Phylogenomic diversity of Vibrio species and other Gammaproteobacteria isolated from Pacific oysters (Crassostrea gigas) during a summer mortality outbreak.

The Pacific oyster (PO), Crassostrea gigas, is an important commercial marine species but periodically experiences large stock losses due to disease events known as summer mortality. Summer mortality has been linked to environmental perturbations and numerous viral and bacterial agents, indicating this disease is multifactorial in nature. In 2013 and 2014, several summer mortality events occurred within the Port Stephens estuary (NSW, Australia). Extensive culture and molecular-based investigations were undertaken and several potentially pathogenic Vibrio species were identified. To improve species identification and genomically characterise isolates obtained from this outbreak, whole-genome sequencing (WGS) and subsequent genomic analyses were performed on 48 bacterial isolates, as well as a further nine isolates from other summer mortality studies using the same batch of juveniles. Average nucleotide identity (ANI) identified most isolates to the species level and included members of the Photobacterium, Pseudoalteromonas, Shewanella and Vibrio genera, with Vibrio species making up more than two-thirds of all species identified. Construction of a phylogenomic tree, ANI analysis, and pan-genome analysis of the 57 isolates represents the most comprehensive culture-based phylogenomic survey of Vibrios during a PO summer mortality event in Australian waters and revealed large genomic diversity in many of the identified species. Our analysis revealed limited and inconsistent associations between isolate species and their geographical origins, or host health status. Together with ANI and pan-genome results, these inconsistencies suggest that to determine the role that microbes may have in Pacific oyster summer mortality events, isolate identification must be at the taxonomic level of strain. Our WGS data (specifically, the accessory genomes) differentiated bacterial strains, and coupled with associated metadata, highlight the possibility of predicting a strain's environmental niche and level of pathogenicity.

Metagenomic Hi-C of a Healthy Human Fecal Microbiome Transplant Donor.

We report the availability of a high-quality metagenomic Hi-C data set generated from a fecal sample taken from a healthy fecal microbiome transplant donor subject. We report on basic features of the data to evaluate their quality.

Evaluation of ddRADseq for reduced representation metagenome sequencing.

BACKGROUND: Profiling of microbial communities via metagenomic shotgun sequencing has enabled researches to gain unprecedented insight into microbial community structure and the functional roles of community members. This study describes a method and basic analysis for a metagenomic adaptation of the double digest restriction site associated DNA sequencing (ddRADseq) protocol for reduced representation metagenome profiling. METHODS: This technique takes advantage of the sequence specificity of restriction endonucleases to construct an Illumina-compatible sequencing library containing DNA fragments that are between a pair of restriction sites located within close proximity. This results in a reduced sequencing library with coverage breadth that can be tuned by size selection. We assessed the performance of the metagenomic ddRADseq approach by applying the full method to human stool samples and generating sequence data. RESULTS: The ddRADseq data yields a similar estimate of community taxonomic profile as obtained from shotgun metagenome sequencing of the same human stool samples. No obvious bias with respect to genomic G + C content and the estimated relative species abundance was detected. DISCUSSION: Although ddRADseq does introduce some bias in taxonomic representation, the bias is likely to be small relative to DNA extraction bias. ddRADseq appears feasible and could have value as a tool for metagenome-wide association studies.

Genomic islands 1 and 2 play key roles in the evolution of extensively drug-resistant ST235 isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa are noscomially acquired, opportunistic pathogens that pose a major threat to the health of burns patients and the immunocompromised. We sequenced the genomes of P. aeruginosa isolates RNS_PA1, RNS_PA46 and RNS_PAE05, which displayed resistance to almost all frontline antibiotics, including gentamicin, piperacillin, timentin, meropenem, ceftazidime and colistin. We provide evidence that the isolates are representatives of P. aeruginosa sequence type (ST) 235 and carry Tn6162 and Tn6163 in genomic islands 1 (GI1) and 2 (GI2), respectively. GI1 disrupts the endA gene at precisely the same chromosomal location as in P. aeruginosa strain VR-143/97, of unknown ST, creating an identical CA direct repeat. The class 1 integron associated with Tn6163 in GI2 carries a blaGES-5-aacA4-gcuE15-aphA15 cassette array conferring resistance to carbapenems and aminoglycosides. GI2 is flanked by a 12 nt direct repeat motif, abuts a tRNA-gly gene, and encodes proteins with putative roles in integration, conjugative transfer as well as integrative conjugative element-specific proteins. This suggests that GI2 may have evolved from a novel integrative conjugative element. Our data provide further support to the hypothesis that genomic islands play an important role in de novo evolution of multiple antibiotic resistance phenotypes in P. aeruginosa.

Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease.

