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July 7, 2019

Complete genome sequencing and genomic characterization of two Escherichia coli strains co-producing MCR-1 and NDM-1 from bloodstream infection.

We previously described the discovery of two Escherichia coli isolates (EC1002 and EC2474) co-harbouring mcr-1 and bla NDM-1 genes, which were recovered from bloodstream infection in China. More importantly, these antibiotic resistance genes were located on different plasmids and signaling the potential spread of pandrug-resistant bacteria. Here, the complete genome sequences of both isolates were determined using Pacbio RS II and Illumina HiSeq2000 systems. The genome of EC1002 consists of a 5,177,501 base pair chromosome and four circular plasmids, while the genome of EC2474 consists of a 5,013,813 base pair chromosome and three plasmids. The plasmid replicon type of pEC1002_NDM and pEC2474_NDM were identified as IncA/C2 and IncF, respectively. The genetic environment of bla NDM-1 in this study was similar to bla NDM-carrying plasmids detected in China, although the overall nucleotide identity and query coverage were variable. The plasmid replicon type of pEC1002_MCR and pEC2474_MCR were identified as IncI2 and IncHI2, respectively. Two different genetic strategies for mcr-1 gene spread were observed in this study and bla NDM-1 genes were also found transferred by two different mobile genetic elements in two plasmids. The findings of this study further support that the diversified transfer mechanisms of bla NDM-1 and mcr-1 present in Enterobacteriaceae.

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July 7, 2019

Automation of PacBio SMRTbell NGS library preparation for bacterial genome sequencing.

The PacBio RS II provides for single molecule, real-time DNA technology to sequence genomes and detect DNA modifications. The starting point for high-quality sequence production is high molecular weight genomic DNA. To automate the library preparation process, there must be high-throughput methods in place to assess the genomic DNA, to ensure the size and amounts of the sheared DNA fragments and final library.The library construction automation was accomplished using the Agilent NGS workstation with Bravo accessories for heating, shaking, cooling, and magnetic bead manipulations for template purification. The quality control methods from gDNA input to final library using the Agilent Bioanalyzer System and Agilent TapeStation System were evaluated.Automated protocols of PacBio 10 kb library preparation produced libraries with similar technical performance to those generated manually. The TapeStation System proved to be a reliable method that could be used in a 96-well plate format to QC the DNA equivalent to the standard Bioanalyzer System results. The DNA Integrity Number that is calculated in the TapeStation System software upon analysis of genomic DNA is quite helpful to assure that the starting genomic DNA is not degraded. In this respect, the gDNA assay on the TapeStation System is preferable to the DNA 12000 assay on the Bioanalyzer System, which cannot run genomic DNA, nor can the Bioanalyzer work directly from the 96-well plates.

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July 7, 2019

Phenotypic and genotypic features of a Salmonella Heidelberg strain isolated in broilers in Brazil and their possible association to antibiotics and short-chain organic acids resistance and susceptibility.

Salmonella enterica serovar Heidelberg is a human pathogen also found in broilers. A strain (UFPR1) has been associated with field reports of resistance to short-chain organic acids (SCOA) in broilers in the South of Brazil, but was susceptible to aBacillus subtilis-based probiotic added in feed in a related study. This work aimed to (i) report clinical symptoms caused by SH UFPR1 in broilers, (ii) study its susceptibility to some antibioticsin vitro, and (iii) SCOAin vivo; and (iv) relate these phenotypic observations with its genome characteristics. Twoin vivotrials used 1-day-old chicks housed for 21?days in 8 sterilized isolated negative pressure rooms with 4 battery cages of 12 birds each. Birds were challenged or not with 107?CFU/bird of SH UFPR1 orally and exposed or not to SCOA in a 2?×?2 factorial design. Zootechnical parameters were unaffected (P?>?0.05), no clinical signs were observed, and few cecal and hepatic histologic and immune-related alterations were seen, in birds challenged with SH. Formic and propionic acids added together in drinking water, fumaric and benzoic acid in feed (Trial 1), and coated calcium butyrate in feed (Trial 2) did not reduce the SH isolation frequencies seen in cecum and liver in broilers after SH challenge (P?>?0.05). SH UFPR1 was susceptible to amikacin, amoxicillin?+?clavulanate, ceftiofur, cephalexin, doxycycline and oxytetracycline; and mildly susceptible to ampicillin?+?sulbactam, cephalothin, ciprofloxacin, enrofloxacin, and gentamycin in anin vitrominimum inhibitory concentration model using Mueller-Hinton agar. The whole genome of SH UFPR1 was sequenced and consisted of a circular chromosome, spanning 4,760,321?bp with 52.18% of GC-content encoding 84 tRNA, 22 rRNA, and 4,427 protein-coding genes. The comparison between SH UFPR1 genome and a multidrug-resistant SL476 strain revealed 11 missing genomic fragments and 5 insertions related tobgt, bgr, andrpoSgenes. The deleted genes codify proteins associated with cell cycle regulation, virulence, drug resistance, cellular adhesion, and salt efflux which collectively reveal key aspects of the evolution and adaptation of SH strains such as organic acids resistance and antibiotic sensitivity and provide information relevant to the control of SH in poultry.

