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July 7, 2019  |  

Genome sequence of Phormia regina Meigen (Diptera: Calliphoridae): implications for medical, veterinary and forensic research.

Blow flies (Diptera: Calliphoridae) are important medical, veterinary and forensic insects encompassing 8 % of the species diversity observed in the calyptrate insects. Few genomic resources exist to understand the diversity and evolution of this group.We present the hybrid (short and long reads) draft assemblies of the male and female genomes of the common North American blow fly, Phormia regina (Diptera: Calliphoridae). The 550 and 534 Mb draft assemblies contained 8312 and 9490 predicted genes in the female and male genomes, respectively; including?>?93 % conserved eukaryotic genes. Putative X and Y chromosomes (21 and 14 Mb, respectively) were assembled and annotated. The P. regina genomes appear to contain few mobile genetic elements, an almost complete absence of SINEs, and most of the repetitive landscape consists of simple repetitive sequences. Candidate gene approaches were undertaken to annotate insecticide resistance, sex-determining, chemoreceptors, and antimicrobial peptides.This work yielded a robust, reliable reference calliphorid genome from a species located in the middle of a calliphorid phylogeny. By adding an additional blow fly genome, the ability to tease apart what might be true of general calliphorids vs. what is specific of two distinct lineages now exists. This resource will provide a strong foundation for future studies into the evolution, population structure, behavior, and physiology of all blow flies.

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July 7, 2019  |  

Emergence of ileS2-carrying, multidrug-resistant plasmids in Staphylococcus lugdunensis.

Of 137 Staphylococcus lugdunensis isolates collected from two nephrology centers in Hong Kong, 10 (7.3%) and 3 (2.2%) isolates had high-level and low-level mupirocin resistance, respectively. Isolates with high-level resistance contained the plasmid-mediated ileS2 gene, while isolates with low-level resistance contained the mutation V588F within the chromosomal ileS gene. All but one of the ileS2-positive isolates belong to the predominating clone HKU1. Plasmids carrying the ileS2 gene were mosaic and also cocarry multiple other resistance determinants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Neuraminidase A-exposed galactose promotes Streptococcus pneumoniae biofilm formation during colonization.

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and ß-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Transfer of the methicillin resistance genomic island among staphylococci by conjugation.

Methicillin resistance creates a major obstacle for treatment of Staphylococcus aureus infections. The resistance gene, mecA, is carried on a large (20 kb to?>?60 kb) genomic island, staphylococcal cassette chromosome mec (SCCmec), that excises from and inserts site-specifically into the staphylococcal chromosome. However, although SCCmec has been designated a mobile genetic element, a mechanism for its transfer has not been defined. Here we demonstrate the capture and conjugative transfer of excised SCCmec. SCCmec was captured on pGO400, a mupirocin-resistant derivative of the pGO1/pSK41 staphylococcal conjugative plasmid lineage, and pGO400::SCCmec (pRM27) was transferred by filter-mating into both homologous and heterologous S. aureus recipients representing a range of clonal complexes as well as S. epidermidis. The DNA sequence of pRM27 showed that SCCmec had been transferred in its entirety and that its capture had occurred by recombination between IS257/431 elements present on all SCCmec types and pGO1/pSK41 conjugative plasmids. The captured SCCmec excised from the plasmid and inserted site-specifically into the chromosomal att site of both an isogenic S. aureus and a S. epidermidis recipient. These studies describe a means by which methicillin resistance can be environmentally disseminated and a novel mechanism, IS-mediated recombination, for the capture and conjugative transfer of genomic islands. © 2016 John Wiley & Sons Ltd.

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July 7, 2019  |  

Co-infection and emergence of rifamycin resistance during a recurrent Clostridium difficile infection.

Clostridium difficile (Peptoclostridium difficile) is a common health care associated infection with a disproportionately high incidence in elderly patients. Disease symptoms range from mild diarrhoea through to life threatening pseudomembranous colitis. Around 20% of patients may suffer recurrent disease which often requires re-hospitalisation of patients.C. difficile was isolated from stool samples from a patient with two recurrent C. difficile infections. PCR-ribotyping, whole genome sequencing and phenotypic assays were used to characterise these isolates.Genotypic and phenotypic screening of C. difficile isolates revealed multiple PCR-ribotypes present, and the emergence of rifamycin resistance during the infection cycle.Understanding both the clinical and bacterial factors that contribute to the course of recurrent infection could inform strategies to reduce recurrence. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Characterization and comparative overview of complete sequences of the first plasmids of Pandoraea across clinical and non-clinical strains.

