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United States Department of Agriculture

Agricultural Research Service


item Svetoch, Edward - ST RES CTR RUSSIA
item Stern, Norman
item Eruslanov, Boris - ST RES CTR RUSSIA
item Kovalev, Yuri - ST RES CTR RUSSIA
item Volodina, Larisa - ST RES CTR RUSSIA
item Perelygin, Vladimir - ST RES CTR RUSSIA
item Mitsevich, Evgeni - ST RES CTR RUSSIA
item Mitsevich, Irina - ST RES CTR RUSSIA
item Pokhilenko, Victor - ST RES CTR RUSSIA
item Borzenkov, Valery - ST RES CTR RUSSIA

Research conducted cooperatively with:
item The Russian State Research Centre For Applied Microbiology

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 14, 2004
Publication Date: January 5, 2005
Citation: Svetoch, E., Stern, N.J., Eruslanov, B.V., Kovalev, Y., Volodina, L., Perelygin, V., Mitsevich, E., Mitsevich, I., Pokhilenko, V., Borzenkov, V. 2005. Isolation of bacillus circulans and paenibacillus polymyxa inhibitory to campylobacter jejuni and characterization of associated bacteriocins. Journal of Food Protection. 68(1):11-17.

Interpretive Summary: Campylobacter is one of the most frequently reported food borne bacterial agents and, is most associated with transmission through poultry products. As the bacteria cannot multiply outside of the chicken intestine, controlling the numbers of the organism will reduce the public exposure and reduce human disease. Numerous bacteria within the chicken intestine compete for available space and nutrients by producing proteins that kill competitors (bacteriocins) within that niche. We isolated a subgroup of spore forming bacteria that produced these proteins. We monitored zones of Campylobacter inhibition surrounding these competitors and selected the most lethal isolates. From these, we used biochemical methods to characterize the associated secreted proteins. Solubility's, molecular weights, amino acid sequences, temperature stability and lethality against Campylobacter were recorded. Four competitor isolates were deposited under the Budapest treaty to protect the intellectual property. These competitors and their corresponding bacteriocins provide a potential tool for controlling Campylobacter colonization during livestock and poultry production. The bacteriocins derived from this study may be useful in creating a new, non-antibiotic treatment of animals carrying human pathogens.

Technical Abstract: We evaluated anti-Campylobacter activity among 365 Bacillus spp. and Paenibacillus spp. isolates from poultry production environments. We measured zones of inhibition surrounding the candidate strains on agar lawns of Campylobacter. One novel antagonistic Bacillus circulans and three Paenibacillus polymyxa strains were identified and deposited under provisions of the Budapest Treaty. The cell-free, ammonium sulfate precipitate from each candidate culture was termed the crude antimicrobial preparation (CAP). Zones of Campylobacter growth inhibition surrounding 10 ml of the CAP were quantified using a spot test. Campylobacter growth resumed when the CAP was pre-incubated with selected protease enzymes, thus demonstrating a peptide characteristic consistent with bacteriocin definition. These peptides were further purified using combinations of CM-Sepharose, Superose, and ion exchange chromatography. Molecular weights of the peptides were estimated by SDS-PAGE electrophoresis. Isoelectric focusing (pI) was used to determine the isoelectric points of the peptides. Amino acid sequences of the bacteriocins and more precise molecular weights by MALDI-TOF analysis were obtained. Each of the characterized bacteriocins had an approximate molecular weight of 3.5 kDa. The bacteriocin from P. polymyxa NRRL B-30507 had a pI of 4.8; the bacteriocin from P. polymyxa NRRL B-30509 had a pI of 7.2; P. polymyxa NRRL B-30508 had a pI of 4.8 and B. circulans NRRL B-30644 had a pI of 7.8. The bacteriocin amino acid sequences were consistent with Class IIa classification. These antagonists and the corresponding bacteriocin products may be useful in the control of Campylobacter in poultry. Normal intestinal bacteria are critical to the health of any host animal. The host derives benefit through the digestive metabolic processes mediated by the native bacterial flora. From the perspective of the intestinal bacteria, competition, and consequent evolution provides nutrients, living space and reproductive potential enabling certain strains and species to gain survival advantage. During bacterial evolution bacteriocin production has occurred to provide ecological advantage. Bacteriocins are secreted to antagonize other organisms within a given competitive niche, and thus provide an ecological advantage. These bacteriocin compounds are typically low molecular weight polypeptides and are classified based on differences in molecular weights (13). These compounds can be easily digested into their component amino acids by host protease enzymes. Bacteriocins may be a significant component explaining the mechanisms involved in the phenomenon manifested by 'competitive exclusion' (18). Use of growth-promoting antibiotics during broiler production, chemicals, and some processing treatments can result in undesirable residues on the processed product. Nurmi originally described the advantages of competitive exclusion gained in controlling Salmonella colonization among newly hatched chicks by using an undefined bacterial flora derived from the feces of healthy adult birds. The procedure provides an attractive alternative to current husbandry practice. A mucosal competitive exclusion (MCE) flora was described. (23) as an anaerobic subculture from the scrapings of the intestinal mucosal linings in healthy adult hens. This undefined flora provided excellent protection against Salmonella colonization in chickens, but only enabled inconsistent advantage in control of Campylobacter colonization (24). Despite the fact that the competitive exclusion process does occur constantly within the intestinal tract of undomesticated birds and contributes to healthy gut ecology, the Food and Drug Administration has been reticent to approve commercial distribution of MCE within the United States primarily because of the products undefined nature. Additionally, the application of a complex, uncharacterized competitive exclusion (CE) microflora may result in only short term benefit, provide inconsistent results, and entail some limited risks (7). A significant reduction in broiler colonization by C. jejuni through the application of carbohydrate supplements together with three identified antagonists: Citrobacter diversus 22, Klebsiella pneumoniae 23 and Escherichia coli 25 was reported (20). There is also evidence of a significant decrease of C. jejuni in intestinal samples from infected broilers after treatment with poultry-isolated cultures of Lactobacillus acidophilus and Streptococcus fecium (17). However, these treatments have not found application within the commercial poultry industry. Diverse biological activities are common among Bacillus spp. This genus can produce pronounced antagonism to pathogenic microorganisms. To create this antagonism, bacilli may manifest amylolytic, cellulolytic, lipolytic, proteolytic, and pectinolytic activities. They can generate lysozyme and are effectively involved in synthesis of numerous amino acids and other biologically active substances (21, 22). With the exception of some isolates [B. anthracis, B. cereus, B. licheniformis, B. subtilis and B. pumilus (26)] members of the genus Bacillus are harmless for warm-blooded host animals and have phylogenetic relatedness to lactobacilli (6). Owing to these desirable characteristics, bacteria within the genus of Bacillus spp. have found wide application as probiotics and are widely used in medicine and veterinary practice (21). Paenibacillus spp. Is a closely related genus of facultative, anaerobic bacteria that form endospores, are Gram-positive, and can fix nitrogen. They are often associated with the rhizobia of plant roots. Our study screened bacteria active against campylobacter among representative isolates of Bacillus spp., and Paenibacillus spp. and provided data on identification, purification and characterization of substances responsible for their antagonism. We describe isolating highly antagonistic cultures of Bacillus spp. and Paenibacillus spp. from the intestine of birds. The native components of characterized antagonists are low molecular weight peptides, i.e. bacteriocins, with anti-Campylobacter activity.

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