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

Abstract

c 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 viable B. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (W␤::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxAB in an earlier version of the reporter phage (W␤::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using W␤::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.

Engineering of Bacteriophages Y2:: dpoL1-C and Y2:: luxAB for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora

“…Detection limits of other luciferase reporter phages ranged from a few cells per milliliter to approximately 10 3 to 10 4 CFU/ml (23,24,26,27,32,40,41). Of these, systems including temperate phages were shown to have lowest detection limits based upon long-term continuous production of the reporter protein (20,27,40). However, Y2::luxAB is a virulent phage, and temperate E. amylovora viruses have not yet been found (43).…”

“…Reporter phages have been designed for specific detection of various Gram-negative and Gram-positive species, such as E. coli (17)(18)(19)(20), Salmonella (21,22), Yersinia pestis (23), Shigella spp. (24), Listeria monocytogenes (25,26), Bacillus anthracis (27,28), and Mycobacterium spp. (29,30).…”

Bacteriophages: Practical Applications for Nature's Biocontrol

“…For example, a rough approximation predicts that it would take 1000 years for one phage to randomly find one bacterium in 1 ml of liquid . Therefore, the introduction of luxAB genes into B. anthracis, using luxAB-based bioluminescent reporter phages would be a novel approach to specifically detect low numbers of anthracis spores (Sharp et al 2016). In order to increase the opportunities for the phage-spores encounter mechanisms, the vertical and horizontal biokinetic interaction can be applied through a programming technology (Chap.…”

“…The subtle changes that temperate phages impart to their hosts in the environment reduce the effectiveness of phage therapy . A novel class of bacterial virulence factors called effector proteins (EPs) are phage encoded among distinct pathovars such as enterohemorrhagic E. coli (EHEC) serotype O157:H7, a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome worldwide, investigated by many researchers using several novel diagnostic approaches such as phage typing, labeling of phage DNA, luxAB-based bioluminescent reporter phages, adenosine triphosphate (ATP), green fluorescent protein (GFP) reporter phages, lacZ-based colorimetric reporter phages, OmniLog™ system, reporter phages ice nucleation, phage amplification, quantum dots, thermal change, changes in conductance, phage components, phage-based limulus amoebocyte lysate assay Goodridge and Griffiths 2002;Jassim et al 2010a, b;Smartt and Ripp 2011;Henry et al 2012;Smartt et al 2012;Schofielda et al 2012;Klumpp and Loessner 2014;Mortari et al 2015;Peltomaa et al 2016;Sharp et al 2016). Phage-encoded virulence factors could then be up-regulated, which could contribute to the pathogenesis of the bacteria.…”

“…Phages can contribute to bacteria virulence through transduction and regulation of phage-encoded virulence factors, alteration of bacterial virulence factors and function in the control of bacterial virulence through in situ prophage induction (Wanger and . The engineered B. anthracis reporter phage (Wb: luxAB-2) which transduces bioluminescence to infected cells was able to detect 1 cfu of B. anthracis spores in 8 h from pure cultures and as low as 10 cfu g −1 in sterile soil (Sharp et al 2016). The lysogenic phage or 'prophage' will drive the adaptive evolution of bacteria to achieve more powerful virulence factors inherited from previously infected bacteria via transduction, i.e., the transfer of genetic material to a bacterial cell via phage infection .…”