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Links of Interest: American Society for Microbiology (ASM)
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Project #1: DNA Fingerprinting of enterotoxigenic Bacillus spp. in dairy products In order to screen contaminated dairy foods for the presence of possible enterotoxigenic spore-formers (i.e., Bacillus spp.), our lab is applying repetitive element palindromic –PCR (rep-PCR) as a means of ‘fingerprinting’ those strains found to harbor at least one of the two enterotoxin gene operons commonly associated with Bacillus cereus. My graduate student Kiev Gracias (M.S. 2003; currently Ed.D. candidate, BSU) has demonstrated a consistent DNA banding pattern on agarose gel electrophoresis among enterotoxigenic strains (all sharing a common band at ~1,230 bp) but one that differs in comparison to nonpathogenic Bacillus spp. (Fig. 1). This work was completed to demonstrate the utility of rep-PCR as a screening tool for toxigenic Bacillus spp. in artificially contaminated foods. Extending Kiev’s work is Robin Cooper (current M.S. student), who is applying Kiev’s optimized rep-PCR conditions to a diverse array of naturally contaminated dairy products, including nonfat dry milk powder, lecithin, whey powder formulations, cocoa powder, and soy protein concentrate. Robin has found the characteristic ‘fingerprint’ of enterotoxigenic Bacillus spp. in a number of foods using rep-PCR and is confirming toxin production in these model food systems with the BCET-RPLA kit and by REA.
Fig. 1. Rep-PCR profile of selected non-enterotoxigenic strains of Bacillus in milk.
Project #2 Nucleic acid sequence-based amplification (NASBA) to monitor enterotoxin gene expression in Bacillus cereus from contaminated milk Bacillus cereus is traditionally considered the most problematic food- related member of the genus Bacillus. Bacillus cereus is an important food-associated pathogen because many strains possess the ability to produce enterotoxins. This organism is responsible for both the emetic and diarrheal syndromes. There are at least two distinct types of enterotoxins encoded by two separate operons. The enterotoxins produced are protein in nature and thus the enterotoxin genes of B. cereus are important to the study of food-related illness. Ingestion of high densities of enterotoxigenic B. cereus may result in the onset of diarrhea and vomiting. Symptoms usually appear 10-14 hours after consumption of contaminated foodstuffs. This process of combining NASBA with Molecular Beacons will provide a quick and easy way to monitor gene expression, the focus of this study. This work was completed by Hope M. Gore (currently at Louisiana State University School of Dentistry, New Orleans), Catherine M. Wakeman (Louisiana Tech University), and Rhiannon M. Hull (currently University of Texas Medical Branch, Galveston).
Fig. 2. NASBA process for transcript-specific RNA amplification in a high gDNA background. NASBA may be linked with a real-time chemistry such as molecular beacons for sensitive, quantitative detection of mRNA in gene expression studies. The advent of emerging nucleic acid-based technologies, including many variations on the common theme of PCR, have allowed for the development of highly specific, sensitive, and rapid detection of food-associated pathogens. Traditional PCR confirms the presence of specific genes in a target organism, but in most cases may not necessarily indicate cell viability. RNA-based methods however, detect and identify viable cells and are useful for ascertaining gene expression. RT-PCR as one such method is potentially very sensitive but also somewhat problematic. Successful RT-PCR requires all residual DNA be eliminated in order to provide accurate detection of specific RNA sequences, but elimination of residual DNA requires steps that also decrease subsequent RNA recovery from cells of interest. Moreover, the complexity of food matrices may inhibit overall RNA yield and/or enzyme activity during RT or amplification steps. Alternatively, a more rapid method for gene expression has been applied in studies of gene expression in bacteria for clinical microbiology. Nucleic acid sequence-based amplification (NASBA) is an isothermic cyclical series of reactions utilizing the RNA sample combined with an enzyme cocktail. (Fig. 2) NASBA begins with first-strand cDNA synthesis. This is done by the use of sequence specific primers and T7 RNA polymerase promoter sequence contained within one primer. RNase H activity digests only the RNA half in the RNA:DNA hetroduplex. Second-strand DNA synthesis then occurs by way of a second sequence-specific primer. Double stranded DNA comprises the sample and in vitro transcription can occur as a result of the T7 RNA polymerase from the T7 promotor. This makes many RNA copies of the original sequence of RNA. NASBA offers speed and sensitivity, but may be improved further if combined with another newly introduced molecular detection system – fluorescently labeled molecular beacon probes. Molecular beacons are short, ssDNA probe molecules that are complimentary to target DNA sequences within the gene (or transcript) under study (Fig. 3). A molecular beacon is a single-stranded oligonucleotide folded upon itself in a hairpin loop fashion. The loop is comprised of complementary bases to the target RNA sequence. The area where it is folded on itself is comprised of a majority guanine and cytosine bases. One end of the oligonucleotide contains a fluorophore and the other end a quencher. When the oligonucleotide is folded upon itself it does not fluoresce, but in the presence of the target RNA sequence the loop attraction to the target sequence is stronger than the C/G bonds holding it upon itself. This results in the quencher and fluorophore being separated and fluorescence occurring. This is directly indicative of the presence of the target RNA sequence.
