Meat and meat products as potential sources of emerging MDR Bacillus cereus: groEL gene sequencing, toxigenic and antimicrobial resistance.

BACKGROUND: Bacillus cereus is implicated in severe foodborne infection in humans. This study intended to assess the occurrence, groEL gene sequencing, biofilm production, and resistance profiles of emerged multidrug resistant (MDR) B. cereus in meat and meat product samples. Moreover, this work highlights the virulence and toxigenic genes (hblABCD complex, nheABC complex, cytK, ces, and pc-plc) and antimicrobial resistance genes (bla1, tetA, bla2, tetB, and ermA). METHODS: Consequently, 200 samples (sausage, minced meat, luncheon, beef meat, and liver; n = 40 for each) were indiscriminately collected from commercial supermarkets in Port Said Province, Egypt, from March to May 2021. Subsequently, food samples were bacteriologically examined. The obtained isolates were tested for groEL gene sequence analysis, antibiotic susceptibility, biofilm production, and PCR screening of toxigenic and resistance genes. RESULTS: The overall prevalence of B. cereus among the inspected food samples was 21%, where the highest predominance was detected in minced meat (42.5%), followed by beef meat (30%). The phylogenetic analysis of the groEL gene exposed that the examined B. cereus strain disclosed a notable genetic identity with other strains from the USA and China. Moreover, the obtained B. cereus strains revealed ß-hemolytic activity, and 88.1% of the recovered strains tested positive for biofilm production. PCR evidenced that the obtained B. cereus strains usually inherited the nhe complex genes (nheA and nheC: 100%, and nheB: 83.3%), followed by cytK (76.2%), hbl complex (hblC and hblD: 59.5%, hblB: 16.6%, and hblA: 11.9%), ces (54.7%), and pc-plc (30.9%) virulence genes. Likewise, 42.9% of the examined B. cereus strains were MDR to six antimicrobial classes and encoded bla1, bla2, ermA, and tetA genes. CONCLUSION: In summary, this study highlights the presence of MDR B. cereus in meat and meat products, posing a significant public health risk. The contamination by B. cereus is common in minced meat and beef meat. The molecular assay is a reliable fundamental tool for screening emerging MDR B. cereus strains in meat and meat products.

Isolation, Identification, and Characterization of Bacillus cereus Group Bacteria Isolated from the Dairy Farm Environment and Raw Milk in Tunisia.

Members of the Bacillus cereus group are well-known opportunistic foodborne pathogens. In this study, the prevalence, hemolytic activity, antimicrobial resistance profile, virulence factor genes, genetic diversity by enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) genotyping, and adhesion potential were investigated in isolates from a Tunisian dairy farm environment and raw milk. A total of 200 samples, including bedding, feces, feed, liquid manure, and raw bovine milk, were examined. Based on PCR test targeting sspE gene, 59 isolates were detected. The prevalence of B. cereus group isolates in bedding, feces, liquid manure, feed, and raw milk was 48%, 37.8%, 20%, 17.1%, and 12.5%, respectively. Out of the tested strains, 81.4% showed ß-hemolytic on blood agar plates. An antimicrobial resistance test against 11 antibiotics showed that more than 50% of the isolates were resistant to ampicillin and novobiocin, while a high sensitivity to other antibiotics tested was observed in most isolates. The distribution of enterotoxigenic genes showed that 8.5% and 67.8% of isolates carried hblABCD and nheABC, respectively. In addition, the detection rate of cytotoxin K (cytk), enterotoxin T (bceT), and ces genes was 72.9%, 64.4%, and 5.1%, respectively. ERIC-PCR fingerprinting genotype analysis allowed discriminating 40 different profiles. The adhesion potential of B. cereus group on stainless steel showed that all isolates were able to adhere at various levels, from 1.5 ± 0.3 to 5.1 ± 0.1 log colony-forming unit (CFU)/cm2 for vegetative cells and from 2.6 ± 0.4 to 5.7 ± 0.3 log CFU/cm2 for spores. An important finding of the study is useful for updating the knowledge of the contamination status of B. cereus group in Tunisia, at the dairy farm level.

