RESUMEN
Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings produce a 1197 Da toxin, named amylosin. Nuclear magnetic resonance (NMR) data showed that amylosin contains a chromophoric polyene structure and the amino acids leucine/isoleucine, proline, aspartic acid/asparagine, glutamic acid/glutamine and tyrosine. A quantitation method for amylosin was developed using commercially available amphotericin B as a reference compound and a known concentration of amylosin determined by NMR with the electronic reference to access in vivo concentration (ERETIC) method. Purified amylosin inhibited motility of boar sperm cells at an exposure concentration of 135 nM and hyperpolarized their cell membrane and depolarized their mitochondria at exposure to concentration of 33-67 nM for 10 min. In a 3-d exposure time only 27 nM of amylosin was needed to provoke the same toxicity functions. Amylosin was cytotoxic to feline lung cells at concentrations of <170 nM. Purified amylosin provoked adenosine 5'-triphosphate (ATP)-independent cation influx into isolated rat liver mitochondria (RLM), inducing swelling of the mitochondria at concentrations of 200 nM K(+) or >250 nM Na(+) medium. In the K(+)- or Na(+)-containing medium, amylosin uncoupled RLM, causing oxidation of pyridine nucleotides (PN), loss of the mitochondrial membrane potential, and suppressed ATP synthesis. Purified amylosin produced cation channels in black-lipid membranes (BLMs) with a selectivity K(+)>Na(+) at a concentration of 26 nM, i.e. the same concentration at which amylosin was toxic to boar sperm cells. The amylosin cation channels were cholesterol- and ATP-independent and more effective with K(+) than with Na(+). We propose that the toxicity of amylosin may be due its ionophoric properties, representing the first K(+)/Na(+) channel-forming substance reported from B. amyloliquefaciens.
Asunto(s)
Bacillus/química , Toxinas Bacterianas/toxicidad , Proteínas de Transporte de Catión/toxicidad , Polienos/toxicidad , Adenosina Trifosfato/metabolismo , Aminoácidos/análisis , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/aislamiento & purificación , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/aislamiento & purificación , Gatos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Pulmón/efectos de los fármacos , Masculino , Espectrometría de Masas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Fluorescente , Resonancia Magnética Nuclear Biomolecular , Polienos/química , Polienos/aislamiento & purificación , Ratas , Motilidad Espermática/efectos de los fármacos , Sus scrofa , Pruebas de ToxicidadRESUMEN
Cereulide producing Bacillus cereus was isolated from randomly chosen commercial infant foods. The cereulide production in infant food formulas was investigated. When the reconstituted foods were inoculated with >10(5) cfu ml(-1) of cereulide producing B. cereus, 2 to 200 microg of cereulide per 100 ml of food accumulated during 24 h of non-refrigerated storage. The amount of cereulide measured in the foods by the accurate chemical assay (LC-MS) matched with that found by sperm micro assay, proving the cereulide was the sole heat stable toxin in the foods and present in its toxic form. The infant formulas containing both cereal and dairy ingredients were the most supportive for cereulide production. Cereulide accumulation was affected by the infant food composition as well as by the handling of the food. Diluting the reconstituted food with water resulted in increased toxin production expressed as mug per volume. More cereulide was accumulated when the food was incubated stationary compared with moderate shaking. The amount of cereulide accumulated within 24 h at room temperature per 100 ml of cereal and dairy or in rice-nondairy reconstituted infant formulas, inoculated with >or=10(5) cfu ml(-1) of B. cereus strain F4810/72, was higher or similar to the amounts reported for foods implicated in emetic type of food poisonings. Thus mishandling and temperature abuse of infant foods may cause food poisoning when emetic B. cereus is present.
Asunto(s)
Bacillus cereus/metabolismo , Seguridad de Productos para el Consumidor , Depsipéptidos/biosíntesis , Contaminación de Alimentos/análisis , Manipulación de Alimentos/métodos , Alimentos Infantiles/análisis , Recuento de Colonia Microbiana , Contaminación de Alimentos/prevención & control , Manipulación de Alimentos/normas , Humanos , Lactante , Temperatura , Factores de TiempoRESUMEN
Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24 strains of B. cereus including two high cereulide producers (600-1,800 ng cereulide mg(-1) wet weight biomass), seven average producers (180-600 ng cereulide mg(-1) wet weight biomass), four low cereulide producers (20-160 ng cereulide mg(-1) wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The 13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234-02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought.
Asunto(s)
Bacillus cereus/clasificación , Bacillus cereus/fisiología , Depsipéptidos/biosíntesis , Variación Genética , Adenilil Ciclasas/genética , Bacillus anthracis/genética , Bacillus cereus/genética , Bacillus cereus/aislamiento & purificación , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Desoxirribonucleasa EcoRI/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Microbiología Ambiental , Microbiología de Alimentos , Enfermedades Transmitidas por los Alimentos/microbiología , Tracto Gastrointestinal/microbiología , Hemólisis , Datos de Secuencia Molecular , Fosfatidilcolinas/metabolismo , ARN Ribosómico 16S/genética , Ribotipificación , Análisis de Secuencia de ADN , Homología de Secuencia , Tirosina/metabolismoRESUMEN
This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.