Impact of Bacillus cereus SPB-10 on Growth Promotion of Wheat (Triticum aestivum L.) Under Arsenic-Contaminated Soil.

This study investigates the impact of bacteria on arsenic reduction in wheat plants, highlighting the potential of microbe-based eco-friendly strategies for plant growth. In the present study, bacterial isolate SPB-10 was survived at high concentration against both form of arsenic (As3+ and As5+). SPB-10 produced 5.2 g/L and 11.3 g/L of exo-polysaccharide at 20 ppm of As3+ and As5+, respectively, whereas qualitative examination revealed the highest siderophores ability. Other PGP attributes such as IAA production were recorded 52.12 mg/L and 95.82 mg/L, phosphate solubilization was 90.23 mg/L and 129 mg/L at 20 ppm of As3+ and As5+, respectively. Significant amount of CAT, APX, and Proline was also observed at 20 ppm of As3+ and As5+ in SPB-10. Isolate SPB-10 was molecularly identified as Bacillus cereus through 16S rRNA sequencing. After 42 days, wheat plants inoculated with SPB-10 had a 25% increase in shoot length and dry weight, and 26% rise in chlorophyll-a pigment under As5+ supplemented T4 treatment than control. Reducing sugar content was increased by 24% in T6-treated plants compared to control. Additionally, SPB-10 enhanced the content of essential nutrients (NPK), CAT, and APX in plant's-leaf under both As3+ and As5+ stressed conditions after 42 days. The study found that arsenic uptake in plant roots and shoots decreased in SPB-10-inoculated plants, with the maximum reduction observed in As5+ treated plants. Bio-concentration factor-BCF was reduced by 90.89% in SPB-10-inoculated treatment T4 after 42 days. This suggests that Bacillus cereus-SPB-10 may be beneficial for plant growth in arsenic-contaminated soil.

Apple cider vinegar exhibits promising antibiofilm activity against multidrug-resistant Bacillus cereus isolated from meat and their products.

Background: Bacillus cereus (B. cereus) biofilm is grown not only on medical devices but also on different substrata and is considered a potential hazard in the food industry. Quorum sensing plays a serious role in the synthesis of biofilm with its surrounding extracellular matrix enabling irreversible connection of the bacteria. Aim: The goal of the current investigation was to ascertain the prevalence, patterns of antimicrobial resistance, and capacity for B. cereus biofilm formation in meat and meat products in Egypt. Methods: In all, 150 meat and meat product samples were used in this study. For additional bacteriological analysis, the samples were moved to the Bacteriology Laboratory. Thereafter, the antimicrobial, antiquorum sensing, and antibiofilm potential of apple cider vinegar (ACV) on B. cereus were evaluated. Results: Out of 150 samples, 34 (22.67%) tested positive for B. cereus. According to tests for antimicrobial susceptibility, every B. cereus isolates tested positive for colistin and ampicillin but negative for ciprofloxacin and imipenem. The ability to form biofilms was present in all 12 multidrug-resistant B. cereus isolates (n = 12); of these, 6 (50%), 3 (25%), and 3 (25%) isolates were weak, moderate, and strong biofilm producers, respectively. It is noteworthy that the ACV demonstrated significant inhibitory effects on B. cereus isolates, with minimum inhibitory concentrations varying between 2 and 8 µg/ml. Furthermore, after exposing biofilm-producing B. cereus isolates to the minimum biofilm inhibitory concentrations 50 of 4 µg/ml, it demonstrated good antibiofilm activity (>50% reduction of biofilm formation). Strong biofilm producers had down-regulated biofilm genes (tasA and sipW) and their regulator (plcR) compared to the control group, according to reverse transcriptase quantitative polymerase chain reaction analysis. Conclusion: Our study is the first report, that spotlights the ACV activity against B. cereus biofilm and its consequence as a strong antibacterial and antibiofilm agent in the food industry and human health risk.

Universal and Naked-Eye Diagnostic Platform for Emetic Bacillus cereus Based on RPA-Assisted CRISPR/Cas12a.

Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10-2 ng µL-1 and 102 CFU mL-1, respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.

Cereulide production capacities and genetic properties of 31 emetic Bacillus cereus group strains.

The highly potent toxin cereulide is a frequent cause of foodborne intoxications. This extremely resistant toxin is produced by Bacillus cereus group strains carrying the plasmid encoded cesHPTABCD gene cluster. It is known that the capacities to produce cereulide vary greatly between different strains but the genetic background of these variations is not clear. In this study, cereulide production capacities were associated with genetic characteristics. For this, cereulide levels in cultures of 31 strains were determined after incubation in tryptic soy broth for 24 h at 24 °C, 30 °C and 37 °C. Whole genome sequencing based data were used for an in-depth characterization of gene sequences related to cereulide production. The taxonomy, population structure and phylogenetic relationships of the strains were evaluated based on average nucleotide identity, multi-locus sequence typing (MLST), core genome MLST and single nucleotide polymorphism analyses. Despite a limited strain number, the approach of a genome wide association study (GWAS) was tested to link genetic variation with cereulide quantities. Our study confirms strain-dependent differences in cereulide production. For most strains, these differences were not explainable by sequence variations in the cesHPTABCD gene cluster or the regulatory genes abrB, spo0A, codY and pagRBc. Likewise, the population structure and phylogeny of the tested strains did not comprehensively reflect the cereulide production capacities. GWAS yielded first hints for associated proteins, while their possible effect on cereulide synthesis remains to be further investigated.

Comparison of the performance of multiple whole-genome sequence-based tools for the identification of Bacillus cereus sensu stricto biovar Thuringiensis.

The Bacillus cereus sensu stricto (s.s.) species comprises strains of biovar Thuringiensis (Bt) known for their bioinsecticidal activity, as well as strains with foodborne pathogenic potential. Bt strains are identified (i) based on the production of insecticidal crystal proteins, also known as Bt toxins, or (ii) based on the presence of cry, cyt, and vip genes, which encode Bt toxins. Multiple bioinformatics tools have been developed for the detection of crystal protein-encoding genes based on whole-genome sequencing (WGS) data. However, the performance of these tools is yet to be evaluated using phenotypic data. Thus, the goal of this study was to assess the performance of four bioinformatics tools for the detection of crystal protein-encoding genes. The accuracy of sequence-based identification of Bt was determined in reference to phenotypic microscope-based screening for the production of crystal proteins. A total of 58 diverse B. cereus sensu lato strains isolated from clinical, food, environmental, and commercial biopesticide products underwent WGS. Isolates were examined for crystal protein production using phase contrast microscopy. Crystal protein-encoding genes were detected using BtToxin_Digger, BTyper3, IDOPS (identification of pesticidal sequences), and Cry_processor. Out of 58 isolates, the phenotypic production of crystal proteins was confirmed for 18 isolates. Specificity and sensitivity of Bt identification based on sequences were 0.85 and 0.94 for BtToxin_Digger, 0.97 and 0.89 for BTyper3, 0.95 and 0.94 for IDOPS, and 0.88 and 1.00 for Cry_processor, respectively. Cry_processor predicted crystal protein production with the highest specificity, and BtToxin_Digger and IDOPS predicted crystal protein production with the highest sensitivity. Three out of four tested bioinformatics tools performed well overall, with IDOPS achieving high sensitivity and specificity (>0.90).IMPORTANCEStrains of Bacillus cereus sensu stricto (s.s.) biovar Thuringiensis (Bt) are used as organic biopesticides. Bt is differentiated from the foodborne pathogen Bacillus cereus s.s. by the production of insecticidal crystal proteins. Thus, reliable genomic identification of biovar Thuringiensis is necessary to ensure food safety and facilitate risk assessment. This study assessed the accuracy of whole-genome sequencing (WGS)-based identification of Bt compared to phenotypic microscopy-based screening for crystal protein production. Multiple bioinformatics tools were compared to assess their performance in predicting crystal protein production. Among them, identification of pesticidal sequences performed best overall at WGS-based Bt identification.

