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A new trans-4-hydroxy-l-proline (trans-Hyp) producing Bacillus cereus HBL-AI, was isolated from the air, which was screened just using l-proline as carbon and energy sources. This strain exhibited 73·4% bioconversion rate from initial l-proline (3 g l -1 ) to trans-Hyp. By sequencing the genome of this bacterium, 6244 coding sequences were obtained. Genome annotation analysis and functional expression were used to identify the proline-4-hydroxylase (BP4H) in HBL-AI. This enzyme belonged to a family of 2-oxoglutarate-related dioxygenases, which required 2-oxoglutarate and O 2 as co-substrates for the reaction. Homologous modelling indicated that the enzyme had two monomers and contained conserved motifs, which included a distorted 'jelly roll' β strand core and the residues (HXDXnH and RXS). The engineering Escherichia coli 3 Δ W3110/pTrc99a-proba-bp4h was constructed using BP4H, which transformed glucose to trans-Hyp in one step with high concentration of 46·2 g l -1 . This strategy provides a green and efficient method for synthesis of trans-Hyp and thus has a great potential in industrial application., (© 2020 The Society for Applied Microbiology.)

Verticillium wilt is a plant vascular disease caused by the soilborne fungus Verticillium dahliae that severely limits cotton production. In a previous study, we screened Bacillus cereus YUPP-10, an efficient antagonistic bacterium, to uncover mechanisms for controlling verticillium wilt. Here, we report a novel antimicrobial cyclodextrin glycosyltransferase (CGTase) from YUPP-10. Compared to other CGTases, six different conserved domains were identified, and six mutants were constructed by gene splicing with overlap extension PCR. Functional analysis showed that domain D was important for hydrolysis activity and domains A1 and C were important for inducing disease resistance. Direct effects of recombinant CGTase on V. dahliae included reduced mycelial growth, spore germination, spore production, and microsclerotia germination. In addition, CGTase also elicited cotton's innate defence reactions. Transgenic Arabidopsis thaliana lines that overexpress CGTase showed higher resistance to verticillium wilt. Transgenic CGTase A. thaliana plants grew faster and resisted disease better. CGTase overexpression enabled a burst of reactive oxygen species production and activated pathogenesis-related gene expression, indicating that the transgenic cotton was better prepared to protect itself from infection. Our work revealed that CGTase could inhibit the growth of V. dahliae, activate innate immunity, and play a major role in the biocontrol of fungal pathogens., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The Bacillus cereus group encompasses beneficial and harmful species in diverse niches and has a much debated taxonomy. Investigating whether selection has led to ecological divergence between phylogenetic clades can help understand the basis of speciation, and has implications for predicting biological safety across this group. Using three most terrestrial species in this group (B. cereus, Bacillus thuringiensis and Bacillus mycoides) we charactererized ecological specialization in terms of resource use, thermal adaptation and fitness in different environmental conditions and tested whether taxonomic species or phylogenetic clade best explained phenotypic variation. All isolates grew vigorously in protein rich media and insect cadavers, but exploitation of soil or plant derived nutrients was similarly weak for all. For B. thuringiensis and B. mycoides, clade and taxonomic species were important predictors of relative fitness in insect infections. Fully psychrotolerant isolates could outcompete B. thuringiensis in insects at low temperature, although psychrotolerance predicted growth in artificial media better than clade. In contrast to predictions, isolates in the Bacillus anthracis clade had sub-optimal growth at 37°C. The common ecological niche in these terrestrial B. cereus species is the ability to exploit protein rich resources such as cadavers. However, selection has led to different phylogenetic groups developing different strategies for accessing this resource. Thus, clades, as well as traditional taxonomic phenotypes, predict biologically important traits., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

Cysteine is a semiessential amino acid and plays an important role in metabolism and protein structure and has also been applied in various industrial fields, such as pharmaceutical, food, cosmetic, and animal feed industries. Metabolic engineering studies have been conducted for the cysteine production through bacterial fermentation, but studies on the cysteine biosynthetic pathway in microorganisms are limited. We report the biochemical characteristics of cystathionine γ-lyase from Bacillus cereus ATCC 14579 ( Bc CGL). We also determined the crystal structure of Bc CGL in complex with the PLP cofactor and identified the substrate binding mode. We observed that the replacement of the conserved Glu321 residue to alanine showed increased activity by providing wider active site entrance and hydrophobic interaction for the substrate. We suggest that the structural differences of the α13-α14 region in CGL enzymes might determine the active site conformation.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing., Competing Interests: Declaration of competing interest The authors declare that no conflict of interest exists., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacillus cereus sensu lato strains (B. cereus group) are widely distributed in nature and have received interest for decades due to their importance in insect pest management, food production and their positive and negative repercussions in human health. Consideration of practical uses such as virulence, physiology, morphology, or ill-defined features have been applied to describe and classify species of the group. However, current comparative studies have exposed inconsistencies between evolutionary relatedness and biological significance among genomospecies of the B. cereus group. Here, the combined analyses of core-based phylogeny and all versus all Average Nucleotide Identity values based on 2116 strains were conducted to update the genomospecies circumscriptions within B. cereus group. These analyses suggested the existence of 57 genomospecies, 37 of which are novel, thus indicating that the taxonomic identities of more than 39% of the analyzed strains should be revised or updated. In addition, we found that whole-genome in silico analyses were suitable to differentiate genomospecies such as B. anthracis, B. cereus and B. thuringiensis. The prevalence of toxin and virulence factors coding genes in each of the genomospecies of the B. cereus group was also examined, using phylogeny-aware methods at wide-genome scale. Remarkably, Cry and emetic toxins, commonly assumed to be associated with B. thuringiensis and emetic B. paranthracis, respectively, did not show a positive correlation with those genomospecies. On the other hand, anthrax-like toxin and capsule-biosynthesis coding genes were positively correlated with B. anthracis genomospecies, despite not being present in all strains, and with presumably non-pathogenic genomospecies. Hence, despite these features have been so far considered relevant for industrial or medical classification of related species of the B. cereus group, they were inappropriate for their circumscription. In this study, genomospecies of the group were accurately affiliated and representative strains defined, generating a rational framework that will allow comparative analysis in epidemiological or ecological studies. Based on this classification the role of specific markers such as Type VII secretion system, cytolysin, bacillolysin, and siderophores such as petrobactin were pointed out for further analysis.

Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Pentachlorophenol (PCP) is a recalcitrant biocide that bioaccumulates in the environment due to its persistent nature and has been listed as a priority pollutant due to its toxicological and health effects. In this study, a novel PCP-degrading Bacillus cereus strain AOA-CPS1 (BcAOA) was isolated from wastewater and characterized for PCP biotransformation in a batch reactor. The degradation kinetics were elucidated via substrate inhibition models, while PCP biotransformation was established by spectrophotometric and GC-MS analysis. BcAOA shared 95% sequence homology with Bacillus cereus strain XS2 and is closely related to some B. cereus strains which are previously reported to degrade PCP and other related pollutants. BcAOA degraded 74% of 350 mg l -1  of PCP within 9 days in a batch culture. The biotransformation of PCP by BcAOA followed the first and zero-order kinetics at low and high PCP concentration, respectively, with biokinetic constants: maximum biotransformation rate (0.0996 mg l -1  h -1 ); substrate inhibition constant (723.75 mg l -1 ); half-saturation constant (171.198 mg l -1 ) and R 2 (0.98). The genes (pcpABCDE, cytochrome P450) encoding the enzymes involved in the biodegradation of PCP were amplified from the genomic DNA of BcAOA. Further, depending upon the genes amplified and identified metabolites using GC-MS, two different PCP biotransformation pathways were proposed in this study. Cloning and expression of the catabolic genes are underway to map out the concise pathway for PCP biotransformation by BcAOA.

Active migration across semi-solid surfaces is important for bacterial success by facilitating colonization of unoccupied niches and is often associated with altered virulence and antibiotic resistance profiles. We isolated an atmospheric contaminant, subsequently identified as a new strain of Bacillus mobilis, which showed a unique, robust, rapid, and inducible filamentous surface motility. This flagella-independent migration was characterized by formation of elongated cells at the expanding edge and was induced when cells were inoculated onto lawns of metabolically inactive Campylobacter jejuni cells, autoclaved bacterial biomass, adsorbed milk, and adsorbed blood atop hard agar plates. Phosphatidylcholine (PC), bacterial membrane components, and sterile human fecal extracts were also sufficient to induce filamentous expansion. Screening of eight other Bacillus spp. showed that filamentous motility was conserved amongst B. cereus group species to varying degrees. RNA-Seq of elongated expanding cells collected from adsorbed milk and PC lawns versus control rod-shaped cells revealed dysregulation of genes involved in metabolism and membrane transport, sporulation, quorum sensing, antibiotic synthesis, and virulence (e.g., hblA/B/C/D and plcR). These findings characterize the robustness and ecological significance of filamentous surface motility in B. cereus group species and lay the foundation for understanding the biological role it may play during environment and host colonization.

