No abstract available.
Salmonella enterica forms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were active in vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F(0)F(1)-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2 × Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.
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.
We have investigated bottom-up chemical synthesis of quaternary ammonium (QA) groups exhibiting antibacterial properties on stainless steel (SS) and filter paper surfaces via nonequilibrium, low-pressure plasma-enhanced functionalization. Ethylenediamine (ED) plasma under suitable conditions generated films rich in secondary and tertiary amines. These functional structures were covalently attached to the SS surface by treating SS with O 2 and hexamethyldisiloxane plasma prior to ED plasma treatment. QA structures were formed by reaction of the plasma-deposited amines with hexyl bromide and subsequently with methyl iodide. Structural compositions were examined by electron spectroscopy for chemical analysis and Fourier transform infrared spectroscopy, and surface topography was investigated with atomic force microscopy and water contact angle measurements. Modified SS surfaces exhibited greater than a 99.9% decrease in Staphylococcus aureus counts and 98% in the case of Klebsiella pneumoniae. The porous filter paper surfaces with immobilized QA groups inactivated 98.7% and 96.8% of S. aureus and K. pneumoniae, respectively. This technique will open up a novel way for the synthesis of stable and very efficient bactericidal surfaces with potential applications in development of advanced medical devices and implants with antimicrobial surfaces.
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.
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.
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.
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.
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.
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.
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.
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.
The levels to which microbial colony forming units are permitted in various waters fit for human contact are carefully regulated. Conventional chemical and physical approaches usually are complex processes with significant limitations due to the generation of toxic side-products. In this contribution a novel plasma reactor--dense medium plasma reactor--is described, and its efficiency for the disinfection of contaminated water is discussed. It has been shown that owing to the intense stirring of the reaction medium (e.g. contaminated water), as a result of the specially designed spinning electrode and gas-flow system, a volume-character discharge is created, which can efficiently kill bacteria. It has been demonstrated that treatment times as low as 20 s are enough for the total inactivation of microorganisms for 200 mL of 10(5) bacteria/mL contaminated water.
Defects in cheese, such as undesirable flavors, gas formation, or white surface haze from calcium lactate crystals, can result from growth of nonstarter lactic acid bacteria (NSLAB). The potential for biofilm formation by NSLAB during cheese manufacturing, the effect of cleaning and sanitizing on the biofilm, and bacterial growth and formation of defects during ripening of the contaminated cheese were studied. Stirred-curd Cheddar cheese was made in the presence of stainless steel chips containing biofilms of either of two strains of erythromycin-resistant NSLAB (Lactobacillus curvatus strain JBL2126 or Lactobacillus fermentum strain AWL4001). During ripening, the cheese was assayed for total lactic acid bacteria, numbers of NSLAB, and percentage of lactic acid isomers. Biofilms of L. curvatus formed during cheese making survived the cleaning process and persisted in a subsequent batch of cheese. The starter culture also survived the cleaning process. Additionally, L. curvatus biofilms present in the vat dislodged, grew to high numbers, and caused a calcium lactate white haze defect in cheese during ripening. On the other hand, biofilms of L. fermentum sloughed off during cheese making but could not compete with other NSLAB present in cheese during ripening. Pulsed-field gel electrophoresis results verified the presence of the two biofilm strains during cheese making and in the ripening cheese. Probable contamination sites in the plant for other NSLAB isolated in the cheese were identified, thus supporting the hypothesis that resident NSLAB biofilms are a viable source of contamination in the dairy environment.
