Salmonella enterica and Escherichia coli in Wheat Flour: Detection and Serotyping by a Quasimetagenomic Approach Assisted by Magnetic Capture, Multiple-Displacement Amplification, and Real-Time Sequencing.

Food safety is a new area for novel applications of metagenomics analysis, which not only can detect and subtype foodborne pathogens in a single workflow but may also produce additional information with in-depth analysis capabilities. In this study, we applied a quasimetagenomic approach by combining short-term enrichment, immunomagnetic separation (IMS), multiple-displacement amplification (MDA), and nanopore sequencing real-time analysis for simultaneous detection of Salmonella and Escherichia coli in wheat flour. Tryptic soy broth was selected for the 12-h enrichment of samples at 42°C. Enrichments were subjected to IMS using beads capable of capturing both Salmonella and E. coli MDA was performed on harvested beads, and amplified DNA fragments were subjected to DNA library preparation for sequencing. Sequencing was performed on a portable device with real-time basecalling adaptability, and resulting sequences were subjected to two parallel pipelines for further analysis. After 1 h of sequencing, the quasimetagenomic approach could detect all targets inoculated at approximately 1 CFU/g flour to the species level. Discriminatory power was determined by simultaneous detection of dual inoculums of Salmonella and E. coli, absence of detection in control samples, and consistency in microbial flora composition of the same flour samples over several rounds of experiments. The total turnaround time for detection was approximately 20 h. Longer sequencing for up to 15 h enabled serotyping for many of the samples with more than 99% genome coverage, which could be subjected to other appropriate genetic analysis pipelines in less than a total of 36 h.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) and Salmonella are of serious concern in low-moisture foods, including wheat flour and its related products, causing illnesses, outbreaks, and recalls. The development of advanced detection methods based on molecular principles of analysis is essential to incorporate into interventions intended to reduce the risk from these pathogens. In this work, a quasimetagenomic method based on real-time sequencing analysis and assisted by magnetic capture and DNA amplification was developed. This protocol is capable of detecting multiple Salmonella and/or E. coli organisms in the sample within less than a day, and it can also generate sufficient whole-genome sequences of the target organisms suitable for subsequent bioinformatics analysis. Multiplex detection and identification were accomplished in less than 20 h and additional whole-genome analyses of different nature were attained within 36 h, in contrast to the several days required in previous sequencing pipelines.

Evaluation of single and combined antimicrobial treatments to inhibit Salmonella in queso fresco.

Hispanic style soft non-fermented cheeses, such as queso fresco (QF) have been linked to outbreaks and recalls. Salmonella is one of the main causes of these incidents. Due to lack of ripening or post-processing antimicrobial treatments, incorporating GRAS antimicrobials to production process may be a suitable approach to minimize microbial risk in QF. The aim of this study was to evaluate the efficiency of nisin (N), caprylic acid (CA) and trans-cinnamaldehyde (CN) as single or combined treatments to reduce Salmonella populations in QF during storage. Batches of QF were inoculated after curding with approx. 4 Log CFU/g of 5-strain cocktails of Salmonella and stored at 8 °C for 20 days. The final Salmonella counts in control samples ranged from 6.96 to 7.14 Log CFU/g. Application of CN at 0.6 g/kg inhibited Salmonella growth during storage, resulting in at least 3 Log CFU/g difference with the untreated controls (p < 0.05). Addition of N (0.5 g/kg) and CA (0.4 g/kg) with CN (0.3 and 0.6 g/kg) further enhanced the antimicrobial activity resulting in complete suppression of growth and even caused a 1 Log CFU/g reduction by the end of the experimental period compared to initial counts. Samples treated with the combined treatment (N, CA, CN) were evaluated in a consumer panel (n = 112). Participants preferred the control and commercial QF to the treated samples. However, treated samples with 0.3 g/kg CN were still within the acceptable range of neutral to like slightly. Results obtained, revealed that combined treatment of N, CA and CN can provide a solution to reduce the count of Salmonella in QF, whether in process or during storage.