UV-C and wet heat resistance of Bacillus thuringiensis biopesticide endospores compared to foodborne Bacillus cereus endospores.

Bacillus endospores (spores) are generally resistant to environmental and food processing-related stress including thermal and non-thermal processing in the food industry, such as pasteurization, and UV-C inactivation. Bacillus thuringiensis insecticidal crystals and spores as the active substances in commercial biopesticides can also be introduced to vegetable foods and their food processing environment due to pre-harvest treatment of edible crops. The resistance of B. thuringiensis biopesticide spores in comparison to the genetically closely related foodborne B. cereus against heat and UV-C treatment is investigated in this study. The results show that B. thuringiensis biopesticide spores with the commercial granulated product formulation are better protected and as such more resistant to both wet heat (D values at 90 °C: 50.1-79.5 min) and UV-C treatment (D values at 0.6 mW/cm2: 7.5-8.9 min) than the pure spore suspension. The enhanced UV-C resistance properties of B. thuringiensis-formulated spores also indicate that the B. thuringiensis spores in powder or granule formulation applied in the field might not be effectively inactivated by solar radiation (UV-A and UV-B) in a short period. Furthermore, the spores of one emetic B. cereus toxin-producing strain (LFMFP 254; a Belgian outbreak strain) were found more resistant to the wet heat at 90 °C (D90-value = 71.2 min) than other tested pure spore suspensions, although the spores of B. cereus 254 did not show different behavior against UV-C treatment. This result suggests that UV-C treatment can be applied as an effective inactivation method against B. cereus 254 spores.

DNA Nanomachine (DNM) Biplex Assay for Differentiating Bacillus cereus Species.

Conventional methods for the detection and differentiation of Bacillus cereus group species have drawbacks mostly due to the complexity of genetic discrimination between the Bacillus cereus species. Here, we describe a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay uses a universal fluorescent reporter and four all-DNA binding fragments, three of which are responsible for "opening up" the folded rRNA while the fourth stand is responsible for detecting single nucleotide variation (SNV) with high selectivity. Binding of the DNM to 16S rRNA results in the formation of the 10-23 deoxyribozyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. This developed biplex assay enables the detection of B. thuringiensis 16S rRNA at fluorescein and B. mycoides at Cy5 channels with a limit of detection of 30 × 103 and 35 × 103 CFU/mL, respectively, after 1.5 h with a hands-on time of ~10 min. The new assay may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis. The DNM proposed here may become an advantageous tool for detecting SNV in clinically significant DNA or RNA samples and can easily differentiate SNV under broadly variable experimental conditions and without prior amplification.

A study of bacteriocin like substances comparison produced by different species of Bacillus related to B. cereus group with specific antibacterial activity against foodborne pathogens.

The antibiotic-resistant bacteria are emerging as a great threat worldwide. For this reason it is important to develop new antibiotic substances. Bacillus is considered as a factory of a wide range of chemical compounds with a variety of activities. Among these substances are bacteriocins which are small peptides showing stability in a wide range of pH and temperatures and having a potent antibacterial activity. Bacillus species can be grouped into families such as B. cereus group based on their genetic similarity. It can be helpful to study the bacteriocins presented in these related species identifying the differences and similarities between them to relate the presence of a given bacteriocin with the producer specie. The aim of this study was to isolate the bacteriocins from three related species of B. cereus group such as B. mycoides, B. weihenstephanensis and B. toyonensis and compare among them and with the bacteriocins isolated from B. velezensis. Besides it was analyzed the bactericidal activity of each isolated bacteriocin. Five different bacteriocins of similar molecular mass and specific against foodborne pathogens were isolated from three Bacillus species related to B. cereus group, that were quite different both in molecular mass and bactericidal activity from that was isolated from B. velezensis. The results indicated that bacteriocins can be distinguished according to Bacillus specie from it has been isolated.

Boosting bacteria differentiation efficiency with multidimensional surface-enhanced Raman scattering: the example of Bacillus cereus.

