RESUMEN
Salmonella enterica serovar Enteritidis strain E40 filaments were developed under conditions of a reduced water activity (a(w)) of 0.95 in tryptic soy broth (TSB) or tryptic soy agar (TSA) supplemented with 8% or 7% NaCl, respectively. Filament formation was accompanied by an increase of biomass without an increase in CFU and was affected by incubation temperature and the physical milieu. The greatest amount of filaments was recovered from TSA with 7% NaCl and incubation at 30°C. Within 2 h of transfer to fresh TSB, filaments started to septate into normal-sized cells, resulting in a rapid increase in CFU. S. Enteritidis E40 filaments were not more tolerant of low- or high-temperature stresses than nonfilamented control cells. However, there was greater survival of filaments in 10% bile salts after 24 to 48 h of incubation, during pH 2.0 acid challenge for 10 min, and under desiccation on stainless steel surfaces at 25°C and 75.5% relative humidity for 7 days. S. Enteritidis E40 filaments invaded and multiplied within Caco-2 human intestinal epithelial cells to a similar degree as control cells when a comparable CFU of filaments and control cells was used. S. Enteritidis E40 filaments established a successful infection in mice via intragastric inoculation. The filaments colonized the gastrointestinal tract and disseminated to the spleen and liver at levels comparable to those attained by control cells, even when animals were inoculated with 10- to 100-fold fewer CFU. To our knowledge this is the first demonstration of virulence of stress-induced Salmonella filaments in vitro and in vivo. Formation of filaments by Salmonella in food products and food processing environments is significant to food safety, because detection and quantitation of the pathogen may be compromised. The finding that these filaments are virulent further enhances their potential public health impact.
Asunto(s)
Respuesta al Choque Térmico , Salmonella enteritidis/crecimiento & desarrollo , Salmonella enteritidis/patogenicidad , Animales , Biomasa , Células CACO-2/microbiología , Recuento de Colonia Microbiana , Medios de Cultivo , Desecación , Femenino , Tracto Gastrointestinal/microbiología , Humanos , Concentración de Iones de Hidrógeno , Ratones , Salmonelosis Animal/microbiología , Salmonella enteritidis/ultraestructura , Cloruro de Sodio/farmacología , Temperatura , Virulencia , AguaRESUMEN
The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease ( approximately 130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.
Asunto(s)
Bacillus cereus/fisiología , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/metabolismo , Bacillus cereus/química , Bacillus cereus/enzimología , Bacillus cereus/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biopelículas/efectos de los fármacos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Endopeptidasa K/farmacología , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Insercional , Operón , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Análisis de Secuencia de ADNRESUMEN
Bacillus cereus ATCC 14579 can respond to nutrient changes by adopting different forms of surface translocation. The B. cereus ATCC 14579 DeltaplcR mutant, but not the wild type, formed dendritic (branched) patterns on EPS [a low-nutrient medium that contains 7.0 g K(2)HPO(4), 3.0 g KH(2)PO(4), 0.1 g MgSO(4).7H(2)O, 0.1 g (NH(4))(2)SO(4), 0.01 g CaCl(2), 0.001 g FeSO(4), 0.1 g NaCl, 1.0 g glucose, and 125 mg yeast extract per liter] containing 0.7% agar. The dendritic patterns formed by sliding translocation of nonflagellated cells are enhanced under low-nutrient conditions and require sufficient production of a biosurfactant, which appears to be repressed by PlcR. The wild-type and complemented strains failed to slide on the surface of EPS agar because of the production of low levels of biosurfactant. Precoating EPS agar surfaces with surfactin (a biosurfactant produced by Bacillus subtilis) or biosurfactant purified from the DeltaplcR mutant rescued the ability of the wild-type and complemented strains to slide. When grown on a nutrient-rich medium like Luria-Bertani agar, both the wild-type and DeltaplcR mutant strains produced flagella. The wild type was hyperflagellated and elongated and exhibited swarming behavior, while the DeltaplcR mutant was multiflagellated and the cells often formed long chains but did not swarm. Thin-layer chromatography and mass spectrometry analyses suggested that the biosurfactant purified from the DeltaplcR mutant was a lipopeptide and had a mass of 1,278.1722 (m/z). This biosurfactant has hemolytic activity and inhibited the growth of several gram-positive bacteria.
