GROWTH AND INHIBITION OF BACTERIAL SPORE-FORMING PATHOGENS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0222798
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Food Science

Non Technical Summary
Seafood in increasingly consumed in the U.S. The ability of a particular foodborne pathogen to grow on raw seafood will be determined as will the process leading to the production of a toxin responsible for foodborne illness.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

bacillus cereus

clostridium perfringens

enterotoxin

seafood

sporulation

microbial growth

microbial inhibitors

spores

Goals / Objectives
Determine the growth potential of Bacillus cereus in fish. Identify strategies for preventing and limiting growth of toxigenic B. cereus in/on fresh seafood. Identify the signal(s) which initiate sporulation and subsequent enterotoxin formation by Clostridium perfringens. Determine the ability of spores of each species to grow in inoculated pack studies. Graduate students will be trained in microbial food safety and results presented at national meetings and submitted for publication in peer-reviewed food microbiology publications for promulgation to food safety professionals.

Project Methods
Cored samples of salmon will be inoculated with active cultures or spores of Bacillus cereus at inoculum levels of 100 to 100 colony forming units per gram. Strains to be used will be those previously isolated from seafood. Samples will be held at temperatures representing mild temperature abuse for 24 and 48 hours at 7 C, 12 C and 20 C. Levels of growth will be determined using a new chromogenic agar following appropriate dilutions. In cases of significant growth, the presence of the hemolysin BL enterotoxin and non-hemolytic enterotoxins will be determined by commercial assay kits. Both enterotoxigenic and emetic toxin types will be used. The role of competitive exclusion will be determined by the co-addition of the predominate bacterial species isolated from salmon. This will be identified by 16S rRNA typing. FDA-approved antimicrobials will be used to determine their effectiveness in preventing the growth of B. cereus. These include lactates and nisin at various concentrations. Other marine species will be assessed for their ability to competitively exclude the growth of this foodborne pathogens as well as the effect of additional temperatures and holding times representing more abusive food service conditions. A separate study will identify the molecule which signals the bacterial foodborne pathogen C. perfringens to sporulate and produce enterotoxin. This molecule will be isolated from spent medium using high-pressure liquid chromatography using various reverse phase columns preceded by low pressure chromatography using Sephadex LH-20, Sephadex G-10 and related columns using eluents to be determined but initially methanol and subsequently aqueous buffers. The identity of the putative peptide will be identified in conjunction with this university's NMR facility. Subsequent research - if time permits - will involve a determination of the mode of action of the isolated molecule. Key milestones will be the ability of B. cereus to grow in large numbers on salmon and, subsequently, separate marine species, in particular those consumed raw. The presence of toxin in such foods will be indicative of the potential hazards of their consumption. In the case of C. perfringens successive milestone will occur at the multiple purification stages with increasing degrees of purity. Knowledge of the events triggering sporulation and toxin of this organism will provide insight on how the process can be inhibited.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:academic professionals, research and instructional; public health authorities Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project allowed for the training of a graduate student in food safety How have the results been disseminated to communities of interest?Published in a peer-reviewed journal What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The presence of enterotoxin genes in isolatesof Bacillus cereus and Bacillus thuriginsis from retail spices was determined. 88% of B. cereus isolates and 91% of B. thuringiensis possessed at last one type of enterotoxin gene: HBL (hemolysin BL) or nonhemolytic enterotoxin enterotoxin (NHE) . None of the isolatesobtained possessed the emetic toxin gene. The NHE enterotoxin was expressed in 98% of B. cereus and 100% of B. thuringiensis isolates. 50% of all isolates produced both enterotoxxins. 97% of B. cereus isolates grew grew at 12C.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Spore prevalence and toxigenicity of Bacillus cereus and Bacillus thuringiensis isolates from U.S. retail spices Journal of Food Protection 78, p. 590

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: academic professionals, research and instructional; public health authorities Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project allowed for the training of an undergradute and graduate student in microbial food safety How have the results been disseminated to communities of interest? The results were presented at a national conference of microbial food safety professionals. What do you plan to do during the next reporting period to accomplish the goals? Determine the ability for growth of B. cereus in spices in a model food.