Background: Bacillus thuringiensis bacteria share similar genetic, physiological, and biochemical characteristics with other members of the Bacillus cereus group. Their diversity and entomopathogenic origin are related to their mobile genetic elements. However, the effects of wide-spread application of B. thuringiensis-based pesticides on genetically related B. cereus group populations present in the environment remain poorly understood., Results: We first identified pBMB76 from B. thuringiensis tenebrionis as a new conjugative plasmid. Mixed mating experiments suggested that pBMB76 may compete with pHT73, another known conjugative plasmid. Applications of single (tenebrionis 4AA1 and kurstaki HD73 carrying pBMB76 and pHT73, respectively) and mixed (4AA1 + HD73) B. thuringiensis strains were performed in confined plot habitats (soil and leaf) over two planting seasons. In total, 684 B. cereus group isolates were randomly selected from different treatment sets, and the transmissibility and occurrence rate of potential conjugative plasmids were surveyed. Results showed that the percentage of isolates with plasmid mobility was markedly enhanced in the B. thuringiensis-sprayed groups. Furthermore, we performed multi-locus sequence typing (MLST) for a subset of 291 isolates, which indicated that the dominant sequence types in the treated habitats were identical or related to the corresponding sprayed formulations., Conclusions: The application of B. thuringiensis strains with conjugal and mobilizing capability drove gene transmissibility within the B. cereus group populations in confined habitats and potentially modified the population structure.

The effect of Plant Growth Promoting Rhizobacteria (Bacillus sp.) and silver nanoparticles on Zea mays was evaluated. The silver nanoparticles were synthesized from Tagetes erecta (Marigold) leaf and flower extracts, whereas PGPR isolated from spinach rhizosphere. The silver nanoparticles (AgNPs) were purified using ultra centrifugation and were characterized using UV-Vis spectroscopy at gradient wavelength and also by High Resolution Transmission Electron microscopy (HRTEM). The average particles size of AgNPs was recorded approximately 60 nm. Almost all potential isolates were able to produce Indole Acetic Acid (IAA), ammonia and Hydrogen cyanide (HCN), solubilized tricalcium phosphate and inhibited the growth of Macrophomina phaseolina in vitro but the isolate LPR2 was found the best among all. On the basis of 16S rRNA gene sequence, the isolate LPR2 was characterized as Bacillus cereus LPR2. The maize seeds bacterized with LPR2 and AgNPs individually showed a significant increase in germination (87.5%) followed by LPR2 + AgNPs (75%). But the maximum growth of root and shoot of maize plant was observed in seeds coated with LPR2 followed by AgNPs and a combination of both. Bacillus cereus LPR2 and silver nanoparticles enhanced the plant growth and LPR2 strongly inhibited the growth of deleterious fungal pathogen. Therefore, LPR2 and AgNPs could be utilized as bioinoculant and growth stimulator, respectively for maize.

Microorganisms that display unique biotechnological characteristics are usually selected for industrial applications. Bacillus cereus NWUAB01 was isolated from a mining soil and its heavy metal resistance was determined on Luria-Bertani agar. The biosurfactant production was determined by screening methods such as drop collapse, emulsification and surface tension measurement. The biosurfactant produced was evaluated for metal removal (100 mg/L of each metal) from contaminated soil. The genome of the organism was sequenced using Illumina Miseq platform. Strain NWUAB01 tolerated 200 mg/L of Cd and Cr, and was also tolerant to 1000 mg/L of Pb. The biosurfactant was characterised as a lipopeptide with a metal-complexing property. The biosurfactant had a surface tension of 39.5 mN/m with metal removal efficiency of 69%, 54% and 43% for Pb, Cd and Cr respectively. The genome revealed genes responsible for metal transport/resistance and biosynthetic gene clusters involved in the synthesis of various secondary metabolites. Putative genes for transport/resistance to cadmium, chromium, copper, arsenic, lead and zinc were present in the genome. Genes responsible for biopolymer synthesis were also present in the genome. This study highlights biosurfactant production and heavy metal removal of strain NWUAB01 that can be harnessed for biotechnological applications.

Gram-positive, spore-forming members of the Bacillus cereus group species complex are widespread in natural environments and display various degrees of pathogenicity. Recently, B. cereus group strain Bacillus mycoides Flugge ATCC 21929 was found to represent a novel lineage within the species complex, sharing a relatively low degree of genomic similarity with all B. cereus group genomes (average nucleotide identity [ANI] < 88). ATCC 21929 has been previously associated with the production of a patented antibiotic, antibiotic 60-6 (i.e., cerexin A); however, the virulence potential and growth characteristics of this lineage have never been assessed. Here, we provide an extensive genomic and phenotypic characterization of ATCC 21929, and we assess its pathogenic potential in vitro. ATCC 21929 most closely resembles Bacillus paramycoides NH24A2 T (ANI and in silico DNA-DNA hybridization values of 86.70 and 34.10%, respectively). Phenotypically, ATCC 21929 does not possess cytochrome c oxidase activity and is able to grow at a range of temperatures between 15 and 43°C and a range of pH between 6 and 9. At 32°C, ATCC 21929 shows weak production of diarrheal enterotoxin hemolysin BL (Hbl) but no production of nonhemolytic enterotoxin (Nhe); at 37°C, neither Hbl nor Nhe is produced. Additionally, at 37°C, ATCC 21929 does not exhibit cytotoxic effects toward HeLa cells. With regard to fatty acid composition, ATCC 21929 has iso-C17:0 present in highest abundance. Based on the characterization provided here, ATCC 21929 T (= PS00077A T = PS00077B T = PSU-0922 T = BHP T ) represents a novel effective B. cereus group species, which we propose as effective species " Bacillus clarus " IMPORTANCE The B. cereus group comprises numerous closely related lineages with various degrees of pathogenic potential and industrial relevance. Species-level taxonomic classification of B. cereus group strains is important for risk evaluation and communication but remains challenging. Biochemical and phenotypic assays are often used to assign B. cereus group strains to species but are insufficient for accurate taxonomic classification on a genomic scale. Here, we show that antibiotic-producing ATCC 21929 represents a novel lineage within the B. cereus group that, by all metrics used to delineate prokaryotic species, exemplifies a novel effective species. Furthermore, we show that ATCC 21929 is incapable of producing enterotoxins Hbl and Nhe or exhibiting cytotoxic effects on HeLa cells at human body temperature in vitro These results provide greater insight into the genomic and phenotypic diversity of the B. cereus group and may be leveraged to inform future public health and food safety efforts., (Copyright © 2020 Méndez Acevedo et al.)

Pathogenicity of Bacillus cereus is associated with the production of various toxins. This study investigated the distribution of toxin genes encoding haemolysin BL (hblA, hblC, and hblD), nonhemolytic enterotoxin (nheA, nheB, and nheC), enterotoxin FM (entFM), cytotoxin K (cytK), and cereulide (ces) in 185 B. cereus strains isolated from draft beer, microbrewed beer, pasteurized beer, grape wine, other fruit wine, refined rice wine, traditional Korean pure liquor, and traditional Korean turbid rice wines. A total of 93.0% (172 isolates) of these isolates possessed at least one toxin gene. The nonhemolytic enterotoxin-encoding genes were highly prevalent in the isolates; the detection rate of enterotoxins was 91.4% for nheC, 81.6% for entFM, 62.7% for nheB, 57.3% for nheA, 53.0% for hblC, 48.6% for cytK, 36.8% for hblA, and 36.2% for hblD. Overall, 54.6% and 33.0% of strains carried the integrated Nhe-encoding gene cluster (nheA, nheB, nheC at the same time) and had the Hbl-encoding gene cluster, respectively. The cereulide synthetase gene was detected in only 2.2% of isolates. Toxin gene distribution patterns could be classified into 8 major profile clusters, and the most prevalent profile was the presence of enterotoxin genes only and no emetic toxin genes. Therefore, B. cereus in fermented alcoholic beverages was predominantly of the diarrhoeagenic type. Our results may provide important basic information when considering microbial standards and regulations for B. cereus in related products., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bacillus cereus 905, one of the plant growth-promoting rhizobacteria (PGPRs), is capable of colonizing wheat roots in a large population size. From previous studies, we learned that the sodA2-encoding manganese-containing superoxide dismutase (MnSOD2) is important for B. cereus 905 to survive in wheat rhizosphere. In this investigation, we demonstrated that deletion of the recA gene, which codes for the recombinase A, significantly reduced MnSOD2 expression at both the mRNA and the protein levels. Through comparison with the wild-type, the ∆recA showed a dramatic decrease in cell survival after exposure to 50 μM paraquat or 15 mM H 2 O 2 . Evidence indicated that the recA gene of B. cereus 905 also notably regulated nutrition utilization efficiency, biofilm formation, and swarming motility. The root colonization examination showed that the ∆recA had a 1000- to 2500-fold reduction in colonization on wheat roots, suggesting that RecA plays an indispensable role in effective colonization on wheat roots by B. cereus 905. Taken together, the recA gene positively regulates MnSOD2 production and nutrition utilization and protects B. cereus 905 cells against paraquat and H 2 O 2 . Besides, biofilm formation and swarming motility of B. cereus 905 are promoted by RecA. Finally, RecA significantly contributes to wheat root colonization of B. cereus 905. Our results showed the important role of RecA during physiological processes in B. cereus 905, especially for colonization on wheat roots. Our findings will point out a research direction to study the colonization mechanisms of B. cereus 905 in the future and provide potential effective strategy to enhance the biocontrol efficacy of PGPR strains. KEY POINTS : • RecA plays an indispensable role in root colonization of B. cereus.

