John L. McKillip

Enterotoxin Production in Natural Isolates of Bacillaceae outside the Bacillus cereus Group

ABSTRACT Thirty-nine Bacillus strains obtained from a variety of environmental and food sources were screened by PCR for the presence of five gene targets (hblC, hblD, hblA, nheA, and nheB) in two enterotoxin operons (HBL and NHE) traditionally harbored by Bacillus cereus. Seven isolates exhibited a positive signal for at least three of the five possible targets, including Bacillus amyloliquefaciens, B. cereus, Bacillus circulans, Bacillus lentimorbis, Bacillus pasteurii, and Bacillus thuringiensis subsp. kurstaki. PCR amplicons were confirmed by restriction enzyme digest patterns compared to a positive control strain. Enterotoxin gene expression of each strain grown in a model food system (skim milk) was monitored by gene-specific reverse transcription-PCR and confirmed with the Oxoid RPLA and Tecra BDE commercial kits. Lecithinase production was noted on egg yolk-polymyxin B agar for all strains except B. lentimorbis, whereas discontinuous beta hemolysis was exhibited by all seven isolates grown on 5% sheep blood agar plates. The results of this study confirm the presence of enterotoxin genes in natural isolates of Bacillus spp. outside the B. cereus group and the ability of these strains to produce toxins in a model food system under aerated conditions at 32°C.

Molecular beacon polymerase chain reaction detection of Escherichia coli O157 :H7 in milk

A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when the hairpin-stem conformation was linearized upon hybridization to the target sequence. The molecular beacon was incorporated into PCR reactions containing DNA extracted from artificially contaminated skim milk. The degree of fluorescence was monitored in PCR reactions containing 10(3), 10(5), and 10(7) CFU of E. coli O157:H7 per ml and was found to correlate with the amount of template in each reaction. Fluorescence significantly increased above background levels by cycle 8, 14, or 14 in reactions containing DNA from the 10(7)-, 10(5)-, or 10(3)-CFU/ml template, respectively (P < 0.05). Molecular beacon PCR demonstrated positive results more rapidly than traditional agarose gel electrophoresis analysis of PCR products. Use of molecular beacons allows real-time monitoring of PCR reactions, and the closed-tube format allows simultaneous detection and confirmation of target amplicons without the need for agarose gel electrophoresis and/or Southern blotting. This is the first report of a stem-and-loop molecular beacon being applied for direct detection of a pathogen in food.

Microbial degradation of gasoline in soil: Effect of season of sampling

In cases where fire debris contains soil, microorganisms can rapidly and irreversibly alter the chemical composition of any ignitable liquid residue that may be present. In this study, differences in microbial degradation due to the season in which the sample is collected was examined. Soil samples were collected from the same site during Fall, Winter, Spring and Summer and the degradation of gasoline was monitored over 30 days. Predominant viable bacterial populations enumerated using real-time PCR and reverse transcriptase polymerase chain reaction (RT-PCR) enumeration revealed the predominant viable bacterial genera to be Alcaligenes, Bacillus, and Flavobacterium. Overall, the compounds most vulnerable to microbial degradation are the n-alkanes, followed by the mono-substituted alkylbenzenes (e.g., toluene, ethylbenzene, propylbenzene and isopropylbenzene). Benzaldehyde (a degradation product of toluene) was also identified as a marker for the extent of biodegradation. Ultimately, it was determined that soil collected during an unusually hot and dry summer exhibited the least degradation with little to no change in gasoline for up to 4 days, readily detectable n-alkanes for up to 7 days and relatively high levels of resilient compounds such as o-xylene, p-xylene and 1,3,5-trimethylbenzene. These results demonstrate, however, that prompt preservation and/or analysis of soil evidence is required in order to properly classify an ignitable liquid residue.

Microbial Degradation of Gasoline in Soil: Comparison by Soil Type

During the investigation of a suspicious fire, debris is often collected from the scene and analyzed for residues of ignitable liquids (e.g., gasoline). In cases where the debris is contaminated with soil, it is known that heterotrophic soil microorganisms can alter the chemical composition of the ignitable liquid residue over time. The effects of soil type and season upon this phenomenon are not known, however. Hence, soil collected from locations under three different uses (residential, agricultural, brownfield) were spiked with gasoline and microbial degradation was monitored for 30 days. The soils were also chemically and biologically characterized. Gas chromatographic profiles showed that residential soil was most active and brownfield soil least active for the microbial degradation of gasoline. The brownfield soil possessed relatively high (497 mg/kg) concentrations of Pb, which may have affected bacterial activity. Predominant viable bacterial populations enumerated using real-time reverse transcriptase polymerase chain reaction (RT-PCR) included members of the Alcaligenes, Acinetobacter, Arthrobacter, Bacillus, Flavobacterium, and Pseudomonas genera. Principal Components Analysis (PCA) was found effective in elucidating trends of microbial degradation among the different soil types and seasons. The results of this study demonstrate the necessity of prompt analysis of forensic evidence for proper identification of possible ignitable liquids.

