Genetic and toxigenic diversity of Bacillus cereus group isolated from powdered foods.

Bacillus cereus is a human pathogenic bacterium that produces emetic and diarrheal foodborne diseases. This study evaluated the genetic and toxigenic diversity in B. cereus group isolates from powdered foods collected in public educational institutions, bakeries and powdered food companies located in Medellín, Colombia. B. cereus was detected in 35 of 305 (11%) powdered food samples and 52 B. cereus were isolated. The presence of ten toxin genes, hblCDAB, nheABC, cytK2, entFM and cesB, was evaluated in the isolates by multiplex PCR. The nheABC operon was found in all isolates (100%), hblCDAB in 22 (42%), hblCDA in 8 (15%) and hblCD in 3 (6%); the cytK2 gene was detected in 32 isolates (62%) and entFM in 32 (62%). Notably, the cesB gene was not detected. According to the presence of toxin genes, fifteen profiles were identified. The predominant toxigenic profile contained all toxin genes but cesB. A large genetic diversity was observed by GTG5 fingerprinting with 46 isolates grouped in seven clusters and the remaining six clustering individually. There was no relationship between toxigenic profiles and genetic clusters, but some genetic clusters seemed to be related to particular powdered food types. In general, the results evidenced high genetic and enterotoxigenic diversity among the B. cereus group isolates.

Enterotoxin Gene Distribution and Genotypes of Bacillus cereussensu lato Isolated from Cassava Starch.

Bacillus cereus is a human pathogenic bacterium found in foods with the potential to cause emesis and diarrhea. This study estimated the presence, toxigenic and genomic diversity of B. cereus s.l. obtained from cassava starch samples collected in bakeries and powdered food companies in Medellín (Colombia). Bacillus cereuss.l. was found in 43 of 75 (57%) cassava starch samples and 98 isolates were obtained. The nheABC, hblCDAB, cytK2, entFM and cesB toxin genes were detected by multiplex PCR and the most frequent operon was nheABC, whereas cesB gene was not found. Twelve toxigenic profiles were determined by the detection of toxin genes, and the most frequent profiles harbored all enterotoxin genes. A broad genomic diversity was detected according to GTG5-PCR fingerprinting results with 76 B. cereus s.l. grouped in sixteen clusters and the 22 isolates clustering separately. No relationship was observed between genomic background and toxigenic profiles. In general, the results showed a high genomic and enterotoxigenic diversity in B. cereus s.l. found in cassava starch. These results should incentive future studies to understand the distribution of B. cereus s.l. isolated on raw materials in comparison with finished products.

Genomic and Toxigenic Heterogeneity of Bacillus cereus sensu lato Isolated from Ready-to-Eat Foods and Powdered Milk in Day Care Centers in Colombia.

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 ß-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.

Use of Bacillus thuringiensis supernatant from a fermentation process to improve bioremediation of chlorpyrifos in contaminated soils.

The aim of this research was to investigate the potential of a nutrient-rich organic waste, namely the cell-free supernatant of Bacillus thuringiensis (BtS) gathered from fermentation, as a biostimulating agent to improve and sustain microbial populations and their enzymatic activities, thereby assisting in the bioremediation of chlorpyrifos-contaminated soil at a high dose (70 mg kg(-1)). Experiments were performed for up to 80 d. Chlorpyrifos degradation and its major metabolic product, 3,5,6-trichloro-2-pyridinol (TCP), were quantified by high-performance liquid chromatography (HPLC); total microbial populations were enumerated by direct counts in specific medium; and fluorescein diacetate (FDA) hydrolysis was measured as an index of soil microbial activity. Throughout the experiment, there was higher chlorpyrifos degradation in soil supplemented with BtS (83.1%) as compared to non-supplemented soil. TCP formation and degradation occurred in all soils, but the greatest degradation (30.34%) was observed in soil supplemented with BtS. The total microbial populations were significantly improved by supplementation with BtS. The application of chlorpyrifos to soil inhibited the enzymatic activity; however, this negative effect was counteracted by BtS, inducing an increase of approximately 16% in FDA hydrolysis. These results demonstrate the potential of B. thuringiensis supernatant as a suitable biostimulation agent for enhancing chlorpyrifos and TCP biodegradation in chlorpyrifos-contaminated soils.

