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July 7, 2019  |  

Complete genome sequence of a Burkholderia mallei isolate originating from a glanderous horse from the Kingdom of Bahrain.

Burkholderia mallei is a zoonotic agent causing glanders, a notifiable disease in equines. During the past decades glanders emerged, and the Kingdom of Bahrain reported outbreaks to the World Organization of Animal Health in 2010 and 2011. This paper presents the complete genome sequence of the Burkholderia mallei strain 11RR2811 Bahrain1. Copyright © 2016 Elschner et al.


July 7, 2019  |  

Complete genome sequences of six Legionella pneumophila isolates from two collocated outbreaks of Legionnaires’ disease in 2005 and 2008 in Sarpsborg/Fredrikstad, Norway.

Here, we report the complete genome sequences of Legionella pneumophila isolates from two collocated outbreaks of Legionnaires’ disease in 2005 and 2008 in Sarpsborg/Fredrikstad, Norway. One clinical and two environmental isolates were sequenced from each outbreak. The genome of all six isolates consisted of a 3.36 Mb-chromosome, while the 2005 genomes featured an additional 68 kb-episome sharing high sequence similarity with the L. pneumophila Lens plasmid. All six genomes contained multiple mobile genetic elements including novel combinations of type-IVA secretion systems. A comparative genomics study will be launched to resolve the genetic relationship between the L. pneumophila isolates. Copyright © 2016 Dybwad et al.


July 7, 2019  |  

Listeria monocytogenes in stone fruits linked to a multistate outbreak: enumeration of cells and whole-genome sequencing.

In 2014, the identification of stone fruits contaminated with Listeria monocytogenes led to the subsequent identification of a multistate outbreak. Simultaneous detection and enumeration of L. monocytogenes were performed on 105 fruits, each weighing 127 to 145 g, collected from 7 contaminated lots. The results showed that 53.3% of the fruits yielded L. monocytogenes (lower limit of detection, 5 CFU/fruit), and the levels ranged from 5 to 2,850 CFU/fruit, with a geometric mean of 11.3 CFU/fruit (0.1 CFU/g of fruit). Two serotypes, IVb-v1 and 1/2b, were identified by a combination of PCR- and antiserum-based serotyping among isolates from fruits and their packing environment; certain fruits contained a mixture of both serotypes. Single nucleotide polymorphism (SNP)-based whole-genome sequencing (WGS) analysis clustered isolates from two case-patients with the serotype IVb-v1 isolates and distinguished outbreak-associated isolates from pulsed-field gel electrophoresis (PFGE)-matched, but epidemiologically unrelated, clinical isolates. The outbreak-associated isolates differed by up to 42 SNPs. All but one serotype 1/2b isolate formed another WGS cluster and differed by up to 17 SNPs. Fully closed genomes of isolates from the stone fruits were used as references to maximize the resolution and to increase our confidence in prophage analysis. Putative prophages were conserved among isolates of each WGS cluster. All serotype IVb-v1 isolates belonged to singleton sequence type 382 (ST382); all but one serotype 1/2b isolate belonged to clonal complex 5.WGS proved to be an excellent tool to assist in the epidemiologic investigation of listeriosis outbreaks. The comparison at the genome level contributed to our understanding of the genetic diversity and variations among isolates involved in an outbreak or isolates associated with food and environmental samples from one facility. Fully closed genomes increased our confidence in the identification and comparison of accessory genomes. The diversity among the outbreak-associated isolates and the inclusion of PFGE-matched, but epidemiologically unrelated, isolates demonstrate the high resolution of WGS. The prevalence and enumeration data could contribute to our further understanding of the risk associated with Listeria monocytogenes contamination, especially among high-risk populations. Copyright © 2016 Chen et al.


July 7, 2019  |  

Genomewide Dam methylation in Escherichia coli during long-term stationary phase.

DNA methylation in prokaryotes is widespread. The most common modification of the genome is the methylation of adenine at the N-6 position. In Escherichia coli K-12 and many gammaproteobacteria, this modification is catalyzed by DNA adenine methyltransferase (Dam) at the GATC consensus sequence and is known to modulate cellular processes including transcriptional regulation of gene expression, initiation of chromosomal replication, and DNA mismatch repair. While studies thus far have focused on the motifs associated with methylated adenine (meA), the frequency of meA across the genome, and temporal dynamics during early periods of incubation, here we conduct the first study on the temporal dynamics of adenine methylation in E. coli by Dam throughout all five phases of the bacterial life cycle in the laboratory. Using single-molecule real-time sequencing, we show that virtually all GATC sites are significantly methylated over time; nearly complete methylation of the chromosome was confirmed by mass spectroscopy analysis. However, we also detect 66 sites whose methylation patterns change significantly over time within a population, including three sites associated with sialic acid transport and catabolism, suggesting a potential role for Dam regulation of these genes; differential expression of this subset of genes was confirmed by quantitative real-time PCR. Further, we show significant growth defects of the dam mutant during long-term stationary phase (LTSP). Together these data suggest that the cell places a high premium on fully methylating the chromosome and that alterations in methylation patterns may have significant impact on patterns of transcription, maintenance of genetic fidelity, and cell survival. IMPORTANCE While it has been shown that methylation remains relatively constant into early stationary phase of E. coli, this study goes further through death phase and long-term stationary phase, a unique time in the bacterial life cycle due to nutrient limitation and strong selection for mutants with increased fitness. The absence of methylation at GATC sites can influence the mutation frequency within a population due to aberrant mismatch repair. Therefore, it is important to investigate the methylation status of GATC sites in an environment where cells may not prioritize methylation of the chromosome. This study demonstrates that chromosome methylation remains a priority even under conditions of nutrient limitation, indicating that continuous methylation at GATC sites could be under positive selection.


