Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages.

Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viableB. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wß::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxABin an earlier version of the reporter phage (Wß::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wß::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 10(5)CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 10(4)CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils.

Rapid Detection of Bacillus anthracis in Complex Food Matrices Using Phage-Mediated Bioluminescence.

Bacillus anthracis, the causative agent of anthrax, is considered a high-priority agent that may be used in a food-related terrorist attack because it can be contracted by ingestion and it also forms spores with heat and chemical resistance. Thus, novel surveillance methodologies to detect B. anthracis on adulterated foods are important for bioterrorism preparedness. We describe the development of a phage-based bioluminescence assay for the detection of B. anthracis on deliberately contaminated foods. We previously engineered the B. anthracis phage Wß with genes encoding bacterial luciferase (luxA and luxB) to create a "light-tagged" reporter (Wß::luxAB) that is able to rapidly detect B. anthracis by transducing a bioluminescent signal response. Here, we investigate the ability of Wß::luxAB to detect B. anthracis Sterne, an attenuated select agent strain, in inoculated food (ground beef) and milk (2%, baby formula, and half and half) matrices after incubation with spores for 72 h at 4°C as per AOAC testing guidelines. The majority of B. anthracis bacilli remained in spore form, and thus were potentially infectious, within each of the liquid matrices for 14 days. Detection limits were 80 CFU/ml after 7 h of enrichment; sensitivity of detection increased to 8 CFU/ml when enrichment was extended to 16 h. The limit of detection in ground beef was 3.2 × 10(3) CFU/g after 7 h of enrichment, improving to 3.2 × 10(2) CFU/g after 16 h. Because the time to result is rapid and minimal processing is required, and because gastrointestinal anthrax can be fatal, the reporter technology displays promise for the protection of our food supply following a deliberate release of this priority pathogen.

Bacillus anthracis diagnostic detection and rapid antibiotic susceptibility determination using 'bioluminescent' reporter phage.

Genetically modified phages have the potential to detect pathogenic bacteria from clinical, environmental, or food-related sources. Herein we assess an engineered 'bioluminescent' reporter phage (Wß::luxAB) as a clinical diagnostic tool for Bacillus anthracis, the etiological agent of anthrax. Wß::luxAB is able to rapidly (within minutes) detect a panel of B. anthracis strains by transducing a bioluminescent phenotype. The reporter phage displays species specificity by its inability, or significantly reduced ability, to detect members of the closely related Bacillus cereus group and other common bacterial pathogens. Using spiked clinical specimens, Wß::luxAB detects B. anthracis within 5 h at clinically relevant concentrations, and provides antibiotic susceptibility information that mirrors the CLSI method, except that data are obtained at least 5-fold faster. Although anthrax is a treatable disease, a positive patient prognosis is dependent on timely diagnosis and appropriate therapy. Wß::luxAB rapidly detects B. anthracis and determines antibiotic efficacy, properties that will help patient outcome.

Transcriptomic responses of juvenile Pacific whiteleg shrimp, Litopenaeus vannamei, to hypoxia and hypercapnic hypoxia.

Estuarine crustaceans are often exposed to low dissolved O2 (hypoxia) accompanied by elevated CO2 (hypercapnia), which lowers water pH. Acclimatory responses to hypoxia have been widely characterized; responses to hypercapnia in combination with hypoxia (hypercapnic hypoxia) are less well known. Here we used oligonucleotide microarrays to characterize changes in global gene expression in the hepatopancreas of Pacific whiteleg shrimp, Litopenaeus vannamei, exposed to hypoxia or hypercapnic hypoxia for 4 or 24 h, compared with time-matched animals held in air-saturated water (normoxia). Unigenes whose expressions were significantly impacted by treatment and/or time were used to build artificial neural networks (ANNs) to identify genes with the greatest sensitivity in pairwise discriminations between treatments at each time point and between times for each treatment. ANN gene sets that discriminated hypoxia or hypercapnic hypoxia from normoxia shared functions of translation, mitochondrial energetics, and cellular defense. GO terms protein modification/phosphorylation/cellular protein metabolism and RNA processing/apoptosis/cell cycling occurred at highest frequency in discriminating hypercapnic hypoxia from hypoxia at 4 and 24 h, respectively. For 75.4% of the annotated ANN genes, exposure to hypercapnic hypoxia for 24 h reduced or reversed the transcriptional response to hypoxia alone. These results suggest that high CO2/low pH may interfere with transcriptionally based acclimation to hypoxia or elicit physiological or biochemical responses that relieve internal hypoxia. Whether these data reflect resilience or sensitivity of L. vannamei in the face of expanding hypoxic zones and rising levels of atmospheric CO2 may be important to understanding the survival of this and other estuarine species.

Phage-based platforms for the clinical detection of human bacterial pathogens.

Bacteriophages (phages) have been utilized for decades as a means for uniquely identifying their target bacteria. Due to their inherent natural specificity, ease of use, and straightforward production, phage possess a number of desirable attributes which makes them particularly suited as bacterial detectors. As a result, extensive research has been conducted into the development of phage, or phage-derived products to expedite the detection of human pathogens. However, very few phage-based diagnostics have transitioned from the research lab into a clinical diagnostic tool. Herein we review the phage-based platforms that are currently used for the detection of Mycobacterium tuberculosis, Yersinia pestis, Bacillus anthracis and Staphylococcus aureus in the clinical field. We briefly describe the disease, the current diagnostic options, and the role phage diagnostics play in identifying the cause of infection, and determining antibiotic susceptibility.