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Cytotoxicity and Genome Characteristics of an Emetic Toxin-Producing Bacillus cereus Group sp. Isolated from Raw Milk

by Jintana Pheepakpraw, Chanita Sinchao, Sawannee Sutheeworapong, Pachara Sattayawat, Aussara Panya, Yingmanee Tragoolpua and Thararat Chitov

Foods 2025, 14(3), 485; https://doi.org/10.3390/foods14030485 - 3 Feb 2025

Viewed by 1868

Abstract

The Bacillus cereus group frequently contaminates milk and dairy products. Some members of this group can produce the heat-stable pre-formed toxin cereulide, which causes emetic foodborne intoxication. This study characterised emetic B. cereus group isolates from raw cow’s milk in the biochemical, genetic, and toxigenic aspects. Of the 158 B. cereus group isolates derived from 99 raw milk samples, 7 (4.43%) harboured cereulide synthetase A (cesA), which encodes a cereulide synthetase associated with the emetic phenotype. Heat-treated culture filtrates from the cesA-positive isolates demonstrated cytotoxicity to HepG2 and Caco-2 cells, resulting in cell viabilities of 32.22–36.57% and 44.41–47.08%, respectively. The cytotoxicity levels were comparable to those of the reference emetic strain, F4810/72 (alternately termed AH187). Genome analysis of a representative isolate, CSB98, revealed the complete ces gene cluster with additional virulence factors such as non-haemolytic enterotoxin, haemolysins and phospholipases, suggesting that the isolate could be both emetic and diarrhoeagenic. CSB98 exhibited a closer relationship to the type strain of B. paranthracis than to that of B. cereus sensu stricto (ATCC 14579). The genomes of CSB98 and AH187 were indistinguishable through OrthoANI analysis, but 13 variants were identified via SNP calling. These results affirm genetic conservation among the emetic traits. Full article

(This article belongs to the Special Issue The Microbial Safety of Milk, Cheese and Dairy Products)

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11 pages, 1316 KB

Open AccessArticle

Characterization of the Toxigenic Potential of Bacillus cereus sensu lato Isolated from Raw Berries and Their Products

by Márcia Oliveira, Marta Carvalho and Paula Teixeira

Foods 2023, 12(21), 4021; https://doi.org/10.3390/foods12214021 - 3 Nov 2023

Cited by 4 | Viewed by 2120

Abstract

Bacillus cereus is estimated to be responsible for 1.4–12% of all food poisoning outbreaks worldwide. The objective of this study was to investigate the toxigenic potential of 181 isolates of B. cereus previously recovered from different types of berries and berry products (strawberries, raspberries, blackberries, and blueberries) by assessing the presence of enterotoxin genes (hblA, hblC, hblD, nheA, nheB, nheC, and cytK) and an emetic toxin cereulide synthetase gene (ces). The cytotoxic activity on Caco-2 cells was also evaluated for the two isolates containing the gene cytK. Twenty-three toxigenic profiles were found. The nheABC (91.7%) and hblACD (89.0%) complexes were the most prevalent among the isolates, while the cytK and ces genes were detected in low percentages, 1.1% and 3.3%, respectively. In addition, the nheABC/hblACD complex and ces genes were detected in isolates recovered throughout the production process of blackberries and strawberries. The cytotoxic activity on Caco-2 cells was also observed to be greater than 60% for isolates containing the cytK gene. Full article

(This article belongs to the Section Food Microbiology)

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15 pages, 2602 KB

Open AccessArticle

Intraspecific Diversity and Pathogenicity of Bacillus thuringiensis Isolates from an Emetic Illness

by Jintana Pheepakpraw, Thida Kaewkod, Maytiya Konkit, Sasiprapa Krongdang, Kanyaluck Jantakee, Rueankaew Praphruet, Sakunnee Bovonsombut, Aussara Panya, Yingmanee Tragoolpua, Niall A. Logan and Thararat Chitov

Toxins 2023, 15(2), 89; https://doi.org/10.3390/toxins15020089 - 18 Jan 2023

Cited by 4 | Viewed by 2755

Abstract

This study describes an emetic food-borne intoxication associated with a Bacillus cereus group species and the characterization of the bacterial isolates from the incident in aspects of molecular tying, genetic factors, cytotoxicity, and pathogenic mechanisms relating to emetic illness. Through the polyphasic identification approach, all seven isolates obtained from food and clinical samples were identified as Bacillus thuringiensis. According to multilocus sequence typing (MLST) analysis, intraspecific diversity was found within the B. thuringiensis isolates. Four allelic profiles were found, including two previously known STs (ST8 and ST15) and two new STs (ST2804 and ST2805). All isolates harbored gene fragments located in the cereulide synthetase (ces) gene cluster. The heat-treated culture supernatants of three emetic B. thuringiensis isolates, FC2, FC7, and FC8, caused vacuolation and exhibited toxicity to Caco-2 cells, with CC₅₀ values of 56.57, 72.17, and 79.94 µg/mL, respectively. The flow cytometry with the Annexin V/PI assay revealed both apoptosis and necrosis mechanisms, but necrosis was the prominent mechanism that caused Caco-2 cell destruction by FC2, the most toxic isolate. Full article