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  |  

Whole genome sequence and comparative genomics of the novel Lyme borreliosis causing pathogen, Borrelia mayonii.

Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and vaccine studies.


July 7, 2019  |  

Genome sequence and comparative pathogenic determinants of multidrug resistant uropathogenic Escherichia coli O25b: H4, A clinical isolate from Saudi Arabia

Escherichia coli serotype O25b:H4 is involved in human urinary tract infections.In this study, we sequenced and analyzed E. coli O25b:H4 isolated from a patient sufferingfrom recurring UTI infections in an intensive care unit at Hera General Hospital inMakkah, Saudi Arabia. We aimed to determine the virulence genes for pathogenesis anddrug resistance of this isolate compared to other E. coli strains. We sequenced and analyzedthe E. coli O25b:H4 Saudi strain clinical isolate using next generation sequencing. Usingthe ERGO genome analysis platform, we performed annotations and identified virulenceand antibiotic resistance determinants of this clinical isolate. The E. coli O25b:H4 genomewas assembled into four contigs representing a total chromosome size of 5.28 Mb, andthree contigs were identified, including a 130.9 kb (virulence plasmid) contig bearing thebla-CTX gene and 32 kb and 29 kb contigs. In comparing this genome to otheruropathogenic E. coli genomes, we identified unique drug resistance and pathogenicityfactors. In this work, whole-genome sequencing and targeted comparative analysis of aclinical isolate of uropathogenic Escherichia coli O25b:H4 was performed. This strainencodes virulence genes linked with extraintestinal pathogenic E. coli (ExPEC) that areexpressed constitutively in E. coli ST131. We identified the genes responsible forpathogenesis and drug resistance and performed comparative analyses of the virulenceand antibiotic resistance determinants with those of other E. coli UPEC isolates. This isthe first report of genome sequencing and analysis of a UPEC strain from Saudi Arabia.


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  |  

Origins of the current seventh cholera pandemic.

Vibrio cholerae has caused seven cholera pandemics since 1817, imposing terror on much of the world, but bacterial strains are currently only available for the sixth and seventh pandemics. The El Tor biotype seventh pandemic began in 1961 in Indonesia, but did not originate directly from the classical biotype sixth-pandemic strain. Previous studies focused mainly on the spread of the seventh pandemic after 1970. Here, we analyze in unprecedented detail the origin, evolution, and transition to pandemicity of the seventh-pandemic strain. We used high-resolution comparative genomic analysis of strains collected from 1930 to 1964, covering the evolution from the first available El Tor biotype strain to the start of the seventh pandemic. We define six stages leading to the pandemic strain and reveal all key events. The seventh pandemic originated from a nonpathogenic strain in the Middle East, first observed in 1897. It subsequently underwent explosive diversification, including the spawning of the pandemic lineage. This rapid diversification suggests that, when first observed, the strain had only recently arrived in the Middle East, possibly from the Asian homeland of cholera. The lineage migrated to Makassar, Indonesia, where it gained the important virulence-associated elements Vibrio seventh pandemic island I (VSP-I), VSP-II, and El Tor type cholera toxin prophage by 1954, and it then became pandemic in 1961 after only 12 additional mutations. Our data indicate that specific niches in the Middle East and Makassar were important in generating the pandemic strain by providing gene sources and the driving forces for genetic events.


July 7, 2019  |  

Complete genome sequence of human pathogen Kosakonia cowanii type strain 888-76T.

Kosakonia cowanii type strain 888-76T is a human pathogen which was originally isolated from blood as NIH group 42. In this study, we report the complete genome sequence of K. cowanii 888-76T. 888-76T has 1 chromosome and 2 plasmids with a total genome size of 4,857,567bp and C+G 56.15%. This genome sequence will not only help us to understand the virulence features of K. cowanii 888-76T but also provide us the useful information for the study of evolution of Kosakonia genus. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.


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 genomic analysis of multidrug-resistance Pseudomonas aeruginosa Guangzhou-Pae617, the host of megaplasmid pBM413.

We previously described the novel qnrVC6 and blaIMP-45carrying megaplasmid pBM413. This study aimed to investigate the complete genome of multidrug-resistance P. aeruginosa Guangzhou-Pae617, a clinical isolate from the sputum of a patient who was suffering from respiratory disease in Guangzhou, China.The genome was sequenced using Illumina Hiseq 2500 and PacBio RS II sequencers and assembled de novo using HGAP. The genome was automatically and manually annotated.The genome of P. aeruginosa Guangzhou-Pae617 is 6,430,493 bp containing 5881 predicted genes with an average G + C content of 66.43%. The genome showed high similarity to two new sequenced P. aeruginosa strains isolated from New York, USA. From the whole genome sequence, we identified a type IV pilin, two large prophages, 15 antibiotic resistant genes, 5 genes involved in the “Infectious diseases” pathways, and 335 virulence factors.The antibiotic resistance and virulence factors in the genome of P. aeruginosa strain Guangzhou-Pae617 were identified by complete genomic analysis. It contributes to further study on antibiotic resistance mechanism and clinical control of P. aeruginosa. Copyright © 2018 Elsevier Ltd. All rights reserved.


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