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July 7, 2019

Probing genomic aspects of the multi-host pathogen Clostridium perfringens reveals significant pangenome diversity, and a diverse array of virulence factors.

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an “open” pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens-associated exotoxins genes including a-toxin (plc), enterotoxin (cpe), and Perfringolysin O (pfo or pfoA), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (tet) and anti-defensins genes (mprF) were consistently detected in silico (tet: 75%; mprF: 100%). However, pre-antibiotic era strain genomes did not encode for tet, thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen.

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July 7, 2019

Safety evaluation of HOWARU®Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM®, Lactobacillus paracasei Lpc-37®, Bifidobacterium animalis subsp. lactis strains Bl-04®, and Bi-07®, and their combination (HOWARU®Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 1012 CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 1012 CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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July 7, 2019

Evolutionary context of non-sorbitol-fermenting Shiga toxin-producing Escherichia coli O55:H7.

In July 2014, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O55:H7 in England involved 31 patients, 13 (42%) of whom had hemolytic uremic syndrome. Isolates were sequenced, and the sequences were compared with publicly available sequences of E. coli O55:H7 and O157:H7. A core-genome phylogeny of the evolutionary history of the STEC O55:H7 outbreak strain revealed that the most parsimonious model was a progenitor enteropathogenic O55:H7 sorbitol-fermenting strain, lysogenized by a Shiga toxin (Stx) 2a-encoding phage, followed by loss of the ability to ferment sorbitol because of a non-sense mutation in srlA. The parallel, convergent evolutionary histories of STEC O157:H7 and STEC O55:H7 may indicate a common driver in the evolutionary process. Because emergence of STEC O157:H7 as a clinically significant pathogen was associated with acquisition of the Stx2a-encoding phage, the emergence of STEC O55:H7 harboring the stx2a gene is of public health concern.

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July 7, 2019

Characterization of Fusobacterium varium Fv113-g1 isolated from a patient with ulcerative colitis based on complete genome sequence and transcriptome analysis.

Fusobacterium spp. present in the oral and gut flora is carcinogenic and is associated with the risk of pancreatic and colorectal cancers. Fusobacterium spp. is also implicated in a broad spectrum of human pathologies, including Crohn’s disease and ulcerative colitis (UC). Here we report the complete genome sequence of Fusobacterium varium Fv113-g1 (genome size, 3.96 Mb) isolated from a patient with UC. Comparative genome analyses totally suggested that Fv113-g1 is basically assigned as F. varium, in particular, it could be reclassified as notable F. varium subsp. similar to F. ulcerans because of partial shared orthologs. Compared with the genome sequences of F. varium ATCC 27725 (genome size, 3.30 Mb) and other strains of Fusobacterium spp., Fv113-g1 possesses many accessary pan-genome sequences with noteworthy multiple virulence factors, including 44 autotransporters (type V secretion system, T5SS) and 13 Fusobacterium adhesion (FadA) paralogs involved in potential mucosal inflammation. Indeed, transcriptome analysis demonstrated that Fv113-g1-specific accessary genes, such as multiple T5SS and fadA paralogs, showed notably increased expression with D-MEM cultivation than with brain heart infusion broth. This implied that growth condition may enhance the expression of such potential virulence factors, leading to remarkable survival against other gut microorganisms and to the pathogenicity to human intestinal epithelium.

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July 7, 2019

Complete genome sequence and comparative genomics of the golden pompano (Trachinotus ovatus) pathogen, Vibrio harveyistrain QT520.