Direct detection of toxigenic Bacillus cereus in dietary complement for children and cassava starch / Detección directa de Bacillus cereus toxigénicos en complementos dietarios para niños y en almidón de yuca / Detecção direta de Bacillus cereus toxigênicos em suplementos alimentares para crianças e amido de mandioca

Bacillus cereus is a food contaminant and a known human pathogen that can cause emetic and diarrheal syndromes. In this study we evaluated the presence of toxigenic B. cereus by multiplex PCR directly in dietary complement for children and cassava starch samples collected on Medellin, Colombia. Of 75 dietary complement for children samples evaluated, 70.7% were contaminated with toxigenic B. cereus and four different toxigenic consortia were detected: I: nheA, hblC, cytK (9.8%), II: nheA, hblC (2%), III: hblC, cytK (41.2%), IV: hblC (47%). Of 75 cassava starch samples, 44% were contaminated with toxigenic B. cereus and four different toxigenic consortia were determined: I: nheA, hblC, cytK (48.5%), II: nheA, hblC, cytK, cesB (3%), III: hblC, cytK (30.3%), IV: hblC (18.2%). In general, in dietary complement for children only enterotoxigenic consortia were detected while in cassava starch the enterotoxigenic consortia predominated over the emetic. Multiplex PCR was useful to detect toxigenic B. cereus contamination allowing direct and simultaneous detection of all toxin genes in foods. This study is the first in Colombia to evaluate toxigenic B. cereus, providing information of importance for microbiological risk evaluation in dried foods.

Bacillus cereus es un contaminante de alimentos conocido por ser patogénico para los humanos, causando síndromes de vómito y diarrea. En este estudio se evaluó la presencia de B. cereus toxigénicos utilizando PCR múltiple directamente en complementos dietarios para niños y en almidón de yuca colectados en Medellín, Colombia. De 75 muestras de complemento dietario para niños, 70,7% estuvieron contaminadas con B. cereus toxigénicos y se detectaron cuatro diferentes consorcios toxigénicos: I: nheA, hblC, cytK (9,8%), II: nheA, hblC (2%), III: hblC, cytK (41.2%), IV: hblC (47%). De 75 muestras de almidón de yuca, 44% estuvieron contaminadas con B. cereus toxigénicos y se determinaron cuatro diferentes consorcios toxigénicos: I: nheA, hblC, cytK (48.5%), II: nheA, hblC, cytK, cesB (3%), III: hblC, cytK (30,3%), IV: hblC (18.2%). En general, en los complementos dietarios para niños sólo se detectaron consorcios enterotoxigénicos, mientras que en el almidón los consorcios enterotoxigénicos predominaron sobre el emético. La PCR múltiple fue de utilidad para detectar contaminación con B. cereus toxigénicos permitiendo la detección directa y simultánea de todos los genes tóxicos en los alimentos. Este estudio es el primero en Colombia en evaluar B. cereus toxigénicos y proporciona información importante para la evaluación de riesgos microbiológicos en los alimentos pulverizados.

Bacillus cereus é um contaminante de alimentos e é conhecido por ser patogénico nos seres humanos ocasionando síndromes de vômitos e diarreia. Neste estudo foi avaliada a presença de B. cereus toxigênicos por PCR multiplex diretamente em complementos da dieta para crianças e amido de mandioca, em amostras coletadas em Medellín, na Colômbia. De 75 amostras dos complementos da dieta para crianças, 70,7% estiveram contaminadas com B. cereus toxigênicos e foram detectados quatro diferentes consórcios: I: nheA, hblC, cytK (9,8%), II: nheA, hblC (2%), III: hblC, cytK (41,2%), IV: hblC (47%). De 75 amostras de amido de mandioca, 44% estiveram contaminadas com B. cereus toxigênicos e quatro consórcios diferentes foram determinados: I: nheA, hblC, cytK (48,5%), II: nheA, hblC, cytK, cesB (3%) III: hblC, cytK (30,3%), IV: hblC (18,2%). Em geral, nos complementos da dieta para crianças foram detectados apenas consórcios enterotoxigênicos, não obstante no amido os consórcios enterotoxigênicos predominaram sobre o emético. A PCR multiplex foi útil para detectar contaminação com B. cereus toxigênico permitindo a detecção direta e simultânea de todos os genes tóxicos em alimentos. Este estudo é o primeiro na Colômbia em avaliar B. cereus toxigênico e providencia informação importante para a avaliação de riscos microbiológicos em alimentos pulverizados.

Detección directa de genes toxigénicos de Bacillus cereus en fécula de maíz y harina de trigo mediante reacción en cadena de la polimerasa múltiple (mPCR) / Direct detection of toxigenic genes of Bacillus cereus in corn starch and wheat flour using multiplex polymerase chain reaction (mPCR)