Haemolysis of erythrocytes from different species (sheep, bovine, swine and human), caused by various combinations of phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC), sphingomyelinase (SMase) and the three-component, pore-forming toxin haemolysin BL (HBL) from Bacillus cereus was analysed. The lytic potency of HBL did not correlate with phospholipid (PL) content, but lysis by the individual or combined enzymes did. SMase alone lysed ruminant erythrocytes, which contain 46-53% sphingomyelin (SM). The cooperative action of PC-PLC and SMase was needed to lyse swine and human erythrocytes (22-31% PC and 28-25% SM). SMase synergistically enhanced haemolysis caused by HBL for all erythrocytes tested, which all contained >25% SM. PC-PLC enhanced HBL haemolysis only in cells containing significant amounts of PC (swine, 22% PC; human, 31% PC). Unexpectedly, PC-PLC inhibited HBL lysis of sheep erythrocytes (<2% PC) and enhanced the discontinuous haemolysis pattern that is characteristic of HBL in sheep blood agar. Inhibition and pattern enhancement was abolished by washing PC-PLC-treated erythrocytes or by adding EDTA, suggesting that enzymic alteration of the membrane is not involved, but that zinc in the active site is required, perhaps to facilitate binding. These observations highlight the potential for cooperative and synergistic interactions among virulence factors in B. cereus infections and dependence of these effects on tissue composition.
Bacillus cereus causes a highly fulminant endophthalmitis which usually results in blindness. We previously concluded that hemolysin BL (HBL), a tripartite necrotizing pore-forming toxin, is a probable endophthalmitis virulence factor because it is highly toxic to retinal tissue in vitro and in vivo. We also determined that B. cereus produces additional retinal toxins that might contribute to virulence. Here we fractionated crude B. cereus culture supernatant by anion-exchange chromatography and found that in vitro retinal toxicity was also associated with phosphatidylcholine-preferring phospholipase C (PC-PLC). The pure enzyme also caused retinal necrosis in vivo. We showed that phosphatidylinositol-specific PLC and sphingomyelinase were nontoxic and that two hemolysins, cereolysin O and a novel hemolysin designated hemolysin IV, were marginally toxic in vitro. The histopathology of experimental septic endophthalmitis in rabbits mimicked the pathology produced by pure HBL, and both HBL and PC-PLC were detected at toxic concentrations in infected vitreous fluid. Bacterial cells were first seen associated with the posterior margin of the lens and eventually were located throughout the lens cortex. Detection of collagenase in the vitreous humor suggested that infiltration was facilitated by the breakdown of the protective collagen lens capsule by that enzyme. This work supports our conclusion that HBL contributes to B. cereus virulence and implicates PC-PLC and collagenase as additional virulence factors.
Haemolysin BL (HBL), a three-component enterotoxic/necrotizing/vascular permeability toxin, is a likely virulence factor of Bacillus cereus diarrhoeal food poisoning and necrotic infections. This paper describes the isolation of two distinct homologous sets of all three HBL components from a single B. cereus isolate, MGBC 145. The proteins of one set (designated HBL, consisting of B, L(1) and L(2)), were about 87-100% identical in N-terminal amino acid sequences to their respective prototype components from strain F837/76, and the proteins of the homologous set (HBL(a), consisting of B(a), L(1a) and L(2a)) were all about 62-65% identical. Only the latter homologues differed immunochemically and physicochemically from the prototypes. HBL and HBL(a) exhibited similar haemolytic and vascular permeability potencies, and the homologues could be interchanged freely. There were no notable differences in activity between the L component homologues. However, components B and B(a) were significantly different. Both were secreted as monomers, but unlike B, B(a) was isolated as a relatively inactive complex that could be reactivated with urea. When B(a) was substituted for B in gel-diffusion assays the distinct discontinuous haemolysis pattern typical of the presence of B did not occur. In suspension assays, excess B inhibited the haemolysis of B-primed cells by L(1) (as previously described), but not that of B(a)-primed cells. Excess B(a) had the opposite effect and enhanced lysis of B(a)-primed cells, but not that of B-primed cells. These differences reveal details about how the toxin components interact on target cell membranes. The authors' observations indicate that HBL represents a new family of multicomponent toxins that was generated by a process of gene and operon duplication that occurred either intracellularly or by horizontal transfer, and raise the possibility of the existence of other related toxins in the genetically diverse B. cereus taxonomic group.
