Search

We conducted comprehensive (untargeted) metabolic profiling of volatile organic compounds (VOCs) emitted in culture by bacterial taxa Francisella tularensis (F. tularensis) subspecies novicida and Bacillus anthracis (B. anthracis) Sterne, surrogates for potential bacterial bioterrorism agents, as well as selective measurements of VOCs from their fully virulent counterparts, F. tularensis subspecies tularensis strain SCHU S4 and B. anthracis Ames. F. tularensis and B. anthracis were grown in liquid broth for time periods that covered logarithmic growth, stationary, and decline phases. VOCs emitted over the course of the growth phases were collected from the headspace above the cultures using solid phase microextraction (SPME) and were analyzed using gas chromatography-mass spectrometry (GC-MS). We developed criteria for distinguishing VOCs originating from bacteria versus background VOCs (originating from growth media only controls or sampling devices). Analyses of collected VOCs revealed methyl ketones, alcohols, esters, carboxylic acids, and nitrogen- and sulfur-containing compounds that were present in the bacterial cultures and absent (or present at only low abundance) in control samples indicating that these compounds originated from the bacteria. Distinct VOC profiles where observed for F. tularensis when compared with B. anthracis while the observed profiles of each of the two F. tularensis and B. anthracis strains exhibited some similarities. Furthermore, the relative abundance of VOCs was influenced by bacterial growth phase. These data illustrate the potential for VOC profiles to distinguish pathogens at the genus and species-level and to discriminate bacterial growth phases. The determination of VOC profiles lays the groundwork for non-invasive probes of bacterial metabolism and offers prospects for detection of microbe-specific VOC biomarkers from two potential biowarfare agents.

A comprehensive laboratory evaluation of the Tetracore RedLine Alert test, a lateral flow immunoassay (LFA) for the rapid presumptive identification of Bacillus anthracis, was conducted at 2 different test sites. The study evaluated the sensitivity of this assay using 16 diverse strains of B. anthracis grown on sheep blood agar (SBA) plates. In addition, 83 clinically relevant microorganisms were tested to assess the specificity of the RedLine Alert test. The results indicated that the RedLine Alert test for the presumptive identification of B. anthracis is highly robust, specific, and sensitive. RedLine Alert is a rapid test that has applicability for use in a clinical setting for ruling-in or ruling-out nonhemolytic colonies of Bacillus spp. grown on SBA medium as presumptive isolates of B. anthracis.

We conducted a comprehensive, multiphase laboratory evaluation of the Anthrax BioThreat Alert(®) test strip, a lateral flow immunoassay (LFA) for the rapid detection of Bacillus anthracis spores. The study, conducted at 2 sites, evaluated this assay for the detection of spores from the Ames and Sterne strains of B. anthracis, as well as those from an additional 22 strains. Phylogenetic near neighbors, environmental background organisms, white powders, and environmental samples were also tested. The Anthrax LFA demonstrated a limit of detection of about 10(6) spores/mL (ca. 1.5 × 10(5) spores/assay). In this study, overall sensitivity of the LFA was 99.3%, and the specificity was 98.6%. The results indicated that the specificity, sensitivity, limit of detection, dynamic range, and repeatability of the assay support its use in the field for the purpose of qualitatively evaluating suspicious white powders and environmental samples for the presumptive presence of B. anthracis spores.

The Rapid Viability Polymerase Chain Reaction (RV-PCR) method was evaluated during the Bio-Response Operational Testing and Evaluation (BOTE), an interagency project to evaluate field-level facility biological remediation, using leading decontamination technologies. The tests were performed using an intentional release (aerosolization) of spores of Bacillus atrophaeus subspecies globigii (BG), as a surrogate for Bacillus anthracis, the etiologic agent for anthrax. Three decontamination methods were assessed including fumigation with vaporized hydrogen peroxide (VHP), fumigation with chlorine dioxide (CD), and a surface treatment process using pH-adjusted bleach.The RV-PCR method was developed to rapidly detect live B. anthracis spores during a bioterrorism event. The method uses a change in realtime PCR response before and after a nine hour incubation step, to determine the presence of viable bacterial spores in the sample; the method was recently verified for air filter, wipe and water samples at the 10-spore level for B. anthracis Ames spores, and was also developed for swab, sponge-stick, and vacuum sock/filter samples. In the method, high throughput sample processing is combined with PCR-based analysis before and after a rapid culture step to speed viability determination, especially for complex surface and environmental samples that present challenges to current culture-based methods. In the BOTE project, a total of 159 surface wipe samples from post-decontamination events were analyzed by splitting the suspension after spore recovery into two equal parts, with one part analyzed by RV-PCR and the other part by culture after concentrating to the same volume. In the BOTE project, the RV-PCR method provided rapid results for post-decontamination samples that were 98% (156/159 samples) consistent with results from culture analysis. The percentage agreement was noteworthy, given the large number of samples containing low spore levels. For the Post-VHP, Post-Bleach, and Post-CD event samples, the percentage agreement was 93% (41/44 samples), 100% (47/47 samples), and 100% (68/68 samples), respectively. The RV-PCR method performed well for the surrogate BG spores exposed to decontaminants at real-world application levels, and with wipe samples containing background debris and indigenous microbial populations.

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis , in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.