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Friday, July 19, 2019

Diversity of the TLR4 immunity receptor in Czech native cattle breeds revealed using the Pacific Biosciences sequencing platform.

The allelic variants of immunity genes in historical breeds likely reflect local infection pressure and therefore represent a reservoir for breeding. Screening to determine the diversity of the Toll-like receptor gene TLR4 was conducted in two conserved cattle breeds: Czech Red and Czech Red Pied. High-throughput sequencing of pooled PCR amplicons using the PacBio platform revealed polymorphisms, which were subsequently confirmed via genotyping techniques. Eight SNPs found in coding and adjacent regions were grouped into 18 haplotypes, representing a significant portion of the known diversity in the global breed panel and presumably exceeding diversity in production populations. Notably, the ancient…

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Friday, July 19, 2019

A high-throughput approach for identification of nontuberculous mycobacteria in drinking water reveals relationship between water age and Mycobacterium avium.

Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTMrpoBgenes, allowed…

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Sunday, July 7, 2019

Insights on the emergence of Mycobacterium tuberculosis from the analysis of Mycobacterium kansasii.

By phylogenetic analysis, Mycobacterium kansasii is closely related to Mycobacterium tuberculosis. Yet, although both organisms cause pulmonary disease, M. tuberculosis is a global health menace, whereas M. kansasii is an opportunistic pathogen. To illuminate the differences between these organisms, we have sequenced the genome of M. kansasii ATCC 12478 and its plasmid (pMK12478) and conducted side-by-side in vitro and in vivo investigations of these two organisms. The M. kansasii genome is 6,432,277 bp, more than 2 Mb longer than that of M. tuberculosis H37Rv, and the plasmid contains 144,951 bp. Pairwise comparisons reveal conserved and discordant genes and genomic regions.…

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Sunday, July 7, 2019

Whole-genome sequencing for comparative genomics and de novo genome assembly.

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as…

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Sunday, July 7, 2019

The complete genome sequence of the emerging pathogen Mycobacterium haemophilum explains its unique culture requirements.

Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear. Using a combination of Illumina, PacBio, and Sanger sequencing, the complete genome sequence of M. haemophilum was determined. Guided by this sequence, experiments were performed to define the basis for the unique growth requirements of M. haemophilum. We found that M. haemophilum, unlike many other mycobacteria, is unable to synthesize iron-binding siderophores known as mycobactins…

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Sunday, July 7, 2019

FASTQSim: platform-independent data characterization and in silico read generation for NGS datasets.

High-throughput next generation sequencing technologies have enabled rapid characterization of clinical and environmental samples. Consequently, the largest bottleneck to actionable data has become sample processing and bioinformatics analysis, creating a need for accurate and rapid algorithms to process genetic data. Perfectly characterized in silico datasets are a useful tool for evaluating the performance of such algorithms.Background contaminating organisms are observed in sequenced mixtures of organisms. In silico samples provide exact truth. To create the best value for evaluating algorithms, in silico data should mimic actual sequencer data as closely as possible.FASTQSim is a tool that provides the dual functionality of…

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Sunday, July 7, 2019

Genomic reconnaissance of clinical isolates of emerging human pathogen Mycobacterium abscessus reveals high evolutionary potential.

Mycobacterium abscessus (Ma) is an emerging human pathogen that causes both soft tissue infections and systemic disease. We present the first comparative whole-genome study of Ma strains isolated from patients of wide geographical origin. We found a high proportion of accessory strain-specific genes indicating an open, non-conservative pan-genome structure, and clear evidence of rapid phage-mediated evolution. Although we found fewer virulence factors in Ma compared to M. tuberculosis, our data indicated that Ma evolves rapidly and therefore should be monitored closely for the acquisition of more pathogenic traits. This comparative study provides a better understanding of Ma and forms the…

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Sunday, July 7, 2019

The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohn’s disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative…

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Sunday, July 7, 2019

Complete genome sequence of Mycobacterium avium subsp. hominissuis strain H87 isolated from an indoor water sample.

Mycobacterium avium subsp. hominissuis is an environmentally acquired bacterium known to cause pulmonary and soft tissue infections, lymphadenitis, and disseminated disease in humans. We report here the complete genome sequence of strain H87, isolated from an indoor water sample, as a single circular chromosome of 5,626,623 bp with a G+C content of 68.8%. Copyright © 2017 Zhao et al.

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Sunday, July 7, 2019

Whole genome characterization of a naturally occurring vancomycin-dependent Enterococcus faecium from a patient with bacteremia.

Vancomycin-dependent enterococci are a relatively uncommon phenotype recovered in the clinical laboratory. Recognition and recovery of these isolates are important, to provide accurate identification and susceptibility information to treating physicians. Herein, we describe the recovery of a vancomycin-dependent and revertant E. faecium isolates harboring vanB operon from a patient with bacteremia. Using whole genome sequencing, we found a unique single nucleotide polymorphism (S186N) in the D-Ala-D-Ala ligase (ddl) conferring vancomycin-dependency. Additionally, we found that a majority of in vitro revertants mutated outside ddl, with some strains harboring mutations in vanS, while others likely containing novel mechanisms of reversion. Copyright ©…

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Sunday, July 7, 2019

Tracing the Enterococci from Paleozoic origins to the hospital.

We examined the evolutionary history of leading multidrug resistant hospital pathogens, the enterococci, to their origin hundreds of millions of years ago. Our goal was to understand why, among the vast diversity of gut flora, enterococci are so well adapted to the modern hospital environment. Molecular clock estimation, together with analysis of their environmental distribution, phenotypic diversity, and concordance with host fossil records, place the origins of the enterococci around the time of animal terrestrialization, 425-500 mya. Speciation appears to parallel the diversification of hosts, including the rapid emergence of new enterococcal species following the End Permian Extinction. Major drivers…

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Sunday, July 7, 2019

One year genome evolution of Lausannevirus in allopatric versus sympatric conditions.

Amoeba-resisting microorganisms raised a great interest during the last decade. Among them, some large DNA viruses present huge genomes up to 2.5?Mb long, exceeding the size of small bacterial genomes. The rate of genome evolution in terms of mutation, deletion, and gene acquisition in these genomes is yet unknown. Given the suspected high plasticity of viral genomes, the microevolution of the 346?kb genome of Lausannevirus, a member of Megavirales, was studied. Hence, Lausannevirus was co-cultured within the amoeba Acanthamoeba castellanii over one year. Despite a low number of mutations, the virus showed a genome reduction of 3.7% after 12?months. Lausannevirus…

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Sunday, July 7, 2019

Complete genome sequence of Mycobacterium chimaera strain CDC2015-22-71.

Mycobacterium chimaera is a nontuberculous mycobacterium species commonly found in the environment. Here, we report the first complete genome sequence of a strain from the investigation of invasive infections following open-heart surgeries that used contaminated LivaNova Sorin Stockert 3T heater-cooler devices. Copyright © 2017 Hasan et al.

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Sunday, July 7, 2019

Identification of novel conjugative plasmids with multiple copies of fosB that confer high-level fosfomycin resistance to vancomycin-resistant Enterococci.

To further characterize the fosB-carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the fosB- and vanA-containing plasmids of Enterococcus faecium (pEMA120) and E. avium (pEA19081) were obtained by single-molecule, real-time sequencing. We found that these two plasmids are essentially identical (99.99% nucleotide sequence identity), which proved the possibility of interspecies transmission. Comparative analysis of the plasmids revealed that the backbone of pEMA120 is 99% similar to a conjugative fosB-negative E. faecium plasmid, pZB18. There is a traE disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact traE. The difference of their transfer frequencies…

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