Whole genome sequencing for genomics-guided investigations of Escherichia coli O157:H7 outbreaks

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies

Closed genome and comparative phylogenetic analysis of the clinical multidrug resistant Shigella sonnei strain 866

Shigella sonneiis responsible for the majority of shigellosis infections in the US with over 500,000 cases reported annually. Here, wepresent the complete genome of the clinical multidrug resistant (MDR) strain 866, which is highly susceptible to bacteriophageinfections. The strain has a circular chromosome of 4.85 Mb and carries a 113 kb MDR plasmid. This IncB/O/K/Z-type plasmid, termedp866, confers resistance to five different classes of antibiotics including ß-lactamase, sulfonamide, tetracycline, aminoglycoside, andtrimethoprim. Comparative analysis of the plasmid architecture and gene inventory revealed that p866 shares its plasmid backbonewith previously described IncB/O/K/Z-typeShigellaspp. andEscherichiacoliplasmids, but is differentiated by the insertion of antibioticresistance cassettes, which we found associated with mobile genetic elements such as Tn3, Tn7, and Tn10. A whole genome-derivedphylogenetic reconstruction showed the evolutionary relationships ofS. sonneistrain 866 and the four establishedShigellaspecies,highlighting the clonal nature ofS. sonnei

Twice-daily application of HIV microbicides alters the vaginal microbiota

Vaginal HIV microbicides offer great promise in preventing HIV transmission, but failures of phase 3 clinical trials, in which microbicide-treated subjects had an increased risk of HIV transmission, raised concerns about endpoints used to evaluate microbicide safety. A possible explanation for the increased transmission risk is that the agents shifted the vaginal bacterial community, resulting in loss of natural protection and enhanced HIV transmission susceptibility. We characterized vaginal microbiota, using pyrosequencing of bar-coded 16S rRNA gene fragments, in samples from 35 healthy, sexually abstinent female volunteer subjects (ages 18 to 50 years) with regular menses in a repeat phase 1 study of twice-daily application over 13.5 days of 1 of 3 gel products: a hydroxyethylcellulose (HEC)-based “universal” placebo (10 subjects), 6% cellulose sulfate (CS; 13 subjects), and 4% nonoxynol-9 (N-9; 12 subjects). We used mixed effects models inferred using Bayesian Markov chain Monte Carlo methods, which showed that treatment with active agents shifted the microbiota toward a community type lacking significant numbers of Lactobacillus spp. and dominated by strict anaerobes. This state of the vaginal microbiota was associated with a low or intermediate Nugent score and was not identical to bacterial vaginosis, an HIV transmission risk factor. The placebo arm contained a higher proportion of communities dominated by Lactobacillus spp., particularly L. crispatus, throughout treatment. The data suggest that molecular evaluation of microbicide effects on vaginal microbiota may be a critical endpoint that should be incorporated in early clinical assessment of microbicide candidates. IMPORTANCE Despite large prevention efforts, HIV transmission and acquisition rates remain unacceptably high. In developing countries, transmission mainly occurs through heterosexual intercourse, where women are significantly more vulnerable to infection than men. Vaginal microbicides are considered to be one of the most promising female-controlled products, in that women themselves insert the microbicides into the vagina to prevent HIV transmission during sexual intercourse. The failure of several microbicides in clinical trials has raised questions concerning the low in vivo efficacy of such anti-HIV molecules. This study was designed to gain insights into the failures of two microbicides by testing the hypothesis that the microbicides negatively affect a critical line of defense against HIV, the vaginal microbiota. The results suggest that in the early assessment of candidate microbicides, culture-independent evaluation of their effect on the vaginal microbiota should be considered and may constitute a critical endpoint

Closed genome and comparative phylogenetic analysis of the clinical multidrug resistant Shigella sonnei strain 866

Shigella sonneiis responsible for the majority of shigellosis infections in the US with over 500,000 cases reported annually. Here, wepresent the complete genome of the clinical multidrug resistant (MDR) strain 866, which is highly susceptible to bacteriophageinfections. The strain has a circular chromosome of 4.85 Mb and carries a 113 kb MDR plasmid. This IncB/O/K/Z-type plasmid, termedp866, confers resistance to five different classes of antibiotics including ß-lactamase, sulfonamide, tetracycline, aminoglycoside, andtrimethoprim. Comparative analysis of the plasmid architecture and gene inventory revealed that p866 shares its plasmid backbonewith previously described IncB/O/K/Z-typeShigellaspp. andEscherichiacoliplasmids, but is differentiated by the insertion of antibioticresistance cassettes, which we found associated with mobile genetic elements such as Tn3, Tn7, and Tn10. A whole genome-derivedphylogenetic reconstruction showed the evolutionary relationships ofS. sonneistrain 866 and the four establishedShigellaspecies,highlighting the clonal nature ofS. sonnei