Whole genome sequencing of diverse Shiga toxin-producing and non-producing Escherichia coli strains reveals a variety of virulence and novel antibiotic resistance plasmids.

The genomes of a diverse set of Escherichia coli, including many Shiga toxin-producing strains of various serotypes were determined. A total of 39 plasmids were identified among these strains, and many carried virulence or putative virulence genes of Shiga toxin-producing E. coli strains, virulence genes for other pathogenic E. coli groups, and some had combinations of these genes. Among the novel plasmids identified were eight that carried resistance genes to aminoglycosides, carbapenems, penicillins, cephalosporins, chloramphenicol, dihydrofolate reductase inhibitors, sulfonamides, tetracyclines and resistance to heavy metals. Two of the plasmids carried six of these resistance genes and two novel IncHI2 plasmids were also identified. The results of this study showed that plasmids carrying diverse resistance and virulence genes of various pathogenic E. coli groups can be found in E. coli strains and serotypes regardless of the isolate's source and therefore, is consistent with the premise that these mobile elements carrying these traits may be broadly disseminated among E. coli.

Identification and taxonomic characterization of Bordetella pseudohinzii sp. nov. isolated from laboratory-raised mice.

Bordetella hinzii is known to cause respiratory disease in poultry and has been associated with a variety of infections in immunocompromised humans. In addition, there are several reports of B. hinzii infections in laboratory-raised mice. Here we sequenced and analysed the complete genome sequences of multiple B. hinzii-like isolates, obtained from vendor-supplied C57BL/6 mice in animal research facilities on different continents, and we determined their taxonomic relationship to other Bordetella species. The whole-genome based and 16S rRNA gene based phylogenies each identified two separate clades in B. hinzii, one was composed of strains isolated from poultry, humans and a rabbit whereas the other clade was restricted to isolates from mice. Distinctly different estimated DNA-DNA hybridization values, average nucleotide identity scores, gene content, metabolic profiles and host specificity all provide compelling evidence for delineation of the two species, B. hinzii - from poultry, humans and rabbit - and Bordetella pseudohinzii sp. nov. type strain 8-296-03T (=NRRL B-59942T=NCTC 13808T) that infect mice.

Tranexamic acid administration and pulmonary embolism in combat casualties with orthopaedic injuries.

In combat casualty care, tranexamic acid (TXA) is administered as part of initial resuscitation effort; however, conflicting data exist as to whether TXA contributes to increased risk of venous thromboembolism (VTE). The purpose of this study is to determine what factors increase risk of pulmonary embolism after combat-related orthopaedic trauma and whether administration of TXA is an independent risk factor for major thromboembolic events. SETTING: United States Military Trauma Centers. PATIENTS: Combat casualties with orthopaedic injuries treated at any US military trauma center for traumatic injuries sustained from January 2011 through December 2015. In total, 493 patients were identified. INTERVENTION: None. MAIN OUTCOME MEASURES: Occurrence of major thromboembolic events, defined as segmental or greater pulmonary embolism or thromboembolism-associated pulseless electrical activity. RESULTS: Regression analysis revealed TXA administration, traumatic amputation, acute kidney failure, and hypertension to be associated with the development of a major thromboembolic event for all models. Injury characteristics independently associated with risk of major VTE were Injury Severity Score 23 or greater, traumatic amputation, and vertebral fracture. The best performing model utilized had an area under curve  = 0.84, a sensitivity=0.72, and a specificity=0.84. CONCLUSIONS: TXA is an independent risk factor for major VTE after combat-related Orthopaedic injury. Injury factors including severe trauma, major extremity amputation, and vertebral fracture should prompt suspicion for increased risk of major thromboembolic events and increased threshold for TXA use if no major hemorrhage is present. LEVEL OF EVIDENCE: III, Prognostic Study.

Draft Genome Sequences of 53 Genetically Distinct Isolates of Bordetella bronchiseptica Representing 11 Terrestrial and Aquatic Hosts.

