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July 7, 2019

ICESag37, a novel integrative and conjugative element carrying antimicrobial resistance genes and potential virulence factors in Streptococcus agalactiae.

ICESag37, a novel integrative and conjugative element carrying multidrug resistance and potential virulence factors, was characterized in a clinical isolate of Streptococcus agalactiae. Two clinical strains of S. agalactiae, Sag37 and Sag158, were isolated from blood samples of new-borns with bacteremia. Sag37 was highly resistant to erythromycin and tetracycline, and susceptible to levofloxacin and penicillin, while Sag158 was resistant to tetracycline and levofloxacin, and susceptible to erythromycin. Transfer experiments were performed and selection was carried out with suitable antibiotic concentrations. Through mating experiments, the erythromycin resistance gene was found to be transferable from Sag37 to Sag158. SmaI-PFGE revealed a new SmaI fragment, confirming the transfer of the fragment containing the erythromycin resistance gene. Whole genome sequencing and sequence analysis revealed a mobile element, ICESag37, which was characterized using several molecular methods and in silico analyses. ICESag37 was excised to generate a covalent circular intermediate, which was transferable to S. agalactiae. Inverse PCR was performed to detect the circular form. A serine family integrase mediated its chromosomal integration into rumA, which is a known hotspot for the integration of streptococcal ICEs. The integration site was confirmed using PCR. ICESag37 carried genes for resistance to multiple antibiotics, including erythromycin [erm(B)], tetracycline [tet(O)], and aminoglycosides [aadE, aphA, and ant(6)]. Potential virulence factors, including a two-component signal transduction system (nisK/nisR), were also observed in ICESag37. S1-PFGE analysis ruled out the existence of plasmids. ICESag37 is the first ICESa2603 family-like element identified in S. agalactiae carrying both resistance and potential virulence determinants. It might act as a vehicle for the dissemination of multidrug resistance and pathogenicity among S. agalactiae.

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July 7, 2019

Widespread distribution of mcr-1-bearing bacteria in the ecosystem, 2015 to 2016.

The recently discovered colistin resistance-encoding element, mcr-1, adds to the list of mobile resistance genes whose products rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics, but also the last line agents of carbapenems and colistin. The relative prevalence of mcr-1-bearing strains in various ecological niches including 1,371 food samples, 480 animal faecal samples, 150 human faecal samples and 34 water samples was surveyed using a novel in-house method. Bacteria bearing mcr-1 were commonly detected in water (71% of samples), animal faeces (51%), food products (36%), and exhibited stable carriage in 28% of human subjects surveyed. Such strains, which exhibited variable antibiotic susceptibility profiles, belonged to various Enterobacteriaceae species, with Escherichia coli being the most dominant in each specimen type. The mcr-1 gene was detectable in the chromosome as well as plasmids of various sizes. Among these, two conjugative plasmids of sizes ca?33 and ca?60 kb were found to be the key vectors that mediated mcr-1 transmission in organisms residing in various ecological niches. The high mcr-1 carriage rate in humans found in this study highlights the importance of continued vigilance, careful antibiotic stewardship, and the development of new antimicrobials.

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July 7, 2019

Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori.

The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 – modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5′-Gm6ACC-3′. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ?modH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility.

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July 7, 2019

Identification of sRNA mediated responses to nutrient depletion in Burkholderia pseudomallei.

The Burkholderia genus includes many species that are known to survive in diverse environmental conditions including low nutrient environments. One species, Burkholderia pseudomallei is a versatile pathogen that can survive in a wide range of hosts and environmental conditions. In this study, we investigated how a nutrient depleted growth environment evokes sRNA mediated responses by B. pseudomallei. Computationally predicted B. pseudomallei D286 sRNAs were mapped to RNA-sequencing data for cultures grown under two conditions: (1) BHIB as a nutrient rich media reference environment and (2) M9 media as a nutrient depleted stress environment. The sRNAs were further selected to identify potentially cis-encoded systems by investigating their possible interactions with their flanking genes. The mappings of predicted sRNA genes and interactions analysis to their flanking genes identified 12 sRNA candidates that may possibly have cis-acting regulatory roles that are associated to a nutrient depleted growth environment. Our approach can be used for identifying novel sRNA genes and their possible role as cis-mediated regulatory systems.

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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

RNA-seq and Tn-seq reveal fitness determinants of vancomycin-resistant Enterococcus faecium during growth in human serum.

The Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq).We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010 nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745.Genes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.

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July 7, 2019

A novel Tn1696-like composite transposon (Tn6404) harboring bla IMP-4 in a Klebsiella pneumoniae isolate carrying a rare ESBL gene bla SFO-1.