Bacillus cereus NJ01 induces SA- and ABA-mediated immunity against bacterial pathogens through the EDS1-WRKY18 module.

Emerging evidence suggests a beneficial role of rhizobacteria in ameliorating plant disease resistance in an environment-friendly way. In this study, we characterize a rhizobacterium, Bacillus cereus NJ01, that enhances bacterial pathogen resistance in rice and Arabidopsis. Transcriptome analyses show that root inoculation of NJ01 induces the expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes in Arabidopsis leaves. Genetic evidence showed that EDS1, PAD4, and WRKY18 are required for B. cereus NJ01-induced bacterial resistance. An EDS1-PAD4 complex interacts with WRKY18 and enhances its DNA binding activity. WRKY18 directly binds to the W box in the promoter region of the SA biosynthesis gene ICS1 and ABA biosynthesis genes NCED3 and NCED5 and contributes to the NJ01-induced bacterial resistance. Taken together, our findings indicate a role of the EDS1/PAD4-WRKY18 complex in rhizobacteria-induced disease resistance.

Molecular and metabolic characterization of petroleum hydrocarbons degrading Bacillus cereus.

Hydrocarbon constituents of petroleum are persistent, bioaccumulated, and bio-magnified in living tissues, transported to longer distances, and exert hazardous effects on human health and the ecosystem. Bioaugmentation with microorganisms like bacteria is an emerging approach that can mitigate the toxins from environmental sources. The present study was initiated to target the petroleum-contaminated soil of gasoline stations situated in Lahore. Petroleum degrading bacteria were isolated by serial dilution method followed by growth analysis, biochemical and molecular characterization, removal efficiency estimation, metabolites extraction, and GC-MS of the metabolites. Molecular analysis identified the bacterium as Bacillus cereus, which exhibited maximum growth at 72 hours and removed 75% petroleum. Biochemical characterization via the Remel RapID™ ONE panel system showed positive results for arginine dehydrolase (ADH), ornithine decarboxylase (ODC), lysine decarboxylase (LDC), o-nitrophenyl-ß-D-galactosidase (ONPG), p-nitrophenyl-ß-D-glucosidase (ßGLU), p-nitrophenyl-N-acetyl-ß-D-glucosaminidase (NAG), malonate (MAL), adonitol fermentation (ADON), and tryptophane utilization (IND). GC-MS-based metabolic profiling identified alcohols (methyl alcohol, o-, p- and m-cresols, catechol, and 3-methyl catechol), aldehydes (methanone, acetaldehyde, and m-tolualdehyde), carboxylic acid (methanoic acid, cis,cis-muconic acid, cyclohexane carboxylic acid and benzoic acid), conjugate bases of carboxylic acids (benzoate, cis,cis-muconate, 4-hydroxybenzoate, and pyruvate) and cycloalkane (cyclohexene). It suggested the presence of methane, methylcyclohexane, toluene, xylene, and benzene degradation pathways in B. cereus.

[Virulence genes and antimicrobial resistance of Bacillus cereus in prepackaged pastries sold in Taizhou from 2020 to 2022].