Enumeration of Sublethally Injured Escherichia coli O157:H7 ATCC 43895 and Escherichia coli Strain B-41560 Using Selective Agar Overlays versus Commercial Methods

Quality control procedures during food processing may involve direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured E. coli B-41560 and O157:H7 strains. Bacteria were propagated in tryptic soy broth (TSB), ground beef slurry, and infant milk formula to a density of 10(6) to 10(8) CFU/ml and then were stressed for 6 min either in lactic acid (pH 4.5) or heat shocked for 3 min at 60°C. Samples were pour plated in basal layers of either tryptic soy agar (TSA), sorbitol MacConkey agar (SMAC), or violet red bile agar (VRB) and were resuscitated for 4 h prior to addition of agar overlays. Other stressed bacteria were plated directly onto Petrifilm and Easygel. Results indicate that selective and nonselective agar overlays recovered significantly higher numbers (greater than 1 log) of acid- and heat-injured E. coli O157:H7 from TSB, ground beef, and infant milk formula compared with direct plating onto selective media, Petrifilm, or Easygel, while no significant differences among these media combinations were observed for stressed E. coli B-41560. Nonstressed bacteria from TSB and ground beef were also recovered at densities significantly higher in nonselective TSA-TSA and in VRB-VRB and SMAC-SMAC compared with Petrifilm and Easygel. These data underscore the need to implement food safety measures that address sublethally injured pathogens such as E. coli O157:H7 in order to avoid underestimation of true densities for target pathogens.

Triplex PCR-based detection of enterotoxigenic Bacillus cereus ATCC 14579 in nonfat dry milk

Although many strains of Bacillaceae are considered nonpathogenic, Bacillus cereus is recognized worldwide as a bacterial pathogen in a variety of foods. The ability of B. cereus to cause gastroenteritis following ingestion of contaminated food is due to the production of enterotoxins. The ubiquity of this genus makes it a persistent problem for quality assurance in food processing environments. The primary objective of this study was to develop and apply a multiplex real‐time PCR‐based assay for rapid and sensitive detection of enterotoxigenic B. cereus. Template DNA was separately extracted from tryptic soy broth (TSB)‐grown and 2.5% Nonfat Dry Milk (NFDM)‐grown B. cereus using a commercial system. Three enterotoxin gene fragments (hblC, nheA, and hblA) were simultaneously amplified in real‐time followed by melting curve analysis to confirm amplicon identity. Resolution of melting curves (characteristic Tm) was achieved for each amplicon (hblC = 74.5 °C; nheA = 78 °C; and hblA = 85.5 °C in TSB and 84 °C in NFDM) with an assay sensitivities of 101 CFU/ml for both TSB and NFDM‐grown B. cereus compared to 104 CFU/ml in either matrix using gel electrophoresis. The results demonstrate the potential sensitivity of real‐time bacterial detection methods in a heterogenous food matrix using real‐time PCR. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Forensic blood evidence analysis using RNA targets and novel molecular tools

In the realm of forensic investigation, messenger RNA (mRNA) is a good source for specimen analysis, owing to tissue-specific expression patterns for many transcripts. Thus, differentiation of bodily fluids collected at a crime scene is possible if the proper target mRNA is identifiable and detectable using molecular methods. With the analysis of menstrual blood evidence, for example, the matrix metalloproteinase (MMP) family of genes encodes proteins involved in extracellular matrix breakdown during tissue remodeling, and in particular MMP11 and MMP7 mRNA have proven useful for forensic blood evidence analysis. Nucleic acid sequence based amplification (NASBA) is a highly sensitive RNA transcription-based amplification method used to detect gene expression. As an emerging real-time technology, NASBA holds promise as a tool to rapidly and sensitively amplify mRNA markers associated with the MMP genes to verify the presence of menstrual blood in a forensic sample, and has a number of advantages compared to reverse transcriptase polymerase chain reaction. The purpose of this review is to overview the current state of mRNA analysis in forensic evidence casework and to highlight areas of research, progress, and development in the near-term future that will lead to reduction of case evidence backlog in crime laboratories in developed countries.

Bacillus spp. as Pathogens in the Dairy Industry

Abstract The bacterial species Bacillus cereus accounts for 1.4%–12% of foodborne illness outbreaks worldwide, a statistic that is certainly an underestimate. This bacterial genus is capable of contaminating a wide range of food products, including rice, chicken, vegetables, spices, and dairy products. B. cereus endospores are partially resistant to pasteurization, dehydration, gamma radiation, and other physical stresses used in food processing, and their adhesive characteristics promote biofilm-forming capability on a variety of substrates in dairy operations. B. cereus and other closely related species produce several types of exotoxins, including at least four hemolysins, three phospholipases, a heat/acid-stable emetic toxin called cereulide, and three well-studied heat-labile enterotoxins that all cause gastroenteritis following ingestion. While a great deal of information on virulence gene presence and expression is known in B. cereus, very little has been done to explore the virulence potential of thermoduric spore formers that may be found in ultrahigh temperature pasteurized milk, and their ability to produce biofilms. Biofilm production is understood to be under similar regulation as toxins and other extracellular virulence determinants. This chapter describes the current status of knowledge with Bacillus spp. relevant to the dairy industry, virulence potential, and biofilm production from the perspective of food safety.