Design of a two functional permeable reactive barrier for synergistic enzymatic and microbial bioremediation of phenol-contaminated waters: laboratory column evaluation : Enzymatic and microbial bioremediation of phenol in a bilayer permeable reactive barrier.

The present study aimed to develop a system using a combination of enzymatic and microbial degradation techniques for removing phenol from contaminated water. In our prior research, the HRP enzyme extracted from horseradish roots was utilized within a core-shell microcapsule to reduce phenolic shock, serving as a monolayer column. To complete the phenol removal process, a second column containing degrading microorganisms was added to the last column in this research. Phenol-degrading bacteria were isolated from different microbial sources on a phenolic base medium. Additionally, encapsulated calcium peroxide nanoparticles were used to provide dissolved oxygen for the microbial population. Results showed that the both isolated strains, WC1 and CC1, were able to completely remove phenol from the contaminated influent water the range within 5 to 7 days, respectively. Molecular identification showed 99.8% similarity for WC1 isolate to Stenotrophomonas rizophila strain e-p10 and 99.9% similarity for CC1 isolate to Bacillus cereus strain IAM 12,605. The results also indicated that columns using activated sludge as a microbial source had the highest removal rate, with the microbial biofilm completely removing 100% of the 100 mg/L phenol concentration in contaminated influent water after 40 days. Finally, the concurrent use of core-shell microcapsules containing enzymes and capsules containing Stenotrophomonas sp. WC1 strain in two continuous column reactors was able to completely remove phenol from polluted water with a concentration of 500 mg/L for a period of 20 days. The results suggest that a combination of enzymatic and microbial degrading systems can be used as a new system to remove phenol from polluted streams with higher concentrations of phenol by eliminating the shock of phenol on the microbial population.

A rapid and ultrasensitive paper sensor for Bacillus cereus Haemolysin BL detection.

Bacillus cereus is a foodborne opportunistic pathogen commonly found in humans and animals. It produces various toxins, causing frequent food safety incidents. Therefore, establishing a fast and accurate method for detecting B. cereus enterotoxin is crucial for disease diagnosis and food safety. In this study, Haemolysin BL comprising Hbl B and L2 was obtained from a prokaryotic expression system and then used to immunize mice for antibody preparation. Paired antibodies 2A10-5C7 against Hbl B and 1E2-10A4 against Hbl L2 were screened using the chessboard method and then used to construct a double-antibody sandwich detection method and a lateral flow immunochromatographic assay (LFIA) to quantify the concentrations of Hbl B and L2 in PBS and milk. The limits of detection for Hbl B and L2 in milk were 0.74 ng mL-1 and 1 ng mL-1 with detection ranges of 1.48-645.5 ng mL-1 and 2.33-391.5 ng mL-1. The spiked recoveries ranged from 82.2% to 105.67% and there was no cross-reactivity with common microbial toxins. The established LFIA was low in cost and rapid and was comparable with commercially available detection kits for food samples.

Late-onset sepsis in newborns caused by Bacillus Cereus: a case report and literature review.

Bacillus cereus is a bacterium capable of causing late-onset neonatal sepsis. By analyzing 11 cases, this study investigates the diagnosis, treatment, and prognosis of Bacillus cereus infections, aiming to provide insights into clinical diagnosis and therapy. The study scrutinized 11 instances of late-onset neonatal sepsis, including two fatalities attributable to Bacillus cereus, one accompanied by cerebral hemorrhage. An examination and analysis of these cases' symptoms, signs, laboratory tests, and treatment processes, along with a review of related literature from 2010 to 2020, revealed a high mortality rate of 41.38% in non-gastrointestinal infections caused by Bacillus cereus. Our findings underscore the critical importance of rapid diagnosis and effective antimicrobial therapy in reducing mortality rates. Once the source of infection is identified, implementing effective infection control measures is essential.

Bacteriological Survey of Insect Products in Japan.

A microbiological study was conducted on 41 insect product samples (29 raw frozen [21 domestic and 8 imported], 10 powdered, and 2 processed), which were commercially available in Japan. The total aerobic count for raw frozen insects was 5.61 log cfu/g (range: 2.52-8.40), whereas the powdered insect count was 2.89 log cfu/g (range: 1.00-4.57). The bacterial count was significantly higher in raw frozen insects (p < 0.05). The coliform count for the raw frozen insects ranged from <1 to 6.90 log cfu/g, and that for the powdered insects ranged from <1 to 1.00 log cfu/g. The number of samples with values above the detection limit was significantly higher in raw frozen insects (p < 0.05). The detection frequencies of aerobic spores (<1-4.63 log cfu/g), anaerobic spores (<0-4.40 log cfu/g), and Bacillus cereus (<1.7-3.83 log cfu/g) showed no sample type-related significant difference. Listeria spp. was isolated from four samples of raw frozen insects, one of which was Listeria monocytogenes. We did not detect any of the following: Salmonella spp., Shiga toxin-producing E. coli (STEC), Campylobacter jejuni/coli, or pathogenic Yersinia. We isolated insect products retailed in Japan harboring food poisoning bacteria, including L. monocytogenes and B. cereus. In particular, raw frozen products displayed high levels of hygienic indicator bacteria.