OBJECTIVE: To investigate the virulence genes and antimicrobial resistance of Bacillus cereus from the pre-packaged pastries in Taizhou city. METHODS: 500 pre-packaged patries were collected in taizhou city market. 97 Bacillus cereus strains were detected from them by GB 4789.14-2014 method and identified with 4 houseking genes, then 13 virulence genes were detected by polymerase chain reaction(PCR)method and the antimicrobial resistance of Bacillus cereus to 19 antibiotics was detected by paper diffusion method. RESULTS: The result showed that the contamination rate of Bacillus cereus was 19.4% in 500 pre-packaged pastries. The detection rate of four housekeeping genes groEL, gyr B, rpoB and Vrr were 100%, 94.8%, 97.9% and 96.9%, respectively, and 89.7% at the same time. The virulence gene test result showed that the detection rate of nheABC, entFM, bceT, cytK and hblABCD were 91.8%, 88.7%, 61.9%, 51.6% and 25.8%, emetic virulence genes had the lowest detection rate, ces and EMl were 4.1%, cer was 5.2%. 97 Bacillus cereus strains show different degrees of drug resistance to 14 antimicrobials, the resistance rates to penicillin, ampicillin, cefotaxime and cotrimoxazole were higher than 95%, but they were completely sensitive to streptomycin, vancomycin and chloramphenicol. CONCLUSION: There is a risk of contamination by diarrhea-type Bacillus cereus and vomiting-type Bacillus cereus in prepackaged pastries in Taizhou. The isolated and identified Bacillus cereus has multiple-drug resistance.

Counts of mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic sporeforming bacteria and persistence of Bacillus cereus spores throughout cocoa powder processing chain.

Sporeforming bacteria are a concern in some food raw materials, such as cocoa powder. Samples (n = 618) were collected on two farms and at several stages during cocoa powder manufacture in three commercial processing lines to determine the impact of each stage on bacterial spore populations. Mesophilic aerobic, mesophilic anaerobic, thermophilic aerobic, and Bacillus cereus spore populations were enumerated in all the samples. Genetic diversity in B. cereus strains (n = 110) isolated from the samples was examined by M13 sequence-based PCR typing, partial sequencing of the panC gene, and the presence/absence of ces and cspA genes. The counts of different groups of sporeforming bacteria varied amongst farms and processing lines. For example, the counts of mesophilic aerobic spore-forming (MAS) populations of cocoa bean fermentation were lower than 1 log spore/g in Farm 1 but higher than 4 log spore/g in Farm 2. B. cereus isolated from cocoa powder was also recovered from cocoa beans, nibs, and samples after roasting, refining, and pressing, which indicated that B. cereus spores persist throughout cocoa processing. Phylogenetic group IV was the most frequent (73%), along with processing. Strains from phylogenetic group III (14 %) did not show the ces gene's presence.

Cellulolytic Bacillus cereus produces a variety of short-chain fatty acids and has potential as a probiotic.

Cellulolytic bacteria ferment dietary fiber into short-chain fatty acids, which play an important role in improving fiber utilization and maintaining intestinal health. Safe and effective cellulolytic bacteria are highly promising probiotic candidates. In this study, we isolated three strains of Bacillus cereus, which exhibited cellulolytic properties, from Kele pig feces. To assess the genetic basis of cellulose degradation by the isolates, whole-genome sequencing was used to detect functional genes associated with cellulose metabolism. Subsequently, we identified that the B. cereus CL2 strain was safe in mice by monitoring body weight changes, performing histopathologic evaluations, and determining routine blood indices. We next evaluated the biological characteristics of the CL2 strain in terms of its growth, tolerance, and antibiotic susceptibility, with a focus on its ability to produce short-chain fatty acids. Finally, the intestinal flora structure of the experimental animals was analyzed to assess the intestinal environment compatibility of the CL2 strain. In this study, we isolated a cellulolytic B. cereus CL2, which has multiple cellulolytic functional genes and favorable biological characteristics, from the feces of Kele pigs. Moreover, CL2 could produce a variety of short-chain fatty acids and does not significantly affect the diversity of the intestinal flora. In summary, the cellulolytic bacterium B. cereus CL2 is a promising strain for use as a commercial probiotic or in feed supplement. IMPORTANCE: Short-chain fatty acids are crucial constituents of the intestinal tract, playing an important and beneficial role in preserving the functional integrity of the intestinal barrier and modulating both immune responses and the structure of the intestinal flora. In the intestine, short-chain fatty acids are mainly produced by bacterial fermentation of cellulose. Therefore, we believe that safe and efficient cellulolytic bacteria have the potential to be novel probiotics. In this study, we systematically evaluated the safety and biological characteristics of the cellulolytic bacterium B. cereus CL2 and provide evidence for its use as a probiotic.