To date, information on plasmid analysis in Pandoraea spp. is scarce. To address the gap of knowledge on this, the complete sequences of eight plasmids from Pandoraea spp. namely Pandoraea faecigallinarum DSM 23572(T) (pPF72-1, pPF72-2), Pandoraea oxalativorans DSM 23570(T) (pPO70-1, pPO70-2, pPO70-3, pPO70-4), Pandoraea vervacti NS15 (pPV15) and Pandoraea apista DSM 16535(T) (pPA35) were studied for the first time in this study. The information on plasmid sequences in Pandoraea spp. is useful as the sequences did not match any known plasmid sequence deposited in public databases. Replication genes were not identified in some plasmids, a situation that has led to the possibility of host interaction involvement. Some plasmids were also void of par genes and intriguingly, repA gene was also not discovered in these plasmids. This further leads to the hypothesis of host-plasmid interaction. Plasmid stabilization/stability protein-encoding genes were observed in some plasmids but were not established for participating in plasmid segregation. Toxin-antitoxin systems MazEF, VapBC, RelBE, YgiT-MqsR, HigBA, and ParDE were identified across the plasmids and their presence would improve plasmid maintenance. Conjugation genes were identified portraying the conjugation ability amongst Pandoraea plasmids. Additionally, we found a shared region amongst some of the plasmids that consists of conjugation genes. The identification of genes involved in replication, segregation, toxin-antitoxin systems and conjugation, would aid the design of drugs to prevent the survival or transmission of plasmids carrying pathogenic properties. Additionally, genes conferring virulence and antibiotic resistance were identified amongst the plasmids. The observed features in the plasmids shed light on the Pandoraea spp. as opportunistic pathogens.

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July 7, 2019  |  

Complete sequence of a F33:A-:B- conjugative plasmid carrying the oqxAB, fosA3, and blaCTX-M-55 elements from a foodborne Escherichia coli strain.

This study reports the complete sequence of pE80, a conjugative IncFII plasmid recovered from an Escherichia coli strain isolated from chicken meat. This plasmid harbors multiple resistance determinants including oqxAB, fosA3, blaCTX-M-55, and blaTEM-1, and is a close variant of the recently reported p42-2 element, which was recovered from E. coli of veterinary source. Recovery of pE80 constitutes evidence that evolution or genetic re-arrangement of IncFII type plasmids residing in animal-borne organisms is an active event, which involves acquisition and integration of foreign resistance elements into the plasmid backbone. Dissemination of these plasmids may further compromise the effectiveness of current antimicrobial strategies.

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July 7, 2019  |  

Complete genome of Vibrio parahaemolyticus FORC014 isolated from the toothfish.

Foodborne illness can occur due to various pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Vibrio parahaemolyticus, and can cause severe gastroenteritis symptoms. In this study, we completed the genome sequence of a foodborne pathogen V. parahaemolyticus FORC_014, which was isolated from suspected contaminated toothfish from South Korea. Additionally, we extended our knowledge of genomic characteristics of the FORC_014 strain through comparative analysis using the complete sequences of other V. parahaemolyticus strains whose complete genomes have previously been reported.The complete genome sequence of V. parahaemolyticus FORC_014 was generated using the PacBio RS platform with single molecule, real-time (SMRT) sequencing. The FORC_014 strain consists of two circular chromosomes (3,241,330 bp for chromosome 1 and 1,997,247 bp for chromosome 2), one plasmid (51,383 bp), and one putative phage sequence (96,896 bp). The genome contains a total of 4274 putative protein coding sequences, 126 tRNA genes and 34 rRNA genes. Furthermore, we found 33 type III secretion system 1 (T3SS1) related proteins and 15 type III secretion system 2 (T3SS2) related proteins on chromosome 1. This is the first reported result of Type III secretion system 2 located on chromosome 1 of V. parahaemolyticus without thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh).Through investigation of the complete genome sequence of V. parahaemolyticus FORC_014, which differs from previously reported strains, we revealed two type III secretion systems (T3SS1, T3SS2) located on chromosome 1 which do not include tdh and trh genes. We also identified several virulence factors carried by our strain, including iron uptake system, hemolysin and secretion system. This result suggests that the FORC_014 strain may be one pathogen responsible for foodborne illness outbreak. Our results provide significant genomic clues which will assist in future understanding of virulence at the genomic level and help distinguish between clinical and non-clinical isolates.