Hemolysin BL (HBL), a diarrheal enterotoxin originally isolated from Bacillus cereus strain F837/76, is composed of three antigenically distinct proteins designated B, L1, and L2. All three components are required for biological activity. Here, we report antigenic and physical variations in HBL components produced by other B. cereus isolates. Reactions of partial identity were observed in double gel immunodiffusion assays using antibodies to highly purified B, L1, and L2 components of F837/76 and culture supernatants of strains F837/76 and S1C. Western blot analysis showed that F837/76 produced one 38-kDa B protein, one 38-kDa L1, and one 43-kDa L1 protein. In strain S1C, two B (38 and 42 kDa), two L1 (38 and 41 kDa), and one L1 (43 kDa) proteins were detected. Further Western blot analysis of 127 B. cereus isolates showed that 90 produced one or more of the three HBL components. Approximately half of these 90 isolates (43/90; 48%) produced protein profiles which differed from that of F837/76. A total of four B, two L1, and three L2 component profiles with proteins of different sizes were observed. Individual strains produced various combinations of single or multiple bands of each component. In addition, some strains produced only one or two of the three HBL components. The public health significance of these strains is unknown, as all three components are required for biological activity. The data presented here demonstrates a high degree of heterogeneity in HBL and provide the basis for further studies to characterize the variations in HBL and to determine the role of the variant components in pathogenicity.
Biofilms are a constant concern in food processing environments. Our overall research focus has been to understand the interaction of factors affecting bacterial attachment and biofilm formation with the ultimate goal of devising strategies to control this problem. This paper briefly describes three areas of biofilm research in which we have been involved. Listeria monocytogenes, a foodborne pathogen, survived for prolonged periods on stainless steel and buna-n rubber, materials commonly used in food-processing equipment. Survival was affected by temperature, relative humidity, attachment surface, and soil. Some components in the rubber inhibited growth of the organism on buna-n, which also affected the efficacy of sanitizers on biofilm inactivation. In cheese manufacture, biofilms of Lactobacillus curvatus could lead to a defect caused by the formation of calcium lactate crystals in Cheddar cheese. This hardy organism persisted in low numbers on equipment surfaces and was difficult to eradicate. We investigated the relative contributions and interactions of mechanical, thermal, and chemical processes in an air-injected clean-in-place method for milking systems. Overall, it is important to study the interactions between bacteria and the surfaces in a specific food processing environment to provide more effective measures for prevention of biofilm formation and for its removal.
The potential for transfer of Escherichia coli O157:H7 from contaminated ground beef to grinding equipment and the inactivation of attached cells during cleaning and sanitizing was examined. Chub-packed ground beef with lean:fat ratios of 75:25, 80:20 or 90:10 was inoculated with 6 log CFU/g or 2 log CFU/g E. coli O157:H7 strain FRIK 910. Samples were consecutively ground in a Hobart meat grinder with stainless steel (SS) chips (1 cm2) glued to the auger housing. Chips were harvested after grinding, detergent washing with or without manual scrubbing and rinsing, sanitizing in a chlorine or peroxyacetic acid sanitizer, and overnight storage. Survival of E. coli O157:H7 was evaluated both by plate count and enrichment in trypticase soy broth. Approximately 3 to 4 log CFU/cm2 were attached to the SS after grinding with all three fat contents. After washing and sanitizing in a chlorine or peroxyacetic acid sanitizer, viable bacteria were infrequently recovered by plate count. Enrichment of chips resulted in a higher survival rate with both sanitizing treatments, indicating that cell numbers below the limit of detection (5 CFU/cm2) or potentially injured organisms remained on the surface. Manual scrubbing during the washing step reduced the recovery rate. The scrubbing step also increased the number of passing scores assigned using an ATP bioluminescence assay of total residual soil on the chips sanitized in chlorine. The overall results indicate that plate counts alone may not be a reliable indicator of sanitation efficacy and may be validated by enrichment assay.