Characterization of a cell wall hydrolase with high activity against vegetative cells, spores and biofilm of Bacillus cereus.

Bacillus cereus is a prevalent foodborne pathogen that induces food poisoning symptoms such as vomiting and diarrhea. Its capacity to form spores and biofilm enables it to withstand disinfectants and antimicrobials, leading to persistent contamination during food processing. Consequently, it is necessary to develop novel and efficient antimicrobial agents to control B. cereus, its spores, and biofilms. Peptidoglycan hydrolases have emerged as a promising and eco-friendly alternative owing to their specific lytic activity against pathogenic bacteria. Here, we identified and characterized a Lysozyme-like cell wall hydrolase Lys14579, from the genome of B. cereus ATCC 14579. Recombinant Lys14579 specifically lysed B. cereus without affecting other bacteria. Lys14579 exhibited strong lytic activity against B. cereus, effectively lysing B. cereus cell within 20 min at low concentration (10 µg/mL). It also inhibited the germination of B. cereus spores and prevented biofilm formation at 12.5 µg/mL. Moreover, Lys14579 displayed good antimicrobial stability with negligible hemolysis in mouse red blood cells and no cytotoxicity against RAW264.7 cells. Notably, Lys14579 effectively inhibited B. cereus in boiled rice and minced meat in a dose-dependent manner. Furthermore, bioinformatics analysis and point mutagenesis experiments revealed that Glu-47 was the catalytic site, and Asp-57, Gln-60, Ser-61 and Glu-63 were active-site residues related with the cell wall lytic activity. Taken together, Lys14579 could be a promising biocontrol agent against vegetative cells, spores, and biofilm of B. cereus in food industry.

Prevalence and genomic characterization of the Bacillus cereus group strains contamination in food products in Southern China.

The Bacillus cereus group, as one of the important opportunistic foodborne pathogens, is considered a risk to public health due to foodborne diseases and an important cause of economic losses to food industries. This study aimed to gain essential information on the prevalence, phenotype, and genotype of B. cereus group strains isolated from various food products in China. A total of 890 strains of B. cereus group bacteria from 1181 food samples from 2020 to 2023 were identified using the standardized detection method. These strains were found to be prevalent in various food types, with the highest contamination rates observed in cereal flour (55.8 %) and wheat/rice noodles (45.7 %). The tested strains exhibited high resistance rates against penicillin (98.5 %) and ampicillin (98.9 %). Strains isolated from cereal flour had the highest rate of meropenem resistance (7.8 %), while strains from sausages were most resistant to vancomycin (16.8 %). A total of 234 out of the 891 B. cereus group strains were randomly selected for WGS analysis, 18.4 % of which displayed multidrug resistance. The species identification by WGS analysis revealed the presence of 10 distinct species within the B. cereus group, with B. cereus species being the most prevalent. The highest level of species diversity was observed in sausages. Notably, B. anthracis strains lacking the anthrax toxin genes were detected in flour-based food products and sausages. A total of 20 antibiotic resistance genes have been identified, with ß-lactam resistance genes (bla1, bla2, BcI, BcII, and blaTEM-116) being the most common. The B. tropicus strains exhibit the highest average number of virulence genes (23.4). The diarrheal virulence genes nheABC, hblACD, and cytK were found in numerous strains. Only 4 of the 234 (1.7 %) sequenced strains contain the ces gene cluster linked to emetic symptoms. These data offer valuable insights for public health policymakers on addressing foodborne B. cereus group infections and ensuring food safety.

Effect of Temperature, pH, and aw on Cereulide Synthesis and Regulator Genes Transcription with Respect to Bacillus cereus Growth and Cereulide Production.