Bacillus cereus (B. cereus) is widespread in nature and considered an important foodborne pathogen, which can lead to emetic syndrome and diarrheal illness. Therefore, appropriate detection methods are needed to effectively monitor this pathogenic bacterium. Competitive annealing mediated isothermal amplification (CAMP) is a novel nucleic-acid-based detection technology that amplifies DNA with high sensitivity and specificity under isothermal conditions. The aim of this study was to develop a real-time CAMP assay for the rapid and simple detection of B. cereus in milk. In this system, a pair of primers was designed to specifically target the entFM gene of B. cereus. Compared with the conventional PCR method, the CAMP assay has higher sensitivity, the same specificity and shorter detection time. The detection limits of the CAMP assay for pure bacterial cultures and artificially contaminated milk samples were all 59 CFU mL -1 . And this detection method showed a wide linear range (from 5.9 × 10 5 to 59 CFU mL -1 ) and satisfactory recovery values (from 75.76% to 106.78%). These results indicate that the developed CAMP assay is a potentially useful method for the detection of B. cereus in milk.

2,6-Dichloro-p-hydroquinone (DiCHQ) aromatic-ring cleavage by DiCHQ 1,2-dioxygenase (CpsA) is very crucial for complete transformation of pentachlorophenol (PCP) to 2-chloromaleylacetate in Bacillus cereus AOA-CPS&#95;1 (BcAOA). The 978 bp gene (cpsA) was detected and amplified in the genome of BcAOA; cloned, overexpressed and purified to homogeneity. CpsA showed a single ≅36.9 kDa protein band on SDS-PAGE and exhibited optimum activity at 30 °C and pH 9.0. CpsA was stable between 20 °C and 40 °C, and also retained about 90% of its activity at 60 °C for 120 min. The enzyme retained about 90% activity between pH 9.0 and 11.5 and 60% activity at pH 13.0. CpsA was found to be Fe 2+ dependent as about 90% increased activity was observed in the presence of FeSO 4 . CpsA showed apparent v max , K m , k cat and k cat /K m of 27.77 ± 0.9 μMs -1 , 0.990 ± 0.03 mM, 4.20 ± 0.04 s -1 and 4.24 ± 0.03 s -1  mM -1 , respectively at pH 9.0. Analysis of the reaction products via GC-MS confirmed 2-chloromaleylacetate as the ring-cleavage product. CpsA 3D structure revealed a conserved 2-His-1-carboxylate facial triad motif (His 9, His 244 and Thr 11), with Fe 3+ at the centre. Findings from this study provide new insights into the involvement of this enzyme in PCP degradation and suggests alternate possible mechanism of ring-cleavage by dioxygenases., Competing Interests: Declaration of competing interest All the authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Bacillus cereus is a foodborne pathogen commonly found in nature and food and can cause food spoilage and health issues. Although the prevalence of B. cereus in foods has been reported worldwide, the extent of contamination in edible fungi, which has become increasingly popular as traditional or functional food, is largely unknown. Here we investigated the prevalence, toxin genes' distribution, antibiotic resistance, and genetic diversity of B. cereus isolated from edible fungi in China., Results: Six hundred and ninety-nine edible fungi samples were collected across China, with 198 (28.3%) samples found to be contaminated by B. cereus, with an average contamination level of 55.4 most probable number (MPN)/g. Two hundred and forty-seven B. cereus strains were isolated from the contaminated samples. Seven enterotoxin genes and one cereulide synthetase gene were detected. The detection frequencies of all enterotoxin genes were ≥ 80%, whereas the positive rate of the cesB gene in B. cereus was 3%. Most isolates were resistant to penicillins, β-lactam/β-lactamase inhibitor combinations, cephems, and ansamycins, but were susceptible to penems, aminoglycosides, macrolides, ketolide, glycopeptides, quinolones, phenylpropanol, tetracyclines, lincosamides, streptogramins, and nitrofurans. Meanwhile, 99.6% of all isolates displayed multiple antimicrobial resistance to three or more classes of antimicrobials. Using genetic diversity analysis, all isolates were defined in 171 sequence types (STs), of which 83 isolates were assigned to 78 new STs., Conclusions: This study provides large-scale insight into the prevalence and potential risk of B. cereus in edible fungi in China. Approximately one-third of the samples were contaminated with B. cereus, and almost all isolates showed multiple antimicrobial resistance. Detection frequencies of all seven enterotoxin genes were equal to or more than 80%. These new findings may indicate a need for proper pre-/post-processing of edible fungi to eliminate B. cereus, thereby preventing the potential risk to public health.

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

YmdB, which can regulate biofilm formation independently, has been reported to exist in Bacillus subtilis. The B. cereus 0-9 genome also encodes a YmdB-like protein, which has measureable phosphodiesterase activity, and 72.35% sequence identity to YmdB protein of B. subtilis 168. In this work, we studied the function of YmdB protein and its encoding gene, ymdB, in B. cereus 0-9. Our results indicated that YmdB protein is critical for the biofilm formation of B. cereus 0-9. In ΔymdB mutant, the transcriptional levels of sinR and hag were up-regulated, and those of genes closely related to biofilm formation, such as sipW, tasA and calY, were down-regulated. Deletion of ymdB gene stimulates the swarming motility of B. cereus 0-9, and enhances it to travel outward, but reduces its ability to form complex spatial structures on the solid surface of MSgg plates. Hence, it is considered that YmdB plays a key role in biofilm formation, and this effect is likely achieved through the function of repressor SinR in B. cereus 0-9. Furthermore, by comparing the amino acid sequences of YmdB by Basic Local Alignment Search Tool (BLAST) in Genebank, we found that YmdB homologues are present in a variety of bacteria (Including Gram-negative bacteria) except B. subtilis and B. cereus. All these bacteria come at different evolutionary distances and belong to different genera. Therefore, we believe that YmdB exists in many types of bacteria and plays an important role in the stress-resistance of bacteria to adapt to the environment. These results can help us to further understand the biocontrol characteristics of B. cereus 0-9.