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July 7, 2019  |  

Information-optimal genome assembly via sparse read-overlap graphs.

In the context of third-generation long-read sequencing technologies, read-overlap-based approaches are expected to play a central role in the assembly step. A fundamental challenge in assembling from a read-overlap graph is that the true sequence corresponds to a Hamiltonian path on the graph, and, under most formulations, the assembly problem becomes NP-hard, restricting practical approaches to heuristics. In this work, we avoid this seemingly fundamental barrier by first setting the computational complexity issue aside, and seeking an algorithm that targets information limits In particular, we consider a basic feasibility question: when does the set of reads contain enough information to allow unambiguous reconstruction of the true sequence?Based on insights from this information feasibility question, we present an algorithm-the Not-So-Greedy algorithm-to construct a sparse read-overlap graph. Unlike most other assembly algorithms, Not-So-Greedy comes with a performance guarantee: whenever information feasibility conditions are satisfied, the algorithm reduces the assembly problem to an Eulerian path problem on the resulting graph, and can thus be solved in linear time. In practice, this theoretical guarantee translates into assemblies of higher quality. Evaluations on both simulated reads from real genomes and a PacBio Escherichia coli K12 dataset demonstrate that Not-So-Greedy compares favorably with standard string graph approaches in terms of accuracy of the resulting read-overlap graph and contig N50.Available at github.com/samhykim/nsgcourtade@eecs.berkeley.edu or dntse@stanford.eduSupplementary data are available at Bioinformatics online.© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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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.

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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.

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July 7, 2019  |  

Genomics-inspired discovery of three antibacterial active metabolites, aurantinins B, C, and D from compost-associated Bacillus subtilis fmb60.

Fmb60 is a wild-type Bacillus subtilis isolated from compost with significant broad-spectrum antimicrobial activities. Two novel PKS clusters were recognized in the genome sequence of fmb60, and then three polyene antibiotics, aurantinins B, C, and D, 1-3, were obtained by bioactivity-guided isolation from the fermentation of fmb60. The structures of aurantinins B-D were elucidated by LC-HRMS and NMR data analysis. Aurantinins C and D were identified as new antimicrobial compounds. The three aurantinins showed significant activity against multidrug-resistant Staphylococcus aureus and Clostridium sporogenes. However, aurantinins B-D did not exhibit any cytotoxicity (IC50 > 100 µg/mL) against LO2 and Caco2 cell lines by MTT assay. Furthermore, using S. aureus as a model bacterium to explore the antibacterial mechanism of aurantinins B-D, it was revealed that the bactericidal activity of aurantinins B-D was related to their ability to disrupt the cell membrane.

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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.

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July 7, 2019  |  

FDA-CDC antimicrobial resistance isolate bank: A publicly-available resource to support research, development and regulatory requirements.

The FDA-CDC Antimicrobial Resistance Isolate Bank was created in July 2015 as a publicly available resource to combat antimicrobial resistance. It is a curated repository of bacterial isolates with an assortment of clinically-important resistance mechanisms that have been phenotypically and genotypically characterized. In the first two years of operation, the Bank offered 14 panels comprising 496 unique isolates and had filled 486 orders from 394 institutions throughout the United States. New panels are being added. Copyright © 2017 American Society for Microbiology.

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July 7, 2019  |  

De novo mutations resolve disease transmission pathways in clonal malaria

Detecting de novo mutations in viral and bacterial pathogens enables researchers to reconstruct detailed networks of disease transmission and is a key technique in genomic epidemiology. However, these techniques have not yet been applied to the malaria parasite, Plasmodium falciparum, in which a larger genome, slower generation times, and a complex life cycle make them difficult to implement. Here, we demonstrate the viability of de novo mutation studies in P. falciparum for the first time. Using a combination of sequencing, library preparation, and genotyping methods that have been optimized for accuracy in low-complexity genomic regions, we have detected de novo mutations that distinguish nominally identical parasites from clonal lineages. Despite its slower evolutionary rate compared with bacterial or viral species, de novo mutation can be detected in P. falciparum across timescales of just 1-2?years and evolutionary rates in low-complexity regions of the genome can be up to twice that detected in the rest of the genome. The increased mutation rate allows the identification of separate clade expansions that cannot be found using previous genomic epidemiology approaches and could be a crucial tool for mapping residual transmission patterns in disease elimination campaigns and reintroduction scenarios.

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