Flower-Shaped PCR Scaffold-Based Lateral Flow Bioassay for Bacillus cereus Endospores Detection.

Bacillus cereus, a foodborne pathogen, produces resilient endospores that are challenging to detect with conventional methods. This study presents a novel Flower-Shaped PCR Scaffold-based Lateral Flow Biosensor (FSPCRS-LFB), which employs an aptamer-integrated PCR scaffold as capture probes, replacing the traditional streptavidin-biotin (SA-Bio) approach. The FSPCRS-LFB demonstrates high sensitivity and cost-efficiency in detecting B. cereus endospores, with a limit of detection (LOD) of 4.57 endospores/mL a visual LOD of 102 endospores/mL, and a LOD of 6.78 CFU/mL for endospore-cell mixtures. In chicken and tea samples, the platform achieved LODs of 74.5 and 52.8 endospores/mL, respectively, with recovery rates of 82.19% to 97.88%. Compared to existing methods, the FSPCRS-LFB offers a 3.7-fold increase in sensitivity while reducing costs by 26% over the SA-Bio strategy and 87.5% over rolling circle amplification (RCA). This biosensor provides a rapid, sensitive and cost-effective solution for point-of-care testing (POCT) of B. cereus endospores, expanding detection capabilities and offering novel approaches for pathogen detection.

Genetic diversity and the presence of circular plasmids in Bacillus cereus isolates of clinical and environmental origin.

The diversity of 61 Bacillus cereus strains isolated from different clinical specimens, food including raw milk and milk products, and water was evaluated. PFGE analysis could discriminate 61 distinct pulsotypes with similarity levels from 25 to 82%, which were divided into 13 clonal complexes. The similarity between clonal complexes was at least 40%. Clinical strains were divided into 10 clonal complexes, while the strains, isolated from milk, food and water were included in 9, 6 and 6 clonal complexes, respectively. Three clonal complexes were dominated by clinical isolates, while they were absent in two complexes. Bacterial isolates from food, being a probable source of alimentary toxoinfection, showed low similarity to isolates from stool specimens. The isolates from both sources were classified together in only 4 out of 13 clonal complexes. The large circular and linear plasmids with the sizes between 50 and 200 kb were detected in 24 (39.3%) and 14 (23%) B. cereus strains, respectively. Thirteen (21.3%) strains contained only one plasmid, two plasmids were found in 6 (9.8%) of strains, and three or more plasmids were obtained in 5 (8.2%) of tested strains. The plasmids were confirmed in 30.8% and 40% of isolates from clinical specimens and food and milk samples, respectively. No clear correlation between the PFGE profiles, the source as well as plasmid content among all tested strains was observed.

Three novel species of the Bacillus cereus group isolated from clinical samples in Japan.

Three Gram-positive bacterial strains, BML-BC004, BML-BC017 and BML-BC059, isolated from blood samples from three inpatients in Japan, were identified as members of Bacillus cereus using matrix-assisted laser desorption ionization time-of-flight MS. The 16S rRNA gene sequences of these three strains were more than 97.1 % similar to 18 type strains belonging to the B. cereus group. Whole-genome comparisons, using average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH), confirmed that the three strains represented three individual distinct species belonging to the B. cereus group. A phylogenetic tree showed that BML-BC004, BML-BC017 and BML-BC059 were located close to B. luti, B. mobilis and B. paramycoides, respectively. Based on these phylogenetic and phenotypic data, including values below the threshold for ANI and dDDH, the three strains should be classified as representing three different novel species of the B. cereus group: Bacillus sanguinis sp. nov., with type strain BML-BC004T (=DSM 111102T=JCM 34122T), Bacillus paramobilis sp. nov., with type strain BML-BC017T (=DSM 111100T=JCM 34124T) and Bacillus hominis sp. nov., with type strain BML-BC059T (=DSM 111101T=JCM 34125T).

Phenotypic and genotypic characterization of the new Bacillus cereus phage SWEP1.