The elimination of spore-forming bacteria is not guaranteed by current pasteurisation processes and is a challenging problem for the dairy industry. Given that Bacillus cereus sensu lato (B. cereus group) is an important foodborne pathogen and spoiler in the dairy industry, this study aimed at evaluating the prevalence and characteristics of B. cereus group in raw and pasteurised milk samples collected in Victoria, Australia. Isolated B. cereus group were tested for antimicrobial susceptibility, biofilm formation and virulence properties. Genetic diversity was assessed using ERIC-PCR. Proteomic profiling using MALDI-TOF MS and chemical profiling using Fourier-transform infrared (FTIR) spectroscopy were also applied for clustering of the isolates. Results showed 42.3% of milk samples contained B. cereus group, with a higher contamination level for pasteurised milk. Virulence studies identified genes nheA, nheB, hblA and nheC in most isolates and cyk gene in 46% of all isolates. Antimicrobial susceptibility testing showed a high prevalence of resistance towards ampicillin, ceftriaxone and penicillin. The biofilm-forming capacity of our isolates showed that most (53.7%) had the ability to form a biofilm. Genetic profiling using ERIC-PCR placed most B. cereus group isolates from pasteurised milk in the same cluster, indicating that they probably originated from a similar source. Raw milk isolates showed greater diversity indicating various sources. FTIR spectroscopy showed high agreement with genetic profiling. In contrast, low agreement between proteomic (MALDI-TOF MS) and genetic typing was observed. The present study showed that the FTIR spectroscopy could be adopted as a rapid tool for the typing of B. cereus group. Overall, the virulence and antimicrobial resistance characteristics, together with the ability of isolates to produce biofilm, indicate the importance of B. cereus group in the Australian dairy industry.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) play a pivotal role in the production of bioactive natural products, such as antibiotics and cytotoxins. Despite biomedical and pharmaceutical importance, the molecular mechanisms and architectures of these multimodular enzyme complexes are not fully understood. Here, we report on an ABC transporter that forms a vital part of the nonribosomal peptide biosynthetic machinery. Emetic Bacillus cereus produces the highly potent, mitochondrial active nonribosomal depsipeptide cereulide, synthesized by the NRPS Ces. The ces gene locus includes, next to the structural cesAB genes, a putative ABC transporter, designated cesCD Our study demonstrates that tethering of CesAB synthetase to the cell membrane by CesCD is critical for peptide assembly. In vivo studies revealed that CesAB colocalizes with CesCD on the cell membrane, suggesting direct involvement of this ABC transporter in the biosynthesis of a nonribosomal peptide. Mutation of cesCD , disrupting the assembly of the CesCD complex, resulted in decreased interaction with CesAB and, as a consequence, negatively affected cereulide biosynthesis. Specific domains within CesAB synthetase interacting with CesC were identified. Furthermore, we demonstrated that the structurally similar BerAB transporter from Bacillus thuringiensis complements CesCD function in cereulide biosynthesis, suggesting that the direct involvement of ABC transporter in secondary metabolite biosynthesis could be a widespread mechanism. In summary, our study revealed a novel, noncanonical function for ABC transporter, which is essential for megaenzyme functionality of NRPS. The new insights into natural product biosynthesis gained may facilitate the discovery of new metabolites with bioactive potential. IMPORTANCE This study revealed a novel, potentially conserved mechanism involved in the biosynthesis of microbial natural products, exemplified by the mitochondrial active depsipeptide cereulide. Similar to other bioactive substances, such as the last-resort antibiotics vancomycin and daptomycin, the antitumor drug cryptophycin or the cholesterol-lowering agent lovastatin, cereulide is synthesized nonribosomally by multienzyme machinery, requiring the concerted actions of multiple proteins to ensure correct product assembly. Given the importance of microbial secondary metabolites in human and veterinary medicine, it is critical to understand how these processes are orchestrated within the host cells. By revealing that tethering of a biosynthetic enzyme to the cell membrane by an ABC transporter is essential for nonribosomal peptide production, our study provides novel insights into synthesis of microbial secondary metabolites, which could contribute to isolation of novel compounds from cryptic secondary metabolite clusters or improve the yield of produced pharmaceuticals., (Copyright © 2020 Gacek-Matthews et al.)

The emergence of B. cereus as an opportunistic food-borne pathogen has intensified the need to distinguish strains of public health concern. The heterogeneity of the diseases associated with B. cereus infections emphasizes the versatility of these bacteria strains to colonize their host. Nevertheless, the molecular basis of these differences remains unclear. Several toxins are involved in virulence, particularly in gastrointestinal disorders, but there are currently no biological markers able to differentiate pathogenic from harmless strains. We have previously shown that CwpFM is a cell wall peptidase involved in B. cereus virulence. Here, we report a sequence/structure/function characterization of 39 CwpFM sequences, chosen from a collection of B. cereus with diverse virulence phenotypes, from harmless to highly pathogenic strains. CwpFM is homology-modeled in silico as an exported papain-like endopeptidase, with an N-terminal end composed of three successive bacterial Src Homology 3 domains (SH3b 1-3 ) likely to control protein-protein interactions in signaling pathways, and a C-terminal end that contains a catalytic NLPC&#95;P60 domain primed to form a competent active site. We confirmed in vitro that CwpFM is an endopeptidase with a moderate peptidoglycan hydrolase activity. Remarkably, CwpFMs from pathogenic strains harbor a specific stretch of twenty residues intrinsically disordered, inserted between the SH3b 3 and the catalytic NLPC&#95;P60 domain. This strongly suggests this linker as a marker of differentiation between B. cereus strains. We believe that our findings improve our understanding of the pathogenicity of B. cereus while advancing both clinical diagnosis and food safety.

DNA glycosylase is responsible for repairing DNA damage to maintain the genome stability and integrity. However, how glycosylase can efficiently and accurately recognize DNA lesions across the enormous DNA genome remains elusive. It has been hypothesized that glycosylase translocates along the DNA by alternating between a fast but low-accuracy diffusion mode and a slow but high-accuracy mode when searching for DNA lesions. However, the slow mode has not been successfully characterized due to the limitation in the spatial and temporal resolutions of current experimental techniques. Using a newly developed scanning fluorescence resonance energy transfer (FRET)-fluorescence correlation spectroscopy (FCS) platform, we were able to observe both slow and fast modes of glycosylase AlkD translocating on double-stranded DNA (dsDNA), reaching the temporal resolution of microsecond and spatial resolution of subnanometer. The underlying molecular mechanism of the slow mode was further elucidated by Markov state model built from extensive all-atom molecular dynamics simulations. We found that in the slow mode, AlkD follows an asymmetric diffusion pathway, i.e., rotation followed by translation. Furthermore, the essential role of Y27 in AlkD diffusion dynamics was identified both experimentally and computationally. Our results provided mechanistic insights on how conformational dynamics of AlkD-dsDNA complex coordinate different diffusion modes to accomplish the search for DNA lesions with high efficiency and accuracy. We anticipate that the mechanism adopted by AlkD to search for DNA lesions could be a general one utilized by other glycosylases and DNA binding proteins., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Bacillus cereus is a well-known foodborne pathogen capable of causing two types of gastrointestinal diseases, diarrhoea and emesis. It is of particular concern for the food industry causing food safety issues, due to the formation of spores, biofilms and diarrhoea and/or emetic toxins. This review reveals the possible link between two food safety issues - toxins and spores - and the role of biofilms. The review highlights genetic determinants that are involved in sporulation, toxin production and biofilm formation based on current research, and evidence showing the possible correlation of spore, toxin and biofilm formation of B. cereus. This is the first review highlighting the potential relationship between toxin production and biofilm formation in B. cereus., Competing Interests: Declaration of competing interest This manuscript is original, and no part of the manuscript has been published before. There are no conflicts of interest to disclose., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Two strains of 64 endophytic bacteria, Bacillus cereus Si-Ps1 and Pseudomonas azotoformans La-Pot3-3, isolated from Citrus sinensis and C. sinensis var. Thomson's leaves, respectively, reduced N-acyl homoserine-based quorum sensing in bioindicator strain Pseudomonas syringae pv. syringae (Pss) B728a and the biofilm production and swarming motility of field isolate Pss 3289. A homolog of aiiA gene encoding an AHL-lactonase was found in B. cereus (Si-Ps1), suggesting that this isolate can degrade the quorum-sensing signal molecules of Pss 3289. The crude extract of endophytic bacterium, B. cereus (Si-Ps1), inhibited Pss 3289 biofilm formation after 48 and 96 h by 55% and 58%, respectively. Similar reductions in biofilm formation were conferred by crude extracts of P. azotoformans (La-Pot3-3). Correspondingly, the number of planktonic cells in cultures treated with these extracts was higher than in control cultures, indicating a direct effect on biofilm formation and not on cell growth. In greenhouse assays, the virulence of Pss 3289 to different citrus cultivars was decreased when coinoculated with these endophytic bacteria., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Both Gram-positive and Gram-negative bacteria can take up exogenous DNA when they are in a competent state either naturally or artificially. However, the thick peptidoglycan layer in Gram-positive bacteria's cell wall is considered as a possible barrier to DNA uptake. In the present work, two transformation techniques have been evaluated in assessing the protocol's ability to introduce foreign DNA, pBBRGFP-45 plasmid which harbors kanamycin resistance and green fluorescent protein (GFP) genes into a Gram-positive bacterium, Bacillus cereus EB2. B. cereus EB2 is an endophytic bacterium, isolated from oil palm roots. A Gram-negative bacterium, Pseudomonas aeruginosa EB35 was used as a control sample for both transformation protocols. The cells were made competent using respective chemical treatment to Gram-positive and Gram-negative bacteria, and kanamycin concentration in the selective medium was also optimized. Preliminary findings using qualitative analysis of colony polymerase chain reaction (PCR)-GFP indicated that the putative positive transformants for B. cereus EB2 were acquired using the second transformation protocol. The positive transformants were then verified using molecular techniques such as observation of putative colonies on specific media under UV light, plasmid extraction, and validation analyses, followed by fluorescence microscopy. Conversely, both transformation protocols were relatively effective for introduction of plasmid DNA into P. aeruginosa EB35. Therefore, this finding demonstrated the potential of chemically prepared competent cells and the crucial step of heat-shock in foreign DNA transformation process of Gram-positive bacterium namely B. cereus was required for successful transformation.

