve and be transferred between reservoirs within the natural environment is poorly understood. Some ribotypes are found both in clinical and environmental settings. Whether these strains are distinct from each another and evolve in the specific environments is not established. The possession of a highly mobile genome has contributed to the genetic diversity and ongoing evolution of C. difficile. Interpretations of genetic diversity have been limited by fragmented assemblies resulting from short-read length sequencing approaches and by a limited understanding of epigenetic regulation of diversity. To address this, single molecule real time (SMRT) sequencing was used in this study as it produces high quality genome sequences, with resolution of repeat regions (including those found in mobile elements) and can generate data to determine methylation modifications across the sequence (the methylome).Chromosomal rearrangements and ribosomal operon duplications were observed in both genomes. The rearrangements occurred at insertion sites within two mobile genetic elements (MGEs), Tn6164 and Tn6293, present only in the M120 and CD105HS27 genomes, respectively. The gene content of these two transposons differ considerably which could impact upon horizontal gene transfer; differences include CDSs encoding methylases and a conjugative prophage only in Tn6164. To investigate mechanisms which could affect MGE transfer, the methylome, restriction modification (RM)  and the CRISPR/Cas systems were characterised for each strain. Notably, the environmental isolate, CD105HS27, does not share a consensus motif for (m4)C methylation, but has one additional spacer  when compared to the clinical isolate M120.These findings show key differences between the two strains in terms of their genetic capacity for MGE transfer. The carriage of horizontally transferred genes appear to have genome wide effects based on two different methylation patterns. The CRISPR/Cas system appears active although perhaps slow to evolve. Data suggests that both mechanisms are functional and impact upon horizontal gene transfer and genome evolution within C. difficile.


July 7, 2019  |  

Complete genome sequence of Streptococcus sp. strain NPS 308.

Streptococcus sp. strain NPS 308, isolated from an 8-year-old girl diagnosed with infective endocarditis, likely presents a novel species of Streptococcus Here, we present a complete genome sequence of this species, which will contribute to better understanding of the pathogenesis of infective endocarditis. Copyright © 2016 Kondo et al.


July 7, 2019  |  

Epigenetic mechanisms in microbial members of the human microbiota: current knowledge and perspectives.

The human microbiota and epigenetic processes have both been shown to play a crucial role in health and disease. However, there is extremely scarce information on epigenetic modulation of microbiota members except for a few pathogens. Mainly DNA adenine methylation has been described extensively in modulating the virulence of pathogenic bacteria in particular. It would thus appear likely that such mechanisms are widespread for most bacterial members of the microbiota. This review will present briefly the current knowledge on epigenetic processes in bacteria, give examples of known methylation processes in microbial members of the human microbiota and summarize the knowledge on regulation of host epigenetic processes by the human microbiota.


July 7, 2019  |  

Genome-guided design of a defined mouse microbiota that confers colonization resistance against Salmonella enterica serovar Typhimurium.

Protection against enteric infections, also termed colonization resistance, results from mutualistic interactions of the host and its indigenous microbes. The gut microbiota of humans and mice is highly diverse and it is therefore challenging to assign specific properties to its individual members. Here, we have used a collection of murine bacterial strains and a modular design approach to create a minimal bacterial community that, once established in germ-free mice, provided colonization resistance against the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm). Initially, a community of 12 strains, termed Oligo-Mouse-Microbiota (Oligo-MM(12)), representing members of the major bacterial phyla in the murine gut, was selected. This community was stable over consecutive mouse generations and provided colonization resistance against S. Tm infection, albeit not to the degree of a conventional complex microbiota. Comparative (meta)genome analyses identified functions represented in a conventional microbiome but absent from the Oligo-MM(12). By genome-informed design, we created an improved version of the Oligo-MM community harbouring three facultative anaerobic bacteria from the mouse intestinal bacterial collection (miBC) that provided conventional-like colonization resistance. In conclusion, we have established a highly versatile experimental system that showed efficacy in an enteric infection model. Thus, in combination with exhaustive bacterial strain collections and systems-based approaches, genome-guided design can be used to generate insights into microbe-microbe and microbe-host interactions for the investigation of ecological and disease-relevant mechanisms in the intestine.


July 7, 2019  |  

Microbial sequence typing in the genomic era.