Bacillus cereus is a food-borne pathogen that can produce cereulide in the growth period, which causes food poisoning symptoms. Due to its resistance to heat, extreme pH, and proteolytic enzymes, cereulide poses a serious threat to food safety. Temperature, pH, and aw can influence cereulide production, but there is still a lack of research with multi-environmental impacts. In this study, the effects of temperature (15~45 °C), pH (5~8), and aw (0.945~0.996) on the emetic reference strain B. cereus F4810/72 growth, cereulide production, relevant ces genes (cesA, cesB, cesP), and transcription regulators genes (codY and abrB) expression at transcription level were studied. B. cereus survived for 4~53 h or grew to 6.85~8.15 log10 CFU/mL in environmental combinations. Cereulide accumulation was higher in mid-temperature, acidic, or high aw environments. Increased temperature resulted in a lower cereulide concentration at pH 8 or aw of 0.970. The lowest cereulide concentration was found at pH 6.5 with an increased aw from 0.970 to 0.996. Water activity had a strong effect on transcriptional regulator genes as well as the cesB gene, and temperature was the main effect factor of cesP gene expression. Moreover, environmental factors also impact cereulide synthesis at transcriptional levels thereby altering the cereulide concentrations. The interaction of environmental factors may result in the survival of B. cereus without growth for a period. Gene expression is affected by environmental factors, and temperature and pH may be the main factors influencing the correlation between B. cereus growth and cereulide formation. This study contributed to an initial understanding of the intrinsic link between the impact of environmental factors and cereulide formation and provided valuable information for clarifying the mechanism of cereulide synthesis in combined environmental conditions.

Genome sequencing and protein modeling unraveled the 2AP biosynthesis in Bacillus cereus DB25.

2-Acetyl-1-pyrroline (2AP) is an important and major flavor aroma compound responsible for the fragrance of basmati rice, cheese, wine, and several other food products. Biosynthesis of 2AP in aromatic rice and a few other plant species is associated with a recessive Betaine aldehyde dehydrogenase 2 (BADH2) gene. However, the literature is scant on the relationship between the functional BADH2 gene and 2AP biosynthesis in prokaryotic systems. Therefore, in the present study, we aimed to explore the functionality of the BADH2 gene for 2AP biosynthesis in 2AP synthesizing rice rhizobacterial isolate Bacillus cereus DB25 isolated from the rhizosphere of basmati rice (Oryza sativa L.). Full-length BcBADH2 sequence was obtained through whole genome sequencing (WGS) and further confirmed through traditional PCR and Sanger sequencing. Then the functionality of the BcBADH2 gene was evaluated in-silico through bioinformatics analysis and protein docking studies and further experimentally validated through enzyme assay. The sequencing and bioinformatics analysis results revealed a full-length 1485 bp BcBADH2 coding sequence without any deletion or premature stop codons. Full-length BcBADH2 was found to encode a fully functional protein of 54.08 kDa with pI of 5.22 and showed the presence of the conserved amino acids responsible for enzyme activity. The docking studies confirmed a good affinity between the protein and its substrate whereas the presence of BcBADH2 enzyme activity confirmed the functionality of BADH2 enzyme in B. cereus DB25. In conclusion, the findings of the present study suggest that B. cereus DB25 is able to synthesize 2AP despite a functional BADH2 gene and there may be a different molecular mechanism responsible for 2AP biosynthesis in bacterial systems, unlike that found in aromatic rice and other eukaryotic plant species.

The potential of lactose to inhibit cereulide biosynthesis of emetic Bacillus cereus in milk.

This study aims to investigate the potential role of lactose on cereulide biosynthesis by emetic Bacillus cereus in dairy matrices. The cereulide yields in whole milk and lactose-free milk were investigated using the emetic reference strain F4810/72. To eliminate the influence of complex food substrates, the LB medium model was further used to characterize the effect of lactose on cereulide produced by F4810/72 and five other emetic B. cereus strains. Results showed that the lactose-free milk displayed a 13-fold higher amount of cereulide than whole milk, but the cereulide level could be reduced by 91 % when the lactose content was restored. The significant inhibition of lactose on cereulide yields of all tested B. cereus strains was observed in LB medium, showing a dose-dependent manner with inhibition rates ranging of 89-98 %. The growth curves and lactose utilization patterns of all strains demonstrated that B. cereus cannot utilize lactose as a carbon source and lactose might act as a signal molecule to regulate cereulide production. Moreover, lactose strongly repressed the expression of cereulide synthetase genes (ces), possibly by inhibiting the key regulator Spo0A at the transcriptional level. Our findings highlight the potential of lactose as an effective strategy to control cereulide production in food.