Cereulide-producing members of Bacillus cereus sensu lato group III (also known as emetic B. cereus ) possess cereulide synthetase, a plasmid-encoded, nonribosomal peptide synthetase encoded by the ces gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by emetic B. cereus has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characterize group III B. cereus sensu lato genomes which possess ces ( ces positive) alongside their closely related, ces -negative counterparts (i) to assess the genomic diversity encompassed by emetic B. cereus and (ii) to identify potential ces loss and/or gain events within the evolutionary history of the high-risk and medically relevant sequence type (ST) 26 lineage often associated with emetic foodborne illness. Using all publicly available ces -positive group III B. cereus sensu lato genomes and the ces -negative genomes interspersed among them ( n  = 159), we show that emetic B. cereus is not clonal; rather, multiple lineages within group III harbor cereulide-producing strains, all of which share an ancestor incapable of producing cereulide (posterior probability = 0.86 to 0.89). Members of ST 26 share an ancestor that existed circa 1748 (95% highest posterior density [HPD] interval = 1246.89 to 1915.64) and first acquired the ability to produce cereulide before 1876 (95% HPD = 1641.43 to 1946.70). Within ST 26 alone, two subsequent ces gain events were observed, as well as three ces loss events, including among isolates responsible for B. cereus sensu lato toxicoinfection (i.e., "diarrheal" illness). IMPORTANCE B. cereus is responsible for thousands of cases of foodborne disease each year worldwide, causing two distinct forms of illness: (i) intoxication via cereulide (i.e., emetic syndrome) or (ii) toxicoinfection via multiple enterotoxins (i.e., diarrheal syndrome). Here, we show that emetic B. cereus is not a clonal, homogenous unit that resulted from a single cereulide synthetase gain event followed by subsequent proliferation; rather, cereulide synthetase acquisition and loss is a dynamic, ongoing process that occurs across lineages, allowing some group III B. cereus sensu lato populations to oscillate between diarrheal and emetic foodborne pathogens over the course of their evolutionary histories. We also highlight the care that must be taken when selecting a reference genome for whole-genome sequencing-based investigation of emetic B. cereus sensu lato outbreaks, since some reference genome selections can lead to a confounding loss of resolution and potentially hinder epidemiological investigations., (Copyright © 2020 Carroll and Wiedmann.)

The aim of this study was to optimize the culture conditions for production, recovery and characterization of polyhydroxyalkanoate (PHA) from potential Bacillus cereus strain BNPI-92. Cost effective carbon sources such as crude oil (CO), rice bran (RB), sugar cane molasses (SCM) and wheat bran (WB) were evaluated for PHA production using the isolate. Higher cell dry weight (CDW) (g/L), PHA concentration (P conc.) (g/L) PHA contents (PC) (%) and PHA synthesis rate (PSR) were obtained from Glucose (60.67% w/v) as a carbon source at 37 °C and pH 7 using Taguchi DOE methods. The polymer was characterized by FTIR and its functional groups (C=O) (1719.41 cm -1 wavenumber) was a characteristic feature of PHB polymer. For 1 HNMR, signals of methyne (CH) (5.28 ppm), methylene (-CH2-) (2.45 ppm) and methyl (CH3) (1.27 ppm) and copolymers were predicted. Copolymers such as P (3HB-co-3 HV) and P(3HB-co-3HHX) i.e. characteristic feature of PHB were obtained from a sample of glucose, WB, and RB, respectively. XRD pattern also confirmed the presence of PHA. The major compounds obtained from GC/MS analysis of the polymer recovered from the isolate BNP-92 were 2-butenoic acid, methyl ester (8.6%), ethyl cyclopropanecarboxylate (45.99%), 1-Undecanol (10.18%) which corresponds to and have produced from PHA after thermal degradation., (Copyright © 2019. Published by Elsevier B.V.)

A multiplex polymerase chain reaction (mPCR) with propidium monoazide (PMA) and internal amplification control (IAC) for the simultaneous detection of waterborne pathogens Salmonella spp., Pseudomonas aeruginosa, Bacillus cereus, and Escherichia coli O157:H7, was developed. This PMA-IAC-mPCR assay used four new specific primers based on the genes for invA, ecfX, cesB, and fliC, respectively. A 16S rRNA primer was chosen for IAC to eliminate false negative results. The photosensitive dye, propidium monoazide (PMA) was used to exclude signals from dead bacteria that could lead to false positive results. In pure culture, the limits of detection (LOD) were 10 1 CFU/ml for P. aeruginosa, 10 2 CFU/ml for both Salmonella spp. and E. coli O157:H7, and 10 3 CFU/ml for B. cereus, respectively. In addition, with a 6-8 h enrichment of all four bacteria that were combined in a mixture that was spiked in water sample matrix, the LOD was 3 CFU/ml for Salmonella spp., 7 CFU/ml for E. coli O157:H7, 10 CFU/ml for B. cereus and 2 CFU/ml for P. aeruginosa. This PMA-IAC-mPCR assay holds potential for application in the multiplex assay of waterborne pathogens.

Bacillus cereus can survive in the form of spores for prolonged periods posing a serious problem for the manufacture of safe shelf-stable foods of optimal quality. Our study aims at increasing knowledge of B. cereus spores focusing primarily on germination mechanisms to develop novel milder food preservation strategies. Major features of B. cereus spores are a core with the genetic material encased by multiple protective layers, an important one being the spores' inner membrane (IM), the location of many important germination proteins. To study mechanisms involved in germination of B. cereus spores, we have examined the organization of germinant receptors (GRs) in spores' IM. Previous studies have indicated that in spores of B. cereus ATCC 14579 the L-alanine responsive GR, GerR, plays a major role in the germination process. In our study, the location of the GerR GR subunit, GerRB, in spores was examined as a C-terminal SGFP2 fusion protein expressed under the control of the gerR operon's promoter. Our results showed that: i) the fluorescence maxima and integrated intensity in spores with plasmid-borne expression of GerRB-SGFP2 were significantly higher than in wild-type spores; ii) western blot analysis confirmed the expression of the GerRB-SGFP2 fusion protein in spores; and iii) fluorescence microscopy visualized GerRB-SGFP2 specific bright foci in ~30% of individual dormant spores if only GerRB-SGFP2 was expressed, but, noticeably, in ~85% of spores upon co-expression with GerRA and GerRC. Our data corroborates the notion that co-expression of GR subunits improves their stability. Finally, all spores displayed bright fluorescent foci upon expression of GerD-mScarlet-I under the control of the gerD promoter. We termed all fluorescent foci observed germinosomes, the term used for the IM foci of GRs in Bacillus subtilis spores. Our data are the first evidence for the existence of germinosomes in B. cereus spores.

Bacillus cereus commonly causes catheter-related bloodstream infections (BSIs) in hospital settings, and occasionally occurs fatal central nervous system (CNS) complications. B. cereus harboring Ba813, a specific chromosomal marker of Bacillus anthracis, has been found in patients with severe infection and nosocomial BSI. However, the bacteriological profile and clinical feature of Ba813 (+) B. cereus are unclear. Fifty-three patients with B. cereus BSI were examined. Isolates were evaluated for Ba813, B. anthracis-related and food poisoning-related virulence, multilocus sequencing typing, and biofilm formation. Patients' clinical records were reviewed retrospectively. The 53 isolates were comprised of 29 different sequence types in two distinct clades. Seventeen of the 53 (32%) B. cereus isolates including five sequence types possessed Ba813 and were classified into Clade-1/Cereus-III lineage which is most closely related to Anthracis lineage. No B. cereus possessed B. anthracis-related virulence genes. Ba813 (+) strains showed a lower prevalence of enterotoxin genes than Clade-2 strains (n = 4), but no difference from Clade-1. Ba813 (+) strains showed significantly lower biofilm formation than Clade-1/non-Cereus-III (n = 22) and Clade-2 strains, respectively. Compared to Clade-1/non-Cereus-III and Clade-2 B. cereus, Ba813 (+) strains were isolated more frequently from elderly patients, patients with indwelling central venous catheter rather than peripheral venous catheter, and patients who remained in the hospital for longer before BSI onset. No significant differences in disease severity or mortality were observed. Though two of the ten Ba813 (-) strains in Clade-1/Cereus III were isolated from the patients with CNS complication, no significant difference was observed in the bacterial profile and clinical characteristics among Clade-1/Cereus III strains. In conclusion, our report suggested that Ba813-harboring B. cereus strains, genetically closely related to B. anthracis, were abundant among B. cereus strains in the hospital setting, and might cause catheter-related nosocomial BSI. However, it did not affect the clinical outcomes., Competing Interests: The authors have declared that no competing interests exist.