First Report of the Root-Knot Nematode, Meloidogyne floridensis, on Tomato in Georgia, USA.

Meloidogyne floridensis, also known as the peach root-knot nematode (RKN), is a new emerging species found to break crop host-resistance to M. incognita (Stanley et al. 2009). It was first described from Florida (Handoo et al. 2004) parasitizing M. incognita-resistant rootstock cultivars of peach (Prunus persica), and tomato (Solanum lycopersicum) (Church 2005). The nematode has recently been reported in California's almond orchards (Westphal et al. 2019) and peach rootstock (cv. Guardian) in South Carolina (Reighard et al. 2019). In a 2018 survey of vegetable fields sampled randomly in South Georgia, RKN was found with a high density (5,264 second-stage juveniles (J2)/100 cm3 of soil) from a tomato field in Ware County, GA. The soil sample consist of 30 soil cores sampled at 20-cm depth across the field in a zig-zag motion. To perform Koch's postulate, 2,000 eggs from a single egg-mass culture were inoculated into deepots filled with mixture of sand and sterilized field soil (1:1 v/v) and grown with tomato cv. Rutgers for 60 days in the greenhouse. A reproduction factor of 21.1 ± 6.1 was obtained confirming the nematode parasitism on tomato (Fig. 1S). For molecular identification, DNA was extracted by smashing three individual females isolated from the galled roots in 50 µl sterile distilled water, followed by a freeze-thaw (95°C, 1 min). Results of PCR analyzes by species-specific primers (Fjav/Rjav, Finc/Rinc and Far/Rar) did not detect the nematode species (Zijlstra et al. 2000). PCR products were obtained and sequenced from two primer sets consisting of the forward NAD5F2 (5'-TATTTTTTGTTTGAGATATATTAG-3') and the reverse NAD5R1 (5'-CGTGAATCTTGATTTTCCATTTTT-3') for amplification of a fragment of the NADH dehydrogenase subunit 5 (NADH5) gene (Janssen et al. 2016), and the forward TRANH (5'-TGAATTTTTTATTGTGATTAA-3') and the reverse MRH106 (5'-AATTTCTAAAGACTTTTCTTAGT-3') for amplification covering a portion of the cytochrome c oxidase subunit II (COII) and large subunit 16SrDNA (16S) gene (Stanton et al. 1997). DNA sequence of NADH5 gene fragment (accession no. MT795954) was 100% identical (532/532 bp) with a M. floridensis isolate from California and South Carolina (accession no. MH729181 and MN072363), while fragment of the COII and 16S genes (accession no. MT787563) was 99.76% identical (421/422 bp) with an isolate from Florida (accession no. DQ228697). The nematode females were also used for morphometric and perennial pattern analysis. Several micrographs with the inverted microscope (ZEISS Axio Vert.A1, Germany) and camera (ZEISS Axiocam 305 color, Germany) were taken from ten J2s for mean, standard deviation and range of body length: 362.7 ± 11.2 (340.4-379) µm, maximum body width: 15 ± 1.3 (12.4-16.4) µm, stylet length: 12.3 ± 1.3 (9.5-14) µm, hyaline tail terminus: 8.9 ± 1.1 (7.5-10.9) µm and tail length: 35.7 ± 4.4 (28.5-39.5) µm. Morphological measurements and configuration of perineal patterns (Fig. 2S) were comparable to previous reports of M. floridensis isolates from Florida (Handoo et al. 2004; Stanley et al. 2009). To the best of our knowledge, this is the first report of M. floridensis in Georgia as the fourth state in the USA after South Carolina, California and Florida. This nematode has been reported to parasitize several vegetable crops, including cucumber, eggplant, tomato, snap bean and squash. Furthermore, RKN resistant cultivars of tomato (harboring Mi-1 gene), pepper (harboring N gene), corn cv. Mp-710 and tobacco cv. NC 95 have been found susceptible to M. floridensis (Stanley et al. 2009), making it a serious threat.