Asunto(s)
Bacillus cereus/metabolismo , Proteínas Bacterianas/fisiología , Tensoactivos/metabolismo , Transactivadores/fisiología , Animales , Bacillus cereus/genética , Bacillus cereus/fisiología , Proteínas Bacterianas/genética , Cromatografía en Capa Delgada , Medios de Cultivo/farmacología , Eritrocitos/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Hemólisis/efectos de los fármacos , Espectrometría de Masas , Mutación , Ovinos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tensoactivos/farmacología , Transactivadores/genéticaRESUMEN
Stenotrophomonas maltophilia WR-C possesses an rpf/diffusible signal factor (DSF) cell-cell communication system. It produces cis-Delta2-11-methyl-dodecenoic acid, a DSF, and seven structural derivatives, which require rpfF and rpfB for synthesis. Acquisition of iron from the environment is important for bacterial growth as well as the expression of virulence genes. We identified a gene homologous to fecA, which encodes a ferric citrate receptor that transports exogenous siderophore ferric citrate from the environment into the bacterial periplasm. Western blot analysis with anti-FecA-His(6) antibody showed that the FecA homologue was induced in the iron-depleted medium supplemented with a low concentration of ferric citrate. Deletion of rpfF or rpfB resulted in reduced FecA expression compared to the wild type. Synthetic DSF restored FecA expression by the DeltarpfF mutant to the wild-type level. Reverse transcription-PCR showed that the fecA transcript was decreased in the DeltarpfF mutant compared to the wild type. These data suggest that DSF affected the level of fecA mRNA. Transposon inactivation of crp, which encodes cyclic AMP (cAMP) receptor protein (CRP) resulted in reduced FecA expression and rpfF transcript level. Putative CRP binding sites were located upstream of the rpfF promoter, indicating that the effect of CRP on FecA is through the rpf/DSF pathway and by directly controlling rpfF. We propose that CRP may serve as a checkpoint for iron uptake, protease activity, and hemolysis in response to environmental changes such as changes in concentrations of glucose, cAMP, iron, or DSF.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , Compuestos Férricos/metabolismo , Regulación Bacteriana de la Expresión Génica , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Stenotrophomonas maltophilia/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Transporte Biológico , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/genética , Elementos Transponibles de ADN , Proteínas de Escherichia coli/química , Datos de Secuencia Molecular , Mutagénesis Insercional , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Análisis de Secuencia de ADN , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/crecimiento & desarrolloRESUMEN
A simple cold plasma technique was developed to functionalize the surfaces of polyamide (PA) and polyester (PET) for the grafting of polyethylene glycol (PEG) with the aim of reducing biofilm formation. The surfaces of PA and PET were treated with silicon tetrachloride (SiCl4) plasma, and PEG was grafted onto plasma-functionalized substrates (PA-PEG, PET-PEG). Different molecular weights of PEG and grafting times were tested to obtain optimal surface coverage by PEG as monitored by electron spectroscopy for chemical analysis (ESCA). The presence of a predominant C-O peak on the PEG-modified substrates indicated that the grafting was successful. Data from hydroxyl group derivatization and water contact angle measurement also indicated the presence of PEG after grafting. The PEG-grafted PA and PET under optimal conditions had similar chemical composition and hydrophilicity; however, different morphology changes were observed after grafting. Both PA-PEG and PET-PEG surfaces developed under optimal plasma conditions showed about 96% reduction in biofilm formation by Listeria monocytogenes compared with that of the corresponding unmodified substrates. This plasma functionalization method provided an efficient way to graft PEG onto PA and PET surfaces. Because of the high reactivity of Si-Cl species, this method could potentially be applied to other polymeric materials.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Listeria monocytogenes/crecimiento & desarrollo , Nylons/química , Poliésteres/química , Polietilenglicoles/química , Cloruros/química , Materiales Biocompatibles Revestidos , Estudios de Evaluación como Asunto , Ensayo de Materiales , Compuestos de Silicona/químicaRESUMEN
The DeltaplcR mutant of Bacillus cereus strain ATCC 14579 developed significantly more biofilm than the wild type and produced increased amounts of biosurfactant. Biosurfactant production is required for biofilm formation and may be directly or indirectly repressed by PlcR, a pleiotropic regulator. Coating polystyrene plates with surfactin, a biosurfactant from Bacillus subtilis, rescued the deficiency in biofilm formation by the wild type.