Introduction. Bacillus cereus harbouring Ba813 , a specific chromosomal marker of Bacillus anthtacis, is found in patients with severe manifestations and causes nosocomial outbreaks. Aim. We assessed the genetic characteristics and virulence of Ba813 (+) B. cereus in a hospital setting. Methodology. Three neutropenic patients with haematological malignancy developed B. cereus bacteraemia within a short period. Fifteen B. cereus were isolated from different sites in a haematology ward. A total of 18 isolates were evaluated for Ba813 - and B. anthracis -related virulence, food poisoning-related virulence, genetic diversity, bacteria motility and biofilm formation. Results. Ba813 (+) B. cereus was detected in 33 % (1/3) of patients and 66 % (9/15) of the hospital environment. The 18 strains were divided into 2 major clusters (clade 1 and clade 2), and 14 strains were classified into clade 1. All Ba813 (+) strains, including four sequence types, were classified into clade 1/the cereus III lineage, which is most closely related to the anthracis lineage. Two strains belonging to clade 1/non-cereus III carried the B. anthracis -associated cap gene, but not Ba813. B. cereus , including Ba813 (+) strains, had significantly lower prevalence of enterotoxin genes than clade 2 strains. In clade 1, B. cereus , Ba813 (+) strains showed significantly higher swimming motility and biofilm formation ability than Ba813 (-) strains. Conclusion. Ba813 (+) B. cereus , which are genetically closely related to B. anthracis , were abundant in a haematological ward. Ba813 (+) B. cereus with high motility and biofilm formation abilities may spread easily in hospital environments, and could become a hospital-acquired infection.

Toxin-antitoxin (TA) systems are prevalent in bacteria and are known to regulate cellular growth in response to stress. As various functions related to TA systems have been revealed, the importance of TA systems are rapidly emerging. Here, we present the crystal structure of putative mRNA interferase BC0266 and report it as a type II toxin MazF. The MazF toxin is a ribonuclease activated upon and during stressful conditions, in which it cleaves mRNA in a sequence-specific, ribosome-independent manner. Its prolonged activity causes toxic consequences to the bacteria which, in turn, may lead to bacterial death. In this study, we conducted structural and functional investigations of Bacillus cereus MazF and present the first toxin structure in the TA system of B. cereus . Specifically, B. cereus MazF adopts a PemK-like fold and also has an RNA substrate-recognizing loop, which is clearly observed in the high-resolution structure. Key residues of B. cereus MazF involved in the catalytic activity are also proposed, and in vitro assay together with mutational studies affirm the ribonucleic activity and the active sites essential for its cellular toxicity.

Bacillus cereus (B. cereus) is widely distributed in the environment. It is one of the most common opportunistic food-borne pathogens associated with food poisoning, not only being majorly reported to cause fatal infections of the gastrointestinal tract, but also responsible for abdominal distress and vomiting. The current study was undertaken to evaluate the biological characteristics and the genetic evolution of B. cereus isolated from infected organs of dead Elaphurus davidianus (E. davidianus). B. cereus was characterized through antibiotic sensitivity tests, mouse lethality assay, whole genome sequencing analysis, and genome annotation. The results revealed that the isolated B. cereus strain was highly resistant to rifampicin, lincomycin, sulfamethoxazole, erythromycin, and ampicillin, with a high pathogenicity phenotype. KEGG annotation revealed that "metabolic pathways" had the largest number of unigenes, followed by "biosynthesis of secondary metabolites" and "biosynthesis of antibiotics". GO analysis resulted in 8039 unigenes categorized. Meanwhile, 54,779 unigenes were annotated and grouped into 23 categories based on COG functional classifications. Moreover, one gene (codY) was found to be related to the host in conformity with the analysis done on PHI-base. Other tests led to the identification of 16 B. cereus virulence factor genes and five resistance types, with potential resistance against bacitracin, penicillin, and fosfomycin. We isolated a highly drug-resistant and pathogenic B. cereus strain from E. davidianus, showing that a variety of antimicrobial drugs should be avoided in clinical treatments. Furthermore, to the best of our knowledge, this is the first study to report whole genome sequencing of a emergence of food-borne B. cereus strain isolated from E. davidianus deer; it will be helpful to extensively investigate the genetic and molecular mechanisms of drug resistance and pathogenesis about B. cereus in both humans and animals., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Members of the Bacillus cereus group are common contaminants of vegetables. One potential source of contamination is the application of B. thuringiensis based biopesticides. Although evidence of the presence of biopesticidal strains on food products is scarce, this information is essential for assessing potential risks associated with the application of these biopesticides. In order to contribute to knowledge about the presence of biopesticidal B. thuringiensis strains in foodstuffs, we investigated the occurrence of B. thuringiensis on tomatoes and bell pepper. We analyzed 99 samples of fresh bell pepper for B. cereus group members, while 426 samples of tomatoes were tested by the competent food control laboratories of the federal states in Germany. The isolates recovered from these samples were further characterized in terms of their capability to produce parasporal crystals as well as enterotoxins. A possible correlation between the B. thuringiensis isolates and biopesticidal strains was investigated by multilocus sequence typing (MLST) and whole genome Single Nucleotide Polymorphism (wgSNP) analyses. The prevalence of B. cereus group members was 41% for bell pepper and 28% for tomato samples. Isolates recovered from these samples were dominated by B. thuringiensis (93% and 99%, respectively). All B. thuringiensis isolates carried the enterotoxin genes nheA, hblD and cytK-2. In a subset of 83 B. thuringiensis isolates analyzed by MLST, 99% of the isolates matched the sequence types (ST) 8 and 15, which are also shared by the biopesticidal strains B. thuringiensis kurstaki ABTS-351 and B. thuringiensis aizawai ABTS-1857. Of the 82 isolates assigned to ST 8 or ST 15, a selection of 42 isolates was further characterized by wgSNP analysis. Of these, seven isolates differed from strain ABTS-351 by ≤4 core SNPs and 18 isolates differed from strain ABTS-1857 by ≤2 core SNPs, indicating a relationship of these isolates with the respective biopesticidal strain. These isolates originated from samples with maximum colony counts of 5.3 × 10 3  cfu/g for bell pepper and 1.0 × 10 5  cfu/g for tomatoes., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study was conducted to reveal the prevalence, molecular characterization, and antibiotic susceptibility of Bacillus cereus isolated from dairy products including powdered infant formula, raw milk, pasteurized milk, ultra-high-temperature milk, and cheese. Five hundred samples collected from 5 provinces in China were analyzed in overall experiments. Multilocus sequence typing, distribution of toxin genes, and antibiotic susceptibility of the isolates were analyzed. Fifty-four B. cereus strains were detected; of these, 13 isolates (26%) were from raw milk, 12 isolates (12%) from pasteurized milk, 10 isolates (10%) from cheese, 12 isolates (8%) from ultra-high-temperature milk, and 7 isolates (7%) from powdered infant formula. These isolates were divided into 24 sequence types (ST); among them, ST24, ST26, ST82, ST142, ST377, ST857, and ST1046 were the main dominant ST. The results of detection of toxin genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, entFM, bceT, hlyII, and cesB) showed that 94.4% isolates carried nheABC genes, whereas only 11.1% of the isolates contained the hblACD gene cluster. In addition, detection rates of cytK, bceT, entFM, hlyII, and cesB genes were 75.9, 77.8, 85.2, 53.7, and 11.1%, respectively. The antibiotic susceptibility test indicated that most of B. cereus isolates were resistant to ampicillin, penicillin, cefepime, cephalothin, and oxacillin, and were susceptible to gentamicin, chloramphenicol, imipenem, tetracycline, ciprofloxacin, trimethoprim-sulfamethoxazole, erythromycin, kanamycin, and cefotetan. Therefore, this study revealed the prevalence and characteristics of B. cereus isolated from dairy products in China, indicating the potential risk and contributing to the effective prevention and control of this pathogen., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Bacillus cereus sensu lato (s.l.) is a group of bacteria commonly found in diverse environments, including foods, with potential to cause emesis and diarrhea. In Colombia, it is one of the main foodborne pathogens. The aim of this study was to determine the genomic and toxigenic heterogeneity of B. cereus s.l. isolated from ready-to-eat foods and powdered milk collected in day care centers of Medellin, Colombia. Of 112 B. cereus s.l. isolates obtained, 94% were β-hemolytic. Toxigenic heterogeneity was established by the presence of nheABC , hblCDAB , cytK2 , entFM , and cesB toxigenic genes. The nheABC operon and entFM gene were most frequently detected in the isolates, whereas the cesB gene was not found. According to the toxin genes content, nine toxigenic profiles were identified. A 44% of isolates had profiles with all genes for nonhemolytic enterotoxin, hemolysin BL, and enterotoxin FM production (profiles II and IV). Pulsed-field gel electrophoresis analysis indicated a high genomic heterogeneity among the B. cereus s.l. , with 68 isolates grouping into 16 clusters and 33 placed separately in the dendrogram. This study provides useful information on the safety of ready-to-eat foods and powdered milk in day care centers where children, a susceptible population, are exposed and it should incentive for more studies to understand the distribution of different toxin-encoding genes among B. cereus s.l. isolates, enabling detailed risk assessment.