Antibacterial applications of metal-organic frameworks and their composites.

Metal-organic frameworks (MOFs) are porous coordination materials composed of multidentate organic ligands and metal ions or metal clusters. MOFs have the great potential to be utilized in antibacterial materials for biological, environmental, and food antimicrobial fields. In recent years, MOFs have been applied to various antibacterial fields due to their sustained release capability, porosity, and structural flexibility in combination with many chemicals and/or materials (such as nanoparticles, antibiotics, phytochemicals, and polymers). This review offers a detailed summary of the antibacterial applications of MOFs and their composites, focusing on the combination types of MOFs composites and the antibacterial effect in different applications. These applications are illustrated by the examples discussed in this review.

First report of the stubby-root nematode Nanidorus minor infecting Paspalum vaginatum, seashore paspalum grass in Georgia, USA.

We found that Nanidorus spp. was pathogenic to seashore paspalum (Paspalum vaginatum) turfgrass as its population increased from 100 to 2,080 nematodes per pot 180 days after inoculation under greenhouse conditions. Morphological measurements of adult females were similar to those described for N. minor. Molecular analysis also confirmed the morphological identification by targeting three different regions of the genomic DNA. Three primer pairs targeting 18S rDNA (360F/932R), 28S rDNA (D2A/D3B) and ITS1 rDNA (BL18/5818) were used in singleplex PCR. Forward and reverse sequences of each individual primer set were then subjected to multiple alignment and the complimentary sequences were assembled into a consensus sequence. Upon nucleotide blast on the NCBI website, they were all confirmed to be N. minor. A one-step multiplex PCR method using specific primers and a fragment size of 190 bp also confirmed the identity of N. minor. To the best of our knowledge, this is the first report of N. minor infecting seashore paspalum turfgrass in Georgia.

Long-Term Survival and Thermal Death Kinetics of Enterohemorrhagic Escherichia coli Serogroups O26, O103, O111, and O157 in Wheat Flour.

Wheat flour has been associated with outbreaks of enterohemorrhagic Escherichia coli (EHEC), but little is known on EHEC's survival during storage and thermal processing. The objective of this study was to determine long-term viability and thermal inactivation kinetics of EHEC serogroups O26, O103, O111, and O157. Wheat flour samples were inoculated with a cocktail of five strains of a single serogroup and stored at 23 and 35°C. Inoculated samples were heated at 55, 60, 65, and 70°C. Viability was determined by plate counting. Decimal reduction time (D) and first decimal reduction time (δ) values were calculated with log-linear and Weibull models, respectively. At 23°C, EHEC counts declined gradually for 84 days and samples tested positive from 84 to 280 days. The thermal resistance (D and δ) values ranged from 7.5 to 8.2 and 3.1 to 5.3 days, respectively, but there were no significant differences among serogroups (P ≤ 0.05). At 35°C, no EHEC was quantifiable by day 7 and no positive samples were detected after 49 days. Heating at 55 and 65°C resulted in δ-value ranges of 15.6 to 39.7 min and 3.0 to 3.9 min, respectively, with no significant difference among serogroups either. Z values were 12.6, 6.7, 10.2, and 13.4°C for O26, O103, O111, and O157, respectively. Thermal death kinetics of EHEC in flour were better described using the Weibull model. Survival and inactivation rates of four serogroups were remarkably similar. These findings indicated that all EHEC serovars tested remained viable for at least 9 months at room temperature and survived for up to 60 min at 70°C in wheat flour.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) and Salmonella have recently caused several gastroenteritis outbreaks and recalls of wheat flour. Because EHEC can cause illness with very low doses and there is very scarce information regarding their ability to survive storage and heating in flour, the present study was undertaken to assess the long-term survival of EHEC serogroups O26, O103, O111, and O157 in flour. These findings are relevant, as we report that EHEC can survive for more than 9 months in wheat flour during storage. In addition, results obtained suggest that thermal inactivation at 65°C for 30 min or 2 months of storage at 35°C may be feasible strategies to mitigate the risk of most EHEC serovars in wheat flour.

Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB.

The aim of the study was to identify and evaluate specific biomarkers to differentiate within Bacillus cereus group species from contaminated food samples with the use of real-time PCR. A total of 120 strains, comprising of 28 reference, 2 type, 78 wild strains of B. cereus and B. thuringiensis along with 12 strains representing 2 bacterial groups - B. mycoides, B. pseudomycoides, B. weihenstephanensis (B. cereus group); B. amyloliquefaciens, B. subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Staphylococcus aureus, Streptococcus pyogenes (non-Bacillus sp.) were identified by applying valid biomarkers (groEL and gyrB). In addition, the presence of B. cereus group was determined in three different artificially contaminated vegetable samples (lettuce, spinach, and kimbap), using prominent biomarkers targeting on chaperonin protein (GroEL) and topoisomerase enzyme protein (gyrB). Direct analysis of samples revealed the specificity towards identification and characterization of the B. cereus group among wild, reference and type strains and the type strain inoculated in vegetables. Our results demonstrated two existing biomarkers groEL and gyrB with a high specificity of 98% and 96% respectively to analyze the total B. cereus group. Further, we also reported the detection limit of groEL and gyrB in food samples was 3.5 and 3.7 log CFU/g respectively. Thus, the developed real-time PCR approach can be a reliable and effective tool for the identification of B. cereus group strains present in environment and food samples. This does not require band isolation, re-amplification, sequencing or sequence identification, thus reducing the time and cost of analysis.

Unique biomarkers as a potential predictive tool for differentiation of Bacillus cereus group based on real-time PCR.

The aim of the study was to develop unique biomarkers for qPCR detection of Bacillus cereus group. Clinical and soil isolates were identified by specifically designed biomarkers - Lipoprotein (OPL-114-lipo), Methyltransferase (MT-17) and S-layer homology domain protein (151-1BC). In order to design biomarkers, we used 120 bacterial strains grouped into B. cereus and non-Bacillus group. The B. cereus group was confirmed by 108 strains of B. cereus and B. thuringiensis (30 reference and 78 wild), along with 3 strains of B. mycoides, B. pseudomycoides, and B. weihenstephanensis; while the non-Bacillus group was composed of 9 Gram-positive and Gram-negative strains. Direct analysis of samples revealed specificity towards identification and characterization of B. cereus group. The newly developed markers OPL-114-lipo and MT-17 showed specificity of 95% and 81%, respectively in identification of B. cereus. They are efficient tools to identify contaminated sources and the degree of bacterial contamination. Environmental and food samples do not require band isolation, re-amplification, sequencing or sequence identification. Thus, reducing the time and cost of analysis. Hence, it will be an alternative approach to traditional culture methods. Commercial food processing industries will be able to employ these biomarkers specific for B. cereus group as a detection tool to reduce economic loss due to B. cereus contamination.

Effect of water hardness on the production and microbicidal efficacy of slightly acidic electrolyzed water.

Slightly acidic electrolyzed water (SAEW) has been proved as an effective sanitizer against microorganisms attached to foods. However, its physical properties and inactivation efficacy are affected by several factors such as water hardness. Therefore, in this study the effect of water hardness on SAEW properties were studied. Pure cultures of foodborne bacteria were used in vitro and in vivo to evaluate the inactivation efficacy of the SAEWs produced. Results obtained showed water hardness to be an important factor in the production of SAEW. Low water hardness may result in the necessity of further optimization of production process. In this study the addition of 5% HCl and 2 M NaCl at 1.5 mL/min flow rate was found to be the best electrolyte concentration for the optimization of SAEW production from low hardness water (34 ± 2 mg/L). Furthermore, the results showed that pre-heating was a better approach compared to post-production heating of SAEW, resulting in higher ACC values and therefor better sanitization efficacy.

