RESUMEN
The impacts of growth media and temperature on production of cereulide, the emetic toxin of Bacillus cereus, were measured for seven well characterised strains selected for diversity of biochemical and genetic properties and sources of origin. All strains carried cereulide synthase gene, ces, on a megaplasmid of ca. 200 kb and all grew up to 48-50 degrees C, but produced cereulide only up to 39 degrees C. On tryptic soy agar five strains, originating from foods, food poisonings and environment, produced highest amounts of cereulide at 23 to 28 degrees C, whereas two strains, from human faeces, produced cereulide similarly from 23 to 39 degrees C, with no clear temperature trend. These two strains differed from the others also by producing more cereulide on tryptic soy agar if supplemented with 5 vol.% of blood, whereas the other five strains produced similarly, independent on the presence of blood. On oatmeal agar only one strain produced major amounts of cereulide. On skim milk agar, raw milk agar, and MacConkey agar most strains grew well but produced only low amounts of cereulide. Three media components, the ratio [K+]:[Na+], contents of glycine and [Na+], appeared of significance for predicting cereulide production. Increase of [K+]:[Na+] (focal variable) predicted (P < 0.001) high cereulide provided that the contents of glycine and [Na+] (additional variables) were kept constant. The results show that growth medium and temperature up and downregulate cereulide production by emetic B. cereus in a complex manner. The relevance of the findings to production of cereulide in the gut and to the safety of amino acids as additives in foods containing live toxinogenic organisms is discussed.
Asunto(s)
Bacillus cereus/metabolismo , Medios de Cultivo/química , Depsipéptidos/biosíntesis , Microbiología de Alimentos , Temperatura , Bacillus cereus/crecimiento & desarrollo , Seguridad de Productos para el Consumidor , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Manipulación de Alimentos/métodos , Enfermedades Transmitidas por los Alimentos/prevención & control , Glicina/metabolismo , Humanos , Potasio/metabolismo , Medición de Riesgo , Sodio/metabolismoRESUMEN
The members of the Bacillus cereus group, Bacillus anthracis, Bacillus thuringiensis, and B. cereus senso stricto, are largely defined by their content of large plasmids, which encode major virulence factors. Here we offer an easy, fast, and reliable protocol for the isolation and detection of large plasmids up to the size of at least 350kb. Furthermore, using this method, we report that Bacillus mycoides contain large plasmids.
Asunto(s)
Bacillus cereus/genética , Plásmidos/aislamiento & purificación , Bacillus cereus/clasificación , Bacillus cereus/crecimiento & desarrollo , Factores de TiempoRESUMEN
Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain. A new observation was that 31 out of the 40 randomly selected B. cereus-like strains could be classified as Bacillus thuringiensis due to crystal production and/or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis.
Asunto(s)
Bacillus cereus/aislamiento & purificación , Bacillus cereus/patogenicidad , Bacillus thuringiensis/aislamiento & purificación , Bacillus thuringiensis/patogenicidad , Microbiología de Alimentos , Bacillus cereus/clasificación , Bacillus cereus/genética , Bacillus thuringiensis/clasificación , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Técnicas de Tipificación Bacteriana , Secuencia de Bases , ADN Bacteriano/genética , Diarrea/microbiología , Farmacorresistencia Bacteriana , Endotoxinas/genética , Enterotoxinas/genética , Genes Bacterianos , Genotipo , Infecciones por Bacterias Grampositivas/microbiología , Proteínas Hemolisinas , Humanos , FenotipoRESUMEN
The toxicity of Bacillus thuringiensis subsp. israelensis to dipteran larvae (mosquitoes and black flies) depends on the presence of the pBtoxis plasmid. In this paper, two antibiotic resistance tagged pBtoxis were transferred by conjugation to other Bacillus cereus group strains. Among 15 potential recipients, only a lepidopteran active B. thuringiensis subspecies kurstaki and a B. cereus strain received the plasmid pBtoxis with a low transfer rate of about 10(-8) transconjugants/recipient. The resulting B. thuringiensis subspecies kurstaki transconjugant was active to both lepidopteran and dipteran targets and the B. cereus transconjugant was active against dipteran insects. Phase contrast microscopy showed that the B. cereus transconjugants could produce only round crystalline inclusion bodies while B. thuringiensis subspecies kurstaki transconjugant could produce both round and bipyramidal crystals during sporulation. SDS-PAGE revealed that all the major mosquitocidal proteins from pBtoxis could express in the two transconjugants, including Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa and Cyt1Aa. However, none of the experiment showed any indications of mobilising abilities of pBtoxis. The limited number of strains, which could receive and maintain pBtoxis using a conjugational helper plasmid, indicates a very narrow host range of the B. thuringiensis subsp. israelensis pBtoxis plasmid.