Surface-enhanced Raman scattering (SERS) is a powerful tool for constructing biomolecular fingerprints, which play a vital role in differentiation of bacteria. Due to the rather subtle differences in the SERS spectra among different bacteria, artificial intelligence is usually adopted and enormous amounts of spectral data are required to improve the differentiation efficiency. However, in many cases, large volume data acquisition on bacteria is not only technical difficult but labour intensive. It is known that surface modification of SERS nanomaterials can bring additional dimensionality (difference) of the SERS fingerprints. Here in this work, we show that the concept could be used to improve the bacteria differentiation efficiency. Ag NPs were modified with 11-mercaptoundecanoic acid, 11-mercapto-1-undecanol, and 1-dodecanethiol to provide additional dimensionality. The modified NPs then were mixed with cell lysate from different strains of Bacillus cereus (B. cereus). Even by applying a simple PCA process to the resulting SERS spectra data, all the three modified Ag NPs showed superior differentiation results compared with bare Ag NPs, which could only separate Staphylococcus aureus (S. aureus) and B. cereus. It is believed that the multidimensional SERS could find great potential in bacteria differentiation.

Enhanced vanillin production from eugenol by Bacillus cereus NCIM-5727.

Biovanillin production by a wild strain of Bacillus cereus NCIM-5727 is studied using eugenol as the precursor aiming to achieve maximum vanillin productivity. Based on shake flask optimization, molar yield and global volumetric productivity of vanillin reached up to 71.2% (6.6 gL-1) and 0.18 g(Lh)-1, respectively, at 36 h by resting cells of B. cereus NCIM-5727 at the optimum cell concentration of 3 gL-1 using eugenol concentration of 10 gL-1 at 37 ºC, buffer pH 7.0, buffer volume 10%, and shaking speed 180 rpm. Furthermore, small-scale optimization in a bioreactor at the controlled aeration rate of 0.5 Lmin-1, agitation rate of 210 rpm, and pH 7.0 enhanced the global volumetric productivity of vanillin up to 0.28 g(Lh)-1 at 25 h of bioconversion. The highest vanillin molar yield (75.2%) is reported using resting cells of B. cereus NCIM-5727 upon eugenol biotransformation and found stable for 10 h.

Evaluation of interaction among indigenous rhizobacteria and Vigna unguiculata on remediation of metal-containing abandoned magnesite mine tailing.

Abandoned magnesite mine heap causing pollution to nearby farmland and water reservoir. Thus the intention of this research was to screening metal mobilizing and absorbing bacteria from the rhizosphere section of V. unguiculata from farmland nearby to magnesite mine. Further, studied their stimulus effect on growth, biomass, and phytoextraction prospective of V unguiculata in mine tailing. The results of the physicochemical properties of mine tailing shows that four metals (Pb, Mn, Cd, and Zn) were crossing the permissible limit. Out of 27 isolates, 2 isolates (MMS15 and MMS17) were identified with maximum metal tolerance for up to 700 mg L-1 (MIC) and metal mobilization (Pb 5.5 and 5.87, Mn 6.6 and 4.88, Cd 1.99 and 2.59, and Zn 6.55 and 6.94 mg kg-1) and biosorption efficiency as Pb 3.74 and 3.74, Mn 4.9 and 4.7, Cd 2.41 and 3.96, and Zn 4.3 and 4.9 mg g-1. These two strains were identified as members of B. cereus and Kosakonia sp. using 16S rRNA technique and labelled strains NDRMN001 and MGR1, respectively. The Kosakonia sp. MGR1 effectively fixes the nitrogen in the rate of 81.94% and B. cereus NDRMN001 solubilizes 69.98 ± 2.31 mg L-1 of soluble phosphate. The experimental group's study results show that the group C (Kosakonia sp. MGR1 and B. cereus NDRMN001) has effectively stimulate the growth, biomass, and phytoextraction potential of V. unguiculata. The results conclude that the optimistic interaction between these two bacteria could be more significant to minimize the metal pollution in magnesite mine tailing.

Identification and characterization of a new cry-like gene found in a Bacillus cereus strain.