BACKGROUND: Clostridium difficile infections (CDI) are a significant health problem to humans and food animals. Clostridial toxins ToxA and ToxB encoded by genes tcdA and tcdB are located on a pathogenicity locus known as the PaLoc and are the major virulence factors of C. difficile. While toxin-negative strains of C. difficile are often isolated from faeces of animals and patients suffering from CDI, they are not considered to play a role in disease. Toxin-negative strains of C. difficile have been used successfully to treat recurring CDI but their propensity to acquire the PaLoc via lateral gene transfer and express clinically relevant levels of toxins has reinforced the need to characterise them genetically. In addition, further studies that examine the pathogenic potential of toxin-negative strains of C. difficile and the frequency by which toxin-negative strains may acquire the PaLoc are needed. RESULTS: We undertook a comparative genomic analysis of five Australian toxin-negative isolates of C. difficile that lack tcdA, tcdB and both binary toxin genes cdtA and cdtB that were recovered from humans and farm animals with symptoms of gastrointestinal disease. Our analyses show that the five C. difficile isolates cluster closely with virulent toxigenic strains of C. difficile belonging to the same sequence type (ST) and have virulence gene profiles akin to those in toxigenic strains. Furthermore, phage acquisition appears to have played a key role in the evolution of C. difficile. CONCLUSIONS: Our results are consistent with the C. difficile global population structure comprising six clades each containing both toxin-positive and toxin-negative strains. Our data also suggests that toxin-negative strains of C. difficile encode a repertoire of putative virulence factors that are similar to those found in toxigenic strains of C. difficile, raising the possibility that acquisition of PaLoc by toxin-negative strains poses a threat to human health. Studies in appropriate animal models are needed to examine the pathogenic potential of toxin-negative strains of C. difficile and to determine the frequency by which toxin-negative strains may acquire the PaLoc.

A draft genome of Escherichia coli sequence type 127 strain 2009-46.

BACKGROUND: Escherichia coli are a frequent cause of urinary tract infections (UTI) and are thought to have a foodborne origin. E. coli with sequence type 127 (ST127) are emerging pathogens increasingly implicated as a cause of urinary tract infections (UTI) globally. A ST127 isolate (2009-46) resistant to ampicillin and trimethoprim was recovered from the urine of a 56 year old patient with a UTI from a hospital in Sydney, Australia and was characterised here. RESULTS: We sequenced the genome of Escherichia coli 2009-46 using the Illumina Nextera XT and MiSeq technologies. Assembly of the sequence data reconstructed a 5.14 Mbp genome in 89 scaffolds with an N50 of 161 kbp. The genome has extensive similarity to other sequenced uropathogenic E. coli genomes, but also has several genes that are potentially related to virulence and pathogenicity that are not present in the reference E. coli strain. CONCLUSION: E. coli 2009-46 is a multiple antibiotic resistant, phylogroup B2 isolate recovered from a patient with a UTI. This is the first description of a drug resistant E. coli ST127 in Australia.

The genome of Clostridium difficile 5.3.

BACKGROUND: Clostridium difficile is the leading cause of infectious diarrhea in humans and responsible for large outbreaks of enteritis in neonatal pigs in both North America and Europe. Disease caused by C. difficile typically occurs during antibiotic therapy and its emergence over the past 40 years is linked with the widespread use of broad-spectrum antibiotics in both human and veterinary medicine. RESULTS: We sequenced the genome of Clostridium difficile 5.3 using the Illumina Nextera XT and MiSeq technologies. Assembly of the sequence data reconstructed a 4,009,318 bp genome in 27 scaffolds with an N50 of 786 kbp. The genome has extensive similarity to other sequenced C. difficile genomes, but also has several genes that are potentially related to virulence and pathogenicity that are not present in the reference C. difficile strain. CONCLUSION: Genome sequencing of human and animal isolates is needed to understand the molecular events driving the emergence of C. difficile as a gastrointestinal pathogen of humans and food animals and to better define its zoonotic potential.

Identification and differentiation of Cryptosporidium species by capillary electrophoresis single-strand conformation polymorphism.

Cryptosporidium species generally lack distinguishing morphological traits, and consequently, molecular methods are commonly used for parasite identification. Various methods for Cryptosporidium identification have been proposed, each with their advantages and disadvantages. In this study, we show that capillary electrophoresis coupled with single-strand conformation polymorphism (CE-SSCP) is a rapid, simple and cost-effective method for the identification of Cryptosporidium species and genotypes. Species could be readily differentiated based on the SSCP mobility of amplified 18S rRNA gene molecules. Clones that differed by single-nucleotide polymorphisms could be distinguished on CE-SSCP mobility. Profiles of species known to have heterogenic copies of 18S rRNA gene contained multiple peaks. Cloning and sequencing of Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium fayeri and Cryptosporidium possum genotype 18S rRNA gene amplicons confirmed that these multiple peaks represented type A and type B 18S rRNA gene copies. CE-SSCP provides a reliable and sensitive analysis for epidemiological studies, environmental detection and diversity screening.

Recovery of diverse genes for class 1 integron-integrases from environmental DNA samples.

Class 1 integrons are an important vector for the spread of antibiotic resistance. The core of this genetic element is highly conserved in all class 1 integrons recovered from clinical contexts. Recently, bacteria containing more divergent class 1 integrons have been isolated from environmental samples, suggesting undiscovered diversity in these elements. We performed a culture-independent survey of the class 1 integron-integrase gene (intI1) from environmental DNA, assessing sequence variation using capillary electrophoresis single-strand conformation polymorphism. This analysis allowed informed selection of environments for further investigation based on the diversity of intI1 targets that were present. IntI1 was common in environmental samples and exhibited previously unsuspected sequence diversity. The method allowed discrimination between clinical and environmental variants of intI1.