Amy C. Lee Wong

Bacillus cereus food poisoning and its toxins

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.

Influence of culture conditions on biofilm formation by Escherichia coli O157:H7.

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.

Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates.

ABSTRACT 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.

Growth and penetration of Salmonella enteritidis, Salmonella heidelberg and Salmonella typhimurium in eggs

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.

Effect of trisodium phosphate on biofilm and planktonic cells of Campylobacter jejuni, Escherichia coli O157: H7, Listeria monocytogenes and Salmonella typhimurium

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.

Attachment of Listeria monocytogenes and Salmonella typhimurium to stainless steel and Buna-N in the presence of milk and individual milk components

The effects of milk and individual milk components on the attachment of Listeria monocytogenes and Salmonella typhimurium to two commonly used materials in the dairy industry were studied. Attachment of both organisms to stainless steel and Buna-N was significantly inhibited by the presence of skim, 2%, whole, or chocolate 2% milk compared to the phosphate-buffered saline (PBS) control. The addition of individual milk components, casein, α-lactalbumin, and β-lactoglobulin to the attachment menstruum significantly reduced attachment. Pretreating surfaces with milk and milk components for 1 h prior to attachment in PBS gave similar results. The presence of lactose did not affect attachment of either organism; however, attachment of S. typhimurium was significantly decreased on pretreated Buna-N. Cells of either organism pretreated with skim milk or β-lactoglobulin prior to attachment in PBS showed significantly less attachment than untreated cells. Pretreating S. typhimurium cells with casein had no effect on attachment to stainless steel. Pretreatment of S. typhimurium with lactose increased attachment to both surfaces while pretreatment had no effect on L. monocytogenes . Attachment of both organisms was significantly reduced in diluted whole milk. Both organisms attached significantly less to surfaces soiled with one or more layers of whole milk.

Efficacy of two cleaning and sanitizing combinations on Listeria monocytogenes biofilms formed at low temperature on a variety of materials in the presence of ready-to-eat meat residue.

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.

Requirement of flhA for Swarming Differentiation, Flagellin Export, and Secretion of Virulence-Associated Proteins in Bacillus thuringiensis

Bacillus thuringiensis is being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen. While phospholipases, hemolysins, and enterotoxins are claimed to be responsible for B. thuringiensis virulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions. This report describes the characterization of a mini-Tn10 mutant of B. thuringiensis that is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C. The mutant strain was determined to carry the transposon insertion in flhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus. Interestingly, the flhA mutant of B. thuringiensis synthesized flagellin but was impaired in flagellin export. Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable in B. thuringiensis, thus suggesting that the flagellar gene expression hierarchy of B. thuringiensis differs from that described for Bacillus subtilis. The flhA mutant of B. thuringiensis was also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant. Since complementation of the mutant with a plasmid harboring the flhA gene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence in B. thuringiensis may be coordinately regulated by flhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.

Current Perspectives on Detection of Staphylococcal Enterotoxins

Staphylococcal food poisoning is one of the most economically important food-borne diseases in the United States, costing approximately

Differential Biofilm Formation and Motility Associated with Lipopolysaccharide/Exopolysaccharide-Coupled Biosynthetic Genes in Stenotrophomonas maltophilia

1.5 billion each year in medical expenses and loss of productivity. The amount of staphylococcal enterotoxin required to cause illness in humans depends on the susceptibility of the individuals. As little as 0.5 to 0.75 ng/ml of enterotoxin A in chocolate milk was shown to be able to cause illness in school children. Many methods have been developed for the detection of enterotoxins: immunological and biological assays. Immunological assays are more sensitive and specific and are the basis for detection of the identified enterotoxins. However, biological assays are useful for the detection of uncharacterized enterotoxins. This article reviews methods currently available for enterotoxin detection, including biological assays, immunodiffusion, radioimmuno-assay, enzyme-linked immunosorbent assay, polymerase chain reaction-based methods, and various commercially available diagnostic kits.