Bacillus thuringiensis is the most successful microbial insecticide against different pests in agriculture and vectors of diseases. Its activity is mostly attributed to the Cry proteins expressed during its sporulation phase. However, these proteins are not exclusive to B. thuringiensis. Some cry genes have been found in other Bacillus species, or even in other genera. In this work, cry genes were searched in 223 acrystalliferous bacillaceous strains. From these strains 13 amplicons were obtained, cloned, and sequenced; however, only 6 amplicons tested positive for cry-like genes, and the 6 isolates showed to be the same strain. We report the characterization of an unusual strain of B. cereus (LBIC-004) which is unable to form protein inclusions during the sporulation phase. LBIC-004 showed a high identity to B. cereus using the sequences of 16S rRNA, gyrB and hag genes; in addition, a unique plasmid pattern of the strain was obtained. A 1953-bp cry gene was identified, coding for a 651 amino acid protein with a molecular weight of 74.9 kDa. This protein showed a predicted three-domain structure, similar to all Cry proteins. However, the amino acid sequence of the protein showed only 41% identity its highest hit: the Cry8Ca1 protein, indicating the uniqueness of this cry-like gene. It was cloned and transferred into a mutant acrystalliferous B. thuringiensis strain which was used in bioassays against Caenorhabditis elegans, Aedes aegypti, Manduca sexta and Phyllophaga sp. The recombinant strain showed no crystal formation and no toxicity to the tested species.

CspB and CspC are induced upon cold shock in Bacillus cereus strain D2.

Bacillus cereus D2, a psychrotrophic strain, plays an essential role in the restoration of heavy metal-contaminated soils, especially at low temperatures. However, the cold shock response mechanisms of this strain are unclear. In this study, the cold shock response of B. cereus D2 was characterized; as per the Arrhenius curve, 10 °C was chosen as the cold shock temperature. Six cold shock-like proteins were found and temporarily named cold shock protein (Csp)1-6; the respective genes were cloned and identified. Quantitative real-time PCR results showed that csp1, csp2, csp3, and csp6 were overexpressed under cold shock conditions. Interestingly, after cloning the respective encoding genes into the pET-28a (+) vector and their subsequent transformation into E. coli BL21 (DE3), the strains expressing Csp2 and Csp6 grew faster at 10 °C, showing a large number of bacteria. These results suggest that Csp2 and Csp6 are the major cold shock proteins in B. cereus D2. Of note, the comparison of amino acid sequences and structures showed that Csp2 and Csp6 belong to the CspB and CspC families, respectively. Additionally, we show that the number of hydrophobic residues is not a determining feature of major Csps, while, on the other hand, the formation of an α-helix in the context of a leucine residue is the most dominant difference between major and other Bacillus and E. coli Csps.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities.

Structure Revision of Isocereulide A, an Isoform of the Food Poisoning Emetic Bacillus cereus Toxin Cereulide.

The emetic Bacillus cereus toxin cereulide presents an enormous safety hazard in the food industry, inducing emesis and nausea after the consumption of contaminated foods. Additional to cereulide itself, seven structurally related isoforms, namely the isocereulides A-G, have already been elucidated in their chemical structure and could further be identified in B. cereus contaminated food samples. The newly performed isolation of isocereulide A allowed, for the first time, 1D- and 2D-NMR spectroscopy of a biosynthetically produced isocereulide, revealing results that contradict previous assumptions of an l-O-Leu moiety within its chemical structure. By furthermore applying posthydrolytical dipeptide analysis, amino acid and α-hydroxy acid analysis by means of UPLC-ESI-TOF-MS, as well as MSn sequencing, the structure of previously reported isocereulide A could be corrected. Instead of the l-O-Leu as assumed to date, one l-O-Ile unit could be verified in the cyclic dodecadepsipeptide, revising the structure of isocereulide A to [(d-O-Leu-d-Ala-l-O-Val-l-Val)2(d-O-Leu-d-Ala-l-O-Ile-l-Val)].

Thermotolerance effect of plant growth-promoting Bacillus cereus SA1 on soybean during heat stress.