A new Bacillus cereus phage, SWEP1, was isolated from black soil. The host lysis activity of phage SWEP1 has a relatively short latent time (20 min) and a small burst size of 83 PFU. The genome of SWEP1 consists of 162,461 bp with 37.77% G+C content. The phage encodes 278 predicted proteins, 103 of which were assigned functionally. No tRNA genes were found. Comparative genomics analysis indicated that SWEP1 is related to Bacillus phage B4 (86.91% identity, 90% query coverage). Phenotypic and genotypic characterization suggested that SWEP1 is a new member of a new species in the genus Bequatrovirus, family Herelleviridae.

Paper-based sensor from pyrrolidinyl peptide nucleic acid for the efficient detection of Bacillus cereus.

Bacillus cereus is one of the most common foodborne pathogens found in various kinds of staple foods such as rice and wheat. A rapid and accurate detection method for this pathogen is highly desirable for the sustainable production of relevant food products. While several classical and molecular-based detection methods are available for the identification of B. cereus, they suffered one or more limitations such as the requirement for a tedious and time-consuming process, less than ideal specificity, and the lack of portability. Herein, we developed the first paper-based sensing device that exhibits high species specificity with sufficiently low limit of detection for the visual detection of specific DNA sequences of B. cereus. The success is attributed to the strategic planning of fabrication in various dimensions including thorough bioinformatics search for highly specific genes, the use of the pyrrolidinyl peptide nucleic acid (PNA) probe whose selectivity advantage is well documented, and an effective PNA immobilization and DNA-binding visualization method with an internal cross-checking system for validating the results. Testing in rice matrices indicates that the sensor is capable of detecting and distinguishing B. cereus from other bacterial species. Hence, this paper-based sensor has potential to be adopted as a practical means to detect B. cereus in food industries.

Impact of antibiotics on fluorescent Pseudomonas group and Bacillus cereus group isolated from soils exposed to effluent or waste from conventional and organic pig farming.

AIMS: To determine if antibiotics associated with conventional pig farming have a direct role in altering the populations of key soil micro-organisms isolated from piggery environments with and without exposure to antibiotics. METHODS AND RESULTS: Fluorescent Pseudomonas sp. and the Bacillus cereus group from soils adjacent to four conventional piggeries (use of antibiotics) exposed to effluent (via irrigation) and two organic piggeries (non-use of antibiotics) were assessed against nine relevant antibiotics using disc diffusion. The focus of the study was not to determine antibiotic resistance (or sensitivity) of isolates based on the manufacturer-defined sensitive break point, instead this point was used as the interpretation point to compare the populations (i.e. farm/organism combination) for the antibiotics tested. Each population was statistically analysed to determine whether the mean diameters were significantly above this selected interpretation point. Bacterial species from both environments did not show a distinct population pattern linked to the antibiotics. CONCLUSIONS: Antibiotics associated with conventional pig farming do not have a direct role in altering the environmental populations of Pseudomonas and Bacillus sp. when assessed by population shifts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that an understanding of the resident soil microbiota, as compared to the transient bacteria of pig origin, is important in addressing the impact of antibiotic usage on the food-chain as a consequence of effluent re-use in, and around, pig farms.

Comparison of the antibiotic resistance between genetically diverse and toxigenic Bacillus cereus sensu lato from milk, pepper and natural habitats.

AIMS: Bacillus cereus sensu lato is a complex group of closely related bacteria, which are commonly present in the natural environment and food products. These organisms may cause food poisoning and spoilage as well as opportunistic infections. Thus far, their resistance to selected antibiotics has been explored only in part, especially in the context of strain source. Therefore, our goal was to compare the resistance of B. cereus sl from milk (environment with the potential impact of antibiotics) with strains from soil and pepper (environment without contact with antibiotics) in relation to their origin, toxicity and phylogenetic relationships. METHODS AND RESULTS: Antibiotic resistance of B. cereus sl was assessed by determining their minimal inhibitory concentrations (MICs) followed by statistical analyses. The phylogeny of the bacteria was investigated by multilocus sequence typing, and toxicity was determined with quantitative reverse-transcription real-time PCR. We found that the isolates from milk were more often multiresistant and exhibited a common resistance pattern to ß-lactams but a varied sensitivity to the tested macrolides, clindamycin, tetracycline and vancomycin. Moreover they displayed often significantly higher average MICs; however, their resistance did not correlate with phylogeny, toxicity, or in most cases, with taxonomic affiliation. CONCLUSION: We conclude that mainly food matrices may serve as an important reservoir of resistant isolates of B. cereus sl and that the use of antibiotics for the treatment of animal diseases must be carefully monitored as it strongly promotes natural selection for multiresistant strains, even among opportunist pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that compared to the isolates from natural habitats, nonpathogenic B. cereus sl isolated from food acquire antibiotic resistance faster, should increase producers and consumers awareness and result in protection of public health.