Asunto(s)
Bacillus cereus/crecimiento & desarrollo , Bacillus cereus/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Transactivadores/metabolismo , Bacillus cereus/genética , Proteínas Bacterianas/genética , Técnicas Bacteriológicas , Medios de Cultivo , Tensoactivos/metabolismo , Transactivadores/genéticaRESUMEN
Stenotrophomonas maltophilia WR-C is capable of forming biofilm on polystyrene and glass. The lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes rmlA, rmlC, and xanB are necessary for biofilm formation and twitching motility. Mutants with mutations in rmlAC and xanB display contrasting biofilm phenotypes on polystyrene and glass and differ in swimming motility.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Lipopolisacáridos/biosíntesis , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/fisiología , Carbohidrato Epimerasas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Datos de Secuencia Molecular , Movimiento , Mutagénesis Insercional , Mutación , Nucleotidiltransferasas/genética , Análisis de Secuencia de ADNRESUMEN
The presence of hemolysin BL (HBL; components L(2), L(1), and B)-encoding genes (hblC, hblD, and hblA) from 339 Bacillus cereus strains isolated in Thailand was determined. PCR analysis showed that all three hbl genes were detected in 222 strains (65.5%). Two, one or no hbl genes were detected in 3 (0.9%), 6 (1.8%), and 108 (31.8%) strains, respectively. Among the 222 strains in which all three hbl genes were detected, 210 (61.9%) displayed discontinuous hemolysis (DH) characteristic of HBL producers, while 12 (3.5%) showed continuous hemolysis (CH) on sheep blood agar. Among strains in which none of the hbl genes was detected, 97 (28.6%) displayed CH while 11 (3.2%) did not show hemolytic activity. Three strains in which two hbl genes were detected showed CH. hblC was present in five of six strains where only one hbl gene was detected, and all of them (designated SS-00-15, TG-00-06, TG-00-14, F-00-25, and NR-01-49) showed DH. The HpaII restriction profiles of PCR fragments amplified from the hblC-A region in these five strains using hblC forward (FHC) and hblA reverse (RHA(2)) primers displayed heterogeneous patterns, which indicated sequence variation. Western blot analysis using polyclonal antibodies (Pab) raised against HBL components purified from strain F837/76 showed that three of the five strains produced all three components, whereas strain TG-00-06 did not give a signal for any component, and strain TG-00-14 produced B and L(1) but not L(2). The production of L(2) by these five strains was further analyzed using the Oxoid RPLA test. Three strains gave high titers (>64) whereas strains TG-00-06 and TG-00-14 showed lower titers of 16 and 32, respectively. The data show that HBL-encoding genes are widely distributed among B. cereus isolated in Thailand and there is a high degree of heterogeneity in both the genes and proteins. This is the first report of a B. cereus strain showing DH in which all three hbl genes and their proteins were not detected by both PCR primers and antibodies derived from prototype and type strains. The data also suggest that the L(2) component from strains TG-00-06 and TG-00-14 may be antigenically very different from that of most B. cereus isolates.