Asunto(s)
Bacillus cereus/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Endotoxinas/biosíntesis , Endotoxinas/genética , Plásmidos , Transformación Bacteriana , Animales , Bacillus cereus/citología , Bacillus cereus/metabolismo , Bacillus cereus/patogenicidad , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Conjugación Genética , Culex/microbiología , Electroforesis en Gel de Poliacrilamida , Proteínas Hemolisinas , Cuerpos de Inclusión/metabolismo , Control Biológico de Vectores , Reacción en Cadena de la Polimerasa , Esporas Bacterianas/citología , Esporas Bacterianas/metabolismo , Virulencia/genéticaRESUMEN
The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.
Asunto(s)
Bacillus cereus/genética , Bacillus thuringiensis/genética , Conjugación Genética , Proteínas de Insectos/metabolismo , Plásmidos/genética , Receptores de Superficie Celular/metabolismo , Bacillus cereus/metabolismo , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas , Electroforesis en Gel de Agar , Expresión Génica , Proteínas de Insectos/genética , Receptores de Superficie Celular/genéticaRESUMEN
Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.
Asunto(s)
Bacillus thuringiensis/genética , ADN Circular , Plásmidos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Colágeno/química , ADN/metabolismo , Elementos Transponibles de ADN , Genes Bacterianos , Modelos Genéticos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Recombinación Genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
In a study of occupational exposure to Bacillus thuringiensis, 20 exposed greenhouse workers were examined for Bacillus cereus-like bacteria in fecal samples and on biomonitoring filters. Bacteria with the following characteristics were isolated from eight individuals: intracellular crystalline inclusions characteristic of B. thuringiensis, genes for and production of B. cereus enterotoxins, and positivity for cry11 as determined by PCR. DNA fingerprints of the fecal isolates were identical to those of strains isolated from the commercial products used. Work processes (i.e., spraying) correlated with the presence of B. thuringiensis in the fecal samples (10(2) to 10(3) CFU/g of feces). However, no gastrointestinal symptoms correlated with the presence of B. thuringiensis in the fecal samples.
Asunto(s)
Bacillus thuringiensis/clasificación , Bacillus thuringiensis/aislamiento & purificación , Toxinas Bacterianas/genética , Heces/microbiología , Exposición Profesional/análisis , Plaguicidas/efectos adversos , Bacillus cereus/genética , Bacillus cereus/aislamiento & purificación , Bacillus thuringiensis/genética , Dermatoglifia del ADN , Enterotoxinas/toxicidad , Humanos , Control Biológico de VectoresRESUMEN
OBJECTIVES: Since the discovery of the insecticidal activity of Bacillus thuringiensis at the beginning of the twentieth century, this bacterium has been used increasingly against various insect pests. In spite of the extensive use of B. thuringiensis, only sporadic clinical case reports have been published. In recent years, the close relationship between B. thuringiensis and the human pathogen Bacillus cereus has been confirmed. In practice, only the insecticidal activity of B. thuringiensis distinguishes the two species. However, both species are composed of thousands of isolates with varying potential for causing adverse effects in humans. The aim of this study was to employ molecular biology methods for assessment of occupational exposure to B. thuringiensis-based biopesticides by determination of specific genetic information including plasmid profiles and random amplified polymorphic DNA (RAPD). METHODS: Faecal samples from 12 persons, working in Danish greenhouses, were collected for microbial analysis. Seven persons were using B. thuringiensis-based insecticides, whereas five persons were employed at greenhouses that did not use B. thuringiensis. The bacteria were isolated on B. cereus-specific solid substrate, and colonies were further identified using the polymerase chain reaction (PCR). The PCR method was used for the identification of the enterotoxin genes HblA and BceT. The expression of enterotoxins was detected with two commercial serological kits. Primers specific for 16S-23S spacer region were used to identify the bacteria as members of the B. cereus group. Several primers towards insecticidal genes have been used in order to further characterize the isolates as subspecies of B. thuringiensis. RESULTS: Two faecal samples from the B. thuringiensis-exposed greenhouse workers were positive for B. cereus-like bacteria. One isolate displayed intracellular crystalline inclusions characteristic of B. thuringiensis, production of and genes for B. cereus enterotoxins and it was PCR-positive for an insecticidal toxin primer set. RAPD profiles of the faecal isolate were identical to that of strains isolated from a commercial product. CONCLUSIONS: The methods applied have verified that the faecal isolate was identical to the B. thuringiensis isolate found in the biopesticide used. This is the first reported case of isolation of a bacterial biopesticide from human faeces. The biopesticide was shown to harbour and express enterotoxin genes. However, there is no evidence that this caused any adverse effects to the person from whom these bacteria were isolated.