The effects of controlled pH and aeration on the growth of Staphylococcus aureus strain FRI-569 and production of enterotoxin H (SEH) were studied. S. aureus FRI-569 did not grow well anaerobically. Aeration permitted better growth of cells especially at a flow rate of 300 cc/min. The optimal pH for S. aureus FRI-569 growth was 7.0, with less growth being observed at pH 6.5 and 7.5 or with no pH control. Very little SEH (< 21 ng/ml) was produced by S. aureus strain FRI-569 under anaerobic incubation. High SEH production was observed with aeration at 300 cc/min. Increasing aeration to 400 cc/min or decreasing it to 200 cc/min resulted in reduced SEH production. Larger amounts of SEH were produced when the pH was controlled at 7.0, while maintaining the pH at 6.5 or 7.5 resulted in lower levels of SEH being produced. However, the amounts were still greater than those observed when the pH was not controlled.
Hemolysin BL (HBL) is a unique membrane-lytic toxin from Bacillus cereus composed of three distinct proteins, designated B, L1, and L2. HBL produces a paradoxical zone phenomenon in gel diffusion assays in sheep blood agar. Lysis does not begin immediately adjacent to the source of diffusion; rather, it begins several millimeters away. Cells near the source and at intersections of lysis zones remain intact longer. Here, we developed a spectrophotometric hemolysis assay system that measures the activities of the individual HBL components and used it to analyze the mechanisms of hemolysis and the paradoxical zone phenomenon. The B component was rate-limiting, and erythrocytes were slowly primed by B at an optimal concentration of about 1.3 nM to rapid lytic action by the combination of the L components (L(1+2)). All of the individual components bound to cells independently, and membrane-associated HBL components were neutralized by specific antibodies, suggesting that lysis was caused by formation of a membrane attack complex on the cell surface. Osmotic protection experiments indicate a colloid osmotic lysis mechanism. Concentrations of the B component above 1.3 nM caused inhibition of L1-mediated lysis, and L1 inhibited the priming reaction of B over a similar concentration range. From analyses of spectrophotometric and diffusion assays we constructed a basic model for the interactions between HBL components and for the paradoxical zone phenomenon in blood agar. In the latter, areas of slow lysis near diffusion sources are caused primarily by the accumulation of inhibitory levels of L1 reached before cells are primed by B.
A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection of a newly identified staphylococcal enterotoxin H (SEH). Peroxidase was conjugated to antibodies specific to the enterotoxin. 2,2'Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)(ABTS) in hydrogen peroxide solution was used as the enzyme substrate. A standard curve of purified SEH was prepared with concentrations ranging from 1.3 to 50 ng/ml. SEH at levels equal to 2.5 ng/ml and higher were detected by this procedure. Culture supernatant from the growth of selected Staphylococcus aureus strains was analyzed by using the ELISA. SEH was produced by three of 20 strains that produced one identified enterotoxin. Ten of 21 strains, previously shown to produce substances that induced emesis in monkeys but not any known enterotoxins (A through E), were also positive for SEH production. The other 11 strains gave negative results in the ELISA, indicating that other unidentified serological types of enterotoxin exist.
Bacillus cereus causes exotoxin-mediated diarrheal food poisoning. Hemolysin BL (HBL) is a well-characterized B. cereus toxin composed of three components (B, L1, and L2) that together possess hemolytic, cytotoxic, dermonecrotic, and vascular permeability activities. Here, we show that HBL causes fluid accumulation in ligated rabbit ileal loops at a dose of 5 micrograms of each component per loop. Maximal fluid responses occurred for combinations of all three components at > or = 25 micrograms of each component per loop. Individual components and binary combinations did not cause significant fluid accumulation at 25 micrograms of each component. Specific antisera to HBL components inhibited the fluid accumulation response of crude culture supernatant from B. cereus F837/76. These antisera were tested against an antiserum to a partially characterized multicomponent diarrheal toxin described previously by Thompson et al. (N. E. Thompson, M. J. Ketterhagen, M. S. Bergdoll, and E. J. Shantz, Infect. Immun. 43:887-894, 1984). Immunoblot and immunoprecipitation analyses indicate that HBL and that toxin are identical. These results confirm previous speculation that HBL is a tripartite enterotoxin that, as for all of its other known activities, requires all three components for maximal activity.