Introducción: Bacillus cereus es una bacteria contaminante de alimentos y patógena en humanos, cuya toxina emética o cereúlida (Ces) causa el síndrome emético y las enterotoxinas hemolítica o hemolisina BL (Hbl), no hemolítica (Nhe) y citotoxina K (CytK), el síndrome diarreico. Objetivo: Determinar la presencia de genes toxigénicos de Bacillus cereus en muestras de ADN obtenido directamente de fécula de maíz y de harina de trigo, mediante reacción en cadena de la polimerasa múltiple. Materiales y métodos: Se determinaron los genes toxigénicos de Bacillus cereus en muestras de ADN extraído directamente de fécula de maíz y harina de trigo, utilizando una reacción en cadena de la polimerasa múltiple específica para los genes cesB, hblC, nheA y cytK. Resultados: De 76 muestras de fécula de maíz, el 60,5% presentó los genes toxigénicos de Bacillus cereus, que fueron agrupados en seis consorcios: I: hblC, cytK (30,4%), II: nheA, hblC, cytK (21,7%), III: hblC (19,6%), IV: nheA (15,2%), V: nheA, hblC (10,9%), VI: nheA, hblC, cytK, cesB (2,2%). De 79 muestras de harina de trigo, el 65,8% presentó los genes toxigénicos de Bacillus cereus, que se agruparon en cuatro consorcios: I: nheA, hblC, cytK (80,8%), II: hblC, cytK (11,5%), III: hblC (5,8%), IV: nheA, hblC (1,9%)...

Introduction: Bacillus cereus is a human pathogen that causes two kinds of foodborne diseases, the emetic syndrome caused by emetic toxin or cereulide (Ces), and the diarrheal syndrome caused by three different enterotoxins, the hemolytic enterotoxin or hemolysin BL (Hbl), the nonhemolytic enterotoxin (Nhe) and the cytotoxin K (CytK). Objective: To determine the presence of toxigenic genes of Bacillus cereus in DNA samples directly obtained from corn starch and wheat flour using multiplex polymerase chain reaction. Material and methods: The presence of toxigenic genes of Bacillus cereus were determinedin DNA samples directly extracted from corn starch and wheat flour, using a multiplex polymerase chain reaction technique specific for cesB, hblC, nheA and cytK genes. Results: From a total of 76 corn starch samples, 60.5% had toxigenic genes of Bacillus cereus and were grouped in six consortia: I: hblC, cytK (30.4%), II: nheA, hblC, cytK (21.7%), III: hblC (19.6%), IV: nheA (15.2%), V: nheA, hblC (10.9%) and VI: nheA, hblC, cytK, cesB (2.2%). From 79 wheat flour samples tested, 65.8% had toxigenic genes of Bacillus cereus and were grouped into four consortia: I: nheA, hblC, cytK (80.8%), II: hblC, cytK (11.5%), III: hblC (5.8%) and IV: nheA, hblC (1.9%)...

The RNPP family of quorum-sensing proteins in Gram-positive bacteria.

Quorum sensing is one of several mechanisms that bacterial cells use to interact with each other and coordinate certain physiological processes in response to cell density. This mechanism is mediated by extracellular signaling molecules; once a critical threshold concentration has been reached, a target sensor kinase or response regulator is activated (or repressed), facilitating the expression of quorum sensing-dependent genes. Gram-positive bacteria mostly use oligo-peptides as signaling molecules. These cells have a special kind of quorum-sensing systems in which the receptor protein interacts directly with its cognate signaling peptide. The receptors are either Rap phosphatases or transcriptional regulators and integrate the protein family RNPP, from Rap, Npr, PlcR, and PrgX. These quorum-sensing systems control several microbial processes, like sporulation, virulence, biofilm formation, conjugation, and production of extracellular enzymes. Insights of the mechanism of protein-signaling peptide binding as well as the molecular interaction among receptor protein, signaling peptide, and target DNA have changed some earlier perceptions. In spite of the increased knowledge and the potential biotechnological applications of these quorum-sensing systems, few examples on engineering for biotechnological applications have been published. Real applications will arise only when researchers working in applied microbiology and biotechnology are aware of the importance of quorum-sensing systems for health and bioprocess applications.

SKPDT is a signaling peptide that stimulates sporulation and cry1Aa expression in Bacillus thuringiensis but not in Bacillus subtilis.

We have identified and characterized in the supernatant of the transition phase of Bacillus thuringiensis var. kurstaki the peptide SKPDT. This peptide was previously identified by in silico analysis by Pottathil and Lazazzera (Front Biosci 8:32-45 2003) as a putative signaling peptide (NprRB) of the Phr family in B. thuringiensis. The chemically synthesized NprRB did not affect the growth kinetics of B. thuringiensis var. kurstaki but stimulated the sporulation, spore release, and transcription of cry1Aa when added to cultures during the transition phase. In fact, when the peptide (100 nM) was added to a culture in transition phase, the transcription of cry1Aa was stimulated almost threefold, mainly from the late promoter BtII, which requires the late-stage sporulation-specific transcription factor sigma (K). On the other hand, NprRB did not have any effect on B. subtilis. Thus, SKPDT seems to be a signaling peptide specific for B. thuringiensis.