Hurdle enhancement of slightly acidic electrolyzed water antimicrobial efficacy on Chinese cabbage, lettuce, sesame leaf and spinach using ultrasonication and water wash.

Slightly acidic electrolyzed water (SAEW) is well known as a good sanitizer against foodborne pathogens on fresh vegetables. However, microbial reductions from SAEW treatment are not enough to ensure produce safety. Therefore, it is necessary to improve its antimicrobial efficiency by combining it with other appropriate approaches. This study examined the microbicidal activity of SAEW (pH 5.2-5.5, oxidation reduction potential 500-600 mV, available chlorine concentration 21-22 mg/l) on Chinese cabbage, lettuce, sesame leaf and spinach, four common fresh vegetables in Korea under same laboratory conditions. Subsequently, effects of ultrasonication and water wash to enhance the sanitizing efficacy of SAEW were studied, separately. Finally, an optimized simple and easy approach consisting of simultaneous SAEW treatment with ultrasonication (3 min) followed by water wash (150 rpm, 1 min) was developed (SAEW + US-WW). This newly developed hurdle treatment significantly enhanced the microbial reductions compared to SAEW treatment alone, SAEW treatment with ultrasonication (SAEW + US) and SAEW treatment followed by water wash (SAEW-WW) at room temperature (23 ± 2 °C). Microbial reductions of yeasts and molds, total bacteria count and inoculated Escherichia coli O157:H7 and Listeria monocytogenes were in the range of 1.76-2.8 log cfu/g on different samples using the new hurdle approach.

Study on the quality and myofibrillar protein structure of chicken breasts during thawing of ultrasound-assisted slightly acidic electrolyzed water (SAEW).

The effects of air thawing (AT), water thawing (WT), slightly acidic electrolyzed water (ET), ultrasound-assisted water thawing (WUT) and ultrasound-assisted slightly acidic electrolyzed water (EUT) on the quality and myofibrillar protein (MP) structure of chicken breasts were investigated. The results showed that WUT and EUT could significantly improve the thawing rate compared with AT, WT, and ET groups. The EUT group not only had lower thawing loss, but also their immobilized and free water contents were similar to fresh sample according to the low-field nuclear magnetic resonance (LF NMR) results. The EUT treatment had no adverse effect on the primary structure of the protein. The secondary and tertiary structures of MP were more stable in the EUT group according to Raman and fluorescence spectra. The muscle fibers microstructure from EUT group was neater and more compact compared with other thawing methods. Therefore, EUT treatment could be considered as a novel potential thawing method in the food industry.

Recent Advances in the Development of Environmentally Benign Treatments to Control Root-Knot Nematodes.

Root-knot nematodes (RKNs), Meloidogyne spp., are sedentary endoparasites that negatively affect almost every crop in the world. Current management practices are not enough to completely control RKN. Application of certain chemicals is also being further limited in recent years. It is therefore crucial to develop additional control strategies through the application of environmentally benign methods. There has been much research performed around the world on the topic, leading to useful outcomes and interesting findings capable of improving farmers' income. It is important to have dependable resources gathering the data produced to facilitate future research. This review discusses recent findings on the application of environmentally benign treatments to control RKN between 2015 and April 2020. A variety of biological control strategies, natural compounds, soil amendments and other emerging strategies have been included, among which, many showed promising results in RKN control in vitro and/or in vivo. Development of these methods continues to be an area of active research, and new information on their efficacy will continuously become available. We have discussed some of the control mechanisms involved and suggestions were given on maximizing the outcome of the future efforts.

Room Temperature Growth of Salmonella enterica Serovar Saintpaul in Fresh Mexican Salsa.