Biofilms of Escherichia coli O157:H7 were developed on stainless steel chips in trypticase soy broth (TSB), 1/5 dilution of TSB, 0.1% Bacto peptone (BP) and a minimal salts medium (MSM) supplemented with 0.04% of one of the following carbon sources: glucose, glycerol, lactose, mannose, succinic acid, sodium pyruvate or lactic acid. It was found that biofilms developed faster and a higher number of adherent cells (ca. 10(6) CFU/cm2) were recovered when the organisms were grown in the low nutrient media. Regardless of the carbon source, biofilms developed in MSM consisted of shorter bacterial cells and thicker extracellular matrix (ECM), with glucose as the best substrate for stable biofilm formation. Fewer bacteria in initial attachment, non-hydrophobicity of bacterial cells, lack of ECM formation and easy detachment of the biofilm bacteria may contribute to poor biofilm formation in TSB. ECM is probably important for the stability of biofilms; however, at 10 degrees C and under anaerobic conditions, ECM seems to be unnecessary.
A staphylococcal enterotoxin which elicited an emetic response in monkeys but did not share antigenic determinants with any of the identified enterotoxins was identified and purified from Staphylococcus aureus FRI-569. The emetic activity of this new enterotoxin was neutralized only by antibodies specific to it and not by antibodies to enterotoxins A, B, C, D, and E or toxic shock syndrome toxin 1. Immunodiffusion assays did not detect cross-reactivity between this new and all the other identified enterotoxins. The purification procedure involved removal of the enterotoxin from culture supernatant fluids by batch adsorption with CG-50 resin, CM-Sepharose FL ion-exchange chromatography, and Sephacryl 100 HR and Bio-Gel P-30 gel filtration. The molecular weight of this enterotoxin, 27,300, determined by gel filtration on Sephacryl 100 HR agreed with the molecular weight, 28,500, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The apparent migration of this enterotoxin determined by SDS-PAGE did not shift in the presence of a disulfide reducing agent, indicating that it is composed of a single-chain protein. The N-terminal amino acid sequence of the enterotoxin was determined to be Glu-Asp-Leu-His-Asp-Lys-Ser-Glu-Leu-Thr-Asp-Leu-Ala-Leu-Ala-Asn-Ala-Tyr- Gly- Gln-Tyr-Asn-His-Pro-Phe-Ile-Lys-Glu-Asn-Ile, which did not match the N-terminal sequences of any known proteins. The isoelectric point of the enterotoxin determined by isoelectric focusing was about 5.7.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacillus cereus is a common cause of highly fulminant posttraumatic and metastatic endophthalmitis. Exotoxins or enzymes likely contribute to the severity of the infection, but specific virulence factors have not been identified. We developed two methods for the identification of B. cereus ocular virulence factors. In an in vitro assay that allows screening of multiple samples, retinal toxicity was estimated by measuring the release of lactate dehydrogenase from retinal buttons treated with B. cereus toxins. The results from this assay were confirmed with a sterile endophthalmitis model in which the histopathologic effect of intravitreally injected toxins was assessed. We tested pure hemolysin BL (HBL), a tripartite dermonecrotic vascular permeability factor of B. cereus, and crude exotoxin (CET) preparations, consisting of concentrated, cell-free B. cereus culture supernatant. In the in vitro assay, both CET and HBL caused rapid release of lactate dehydrogenase and retinal disintegration. In vivo, the toxins caused endophthalmitis clinically characteristic of B. cereus within 4 h. Histological changes included rapid retinal necrosis and detachment, choroidal edema, detachment and disruption of the retinal pigment epithelium, and rapid infiltration of polymorphonuclear leukocytes. Neutralization of HBL in CET preparations inhibited toxicity in vitro by 54%, and pure HBL was less toxic than CET with equal HBL contents in both methods. The results suggest that B. cereus ocular virulence is multifactorial and that HBL contributes to virulence.