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria., Competing Interests: The authors have declared that no competing interests exist.

Bacterial endospores are exposed to a broad variety of sublethal and lethal stresses in the food production chain. Generally, these stresses will not completely eliminate the existing spore populations, and thus constitute a selection pressure on the spores. One stress that is frequently used in the food production chains to disinfect (food) contact surfaces is UV-C. At a wavelength of 254 nm, UV-C has germicidal properties. The aim of this research is to investigate the impact of UV-C stress on the evolution of endospore recalcitrance and germination in B. cereus. A directed evolution experiment was set up in which B. cereus was repeatedly subjected to a cycle of sporulation, sporicidal UV-C treatment, germination and outgrowth. We show here that three independent lineages of UV-C cycled B. cereus spores reproducibly acquired a 30-fold or higher increase in UV-C resistance at 164 mJ/cm 2 . Surprisingly, the UV-C resistant spores of the clones isolated from each of the lineages also became significantly more sensitive to wet heat as a normally non-lethal heat treatment at 70 °C for 15 min resulted in an average 1.8 log cfu/mL reduction. From time-lapse phase contrast microscopy analysis, UV-C resistant mutant spores also showed a distinctive heterogeneity in refractility and a severe germination defect compared to the wild type. However, UV-C resistance of the corresponding vegetative cells was not altered. In conclusion, this work shows that UV-C resistance of endospores is an adaptive trait that can readily be improved, although at an apparent cost for heat resistance and germination efficiency. As such, these results provide novel insights in the evolvability of, and correlation between, some endospore properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Denaturation bubble-mediated strand exchange amplification (SEA) is a novel, rapid isothermal nucleic acid amplification has been applied for point-of-care molecular diagnostic in food safety, meat adulteration, forest disease and animal disease. Nevertheless, the absence of specialized strategy for SEA primers design led to long-time of primer screening progress before SEA reaction execution, which would largely increase the time consuming when SEA is utilized for detecting other new targets. In this present work, we investigated the impact of the following primers' attributes on SEA efficiency, including Tm value, 3' end G/C content, self-complementary and 3' complementary, according to which we demonstrated that optimal Tm value and reaction temperature were all 61 °C, while 3'-terminal nucleotide should be G/C, as the SEA reaction induced by the primers possessing these attributes exhibited significantly lower threshold time (Tt) value. Moreover, self-complementary and 3' complementary of primers should be avoided. Besides, we also discussed the consideration priority order of these factors, which was self-complementary and 3' complementary, Tm value and 3' end G/C content in turn. Because the SEA primer design strategy is first presented, our work will greatly promote the application of SEA in point-of-care test., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Salmonella enterica, Listeria monocytogenes, Shigella flexneri, Escherichia coli O157:H7, Vibrio parahaemolyticus, Staphylococcus aureus, Vibrio cholerae, Clostridium botulinum type A, Bacillus cereus, Clostridium perfringens Alpha toxin, and Yersinia enterocolitica are 11 common foodborne pathogens. Traditional bacterial culture methods for detecting pathogens are time-consuming and labor-intensive. Multiplex PCR technology, which can detect multiple targets in a single tube, has been increasingly applied to microbial detection due to its high specificity, sensitivity, and fast response. This paper is to establish a multiplex PCR technology mediated by a common primer for the detection of these 11 common foodborne pathogens in order to achieve the goal of nondirectional screening for these 11 common foodborne pathogens. The specificity of the established CP-MPCR detection system was first verified by 100 clinical isolates. The sensitivity of the CP-MPCR detection system was then detected by using cultured bacteria preparations and has been confirmed with a high sensitivity of 10 3 to 10 4 CFU/mL, among them, the sensitivity of the CP-MPCR for Vibrio cholerae and S. flexneri can even achieve 10 2 CFU/mL. Sixty anal swab samples collected from Suzhou CDC and 16 enrichment cultured solutions of food samples collected from the Suzhou Food Inspection and Testing Center were tested using the CP-MPCR system. A total of 32 positive results were detected. PRACTICAL APPLICATION: Food poisoning incidents occur frequently around the world, mainly because of the contamination of food by pathogenic bacteria and serious harm to human health. The method provided in this study can detect 11 foodborne pathogens in food, which can effectively prevent the spread of pathogenic microorganisms. At the same time, for the food poisoning incident that has already occurred, this method can be used for diagnosis to find out the cause., (© 2020 Institute of Food Technologists®.)

Replacing agrochemicals with plant growth promoting bacteria (PGPB) may offset some of the environmental impacts of food production. The objectives of this study were to (1) isolate and characterize bacterial strains from tomato rhizosphere, including root, shoot and leaf, (2) select and identify the most promising PGPB strains, (3) verify the phytostimulatory activity and mineral uptake potential of selected strains. Bacterial strains isolated from tomato rhizosphere, were screened for phosphorous (P) solubilization, production of indole acetic acid (IAA), amylase activity, antibiotic resistance, and quick test strip (QTS) for biochemical characterization. The tested strains, positive for all five of these assays were selected for molecular identification and subjected to greenhouse growth trails with tomato and mung bean. Two strains were selected and identified as Bacillus cereus (B. cereus) isolated from rhizosphere and Klebsiella variicola (K. variicola) isolated from root endosphere using 16s rRNA sequences. Both strains produced IAA, gibberellic acid (GA3) and kinetin, however B. cereus showed potential GA3 and IAA production as compared to K. variicola. In tomato, only one growth variable (shoot length) was increased over the control by one of the selected bacterial strains (B. cereus). In mung bean, inoculation with either strain B. cereus or K. variicola increased shoot length and dry weight. Moreover, our results showed that the use of PGPB significantly increased plant growth and Fe, Zn, Ca, Mg, Cu, Na and K contents of plants. It seems that evaluated strains had a higher ability in boosting plant growth and higher yield., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bacterial endophytes are well known inhabitants of living plant system and perform important assignments in maintaining plant growth and health. Currently, limited reports are available on the endophytes of pearl millet (Pennisetum glaucum) reflecting antagonistic and plant growth promoting (PGP) attributes. Therefore, the major objectives of current investigation were to identify antagonistic strains of endophytic Bacillus from pearl millet and further illustrate their PGP capabilities. In this study, 19 endophytic Bacillus strains (EPP5, EPP21, EPP30, EPP32, EPP35, EPP42, EPP49, EPP55, EPP62, EPP65, EPP70, EPP71, EPP74, EPP78, EPP83, EPP86, EPP93, EPP100, and EPP102) displaying antagonistic activity towards Rhizoctonia solani (RS), Sclerotium rolfsii (SR), and Fusarium solani (FS) were isolated from different sections (root, leaf, stem, and root) of pearl millet. Phenotypic (shape, colony, gram staining reaction, endospore formation, and motility) and biochemical features (catalase, oxidase, citrate, gelatinase, urease, Voges Proskauer's, methyl red, indole, and nitrate reduction), along with the similarly comparison of 16S rRNA gene sequence with type strains identified eight antagonistic endophyhtes as B. amyloliquefaciens (EPP35, EPP 42, EPP62, and EPP 102), Bacillus subtilis subsp. subtilis (EPP65), and Bacillus cereus (EPP5, EPP71, and EPP74). The production of indole acetic acid and siderophores was varied among the isolated endophytes. Besides displaying enzymatic activities, these isolates varied in solubilizing capabilities of phosphate, potassium, and zinc. The presence of three antimicrobial peptide genes (ituD, bmyC, and srfA) also confirmed their antifungal nature. Further, single treatment of three promising strains (EPP5, EPP62, and EPP65) offered protection ranging from 35.68 to 45.74% under greenhouse conditions. However, microbial consortium (EPP5+ EPP62 + EPP65) provided the highest protection (71.96%) against root rot and wilt infection with significant increase in plant biomass. Overall, the current study indicated that pearl millet plant harbors various species of endophytic Bacillus that possess excellent biocontrol and growth promotion activities.