Vibrio harveyi is a Gram-negative, halophilic bacterium that is an opportunistic pathogen of commercially farmed marine vertebrate species. To understand the pathogenicity of this species, the genome of V. harveyi QT520 was analyzed and compared to that of other strains. The results showed the genome of QT520 has two unique circular chromosomes and three endogenous plasmids, totaling 6,070,846 bp with a 45% GC content, 5,701 predicted ORFs, 134 tRNAs and 37 rRNAs. Common virulence factors, including ACF, IlpA, OmpU, Flagellin, Cya, Hemolysin and MARTX, were detected in the genome, which are likely responsible for the virulence of QT520. The results of genomes comparisons with strains ATCC 33843 (392 (MAV)) and ATCC 43516 showed that greater numbers genes associated with types I, II, III, IV and VI secretion systems were detected in QT520 than in other strains, suggesting that QT520 is a highly virulent strain. In addition, three plasmids were only observed in the complete genome sequence of strain QT520. In plasmid p1 of QT520, specific virulence factors (cyaB, hlyB and rtxA) were identified, suggesting that the pathogenicity of this strain is plasmid-associated. Phylogenetic analysis of 12 complete Vibrio sp. genomes using ANI values, core genes and MLST revealed that QT520 was most closely related to ATCC 33843 (392 (MAV)) and ATCC 43516, suggesting that QT520 belongs to the species V. harveyi. This report is the first to describe the complete genome sequence of a V. harveyi strain isolated from an outbreak in a fish species in China. In addition, to the best of our knowledge, this report is the first to compare the V. harveyi genomes of several strains. The results of this study will expand our understanding of the genome, genetic characteristics, and virulence factors of V. harveyi, setting the stage for studies of pathogenesis, diagnostics, and disease prevention.

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July 7, 2019

Pathogenicity and whole genome sequence analysis of a Pseudorabies virus strain FJ-2012 isolated from Fujian, Southern China.

The outbreaks of pseudorabies have been frequently reported in Bartha-K61-vaccinated farms in China since 2011. To study the pathogenicity and evolution of the circulating pseudorabies viruses in Fujian Province, mainland China, we isolated and sequenced the whole genome of a wild-type pseudorabies virus strain named “FJ-2012.” We then conducted a few downstream bioinformatics analyses including phylogenetic analysis and pathogenic analysis and used the virus to infect 6 pseudorabies virus-free piglets. FJ-2012-infected piglets developed symptoms like high body temperature and central nervous system disorders and had high mortality rate. In addition, we identified typical micropathological changes such as multiple gross lesions in infected piglets through pathological analysis and conclude that the FJ-2012 genome is significantly different from known pseudorabies viruses, in which insertions, deletions, and substitutions are observed in multiple immune and virulence genes. In summary, this study shed lights on the molecular basis of the prevalence and pathology of the pseudorabies virus strain FJ-2012. The genome of FJ-2012 could be used as a reference to study the evolution of pseudorabies viruses, which is critical to the vaccine development of new emerging pseudorabies viruses.

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July 7, 2019

Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus.

Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses.

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July 7, 2019

Timing, rates and spectra of human germline mutation.

Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. The mutation rate increased with paternal age in all families, but the number of additional mutations per year differed by more than twofold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency in germline mutation spectra between the sexes and at different paternal ages. In parental germ line, 3.8% of mutations were mosaic, resulting in 1.3% of mutations being shared by siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations.

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July 7, 2019

Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms.

CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two separate genes. Sequencing of three overlapping bacterial artificial chromosomes (BACs) spanning the entire canine CD1 locus revealed canCD1A8.2 and canCD1A8.1 to be located in tandem between canCD1A7 and canCD1C, and canCD1A8.1 was consequently renamed canCD1A9. Green fluorescent protein (GFP)-fused canine CD1 transcripts were recombinantly expressed in 293T cells. All proteins showed a highly positive GFP expression except for canine CD1d and a splice variant of canine CD1a8 lacking exon 3. Probing with a panel of anti-CD1 monoclonal antibodies (mAbs) showed that Ca13.9H11 and Ca9.AG5 only recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our findings have refined the structure of the canine CD1 locus and available antibody specificity against canine CD1 proteins. These are important fundamentals for future investigation of the role of canine CD1 in lipid immunity.

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July 7, 2019

Plasmid characterization and chromosome analysis of two netF+ Clostridium perfringens isolates associated with foal and canine necrotizing enteritis.

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.

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