Salsa-associated outbreaks, including the large multistate outbreak in the United States in 2008 caused by jalapeño and serrano peppers contaminated with Salmonella Saintpaul, have raised concerns about salsa as a potential vehicle for transmission. Despite these events, there has been relatively limited research on the potential growth of pathogenic bacteria in salsa. The aim of this study was to characterize the survival and growth of Salmonella, including the outbreak strain of Salmonella Saintpaul (E2003001236), in freshly made salsa and its main ingredients. Chopped tomatoes, jalapeño peppers, cilantro, and onions were tested individually or mixed according to different salsa recipes. Samples were inoculated with five Salmonella serotypes at 3 log CFU/g: Saintpaul (various strains), Typhimurium, Montevideo, Newport, or Enteritidis. Samples were then stored at room temperature (23°C) for up to 12 h or 3 days. The Salmonella Saintpaul levels reached approximately 9 log CFU/g after 2 days in tomato, jalapeño pepper, and cilantro. Growth was slower in onions, reaching 6 log CFU/g by day 3. Salsa recipes, with or without lime juice, supported the growth of Salmonella Saintpaul, and final levels were approximately 7 log CFU/g after 3 days at 23°C. In contrast, the counts of Salmonella Typhimurium, Salmonella Montevideo, Salmonella Newport, and Salmonella Enteritidis increased only 2 log CFU/g after 3 days in any of the salsas. Other Salmonella Saintpaul strains were able to grow in salsas containing 10% lime juice, but their final levels were less than 5 log CFU/g. These findings indicate the enhanced ability of the Salmonella Saintpaul outbreak strain to grow in salsa compared with other Salmonella strains. Recipe modifications including but not limited to adding lime juice (at least 10%) and keeping fresh salsa at room temperature for less than 12 h before consumption are strategies that can help mitigate the growth of Salmonella in salsa.

Salmonella and Enterohemorrhagic Escherichia coli Serogroups O45, O121, O145 in Wheat Flour: Effects of Long-Term Storage and Thermal Treatments.

Salmonella and enterohemorrhagic Escherichia coli (EHEC) are of serious concern in wheat flour and its related products but little is known on their survival and thermal death kinetics. This study was undertaken to determine their long-term viability and thermal inactivation kinetics in flour. Inoculation was performed using mixtures of EHEC serogroups O45, O121, O145 and Salmonella followed by storage at room temperature (23°C) or 35°C (for Salmonella). Plate counting on tryptic soy agar (TSA) and enrichment were used to assess long-term survival. For thermal studies, wheat flour samples were heated at 55, 60, 65, and 70°C and cell counts of EHEC and Salmonella were determined by plating. The δ-values were calculated using the Weibull model. At room temperature, EHEC serovars and Salmonella were quantifiable for 84 and 112 days, and were detectable for the duration of the experiment after 168 and 365 days, respectively. The δ-values were 2.0, 5.54, and 9.3 days, for EHEC O121, O45, and O145, respectively, and 9.7 days for Salmonella. However, the only significant difference among all values was the δ-value for Salmonella and serogroup O121 (p ≤ 0.05). At 35°C, Salmonella counts declined to unquantifiable levels after a week and were not detected upon enrichment after 98 days. Heat treatment of inoculated wheat flour at 55, 60, 65, and 70°C resulted in δ-value ranges of 20.0-42.9, 4.9-10.0, 2.4-3.2, and 0.2-1.6 min, respectively, for EHEC. The δ-values for Salmonella at those temperatures were 152.2, 40.8, 17.9, and 17.4 min, respectively. The δ-values obtained for Salmonella at each temperature were significantly longer than for EHEC (p ≤ 0.05). Weibull model was a good fit to describe the thermal death kinetics of Salmonella and EHEC O45, O121 and O145 in wheat flour. HIGHLIGHTS -EHEC and Salmonella can survive for extended periods of time in wheat flour.-Long-term storage inactivation curves of EHEC and Salmonella were similar.-EHEC was more sensitive to heat than Salmonella.-Weibull model was a good fit to describe thermal death kinetics of EHEC and Salmonella.-Flour storage at 35°C may be a feasible method for microbial reduction.