Genetic determinants of a clinical Klebsiella pneumoniae isolate (KP1814) coproducing IMP-4 and a rare ESBL gene SFO-1 was investigated. KP1814 belongs to a novel sequence type (ST) assigned to ST2270. WGS identified four circular DNA sequences in KP1814, including two multidrug-resistance (MDR) plasmids, one virulence plasmid, and one circular form. The MDR plasmid pKP1814-1 (299.9 Kb) is untypeable, and carries two large mosaic multiresistance regions (MRRs). bla SFO-1 and bla IMP-4 co-exists on MRR1, and bla SFO-1 is associated with an IS/Tn-independent genetic context. bla IMP-4 is carried by a novel In804-like integron (intlI-bla IMP-4-Kl.pn.I3-qacG2-aacA4-catB3?) associated with a novel Tn1696-like transposon (designed Tn6404) flanked by IS5075. The other MDR plasmid pKP1814-3 is a 95,701-bp IncFII plasmid, and is a hybrid of a Shigella flexneri plasmid pSF07201 and an E. coli plasmid pCA08. All resistance genes of pKP1814-3 were detected in a ~16-kb IS26-flanked composite transposon carried by a Tn5396 transposon. The circular form (18.3 Kb) was composed of two parts belonging to pKP1814-1 and pKP1814-3, respectively. The plasmid pKP1814-2, carrying multiple virulence factors, encodes IncFIBK and IncFIIK replicons with a size of 187,349?bp. The coexistence of MDR and virulence plasmids largely enhances the bacterial fitness in the host and environment.

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July 7, 2019

Complete genome sequences of two strains of the meat spoilage bacterium Brochothrix thermosphacta isolated from ground chicken.

Brochothrix thermosphacta is an important meat spoilage bacterium. Here we report the genome sequences of two strains of B. thermosphacta isolated from ground chicken. The genome sequences were determined using long-read PacBio single-molecule real-time (SMRT) technology and are the first complete genome sequences reported for B. thermosphacta.

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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

Genome-wide epigenetic studies in chicken: A review

Over the years, farmed birds have been selected on various performance traits mainly through genetic selection. However, many studies have shown that genetics may not be the sole contributor to phenotypic plasticity. Gene expression programs can be influenced by environmentally induced epigenetic changes that may alter the phenotypes of the developing animals. Recently, high-throughput sequencing techniques became sufficiently affordable thanks to technological advances to study whole epigenetic landscapes in model plants and animals. In birds, a growing number of studies recently took advantage of these techniques to gain insights into the epigenetic mechanisms of gene regulation in processes such as immunity or environmental adaptation. Here, we review the current gain of knowledge on the chicken epigenome made possible by recent advances in high-throughput sequencing techniques by focusing on the two most studied epigenetic modifications, DNA methylation and histone post-translational modifications. We discuss and provide insights about designing and performing analyses to further explore avian epigenomes. A better understanding of the molecular mechanisms underlying the epigenetic regulation of gene expression in relation to bird phenotypes may provide new knowledge and markers that should undoubtedly contribute to a sustainable poultry production.

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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

On the (im)possibility of reconstructing plasmids from whole-genome short-read sequencing data.

To benchmark algorithms for automated plasmid sequence reconstruction from short-read sequencing data, we selected 42 publicly available complete bacterial genome sequences spanning 12 genera, containing 148 plasmids. We predicted plasmids from short-read data with four programs (PlasmidSPAdes, Recycler, cBar and PlasmidFinder) and compared the outcome to the reference sequences. PlasmidSPAdes reconstructs plasmids based on coverage differences in the assembly graph. It reconstructed most of the reference plasmids (recall=0.82), but approximately a quarter of the predicted plasmid contigs were false positives (precision=0.75). PlasmidSPAdes merged 84?% of the predictions from genomes with multiple plasmids into a single bin. Recycler searches the assembly graph for sub-graphs corresponding to circular sequences and correctly predicted small plasmids, but failed with long plasmids (recall=0.12, precision=0.30). cBar, which applies pentamer frequency analysis to detect plasmid-derived contigs, showed a recall and precision of 0.76 and 0.62, respectively. However, cBar categorizes contigs as plasmid-derived and does not bin the different plasmids. PlasmidFinder, which searches for replicons, had the highest precision (1.0), but was restricted by the contents of its database and the contig length obtained fromde novoassembly (recall=0.36). PlasmidSPAdes and Recycler detected putative small plasmids (<10?kbp), which were also predicted as plasmids by cBar, but were absent in the original assembly. This study shows that it is possible to automatically predict small plasmids. Prediction of large plasmids (>50?kbp) containing repeated sequences remains challenging and limits the high-throughput analysis of plasmids from short-read whole-genome sequencing data.

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July 7, 2019

Letting go: bacterial genome reduction solves the dilemma of adapting to predation mortality in a substrate-restricted environment.

Resource limitation and predation mortality are major determinants of microbial population dynamics, and optimization for either aspect is considered to imply a trade-off with respect to the other. Adaptation to these selective factors may, moreover, lead to disadvantages at rich growth conditions. We present an example of a concomitant evolutionary optimization to both, substrate limitation and predation in an aggregate-forming freshwater bacterial isolate, and we elucidate an underlying genomic mechanism. Bacteria were propagated in serial batch culture in a nutrient-restricted environment either with or without a bacterivorous flagellate. Strains isolated after 26 growth cycles of the predator-prey co-cultures formed as much total biomass as the ancestor at ancestral growth conditions, albeit largely reallocated to cell aggregates. A ~273?kbp genome fragment was lost in three strains that had independently evolved with predators. These strains had significantly higher growth yield on substrate-restricted media than others that were isolated from the same treatment before the excision event. Under predation pressure, the isolates with the deletion outcompeted both, the ancestor and the strains evolved without predators even at rich growth conditions. At the same time, genome reduction led to a growth disadvantage in the presence of benzoate due to the loss of the respective degradation pathway, suggesting that niche constriction might be the price for the bidirectional optimization.

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