Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations

The Moraxella adhesin UspA1 binds to its human CEACAM1 receptor by a deformable trimeric coiled-coil

Moraxella catarrhalis is a ubiquitous human-specific bacterium commonly associated with upper and lower respiratory tract infections, including otitis media, sinusitis and chronic obstructive pulmonary disease. The bacterium uses an autotransporter protein UspA1 to target an important human cellular receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Using X-ray crystallography, we show that the CEACAM1 receptor-binding region of UspA1 unusually consists of an extended, rod-like left-handed trimeric coiled-coil. Mutagenesis and binding studies of UspA1 and the N-domain of CEACAM1 have been used to delineate the interacting surfaces between ligand and receptor and guide assembly of the complex. However, solution scattering, molecular modelling and electron microscopy analyses all indicate that significant bending of the UspA1 coiled-coil stalk also occurs. This explains how UspA1 can engage CEACAM1 at a site far distant from its head group, permitting closer proximity of the respective cell surfaces during infection

Entry of Yersinia pestis into the Viable but Nonculturable State in a Low-Temperature Tap Water Microcosm

Yersinia pestis, the causative agent of plague, has caused several pandemics throughout history and remains endemic in the rodent populations of the western United States. More recently, Y. pestis is one of several bacterial pathogens considered to be a potential agent of bioterrorism. Thus, elucidating potential mechanisms of survival and persistence in the environment would be important in the event of an intentional release of the organism. One such mechanism is entry into the viable but non-culturable (VBNC) state, as has been demonstrated for several other bacterial pathogens. In this study, we showed that Y. pestis became nonculturable by normal laboratory methods after 21 days in a low-temperature tap water microcosm. We further show evidence that, after the loss of culturability, the cells remained viable by using a variety of criteria, including cellular membrane integrity, uptake and incorporation of radiolabeled amino acids, and protection of genomic DNA from DNase I digestion. Additionally, we identified morphological and ultrastructural characteristics of Y. pestis VBNC cells, such as cell rounding and large periplasmic spaces, by electron microscopy, which are consistent with entry into the VBNC state in other bacteria. Finally, we demonstrated resuscitation of a small number of the non-culturable cells. This study provides compelling evidence that Y. pestis persists in a low-temperature tap water microcosm in a viable state yet is unable to be cultured under normal laboratory conditions, which may prove useful in risk assessment and remediation efforts, particularly in the event of an intentional release of this organism

Inactivation of the Moraxella catarrhalis Superoxide Dismutase SodA Induces Constitutive Expression of Iron-Repressible Outer Membrane Proteins

Many pathogens produce one or more superoxide dismutases (SODs), enzymes involved in the detoxification of endogenous and exogenous reactive oxygen species that are encountered during the infection process. One detectable cytoplasmic SOD was identified in the human mucosal pathogen Moraxella catarrhalis, and the gene responsible for the SOD activity, sodA, was isolated from a recent pediatric clinical isolate (strain 7169). Sequence analysis of the cloned M. catarrhalis 7169 DNA fragment revealed an open reading frame of 618 bp encoding a polypeptide of 205 amino acids with 48 to 67% identity to known bacterial manganese-cofactored SODs. An isogenic M. catarrhalis sodA mutant was constructed in strain 7169 by allelic exchange. In contrast to the wild-type 7169, the 7169::sodK20 mutant was severely attenuated for aerobic growth, even in rich medium containing supplemental amino acids, and exhibited extreme sensitivity to the redox-active agent methyl viologen. The ability of recombinant SodA to rescue the aerobic growth defects of E. coli QC774, a sodA sodB-deficient mutant, demonstrated the functional expression of SOD activity by cloned M. catarrhalis sodA. Indirect SOD detection assays were used to visualize both native and recombinant SodA activity in bacterial lysates. This study demonstrates that M. catarrhalis SodA plays a critical role in the detoxification of endogenous, metabolically produced oxygen radicals. In addition, the outer membrane protein (OMP) profile of 7169::sodK20 was consistent with iron starvation in spite of growth under iron-replete conditions. This novel observation indicates that M. catarrhalis strains lacking SodA constitutively express immunogenic OMPs previously described as iron repressible, and this potentially attenuated mutant strain may be an attractive vaccine candidate

Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations

Cohort Characteristics.

<p>Differences between patients who died and those who survived were assessed by chi-squared for categorical data and Wilcoxon rank-sum tests for continuous data. IQR = inter quartile range.</p

30-day mortality according to biomarker levels on admission.

<p>Mortality was lower in patients with normal NT-proBNP and Troponin T levels than patients with elevated NT-proBNP alone (9/86, p = 0.0002) and both elevated NT-proBNP and Troponin T (14/62, p<0.0001).</p

1 year Kaplan-Meier survival curve for patients following community-acquired pneumonia stratified according to NT-proBNP level.

<p>Survival was worse in patients with high NT-proBNP levels (>220 pmol/L) compared to patients with normal NT-proBNP levels (≤220 pmol/L) (log-rank test, p<0.0001).</p