Eggs and egg dishes are important vehicles for Salmonella infections. Salmonella enteritidis, Salmonella typhimurium and Salmonella heidelberg, which can be isolated from chicken ovaries and feces, have been implicated in approximately 50% of the foodborne salmonellosis outbreaks in the United States. In this study, the growth of these three organisms, inoculated into yolks and albumen, was compared at 4, 10 and 25 degrees C. Regardless of whether 10(2) cfu/g or 10(4) cfu/g was inoculated into the yolk or albumen, populations of all strains increased 3 logs or more in number in one day when incubated at 25 degrees C. Maximum numbers of Salmonella ranged from 10(8) to 10(10) cfu/g. All strains grew at 10 degrees C, but peak numbers were lower and occurred later than those at 25 degrees C. Populations of the three Salmonella strains inoculated into eggs stored at 4 degrees C grew sporadically; in some test groups populations declined. The potential for Salmonella in contaminated feces to establish in the interior of eggs was examined by monitoring shell penetration. At 25 degrees C, all three Salmonella strains penetrated the shell in 3 days, but at 4 degrees C, only S. typhimurium was found in one membrane sample. When hatchery conditions were simulated by incubating eggs at 35 degrees C for 30 min followed by storage at 4 degrees C, penetration was enhanced. Penetration was observed by day 1-3 when eggs were exposed to 10(4) cfu Salmonella/g feces. Increasing the inoculum to 10(6) cfu/g feces resulted in 50-75% of the contents of eggs to be contaminated by day 1. All Salmonella-positive samples were detected by enrichment. Results of this study indicate that S. enteritidis, S. typhimurium, or S. heidelberg present in feces can penetrate to the interior of eggs and grow during storage.
The usefulness of two commercial immunoassays for the detection of diarrheal enterotoxin of Bacillus cereus is unclear because the identity of the enterotoxin(s) has not been proven and the kits detect different proteins. We found that the Bacillus cereus Enterotoxin-Reversed Passive Latex Agglutination kit (Oxoid) detects the L2 component from hemolysin BL, and the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra) detects two apparently nontoxic proteins.
Trisodium phosphate (TSP) has been approved by the United States Department of Agriculture as a post-chill antimicrobial treatment for raw poultry. This study examines the effectiveness of TSP against planktonic (suspended) and biofilm (attached) cells of Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium at room temperature (RT) and 10 degrees C. At either temperature E. coli O157:H7 was the most sensitive to TSP treatments; 10(6) cfu/ml of planktonic or 10(5) cfu/cm2 of biofilm cells were eliminated by a 30 s treatment with 1% TSP. Campylobacter jejuni was slightly less sensitive. Listeria monocytogenes was the most resistant to the effect of TSP, requiring exposure to 8% TSP for 10 min (RT) or 20 min (10 degrees C) to reduce biofilm bacteria by at least one log. Biofilm cells of S. typhimurium and Listeria monocytogenes were more resistant than planktonic cells. Salmonella typhimurium was more sensitive to treatments using TSP at 10 degrees C than at RT. In contrast, L. monocytogenes was more resistant to TSP at 10 degrees C. Trisodium phosphate appears to be an effective treatment for reducing populations of C. jejuni, E. coli O157:H7 and S. typhimurium. This product has the potential to be used for reduction of bacterial counts on other food products besides raw poultry or on food and non-food contact surfaces.
Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin BL in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains. In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood. Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL. The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates.
Oral inoculation of lactose utilizing cecal bacteria plus 2.5% lactose treatments were tested in young chicks for protective efficacy against infection by Salmonella enteritidis. One-day-old chicks were treated with cecal bacteria upon arrival and challenged orally on day 3 with 10(4)-10(6) cfu S. enteritidis. A single culture identified as Escherichia coli O75:H10 was found significantly more protective than all other isolates tested. This isolate excreted a metabolite(s) in vitro that was inhibitory towards the growth of S. enteritidis. The results of this study indicate that discovery of protective strains can be facilitated by screening isolates in vitro for lactose utilization and growth inhibition of S. enteritidis before administration of treatment.