[Prokaryotic expression and biological activities of the hemolysin BL subunit of a pathogenic Bacillus cereus of cattle origin].

Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.

Virulence-related genotypic differences among Bacillus cereus ocular and gastrointestinal isolates and the relationship to endophthalmitis pathogenesis.

Background: Bacillus cereus (Bc) can cause self-limiting gastrointestinal infections, but when infecting the eye, can cause rapid and irreversible blindness. This study investigated whether clinical ocular and gastrointestinal Bc isolates differed in terms of virulence-related genotypes and endophthalmitis virulence. Methods: Twenty-eight Bc ocular, gastrointestinal, and laboratory reference isolates were evaluated. Hemolysis assays were performed to assess potential differences in hemolytic activity. The presence of twenty Bc virulence-related genes was assessed by PCR. A subset of ocular and gastrointestinal isolates differing in PCR positivity for 5 virulence genes was compared to strain ATCC14579 in an experimental murine model of endophthalmitis. At 8 hours post infection, retinal function was evaluated by electroretinography, and intraocular bacterial concentrations were determined by plate counts. Results: Gastrointestinal Bc isolates were more hemolytic than the Bc ocular isolates and ATCC14579 (p < 0.0001). Bc ocular isolates were more frequently PCR-positive for capK, cytK, hblA, hblC, and plcR compared to the gastrointestinal isolates (p ≤ 0.0002). In the endophthalmitis model, mean A-wave retention did not differ significantly between eyes infected with ATCC14579 and eyes infected with the selected ocular or gastrointestinal isolates (p ≥ 0.3528). Similar results were observed for mean B-wave retention (p ≥ 0.0640). Only one diarrheal isolate showed significantly greater B-wave retention when compared to ATCC14579 (p = 0.0303). No significant differences in mean A-wave (p ≥ 0.1535) or B-wave (p ≥ 0.0727) retention between the selected ocular and gastrointestinal isolates were observed. Intraocular concentrations of ATCC14579 were significantly higher than the selected ocular isolate and 3 of the gastrointestinal isolates (p ≤ 0.0303). Intraocular concentrations of the selected ocular isolate were not significantly different from the gastrointestinal isolates (p ≥ 0.1923). Conclusions: Among the subset of virulence-related genes assessed, 5 were significantly enriched among the ocular isolates compared to gastrointestinal isolates. While hemolytic activity was higher among gastrointestinal isolates, retinal function retention and intraocular growth was not significantly different between the selected ocular and gastrointestinal isolates. These results suggest that Bc strains causing gastrointestinal infections, while differing from ocular isolates in hemolytic activity and virulence-related gene profile, are similarly virulent in endophthalmitis.

Pathological changes of highly pathogenic Bacillus cereus on Pelodiscus sinensis.