Differentiation of Bacillus thuringiensis From Bacillus cereus Group Using a Unique Marker Based on Real-Time PCR.

The efficiency of a novel biomarker (the transcriptional regulator, XRE) was tested and evaluated in differentiating Bacillus thuringiensis from Bacillus cereus group species in environmental and spiked samples based on PCR and real-time PCR. Totally 120 strains, representing two bacterial groups, B. cereus group and non-Bacillus sp., were used to evaluate the performance of XRE and crystal protein (cry2, an existing biomarker). Further, three diverse samples (kimbap, lettuce, and spinach) were inoculated with B. thuringiensis and prominent biomarkers XRE and cry2 were used as targets. Direct analysis of the detection results for the pure cultures of B. cereus group wild-types, references and type strains revealed an accuracy rate of 97.5% targeting XRE, and 83.3% targeting cry2. The real-time PCR was constructed with a R 2-value of 0.993. For the artificially contaminated samples, a concentration of 103 CFU/g of B. thuringiensis in spiked food samples could be detected using real-time PCR targeting XRE. A good performance was obtained with XRE in discriminating B. thuringiensis from B. cereus groups, as well as detecting B. thuringiensis in spiked food samples with PCR or real-time PCR. Therefore, this real-time PCR targeting XRE can be used as a dependable and promising tool to identify B. thuringiensis in foods.

A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus .

Dynabeads protein G antibody conjugates combined with modified brain heart infusion broth for the enrichment and separation of Bacillus cereus in artificially contaminated vegetables.

A modified brain heart infusion (MBHI) broth and a protocol of immunomagnetic separation (IMS) using antibody-coated Dynabeads® protein G were developed for the enrichment and separation of Bacillus cereus in artificially contaminated vegetable samples. The MBHI consisted of BHI and 0.34 g/L magnesium sulfate, 12.08 g/L sodium pyruvate, 1.82 g/L yeast extract, and polymyxin B. The amount of immunomagnetic beads (IMBs) and immunoreaction time were optimized. The capture efficiency was 58.32% with 0.4 mg IMBs when the immunoreaction time was 20 min. Capture of B. cereus by IMBs did not interfere with competing flora. Pre-enrichment IMS was validated with four B. cereus strains in artificially contaminated baby sprouts, bean sprouts, lettuce, and spinach at two levels (∼0.1 and ∼1 CFU/g). We were able to detect and isolate B. cereus in 40/40 samples of vegetables contaminated at 0.1 CFU/g with IMS after 6 h of enrichment in MBHI.

Enterotoxigenic profiling of emetic toxin- and enterotoxin-producing Bacillus cereus, Isolated from food, environmental, and clinical samples by multiplex PCR.

Bacillus cereus comprises the largest group of endospore-forming bacteria and can cause emetic and diarrheal food poisoning. A total of 496 B. cereus strains isolated from various sources (food, environmental, clinical) were assessed by a multiplex PCR for the presence of enterotoxin genes. The detection rate of nheA, entFM, hblC, and cytK enterotoxin genes among all B. cereus strains was 92.33%, 77.21%, 59.47%, and 47.58%, respectively. Enterotoxigenic profiles were determined in emetic toxin- (8 patterns) and enterotoxin-producing strains (12 patterns). The results provide important information on toxin prevalence and toxigenic profiles of B. cereus from various sources. Our findings revealed that B. cereus must be considered a serious health hazard and Bacillus thuringiensis should be considered of a greater potential concern to food safety among all B. cereus group members. Also, there is need for intensive and continuous monitoring of products embracing both emetic toxin and enterotoxin genes.