Campylobacter enteritis in humans has been linked to consumption of chicken. Reducing Campylobacter jejuni colonization in chickens can potentially reduce Campylobacter infections in humans. In this study, the reduction of C. jejuni colonization in chicks by oral administration of defined competitive exclusion (CE) cultures, 2.5% dietary carbohydrates, or CE cultures and carbohydrates was examined. Prevention, elimination, or direct challenge of Campylobacter infection was simulated by administering treatments before, after, or at the same time as that of the Campylobacter inoculation. Additionally, the effect of maintaining high levels of protective bacteria was evaluated by administering CE cultures on days 1 and 4 (booster treatment). All treatments reduced C. jejuni colonization. Protection by aerobically grown CE cultures was not statistically different from that by anaerobically grown CE cultures. A combination of Citrobacter diversus 22, Klebsiella pneumoniae 23, and Escherichia coli 25 (CE 3) was the most effective CE treatment. Maintaining high numbers of CE isolates by administering CE boosters did not increase protection. The greatest reduction of Campylobacter colonization was observed in schemes to prevent or eliminate C. jejuni infection. C. jejuni was not detected in the ceca of birds receiving the prevention treatment, CE 3 with mannose, representing a 62% reduction in the colonization rate. The protection factor (PF), a value combining the colonization rate and the level of infection, for CE 3 with mannose was high (> 13.2). Fructo-oligosaccharides alone strongly prevented Campylobacter colonization. Only 8% of the chicks in this group were colonized, with a PF of > 14.3.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacillus cereus causes diarrheal and emetic food poisoning syndromes as well as a variety of mild to severe infections. A dermonecrotic vascular permeability (VP) factor has been implicated as a virulence factor in these illnesses. Hemolysin BL was previously identified as a unique tripartite hemolysin possessing VP activity. In this study, a high-yield purification scheme, which allowed quantitative characterization of hemolysin BL activity and determination of the molecular weight, pI, and N-terminal sequence of each component, was developed. Milligram quantities of the B, L1, and L2 components were highly purified by a combination of anion-exchange and hydroxylapatite chromatographies. The combined components had VP activity at low doses and were necrotic at higher doses. The toxin exhibited an unusual dose-response zone phenomenon in turbidometric hemolysis assays. Activity increased at doses up to 200 ng/ml, then decreased at doses up to 350 ng/ml, and was constant at doses up to at least 2,500 ng/ml. This behavior may provide an explanation for the unusual discontinuous pattern typical of hemolysin BL in gel diffusion assays. At high concentrations of one or two components, the presence of low amounts of the complementary component(s) resulted in full hemolytic activity. Erythrocytes were protected from lysis by Zn2+ at micromolar concentrations but not by Ca2+ and Mg2+ at concentrations up to 25 mM. These data provide guidelines for future work on this toxin and indicate that hemolysin BL is the dermonecrotic VP factor implicated as a B. cereus virulence factor.
The kinetics of toxic shock syndrome toxin 1 (TSST-1) production by Staphylococcus aureus was studied in a fermentor in which aeration rate, atmospheric composition, pH, and temperature were controlled. The toxin was synthesized at a maximal rate during the exponential phase. High bacterial populations were not necessarily accompanied by high TSST-1 yields. Aerobiosis increased TSST-1 production, but excessive aeration had an adverse effect. Addition of CO2 enhanced TSST-1 yield by increasing toxin production rate and efficiency. Cultures with no pH control made more TSST-1 than those maintained at pH 5.5 to 7.5. Maximum TSST-1 yields were obtained when cultures were supplied with air (20 cm3/min) and CO2 (5 cm3/min) via a sintered glass sparger.