Florid Bacillus cereus Infection of the Placenta Associated With Intrauterine Fetal Demise.

Bacillus cereus is a gram-positive, rod-shaped bacterium that is commonly implicated in foodborne illness but has also become increasingly recognized as a source of serious non-gastrointestinal infections, including sepsis, meningitis, and pneumonia. Non-gastrointestinal B. cereus infections have been identified in children, especially in neonates; however, there are no previously described cases of fetal demise associated with B. cereus placental infection. We present a case of acute chorioamnionitis-related intrauterine fetal demise of twin A at 17 weeks gestation, noted two days after selective termination of twin B. Histological examination revealed numerous gram-positive bacilli in placental tissue, as well as fetal vasculature, in the setting of severe acute necrotizing chorioamnionitis and subchorionitis, intervillous abscesses, acute villitis, and peripheral acute funisitis. Cultures of maternal blood and placental tissue both yielded growth of B. cereus. This case underscores the importance of B. cereus as a human pathogen, and specifically demonstrates its potential as an agent of severe intraamniotic and placental infection with poor outcomes for the fetus.

Coexistence of endonuclease and exonuclease activities in a novel RecJ from Bacillus cereus.

BACKGROUND: All RecJ proteins are known to date only perform exonuclease activity. The present study reports that a novel RecJ protein obtained from Bacillus cereus isolated from marine sediments has both endonuclease and exonuclease activities. METHODS: Analysis of the BcRecJ expression induction in E. coli BL21 revealed that the BcRecJ protein cleaved plasmids and genomic DNA in the host cell, and led to cell death and decreased the DNA content. Further, the BcRecJ protein had the ability to degrade supercoiled plasmid DNA into circular or linear forms in vitro. Meanwhile, the BcRecJ protein loaded with an S-modified guide facilitated plasmid linearization and reduced smear formation. RESULTS: The results suggested that this novel BcRecJ protein was different from any reported RecJs and had a longer C-terminus. Testing the BcRecJ mutants indicated that the endonuclease activity was affected by two residues of BcRecJ (D561, E637) after testing the BcRecJ mutants. CONCLUSION: The discovery of the type of protein is a new breakthrough for the RecJ proteins, which has both endonuclease and exonuclease activities.

Isolation of a Bacillus cereus strain HBL-AI and its application for production of Trans-4-hydroxy-l-proline.

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 O2 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' ß 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.

The cytotoxic potential of Bacillus cereus strains of various origins.

B. cereus is a human pathogen associated with food poisoning leading to gastrointestinal disorders, as well as local and severe systemic infections. The pathogenic spectrum of B. cereus ranges from strains used as probiotics in humans to lethal highly toxic strains. In this study, we gathered a collection of 100 strains representative of the pathological diversity of B. cereus in humans, and characterized these strains for their cytotoxic potential towards human cells. We analyzed the correlation between cytotoxicity to epithelial and macrophage cells and the combination of 10 genes suspected to play a role during B. cereus virulence. We highlight genetic differences among isolates and studied correlations between genetic signature, cytotoxicity and strain pathological status. We hope that our findings will improve our understanding of the pathogenicity of B. cereus, thereby making it possible to improve both clinical diagnosis and food safety.

Characterization of Bacillus cereus sensu lato isolates from milk for consumption; phylogenetic identity, potential for spoilage and disease.

This study addresses the biodiversity of Bacillus cereus group population present along the value chain of milk for consumption. The B. cereus population did not grow and remained mainly unaltered during storage of milk at 4 °C while storage at a suboptimal temperature at 8 °C (representative of a broken cold chain) caused a major shift in its composition. Mesophilic strains dominated the B. cereus population in raw milk and after storage at 4 °C, while psycrotrophic strains dominated after storage at 8 °C. All psycrotrophic and mesophilic isolates (n = 368) demonstrated high spoilage potentials of the milk components. Fifteen out of 20 mesophilic isolates but only two out of 40 psychrotrophic isolates, exhibited vero cell toxicity. No genes encoding the emetic toxin cereulide were detected in the genomes of 100 milk isolates while 14 of them harbored the enterotoxin genes cytK1/cytK2. Both psycrotrophic and mesophilic isolates carried the enterotoxin genes nheA and hblA. Together, the results provide insight into the composition and properties, of the B. cereus population present in milk along the value chain and during storage at optimal refrigerated temperature and at suboptimal temperature. This knowledge is useful in the dairy industry's work to assure high quality products and for risk assessment.