(This article belongs to the Special Issue Foodborne Toxins: Advanced Detection and Toxicity)

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18 pages, 3588 KB

Open AccessArticle

Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus

by Eva Maria Kalbhenn, Markus Kranzler, Agnieszka Gacek-Matthews, Gregor Grass, Timo D. Stark, Elrike Frenzel and Monika Ehling-Schulz

Int. J. Mol. Sci. 2022, 23(19), 11479; https://doi.org/10.3390/ijms231911479 - 29 Sep 2022

Cited by 7 | Viewed by 2677

Abstract

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis. Full article

(This article belongs to the Special Issue Bacterial Regulatory Proteins 2.0)

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13 pages, 2836 KB

Open AccessArticle

Influence of the Phagemid PfNC7401 on Cereulide-Producing Bacillus cereus NC7401

by Peiling Geng, Yunfei Gong, Xiaofu Wan and Xiaomin Hu

Microorganisms 2022, 10(5), 953; https://doi.org/10.3390/microorganisms10050953 - 30 Apr 2022

Viewed by 2134

Abstract

A phagemid-cured strain, NC7401-∆Pf, was constructed to survey the biological function of the plasmidal prophage PfNC7401 in cereulide-producing Bacillus cereus NC7401. The transcriptome analysis between the mutant and the wild strains revealed a series of differentially expressed genes mainly involved in different function classifications, including the two-component signal transduction system, bacterial structure, transporters, related antibiotic response, purine biosynthesis, non-ribosomal peptide synthetases (NRPS) and related secondary metabolites, and aromatic or other amino acid synthesis. BIOLOG and phenotypic experiment analyses confirmed that PfNC7401 may affect phage immunity and the metabolism of several amino acids, including L-Alanine, which was suggested to be related to one precursor (D-Alanine) of cereulide synthesis. However, neither the transcription levels of the cereulide production-related genes (e.g., ilvB, cesA, cesB, and cesH) nor the cereulide production nor cell cytotoxicity were affected by the presence or absence of PfNC7401, corresponding with the transcriptome data, in which only four genes unrelated to cereulide synthesis on the plasmid-carrying ces gene cluster were affected by the curing of PfNC7401. Full article

(This article belongs to the Special Issue Adaptation Mechanisms of Microbial Pathogens to Their Host Niche)

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10 pages, 1408 KB

Open AccessArticle

Evaluation of the Toxicity and Toxicokinetics of Cereulide from an Emetic Bacillus cereus Strain of Milk Origin

by Yifang Cui, Yuan Liu, Xiaoye Liu, Xi Xia, Shuangyang Ding and Kui Zhu

Toxins 2016, 8(6), 156; https://doi.org/10.3390/toxins8060156 - 6 Jun 2016

Cited by 42 | Viewed by 8183

Abstract

Bacillus cereus is an opportunistic foodborne agent causing food poisoning and many infectious diseases. The heat-stable emetic toxin cereulide is one of the most prevalent toxins produced by pathogenic B. cereus, resulting in symptoms such as emesis and liver failure. In the present work, the toxicity and toxicokinetics of cereulide from an emetic B. cereus isolate (CAU45) of raw milk were evaluated. The production of cereulide was tested by a cytotoxicity test and enzyme immunoassay, and confirmed by the presence of the ces (cereulide synthetase) gene and the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. All results showed that the amount and toxicity of cereulide produced by CAU45 was 7 to 15.3 folds higher than the reference emetic B. cereus DSMZ 4312. Cereulide in plasma was collected at different time points after a single intravenous injection to evaluate its toxicokinetics in rabbits. The maximum concentration of cereulide was achieved in 2.6 ± 3.4 h after administration, with the elimination half-life of 10.8 ± 9.1 h, which expands our understanding of the toxic effects of cereulide. Together, it suggests that urgent sanitary practices are needed to eliminate emetic toxins and emetic B. cereus in raw milk. Full article

(This article belongs to the Section Bacterial Toxins)

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