A MarR family transcriptional regulator and subinhibitory antibiotics regulate type VI secretion gene clusters in Burkholderia pseudomallei.

Burkholderia pseudomallei, the aetiological agent of melioidosis, is an inhabitant of soil and water in many tropical and subtropical regions worldwide. It possesses six distinct type VI secretion systems (T6SS-1 to T6SS-6), but little is known about most of them, as they are poorly expressed in laboratory culture media. A genetic screen was devised to locate a putative repressor of the T6SS-2 gene cluster and a MarR family transcriptional regulator, termed TctR, was identified. The inactivation of tctR resulted in a 50-fold increase in the expression of an hcp2-lacZ transcriptional fusion, indicating that TctR is a negative regulator of the T6SS-2 gene cluster. Surprisingly, the tctR mutation resulted in a significant decrease in the expression of an hcp6-lacZ transcriptional fusion. B. pseudomallei K96243 and a tctR mutant were grown to logarithmic phase in rich culture medium and RNA was isolated and sequenced in order to identify other genes regulated by TctR. The results identified seven gene clusters that were repressed by TctR, including T6SS-2, and three gene clusters that were significantly activated. A small molecule library consisting of 1120 structurally defined compounds was screened to identify a putative ligand (or ligands) that might bind TctR and derepress transcription of the T6SS-2 gene cluster. Seven compounds, six fluoroquinolones and one quinolone, activated the expression of hcp2-lacZ. Subinhibitory ciprofloxacin also increased the expression of the T6SS-3, T6SS-4 and T6SS-6 gene clusters. This study highlights the complex layers of regulatory control that B. pseudomallei utilizes to ensure that T6SS expression only occurs under very defined environmental conditions.

A Standard Method To Inactivate Bacillus anthracis Spores to Sterility via Gamma Irradiation.

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 × 1010 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D10) values and irradiation doses required to ensure sterility (DSAL) to the point at which the probability of detecting a viable spore is 10-6 Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.IMPORTANCE The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.

Finished Genome Sequence of Bacillus cereus Strain 03BB87, a Clinical Isolate with B. anthracis Virulence Genes.

Bacillus cereus strain 03BB87, a blood culture isolate, originated in a 56-year-old male muller operator with a fatal case of pneumonia in 2003. Here we present the finished genome sequence of that pathogen, including a 5.46-Mb chromosome and two plasmids (209 and 52 Kb, respectively).

Biolog phenotype microarrays.

Phenotype microarrays nicely complement traditional genomic, transcriptomic, and proteomic analysis by offering opportunities for researchers to ground microbial systems analysis and modeling in a broad yet quantitative assessment of the organism's physiological response to different metabolites and environments. Biolog phenotype assays achieve this by coupling tetrazolium dyes with minimally defined nutrients to measure the impact of hundreds of carbon, nitrogen, phosphorous, and sulfur sources on redox reactions that result from compound-induced effects on the electron transport chain. Over the years, we have used Biolog's reproducible and highly sensitive assays to distinguish closely related bacterial isolates, to understand their metabolic differences, and to model their metabolic behavior using flux balance analysis. This chapter describes Biolog phenotype microarray system components, reagents, and methods, particularly as they apply to bacterial identification, characterization, and metabolic analysis.

Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including shiga toxin encoding phage stx2.

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.

Raman chemical imaging spectroscopy reagentless detection and identification of pathogens: signature development and evaluation.

An optical detection method, Raman chemical imaging spectroscopy (RCIS), is reported, which combines Raman spectroscopy, fluorescence spectroscopy, and digital imaging. Using this method, trace levels of biothreat organisms are detected in the presence of complex environmental backgrounds without the use of amplification or enhancement techniques. RCIS is reliant upon the use of Raman signatures and automated recognition algorithms to perform species-level identification. The rationale and steps for constructing a pathogen Raman signature library are described, as well as the first reported Raman spectra from live, priority pathogens, including Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Francisella tularensis, Brucella abortus, and ricin. Results from a government-managed blind trial evaluation of the signature library demonstrated excellent specificity under controlled laboratory conditions.