An outbreak of a disease with a high mortality rate occurred in a Chinese Softshell Turtle (Pelodiscus sinensis) farm in Hubei Province. This study isolated a highly pathogenic Bacillus cereus strain (Y271) from diseased P. sinensis. Y271 has ß hemolysis, containing both Hemolysin BL (hblA, hblC, and hblD), Non-hemolytic enterotoxin, NHE (nheA, nheB, and nheC), and Enterotoxin FM (entFM) genes. Y271 is highly pathogenic against P. sinensis with an LD50 = 6.80 × 103 CFU/g weight. B. cereus was detected in multiple tissues of the infected P. sinensis. Among them, spleen tissue showed the highest copy number density (1.54 ± 0.12 × 104 copies/mg). Multiple tissues and organs of diseased P. sinensis exhibited significant pathological damage, especially the spleen, liver, kidney, and intestine. It showed obvious tissue structure destruction, lesions, necrosis, red blood cells, and inflammatory cell infiltration. B. cereus proliferating in the spleen, liver, and other tissues was observed. The intestinal microbiota of the diseased P. sinensis was altered, with a greater abundance of Firmicutes, Fusobacterium, and Actinomyces than in the healthy group. Allobaculum, Rothia, Aeromonas, and Clostridium abundance were higher in the diseased group than in the healthy group. The number of unique microbial taxa (472) in the disease group was lower than that of the healthy group (705). Y271 was sensitive to multiple drugs, including florfenicol, enrofloxacin, neomycin, and doxycycline. B. cereus is the etiological agent responsible for the massive death of P. sinensis and reveals its potential risks during P. sinensis cultivation.

Expression, Characterisation, Homology Modelling and Molecular Docking of a Novel M17 Family Leucyl-Aminopeptidase from Bacillus cereus CZ.

Leucyl-aminopeptidase (LAP), an important metallopeptidase, hydrolyses amino acid residues from the N-terminus of polypeptides and proteins, acting preferentially on the peptide bond formed by N-terminus leucine. A new leucyl-aminopeptidase was found in Bacillus cereus CZ. Its gene (bclap) contained a 1485 bp ORF encoding 494 amino acids with a molecular weight of 54 kDa. The bcLAP protein was successfully expressed in E. coli BL21(DE3). Optimal activity is obtained at pH 9.0 and 58 °C. The bcLAP displays a moderate thermostability and an alkaline pH adaptation range. Enzymatic activity is dramatically enhanced by Ni2+. EDTA significantly inhibits the enzymatic activity, and bestatin and SDS also show strong inhibition. The three-dimensional model of bcLAP monomer and homohexamer is simulated byPHYRE2 server and SWISS-MODEL server. The docking of bestatin, Leu-Trp, Asp-Trp and Ala-Ala-Gly to bcLAP is performed using AutoDock4.2.5, respectively. Molecular docking results show that the residues Lys260, Asp265, Lys272, Asp283, Asp342, Glu344, Arg346, Gly372 and His437 are involved in the hydrogen bonding with the ligands and zinc ions. There may be two nucleophilic catalytic mechanisms in bcLAP, one involving His 437 or Arg346 and the other involving His437 and Arg346. The bcLAP can hydrolyse the peptide bonds in Leu-Trp, Asp-Trp and Ala-Ala-Gly.

Utilizing Extraepitopic Amino Acid Substitutions to Define Changes in the Accessibility of Conformational Epitopes of the Bacillus cereus HlyII C-Terminal Domain.

Hemolysin II (HlyII)-one of the pathogenic factors of Bacillus cereus, a pore-forming ß-barrel toxin-possesses a C-terminal extension of 94 amino acid residues, designated as the C-terminal domain of HlyII (HlyIICTD), which plays an important role in the functioning of the toxin. Our previous work described a monoclonal antibody (HlyIIC-20), capable of strain-specific inhibition of hemolysis caused by HlyII, and demonstrated the dependence of the efficiency of hemolysis on the presence of proline at position 324 in HlyII outside the conformational antigenic determinant. In this work, we studied 16 mutant forms of HlyIICTD. Each of the mutations, obtained via multiple site-directed mutagenesis leading to the replacement of amino acid residues lying on the surface of the 3D structure of HlyIICTD, led to a decrease in the interaction of HlyIIC-20 with the mutant form of the protein. Changes in epitope structure confirm the high conformational mobility of HlyIICTD required for the functioning of HlyII. Comparison of the effect of the introduced mutations on the effectiveness of interactions between HlyIICTD and HlyIIC-20 and a control antibody recognizing a non-overlapping epitope enabled the identification of the amino acid residues N339 and K340, included in the conformational antigenic determinant recognized by HlyIIC-20.