BACKGROUND: Incidences of heat stress due to the changing global climate can negatively affect the growth and yield of temperature-sensitive crops such as soybean variety, Pungsannamul. Increased temperatures decrease crop productivity by affecting biochemical, physiological, molecular, and morphological factors either individually or in combination with other abiotic stresses. The application of plant growth-promoting endophytic bacteria (PGPEB) offers an ecofriendly approach for improving agriculture crop production and counteracting the negative effects of heat stress. RESULTS: We isolated, screened and identified thermotolerant B. cereus SA1 as a bacterium that could produce biologically active metabolites, such as gibberellin, indole-3-acetic acid, and organic acids. SA1 inoculation improved the biomass, chlorophyll content, and chlorophyll fluorescence of soybean plants under normal and heat stress conditions for 5 and 10 days. Heat stress increased abscisic acid (ABA) and reduced salicylic acid (SA); however, SA1 inoculation markedly reduced ABA and increased SA. Antioxidant analysis results showed that SA1 increased the ascorbic acid peroxidase, superoxide dismutase, and glutathione contents in soybean plants. In addition, heat stress markedly decreased amino acid contents; however, they were increased with SA1 inoculation. Heat stress for 5 days increased heat shock protein (HSP) expression, and a decrease in GmHSP expression was observed after 10 days; however, SA1 inoculation augmented the heat stress response and increased HSP expression. The stress-responsive GmLAX3 and GmAKT2 were overexpressed in SA1-inoculated plants and may be associated with decreased reactive oxygen species generation, altered auxin and ABA stimuli, and enhanced potassium gradients, which are critical in plants under heat stress. CONCLUSION: The current findings suggest that B. cereus SA1 could be used as a thermotolerant bacterium for the mitigation of heat stress damage in soybean plants and could be commercialized as a biofertilizer only in case found non-pathogenic.

Identification of the bacteria and their metabolic activities associated with the microbial spoilage of custard cream desserts.

The famous French dessert "ile flottante" consists of a sweet egg white foam floating on a vanilla custard cream, which contains highly nutritive raw materials, including milk, sugar and egg. Spoilage issues are therefore a key concern for the manufacturers. This study explored the bacterial diversity of 64 spoiled custard cream desserts manufactured by 2 French companies. B. cereus group bacteria, coagulase negative Staphylococcus, Enterococcus and Leuconostoc spp. were isolated from spoiled products. Thirty-one bacterial isolates representative of the main spoilage species were tested for their spoilage abilities. Significant growth and pH decrease were observed regardless of species. While off-odours were detected with B. cereus group and staphylococci, yoghurt odours were detected with Enterococcus spp. and Leuconostoc spp. B. cereus group bacteria produced various esters and several compounds derived from amino acid and sugar metabolism. Most Staphylococci produced phenolic compounds. Enterococcus spp. and Leuconostoc spp. isolates produced high levels of compounds derived from sugar metabolism. Each type of spoilage bacteria was associated with a specific volatile profile and lactic acid was identified as a potential marker of spoilage of custard cream-based desserts. These findings provide valuable information for manufacturers to improve food spoilage detection and prevention of chilled desserts made with milk and egg.

Contribution of Bacillus cereus ERBP in ozone detoxification by Zamioculcas zamiifolia plants: Effect of ascorbate peroxidase, catalase and total flavonoid contents for ozone detoxification.

Eighteen plant species were screened for ozone (O3) removal in a continuous system. Zamioculcas zamiifolia had the highest O3 removal efficiency. To enhance O3 removal by Z. zamiifolia, adding a compatible endophytic bacteria, Bacillus cereus ERBP into Z. zamiifolia was studied. After operating under an O3 continuous system (150-250 ppb) at a flow rate of 0.3 L min-1 for 80 h, inoculated plants (74%) exhibited higher O3 removal efficiency than non-inoculated ones (53%). In addition, after O3 exposure (80 h), the population of B. cereus ERBP in inoculated plants was significantly increased in both shoots approximately 35 folds and leaves 13 folds compared to inoculated plants without O3 exposure. The results also showed that B. cereus ERBP had the ability to protect Z. zamiifolia against O3 stress conditions. The increase in B. cereus ERBP populations was attributed to the significant increase in ascorbate peroxidase (APX) and catalase (CAT) activity. In addition, increasing B. cereus ERBP populations led to raise total flavonoid contents which is one of antioxidant compounds. Increasing APX, CAT activities, and total flavonoid contents can enhance O3 detoxification in plant tissues. The mechanism of B. cereus ERBP for enhancing O3 phytoremediation was proposed in this study. The results suggested that B. cereus ERBP was a potential tool for alleviating O3 stress on Z. zamiifolia and enhancing O3 phytoremediation efficiency.