Next-generation sequencing (NGS), also known as high-throughput sequencing, is changing the field of microbial genomics research. NGS allows for a more comprehensive analysis of the diversity, structure and composition of microbial genes and genomes compared to the traditional automated Sanger capillary sequencing at a lower cost. NGS strategies have expanded the versatility of standard and widely used typing approaches based on nucleotide variation in several hundred DNA sequences and a few gene fragments (MLST, MLVA, rMLST and cgMLST). NGS can now accommodate variation in thousands or millions of sequences from selected amplicons to full genomes (WGS, NGMLST and HiMLST). To extract signals from high-dimensional NGS data and make valid statistical inferences, novel analytic and statistical techniques are needed. In this review, we describe standard and new approaches for microbial sequence typing at gene and genome levels and guidelines for subsequent analysis, including methods and computational frameworks. We also present several applications of these approaches to some disciplines, namely genotyping, phylogenetics and molecular epidemiology. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019  |  

Complete genome sequence of multiple-antibiotic-resistant Streptococcus parauberis strain SPOF3K, isolated from diseased olive flounder (Paralichthys olivaceus).

Here, we report the complete genome sequence of multiple-antibiotic-resistant Streptococcus parauberis strain SPOF3K, isolated from the kidney of a diseased olive flounder in South Korea in 2013. Sequencing using a PacBio platform yielded a circular chromosome of 2,128,740?bp and a plasmid of 23,538?bp, harboring 2,123 and 24 protein-coding genes, respectively. Copyright © 2018 Lee et al.


July 7, 2019  |  

Strategies for high-altitude adaptation revealed from high-quality draft genome of non-violacein producing Janthinobacterium lividum ERGS5:01.

A light pink coloured bacterial strain ERGS5:01 isolated from glacial stream water of Sikkim Himalaya was affiliated to Janthinobacterium lividum based on 16S rRNA gene sequence identity and phylogenetic clustering. Whole genome sequencing was performed for the strain to confirm its taxonomy as it lacked the typical violet pigmentation of the genus and also to decipher its survival strategy at the aquatic ecosystem of high elevation. The PacBio RSII sequencing generated genome of 5,168,928 bp with 4575 protein-coding genes and 118 RNA genes. Whole genome-based multilocus sequence analysis clustering, in silico DDH similarity value of 95.1% and, the ANI value of 99.25% established the identity of the strain ERGS5:01 (MCC 2953) as a non-violacein producing J. lividum. The genome comparisons across genus Janthinobacterium revealed an open pan-genome with the scope of the addition of new orthologous cluster to complete the genomic inventory. The genomic insight provided the genetic basis of freezing and frequent freeze-thaw cycle tolerance and, for industrially important enzymes. Extended insight into the genome provided clues of crucial genes associated with adaptation in the harsh aquatic ecosystem of high altitude.


July 7, 2019  |  

Whole-genome sequence of phage-resistant strain Escherichia coli DH5a.

The genomes of many strains of Escherichia coli have been sequenced, as this organism is a classic model bacterium. Here, we report the genome sequence of Escherichia coli DH5a, which is resistant to a T4 bacteriophage (CCTCC AB 2015375), while its other homologous E. coli strains, such as E. coli BL21, DH10B, and MG1655, are not resistant to phage invasions. Thus, understanding of the genome of the DH5a strain, along with comparative analysis of its genome sequence along with other sequences of E. coli strains, may help to reveal the bacteriophage resistance mechanism of E. coli. Copyright © 2018 Chen et al.


July 7, 2019  |  

Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans.

We report the whole genome sequence of the serotype e Cbm+ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra-oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall-anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram-positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.


July 7, 2019  |  

Emerging mechanisms of antimicrobial resistance in bacteria and fungi: advances in the era of genomics.

Bacteria and fungi continue to develop new ways to adapt and survive the lethal or biostatic effects of antimicrobials through myriad mechanisms. Novel antibiotic resistance genes such as lsa(C), erm(44), VCC-1, mcr-1, mcr-2, mcr-3, mcr-4, bla KLUC-3 and bla KLUC-4 were discovered through comparative genomics and further functional studies. As well, mutations in genes that hitherto were unknown to confer resistance to antimicrobials, such as trm, PP2C, rpsJ, HSC82, FKS2 and Rv2887, were shown by genomics and transcomplementation assays to mediate antimicrobial resistance in Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Saccharomyces cerevisae, Candida glabrata and Mycobacterium tuberculosis, respectively. Thus, genomics, transcriptomics and metagenomics, coupled with functional studies are the future of antimicrobial resistance research and novel drug discovery or design.


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