Bordetella bronchiseptica infects a variety of mammalian and avian hosts. Here, we report the genome sequences of 53 genetically distinct isolates acquired from a broad range of terrestrial and aquatic animals. These data will greatly facilitate ongoing efforts to better understand the evolution, host adaptation, and virulence mechanisms of B. bronchiseptica.

Genome Sequences of Nine Bordetella holmesii Strains Isolated in the United States.

An increasing number of pertussis-like cases are attributed to the emergent pathogen Bordetella holmesii. The genomes of 9 clinical isolates show that they are clonal, lack the virulence factors encoded by B. pertussis, and are more similar to nonpertussis bordetellae. New markers for B. holmesii can be developed using these sequences.

Pathogenomic inference of virulence-associated genes in Leptospira interrogans.

Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.

Optimizing read mapping to reference genomes to determine composition and species prevalence in microbial communities.

The Human Microbiome Project (HMP) aims to characterize the microbial communities of 18 body sites from healthy individuals. To accomplish this, the HMP generated two types of shotgun data: reference shotgun sequences isolated from different anatomical sites on the human body and shotgun metagenomic sequences from the microbial communities of each site. The alignment strategy for characterizing these metagenomic communities using available reference sequence is important to the success of HMP data analysis. Six next-generation aligners were used to align a community of known composition against a database comprising reference organisms known to be present in that community. All aligners report nearly complete genome coverage (>97%) for strains with over 6X depth of coverage, however they differ in speed, memory requirement and ease of use issues such as database size limitations and supported mapping strategies. The selected aligner was tested across a range of parameters to maximize sensitivity while maintaining a low false positive rate. We found that constraining alignment length had more impact on sensitivity than does constraining similarity in all cases tested. However, when reference species were replaced with phylogenetic neighbors, similarity begins to play a larger role in detection. We also show that choosing the top hit randomly when multiple, equally strong mappings are available increases overall sensitivity at the expense of taxonomic resolution. The results of this study identified a strategy that was used to map over 3 tera-bases of microbial sequence against a database of more than 5,000 reference genomes in just over a month.

Whole genome analysis of Leptospira licerasiae provides insight into leptospiral evolution and pathogenicity.

The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae) that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010(T) and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT). Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for its infectiousness and its unique antigenic characteristics.

Monitoring the long-term molecular epidemiology of the pneumococcus and detection of potential 'vaccine escape' strains.

BACKGROUND: While the pneumococcal protein conjugate vaccines reduce the incidence in invasive pneumococcal disease (IPD), serotype replacement remains a major concern. Thus, serotype-independent protection with vaccines targeting virulence genes, such as PspA, have been pursued. PspA is comprised of diverse clades that arose through recombination. Therefore, multi-locus sequence typing (MLST)-defined clones could conceivably include strains from multiple PspA clades. As a result, a method is needed which can both monitor the long-term epidemiology of the pneumococcus among a large number of isolates, and analyze vaccine-candidate genes, such as pspA, for mutations and recombination events that could result in 'vaccine escape' strains. METHODOLOGY: We developed a resequencing array consisting of five conserved and six variable genes to characterize 72 pneumococcal strains. The phylogenetic analysis of the 11 concatenated genes was performed with the MrBayes program, the single nucleotide polymorphism (SNP) analysis with the DNA Sequence Polymorphism program (DnaSP), and the recombination event analysis with the recombination detection package (RDP). RESULTS: The phylogenetic analysis correlated with MLST, and identified clonal strains with unique PspA clades. The DnaSP analysis correlated with the serotype-specific diversity detected using MLST. Serotypes associated with more than one ST complex had a larger degree of sequence polymorphism than a serotype associated with one ST complex. The RDP analysis confirmed the high frequency of recombination events in the pspA gene. CONCLUSIONS: The phylogenetic tree correlated with MLST, and detected multiple PspA clades among clonal strains. The genetic diversity of the strains and the frequency of recombination events in the mosaic gene, pspA were accurately assessed using the DnaSP and RDP programs, respectively. These data provide proof-of-concept that resequencing arrays could play an important role within research and clinical laboratories in both monitoring the molecular epidemiology of the pneumococcus and detecting 'vaccine escape' strains among vaccine-candidate genes.