Inactivation of Bacillus and Clostridium Spores in Coconut Water by Ultraviolet Light.

Bacterial spores are generally more resistant than vegetative bacteria to ultraviolet (UV) inactivation. The UV sensitivity of these spores must be known for implementing UV disinfection of low acid liquid foods. UV inactivation kinetics of bacterial spores in coconut water (CW) and distilled sterile water was studied. Populations of Bacillus cereus and Clostridium sporogenes dormant spores were reduced by more than 5.5 log10 at the UV-C photon fluence of 1142 µE·m-2 and 1919 µE·m-2 respectively. C. sporogenes spores showed higher UV-C resistance than B. cereus, with the photon fluence 300 µE·m-2 required for one log inactivation (D10) and 194 µE·m-2, respectively. No significant difference was observed in D10 values of spores suspended in the two fluid types (p > 0.05). The inactivation kinetics of microorganisms were described by log linear models with low root mean square error and high coefficient of determination (R2 > 0.98). This study clearly demonstrated that high levels of inactivation of bacterial spores can be achieved in CW. The baseline data generated from this study will be used to conduct spore inactivation studies in continuous flow UV systems. Further proliferation of the technology will include conducting extensive pilot studies.

Heavy metal bioaccumulation and cation release by growing Bacillus cereus RC-1 under culture conditions.

In an effort to explore the detoxifying mechanisms of B. cereus RC-1 under heavy metal stress, the bioaccumulation by growing cells under varying range of pH, culture time and initial metal concentration were investigated from a perspective of cation release. The maximum removal efficiencies were 16.7%, 38.3%, 81.4% and 40.3% for Cu2+, Zn2+, Cd2+ and Pb2+, respectively, with initial concentrations of 10 mg/L at pH 7.0. In presence of Cu2+ or Zn2+, large quantities of cations were released into the medium in descending order of Na+>K+>Ca2+>Mg2+, while bioremoval of the two essential metals Cd2+ and Pb2+ was accompanied with cellular Na+ and Mg2+ uptake from the medium, respectively. The relative mean contributions of intracellular accumulation to the total removal were approximately 19.6% for Cu2+, 12.8% for Zn2+, 51.1% for Cd2+, and only 4.6% for Pb2+. Following exposure at high concentration, B. cereus RC-1 could keep intracellular Cd2+ concentrations constant, possibly by means of a Cd-efflux system whose activity coincided with uptake of Na+, and reduce intracellular Pb2+ concentration due to the effect of Mg2+ on limiting Pb2+ access to the cells. Cellular morphology, surface functional groups and intracellular trace elements were further investigated by SEM-EDX, TEM-EDX, FTIR and ICP-MS analysis. The phenomena that removal of Cd2+ and Pb2+ coincided with uptake of Na+ and Mg2+, respectively, inspires a novel research perspective towards the study of protective mechanism of bacterial cells against the toxicity of heavy metals.

Propidium Monoazide Quantitative Real-Time Polymerase Chain Reaction for Enumeration of Some Viable but Nonculturable Foodborne Bacteria in Meat and Meat Products.

Foodborne infections due to bacterial pathogens are increasing worldwide. Given the surreptitious nature of viable but nonculturable (VBNC) bacteria, they largely remain a threat to public health and food safety due to their non-detectability through conventional plate count techniques. Hence, species-specific quantitative real-time polymerase chain reaction (PCR) (qPCR) alone and combined with the use of propidium monoazide (PMA) was used along with the plate count method to quantify VBNC Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, and Enterobacteriaceae in fresh and processed meat samples. The major bacterial pathogen isolated was S. aureus (93%) followed by Enterobacteriaceae (80.33%), C. perfringens (26.33%), and B. cereus (21.33%); their total viable counts were mostly recorded in raw meat than examined meat products. PMA quantitative real-time PCR (PMA qRT-PCR) could detect and quantify VBNC bacteria in 90.48% of culture-negative samples. It affirmed the presence of VBNC S. aureus (n = 10), B. cereus (n = 8), C. perfringens (n = 6), and Enterobacteriaceae (n = 12) in either single or mixed bacterial contamination. The log10 mean values of VBNC bacterial counts were highly reported for C. perfringens and S. aureus (9.60 ± 0.449 and 8.27 ± 0.453 CFU/g, respectively) followed by Enterobacteriaceae (6.95 ± 0.564 CFU/g) and B. cereus (6.69 ± 0.749 CFU/g). Sequencing of rpoB gene of Enterobacteriaceae enabled the identification of Klebsiella pneumoniae complex, Enterobacter cloacae complex, and Salmonella Typhi, which have been reported to be capable of entry into the VBNC state. To our knowledge, this is the first report at least in Egypt that records the presence of VBNC cells in meat samples representing a strong threat to public health and food safety. Moreover, PMA qRT-PCR allowed a quick and unequivocal way of enumeration of VBNC foodborne bacteria.