The Bacillus cereus group comprises numerous closely related species, including bioterrorism agent B. anthracis , foodborne pathogen B. cereus , and biopesticide B. thuringiensis Differentiating organisms capable of causing illness or death from those used in industry is essential for risk assessment and outbreak preparedness. However, current species definitions facilitate species-phenotype incongruences, particularly when horizontally acquired genes are responsible for a phenotype. Using all publicly available B. cereus group genomes ( n  = 2,231), we show that current species definitions lead to overlapping genomospecies clusters, in which 66.2% of genomes belong to multiple genomospecies at a conventional 95 average nucleotide identity (ANI) genomospecies threshold. A genomospecies threshold of ≈92.5 ANI is shown to reflect a natural gap in genome similarity for the B. cereus group, and medoid genomes identified at this threshold are shown to yield resolvable genomospecies clusters with minimal overlap (six of 2,231 genomes assigned to multiple genomospecies; 0.269%). We thus propose a nomenclatural framework for the B. cereus group which accounts for (i) genomospecies using resolvable genomospecies clusters obtained at ≈92.5 ANI, (ii) established lineages of medical importance using a formal collection of subspecies names, and (iii) heterogeneity of clinically and industrially important phenotypes using a formalized and extended collection of biovar terms. We anticipate that the proposed nomenclature will remain interpretable to clinicians, without sacrificing genomic species definitions, which can in turn aid in pathogen surveillance; early detection of emerging, high-risk genotypes; and outbreak preparedness. IMPORTANCE Historical species definitions for many prokaryotes, including pathogens, have relied on phenotypic characteristics that are inconsistent with genome evolution. This scenario forces microbiologists and clinicians to face a tradeoff between taxonomic rigor and clinical interpretability. Using the Bacillus cereus group as a model, a conceptual framework for the taxonomic delineation of prokaryotes which reconciles genomic definitions of species with clinically and industrially relevant phenotypes is presented. The nomenclatural framework outlined here serves as a model for genomics-based bacterial taxonomy that moves beyond arbitrarily set genomospecies thresholds while maintaining congruence with phenotypes and historically important species names., (Copyright © 2020 Carroll et al.)

The novel anti-fungal cyclic lipopeptide 'Kannurin' and its three structural variants produced by Bacillus cereus AK1 were previously reported from our laboratory. The present study reports unexplored structural variants of Kannurin those have functional benefits. Due to the difference in β-hydroxy fatty acid tail length, they are designated here as Kannurin A (m/z 994.67 ± 0.015), B (m/z 1008.68 ± 0.017), C (m/z 1022.69 ± 0.021), D (m/z 1036.70 ± 0.01), C L (m/z 1040.71 ± 0.02) and D L (m/z 1054.72 ± 0.01). The isoform A (m/z 994.67 ± 0.015) is the shortest cyclic form of Kannurin identified so far. In addition, C L (m/z 1040.71 ± 0.02) and D L (m/z 1054.72 ± 0.01) are the rare natural linear forms. The results of the antimicrobial assays deduced that the difference in lipid tail length of the isoforms contributes tremendous differences in their antimicrobial properties. The isoforms with short lipid tails (A and B) are more selective and potent towards bacteria, whereas the isoforms with long lipid tails (C and D) are more potent against fungi. The molecular dynamics studies and electron microscopic observations supported with circular dichroic spectroscopy analysis showed the structural confirmation and formation of aggregates of Kannurin in solution. The molecular dynamics simulation studies revealed that a single molecule of Kannurin makes enormous intra-molecular interactions and structural re-arrangements to attain stable lowest energy state in solution. When they reach a particular concentration (CMC) especially in aqueous environment, tends to form structural aggregates called 'micelles'. With the structural information and activity relationship described in this study, it is trying to point out the sensitive structural entities that can be modified to improve the efficacy and target specificities of lipopeptide class of antibiotics.

An alkaliphile bacterial strain designated as CH11 was isolated from the sediments of Chilika Lake, Odisha. The isolate showed stupendous growth and production of α-amylase at pH 10.0. Through 16S rRNA gene based molecular technique this isolate was identified as Bacillus cereus strain SP-CH11 having GenBank Accession No. KT992791. Homogenous ~55 kDa extracellular α-amylase was extracted with 241.304, 26.26 and 3.2-fold acceleration in specific activity, purification fold and yield respectively. The alkaline α-amylase AA11 was further characterized. At pH 9.0 the purified enzyme AA11 was highly stable while retaining 88-100% functional viability at temperature range from 35 to 65 °C, confirming its thermostability nature. It showed stability with powdered and liquid detergents at 7 mg/mL and 100-fold dilutions respectively. AA11 efficiently removed the starch stain from cotton fabrics. The findings of this study indicate that the isolate CH11 is a source of novel alkaline α-amylase that has promising application in food and detergent industries., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rhizosphere interactions between microorganisms and plants have great influence on plant health. Bacillus cereus C1L, an induced systemic resistance (ISR)-eliciting rhizobacterium from Lilium formosanum , can protect monocot and dicot plants from disease challenges. To identify the ISR-involved bacterial genes, the systemic protection effect of transposon-tagged mutants of B. cereus C1L against southern corn leaf blight (SCLB) was surveyed, and a mutant of the ptsG gene encoding glucose-specific permease of the phosphotransferase system was severely impaired in the abilities of disease suppression and root colonization. The ptsG mutant lost the preferential utilization of glucose and showed reduction of glucose-assisted growth in minimal medium. A promoter-based reporter assay revealed that ptsG expression could be activated by certain sugar constituents of maize root exudates, among which B. cereus C1L exhibited the highest chemotactic response toward glucose, whereas neither of them could attract the ptsG mutant. Additionally, ptsG deficiency almost completely abolished glucose uptake of B. cereus C1L. Metabolite analysis indicated that the lack of ptsG undermined glucose-induced accumulation of acetoin and 2,3-butanediol in B. cereus C1L, both eliciting maize ISR against SCLB. Pretreatments with B. cereus C1L, ptsG mutant, acetoin, and 2,3-butanediol enhanced defense-related reactive oxygen species accumulation and callose deposition at different levels that were positively correlated to their ISR-eliciting activities. Thus, glucose uptake-mediating ptsG participates in ISR elicitation by endowing B. cereus C1L with the full capacities for root colonization and beneficial glucose metabolite production, providing a clue regarding how ISR-mediating rhizobacteria create a mutually beneficial relationship with various plant species.

Bacterial physiology and adaptation are influenced by the exopolysaccharides (EPS) they produce. These polymers are indispensable for the assembly of the biofilm extracellular matrix in multiple bacterial species. In a previous study, we described the profound gene expression changes leading to biofilm assembly in B. cereus ATCC14579 (CECT148). We found that a genomic region putatively dedicated to the synthesis of a capsular polysaccharide (eps2) was overexpressed in a biofilm cell population compared to in a planktonic population, while we detected no change in the transcript abundance from another genomic region (eps1) also likely to be involved in polysaccharide production. Preliminary biofilm assays suggested a mild role for the products of the eps2 region in biofilm formation and no function for the products of the eps1 region. The aim of this work was to better define the roles of these two regions in B. cereus multicellularity. We demonstrate that the eps2 region is indeed involved in bacterial adhesion to surfaces, cell-to-cell interaction, cellular aggregation and biofilm formation, while the eps1 region appears to be involved in a kind of social bacterial motility. Consistent with these results, we further demonstrate using bacterial-host cell interaction experiments that the eps2 region is more relevant to the adhesion to human epithelial cells and the zebrafish intestine, suggesting that this region encodes a bacterial factor that may potentiate gut colonization and enhance pathogenicity against humans.

Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus) are major human food-borne pathogens that may produce a variety of toxins and cause diarrhea, food poisoning, and even death. In order to monitor and prevent the spread of these pathogens, a multiplex loop-mediated isothermal amplification (multi-LAMP) assay was developed to simultaneously and rapidly detect B. cereus and S. aureus. The sensitivity and specificity of the loop-mediated isothermal amplification (LAMP) reactions were determined via electrophoresis. The multi-LAMP showed 100% inclusivity and exclusivity, the sensitivity was 10 fg/μL and was 10 times more sensitive than that of polymerase chain reaction (PCR), the results were consistent with those of conventional PCR assay, and the entire assay should be finished within 40 min. This multi-LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples and food samples. To our knowledge, this is the first study to report the application of multi-LAMP to detect B. cereus and S. aureus.