Impacts
What was accomplished under these goals? The levels of the spore-forming, foodborne pathogen Bacillus cereus in U.S. retail spices was determined. More than 200 retail spices were examined for levels and toxigenicity of the pathogen. Approximately 30% were positive at levels ranging from 3 to 4600/gm. All isolates possessed one or more entertotoxin genes. The heat resistance of spores produced by this organism was determined. The widespred presence andheat resistance of spores produced byB. cereus demonstrates the importance of consumer awareness to properly handle foods to which large amounts of spices have been added.

Publications

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: academic professionals, research and instructional; public health authorities Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project allowed for the training of undergaduates in microbial food safety. How have the results been disseminated to communities of interest? The results of the work have been published in a peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals? Control of the growth of foodborne pathogens on food and the control of events leading to enterotoxin formation will continue.

Impacts
What was accomplished under these goals? The ability of enterotoxigenic Bacillus cereus to grow on wild fresh salmon at abusive temperatures was determined. A five-fold increase in CFU per gram was observed after 26 and 22 hr at 16C and 20C respectively, while greater than a four-fold increase occured at 12C after 48 hr. Enterotoxin was detected when levels of Bacillu cereus were in excess of 8 log 10 CFU per gram. Nisin, at concentrations of 1 and 15 micrograms per gram of salmon, reduced levels of Bacillus cereus 2.5- and 25-fold respectively. The results indicate the potential for growth of a foodborne pathogen on a marine food which increasingly consumed raw.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Labbe, R. and Rahmati, T. 21012. Growth of enterotoxigenic Bacillus cereus on salmon (Oncorhynchus nerka.) Journal of Food Protection 75:1153-1156.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Growth potential of Bacillus cereus in wild salmon was investigated using an isolate able to produce enterotoxin at elevated levels. Sterile salmon sections were held at abusive tempeatures of 12, 16 and 20C. A five-fold increase in log CFU per gram was observed after 26 and 22 hr at 16 and 20C respectively, while less than a 4-log CFU/g increase occurred on salmon held at 12C for 48 hr. Genertation times of 169 53 and 46 min were observed at 12, 16, and 20C. Nisin at concentrations of 1 and 15 micrograms reduced levels of B. cereus 2.5- and 25-fold respectively. PARTICIPANTS: R. Labbe, K. Shetty, T. Rahmati, K. Johnson, M. Pinto TARGET AUDIENCES: academic professionals, research and instructional; public health authorities PROJECT MODIFICATIONS: Nothing significant to report during this reporting period

Impacts
Results of the research, supported in part by this project, indicate that fresh salmon can serve as an excellent substrate for enterotoxigenic Bacillus cereus and that this organism can reach levels associated with foodborne illnes following moderate temperatue abuse.

Publications

• Ankolekar, C., Johnson, K., Pinto, M., Labbe, and Shetty, K. 2012. Fermentation of whole apple juice using Lactobacillus acidophilus for potential dietary management of hypoglycemia, hypothension and modulation of beneficial bacteria. Journal of Food Biochemistry 36 718.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: The physical characteristics of spores of foodborne isolates of the Bacillus cereus Group were investigated. A total of 35 foodborne, diarrheal-type and 12 food poisoning, emetic-type isolates of B. cereus senso stricto were examined by electron microscopy. All possessed spore appendages. Ten of 12 foodborne isolates of Bacillus thuringiensis from rice possessed appendages but the one Bacillus mycoides isolated from rice did not. The length of the appendages ranged from 0.45-3.8 micrometers. Spores were moderately to highly hydrophobic and each had a net negative charge. PARTICIPANTS: R. Labbe PI C. Ankolekar, graduate student TARGET AUDIENCES: academic professionals, research and instructional; public health authorities PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Enterotoxigenic strains of Bacillus cereus are involved in foodborne illness. Findings indicate that the presence of spore appendages may be involved in adherence to surfaces, foods or epithelial cells. The isolates investigated were originally obtained from retail foods. Methods employed here for physical examination of spores can be used for examination of spores of other toxigenic bacterial species.

Publications

• Ankolekar, C. and Labbe, R. 2010. Physical characteristics of spores of food-associated isolates of the Bacillus cereus group.