Efficacy Evaluation of Control Measures on the Reduction of Staphylococcus aureus in Salad and Bacillus cereus in Fried Rice Served at Restaurants.

The objective of this study was to assess the effect of washing on Staphylococcus aureus reduction in salads and the effect of reheating on Bacillus cereus vegetative cells and spores reduction in fried rice at restaurants using the stochastic food safety objective (FSO) tool. The leaf vegetable was inoculated with S. aureus and washed with tap water, 100 ppm of NaClO, or 30 ppm of slightly acidic electrolyzed water (SAEW) for either 60 s or 5 min. The washing effect of 30 ppm SAEW was greater than that of 100 ppm NaClO. Based on the FSO concept, washing leaf vegetables with 30 ppm SAEW for 5 min was the most efficient control measure for S. aureus in salads. In addition, the salad should be consumed within 4 h at 25°C and 2 h at 35°C after 5 min of washing with 100 ppm NaClO or 30 ppm SAEW. The fried rice was first inoculated with B. cereus vegetative cells or spores and was then reheated in a frying pan at medium (internal temperature of fried rice: 69.2°C-78.8°C) or high heat (internal temperature of fried rice:103.8°C-121.4°C) or in a microwave oven (internal temperature of fried rice:86.3°C-90.6°C) for 3 or 4 min. Based on the FSO, reheating rice in a microwave oven was the most efficient control measure for B. cereus vegetative cells and spores in fried rice. The holding time for fried rice can be extended up to 6 h at 25°C, 3 h at 35°C, and 2 h at 45°C with reheating. Microbiological hazards in salads and fried rice can be controlled by washing with a sanitizer and reheating, respectively and then by controlling of holding temperature before being served at restaurants.

Factors of detection of Bacillus cereus strains in eye cream.

Bacillus cereus has been associated with clinical infections and is also the cause of post-traumatic endophthalmitis as well as endogenous eye infections, which can result in blindness. Cosmetics, although preserved, can be contaminated during manufacture or use and thus cause serious health issues. OBJECTIVE: We investigated the detection factors of Bacillus in eye cream preserved with parabens, including the non-ionic surfactants used as neutralizers such as Tween 80, a blend of Tween 60 and Span 80, Tween 20 and selective media. METHODS: Eye-cream samples were first mixed with neutralizers and individually inoculated with B. cereus strains, B. mycoides, B. subtilis or B. thuringiensis at a final concentration of 5 log CFU per g. The inoculated samples with and without neutralizers were analysed after 30 min and during 84-day storage at room temperature. Presumptive colonies of Bacillus were enumerated on the varieties of Bacillus agar by spiral-plating techniques and most probable number (MPN) method. RESULTS: The recovery counts of all Bacillus strains were between 4.10 and 4.58 log CFU per g in samples with Tween 80 and from 3.62 to 4.53 CFU per g in samples with TS after 30 min. Tween 20 was the least effective neutralizer. The challenged organisms, in samples without neutralizer, B. subtilis ATCC 15563 and B. cereus 4227A were detected at 1.83 and 1.49 log CFU per g after 30 min, respectively. CONCLUSION: This study showed that Tween 80 was the best neutralizer for reducing the antimicrobial effect of parabens. BACARA® and R&F plating media showed typical reaction of Bacillus cereus strains.