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United States Department of Agriculture

Agricultural Research Service

Research Project: INTERVENTIONS AND METHODOLOGIES TO REDUCE HUMAN FOOD-BORNE BACTERIAL PATHOGENS IN CHICKENS
2010 Annual Report


1a.Objectives (from AD-416)
The purpose of the Poultry Microbiological Safety Research Unit is to further reduce or eliminate bacterial pathogen contamination in poultry operations and the following objectives are to:.
1)Assess the effectiveness and further development of bacteriocins (anti-bacterial peptides) and bacteriophage by in vitro bacterial growth inhibition in culture and in vivo experimentation via challenge in chickens..
2)Reduce bacterial populations in chicken litter by monitoring poultry houses for bacterial pathogens carried by chickens during use of intervention technologies. Decontamination techniques, such as in-house foam applied disinfectants will be examined for reduction of Campylobacter spp., Salmonella spp., Clostridium perfringens and Listeria monocytogenes..
3)Improve cultural methods for Campylobacter spp. in poultry in further support of FSIS needs, including the improvement of recovery. Use microarray expression analysis under various cultural conditions to identify nutrients necessary for optimal growth, colonization and culture of Campylobacter spp.


1b.Approach (from AD-416)
New antimicrobial agents will be developed that are usable by the poultry farmer on a large scale to reduce Campylobacter spp. and salmonellae by applying in feed at the appropriate times before slaughter. Also, new means for the drug-free production sector to control clostridial disease without relying on antibiotics are the major expected approaches of Objective 1. Providing a science based source of data to better dispose of spent poultry litter without major energy or financial inputs is the approach for Objective 2. A more reliable and quantitative method to routinely culture Campylobacter spp. from food and environmental samples is the approach of Objective 3.


3.Progress Report
Enhanced bacteriocin production. Novel peptides produced by bacteriocin producing bacteria stimulate the production of bacteriocins in fermentations of at least 5 liters. The bacteriocin producer bacteria are cultured in the presence of a novel inducer bacterium and a peptide having a carboxyl terminal sequence of VKGLT in a limited nutrient medium concentration to achieve an increase in bacteriocin production. Additional novel bacteriocins produced by Bacillus or Paenibacillus B-30507, B-30508, B-30509 and B-30644 were used to reduce levels of pathogenic bacterial colonization in poultry.

Antagonists against Clostridium perfringens. Investigations continued by identifying bacteriophage enzymes that lyse Clostridium perfringens. Additional lytic genes were identified from presumed prophage and host encoded bacterial DNA. Two lysin genes were identified from a Russian Clostridium perfringens bacteriophage. One encoded an amidase while a second gene was identified as a lysozyme-endopeptidase potentially having two enzymatic domains.

Improved cultural methods for pathogen recovery. Phenotype microarrays were utilized to evaluate the ability of Campylobacter jejuni strain 11168 to oxidize 190 different substrates as sole carbon sources at 37oC and 42oC. Our improved understanding of the metabolic pathways and nutritional requirements of C. jejuni is being used to develop enhanced media for detection and isolation of the pathogen.

Sampling protocols for detecting Salmonella on poultry differ among various countries. In the U.S. the whole carcass is rinsed with 400 ml of 1% buffered peptone water, whereas in the European Union 25-g samples composed of neck-skin from three carcasses are evaluated. In a separate study a whole carcass rinse and a neck-skin excision procedure for Salmonella isolation from the same broiler carcass was evaluated. No significant difference (P > 0.05) in Salmonella prevalence was found between the samples processed by the two methods, but both procedures produced many false-negative Salmonella results, which could potentially hinder efforts to globally standardize one particular method.


4.Accomplishments
1. Antimicrobial activities of bacteriocins against pathogenic bacteria. Recognition of the dramatic rise in antibacterial resistance or drug resistance (DR) has caused substantial concern. ARS researchers in Athens, GA together with collaborators in Russia have been exploring and identifying bacteriocins (BCN) to provide potential alternatives and create alternative antimicrobial treatments. Bacteriocins are non-toxic peptides produced by safe bacteria that kill pathogenic bacteria. Paenibacillus polymyxa NRRL B-30509 was used to produce BCN B-602 and Enterococcus faecium NRRL B-30746 produced BCN E 50-52. Researchers tested 64 DR isolates from hospitals and determined the minimal inhibitory concentrations (MIC; mg/ml) to quantify susceptibility and resistance to antibiotic classes and BCN. High resistance of these isolates to the different classes of antibiotics was observed; whereas, these same isolates were very susceptible to BCN. The ranges of susceptibility of the clinical isolates were from =0.025 to 6.4 mg BCN/ml for Acinetobacter baumannii, