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.

Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on fresh-cut celery.

Illnesses from Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella have been associated with the consumption of numerous produce items. Little is known about the effect of consumer handling practices on the fate of these pathogens on celery. The objective of this study was to determine pathogen behavior at different temperatures under different storage conditions. Commercial fresh-cut celery was inoculated at ca. 3logCFU/g onto either freshly cut or outer uncut surfaces and stored in either sealed polyethylene bags or closed containers. Samples were enumerated following storage for 0, 1, 3, 5, and 7 days when held at 4 °C or 12 °C, and after 0, 8, and 17 h, and 1, and 2 days when held at 22 °C. At 4 °C, all populations declined by 0.5-1.0logCFU/g over 7 days. At 12 °C, E. coli O157:H7 and Salmonella populations did not change, while L. monocytogenes populations increased by ca. 0.5logCFU/g over 7 days. At 22 °C, E. coli O157:H7, Salmonella, and L. monocytogenes populations increased by ca. 1, 2, or 0.3logCFU/g, respectively, with the majority of growth occurring during the first 17 h. On occasion, populations on cut surfaces were significantly higher than those on uncut surfaces. Results indicate that populations are reduced under refrigeration, but survive and may grow at elevated temperatures.

Acceleration performance of individual European sea bass Dicentrarchus labrax measured with a sprint performance chamber: comparison with high-speed cinematography and correlates with ecological performance.

Locomotor performance can influence the ecological and evolutionary success of a species. For fish, favorable outcomes of predator-prey encounters are often presumably due to robust acceleration ability. Although escape-response or "fast-start" studies utilizing high-speed cinematography are prevalent, little is known about the contribution of relative acceleration performance to ecological or evolutionary success in a species. This dearth of knowledge may be due to the time-consuming nature of analyzing film, which imposes a practical limit on sample sizes. Herein, we present a high-throughput potential alternative for measuring fish acceleration performance using a sprint performance chamber (SPC). The acceleration performance of a large number of juvenile European sea bass (Dicentrarchus labrax) from two populations was analyzed. Animals from both hatchery and natural ontogenies were assessed, and animals of known acceleration ability had their ecological performance measured in a mesocosm environment. Individuals from one population also had their acceleration performance assessed by both high-speed cinematography and an SPC. Acceleration performance measured in an SPC was lower than that measured by classical high-speed video techniques. However, short-term repeatability and interindividual variation of acceleration performance were similar between the two techniques, and the SPC recorded higher sprint swimming velocities. Wild fish were quicker to accelerate in an SPC and had significantly greater accelerations than all groups of hatchery-raised fish. Acceleration performance had no significant effect on ecological performance (as assessed through animal growth and survival in the mesocosms). However, it is worth noting that wild animals did survive predation in the mesocosm better than farmed ones. Moreover, the hatchery-originated fish that survived the mesocosm experiment, when no predators were present, displayed significantly increased acceleration performance during their 6 mo in the mesocosm; this performance was found to be inversely proportional to growth rate.