Asunto(s)
Bacillus cereus/química , Bacillus cereus/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , ADN Bacteriano/análisis , Secuencia de Bases , Western Blotting/métodos , Genotipo , Proteínas Hemolisinas , Hemólisis , Fenotipo , Reacción en Cadena de la Polimerasa/métodosRESUMEN
The genus Bacillus includes members that demonstrate a wide range of diversity from physiology and ecological niche to DNA sequence and gene regulation. The species of most interest tend to be known for their pathogenicity and are closely linked genetically. Bacillus anthracis causes anthrax, and Bacillus thuringiensis is widely used for its insecticidal properties but has also been associated with foodborne disease. Bacillus cereus causes two types of food poisoning, the emetic and diarrheal syndromes, and a variety of local and systemic infections. Although in this review we provide information on the genus and a variety of species, the primary focus is on the B. cereus strains and toxins that are involved in foodborne illness. B. cereus produces a large number of potential virulence factors, but for the majority of these factors their roles in specific infections have not been established. To date, only cereulide and the tripartite hemolysin BL have been identified specifically as emetic and diarrheal toxins, respectively. Nonhemolytic enterotoxin, a homolog of hemolysin BL, also has been associated with the diarrheal syndrome. Recent findings regarding these and other putative enterotoxins are discussed.
Asunto(s)
Bacillus cereus/aislamiento & purificación , Enfermedades Transmitidas por los Alimentos/epidemiología , Bacillus cereus/clasificación , Bacillus cereus/metabolismo , Bacillus cereus/patogenicidad , Técnicas de Tipificación Bacteriana , Enterotoxinas , Enfermedades Transmitidas por los Alimentos/etiología , Enfermedades Transmitidas por los Alimentos/patología , Humanos , Filogenia , Especificidad de la EspecieRESUMEN
Biofilms in the food-processing industry are a serious concern due to the potential for contamination of food products, which may lead to decreased food quality and safety. The effect of two detergent and sanitizer combinations on the inactivation of Listeria monocytogenes biofilms was studied. Combination A uses a chlorinated-alkaline, low-phosphate detergent, and dual peracid sanitizer. Combination B uses a solvated-alkaline environmental sanitation product and hypochlorite sanitizer. The survival of bacterial biofilms placed at 4 and 10 degrees C and held for up to 5 days was also addressed. To simulate conditions found in a ready-to-eat meat-processing environment, biofilms were developed in low-nutrient conditions at 10 degrees C (with and without meat and fat residue) on a variety of materials found in a plant setting. Included were two types of stainless steel, three materials for conveyor use, two rubber products, a wall, and floor material. Biofilms developed on all surfaces tested; numbers at day 2 ranged from 3.2 log on silicone rubber to 4.47 log CFU/cm2 on Delrin, an acetal copolymer. Biofilm survival during storage was higher at 4 degrees C (36.3 to 1,621%) than 10 degrees C (4.5 to 83.2%). Small amounts of meat extract, frankfurters, or pork fat reduced biofilm formation initially; with time, the biofilm cell number and survival percentage increased. Cleaning efficacy was surface dependent and decreased with residue-soiled surfaces; biofilms developed on the brick and conveyor material were most resistant. Both detergents significantly (P < 0.05) removed or inactivated biofilm bacteria. The sanitizers further reduced biofilm numbers; however, the reduction was not significant in most cases for the dual peracid. Using a benchmark efficacy of >3-log reduction, combination A was only effective on 50.0% of the samples, Combination B, at 86.1%, was more effective.
Asunto(s)
Biopelículas/efectos de los fármacos , Desinfectantes/farmacología , Contaminación de Alimentos/prevención & control , Microbiología de Alimentos , Industria de Procesamiento de Alimentos/normas , Listeria monocytogenes/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Recuento de Colonia Microbiana , Seguridad de Productos para el Consumidor , Higiene , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/fisiología , Temperatura , Factores de Tiempo , Resultado del TratamientoRESUMEN
DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.