Vancomycin-modified poly-l-lysine magnetic separation combined with multiplex polymerase chain reaction assay for efficient detection of Bacillus cereus in milk.

In this study, a new vancomycin (Van)-modified poly-l-lysine (PLL) magnetic bead (MB) technique was developed for isolation of gram-positive bacteria. The method combines magnetic separation with a multiplex PCR (mPCR) assay and gel electrophoresis for easy and rapid detection of Bacillus cereus. Vancomycin was used as a molecular ligand between the MB and the d-alanyl-d-alanine moieties on the cell wall surface of B. cereus. The PLL served as a flexible molecular tether between the MB and Van that reduced steric hindrance maintaining the biological activity of Van. The MB-PLL-Van capture nanoprobes exhibited excellent capture and isolation efficiency for B. cereus in spiked milk matrix samples without interference from the complex food matrix. The subsequent mPCR assay showed high specificity for the 4 target genes in B. cereus, the entFM, cesB, cer, and 16S rRNA genes, that were used to achieve efficient genotyping and detection. Under optimum conditions, the limit of detection reached 103 cfu/mL, with a dynamic range of detection at 103 to 107 cfu/mL in pure culture. Application of the MB-PLL-Van mediated mPCR assay for B. cereus in milk matrix samples achieved results similar to those of the pure culture. In addition, with a 6-h pre-enrichment of B. cereus that was spiked in milk matrix samples, the limit of detection reached 101 cfu/mL. The MB-PLL-Van mediated mPCR assay developed in this study could be used as a universal technology platform for the efficient enrichment and genotyping of gram-positive bacteria.

Genotyping, Antimicrobial Susceptibility and Biofilm Formation of Bacillus cereus Isolated from Powdered Food Products in China.

This study was conducted to reveal the genotyping, antimicrobial susceptibility, and biofilm formation of Bacillus cereus isolated from powdered food products in China. Five hundred powdered food samples were collected from five provinces in China: 100 samples each of powdered infant formula (PIF), soy milk powder (SMP), lotus root powder (LRP), walnut powder (WP), and rice flour (RF). The genotyping of isolates was analyzed using multilocus sequence typing; meanwhile, antimicrobial susceptibility, and ability of biofilms formation on stainless steel tube of isolates were evaluated. Forty-two B. cereus strains were detected with an overall contamination rate of 8.4%, as well as, the highest B. cereus contamination rate was found in SMP (10%), followed by LRP (9%), WP (9%), RF (8%), and PIF (6%). These isolates were divided into 22 sequence types (STs); among them, ST32 (4/42, 9.5%) was the predominant ST. Phylogenetic relationships showed that the 42 strains of B. cereus were divided into three groups (group I, group II, and group III). Antimicrobial susceptibility testing indicated that all isolates were susceptible to tetracycline, gentamicin, erythromycin, and chloramphenicol, while resistant to ampicillin, cefepime, oxacillin, and rifampin. The analysis of ability of biofilm formation on stainless steel tube showed optical density (OD)595 value of 66.7% of B. cereus isolates was greater than 1. The OD595 level of isolates belonging to group III was higher compared with the other two groups, and OD595 values of B. cereus HB1 and HN5 were greater than 2. These findings improved the understanding of the characteristics of B. cereus isolated from powdered food products in China, and provided a theoretical basis for the prevention and control of B. cereus in food industry.

An Investigation on the Occurrence and Molecular Characterization of Bacillus cereus in Meat and Meat Products in China.

Bacillus cereus is a common foodborne pathogen that can cause both gastrointestinal and nongastrointestinal diseases. In this study, we collected 603 meat and meat products from 39 major cities in China. The positive contamination rate of B. cereus in the collected samples was 26.37% (159/603), and the contamination level in 5.03% (8/159) positive samples exceeded 1100 most probable number/g. The detection rates of virulence genes were 89.7% for the nheABC gene group, 37.1% for the hblACD gene cluster, 82.3% for cytK-2, and 2.9% for cesB. Notably, all isolates presented with multiple antibiotic resistance, and 99.43% of isolates were resistant to five classes of antibiotics. In addition, the multilocus sequence typing results indicated that all isolates were rich in genetic diversity. Collectively, we conducted a systematic investigation on the prevalence and characterization of B. cereus in meat and meat products in China, providing crucial information for assessing the risk of B. cereus occurrence in meat and meat products.

Groundwater promotes emergence of asporogenic mutants of emetic Bacillus cereus.

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.