Fig. 3. Molecular beacon structure and hybridization. www.molecular-beacons.org
Project #3
Multiplex PCR and melt-curve profile analysis on enterotoxigenic
Bacillus spp. This work was initiated by Huang Ying (M.S. , 2003, currently a Ph.D. candidate at University of Connecticut, Department of Molecular & Cell Biology). Nucleic acid based assays such as traditional polymerase chain reaction (PCR) that contain only one primer set is useful for one component of either the HBL or NHE operon. However, since all three components of either HBL or NHE are required for maximal activity, a multiplex PCR assay, which allows simultaneous detection of two amplicons on separate operons, increases assay versatility and reduces the chances of false negative results. The operons involved in enterotoxigenic Bacillus spp. are shown in Fig. 4. Phelps and McKillip (2002) found only seven isolates exhibited a possible signal for at least three of the five target genes (hblC, hblD, hblA, nheA, and nheB) by DNA-based PCR, their study suggest that in some cases only one or two of the three enterotoxin genes of each operon may be detectable simultaneously. Based on their study, we assumed that one or more hbl and/or nhe genes may be detectable in B. cereus in virtually any combination, and the presence of one or more genes in either operon indicates enterotoxigenic potential of the B. cereus being screened. The fact that very few of the strains being screened will show detectable amplicons from all 5 gene targets defines the rationale for and thus more accurately allows for shelf life predictions on suspect foods.
Fig. 4A. HBL operon of enterotoxigenic Bacillus cereus
Fig. 4B. NHE operon of enterotoxigenic Bacillus cereus The multiplex PCR detection assay for enterotoxigenic Bacillus cereus is being extended now to a real-time detection format using SYBRÒ Green I & melt curve analysis. This work is ongoing, and is being completed by Dr. McKillip. Check back for progress and quarterly updates on this website. Project #4: Sporocidal effects of FreshFxTM www.sterifx.com This work is part of an ongoing collaboration with SteriFx, Inc., Shreveport, LA to perform contract studies on the efficacy of a novel sterilant for treating spores in and on a variety of materials and surfaces. This work is being performed by Dr. McKillip. The ability of FreshFxTM to demonstrate predicted sporocidal activity on living tissue and in complex matrices such as food systems would significantly expand its marketability and utility to other, unforeseen areas. A recent report in Food Technology addressed the state of the science and outlined critical research needs regarding spores and other dormant microbes (R. Newsome. 2003. Dormant Microbes: Research Needs. Food Tech. pp. 38-42, June). In addition to the paramount need to control spore viability and germination, the priorities were itemized as: a fundamental knowledge of spore physiology, including resistance to germicidal agents, sampling issues, and detection and enumeration methodologies. Application of FreshFxTM as a sporocidal agent on skin and its demonstration in dry dairy products and functional ingredients addresses a need from the standpoints of both clinical and industrial microbiology. Whether it be applying this novel agent in homeland defense as an antagonist in bioterrorism threats, or as a step in the processing of spray-dried infant milk formulations containing high levels (~105 spores/g) of spores that could elicit severe gastroenteritis if consumed, FreshFxTM has importance to the consumer/general public. This study will set out to quantitate, in a laboratory setting, the sporocidal activity of this compound when applied to living tissue and in a series of powdered dairy products and functional ingredients. Objectives The objectives of this contract study are twofold: 1.) To determine the level of Fx and exposure time of this agent needed to inactivate spores of Bacillus subtilis applied to living tissue using a mouse model. 2.) To determine the effectiveness Fx at ‘4X’ concentration for 15 min. exposure time at inactivating spores of potentially enterotoxigenic Bacillus spp. in nonfat dry milk powder, lecithin powder, and 2 formulations of cocoa powder.
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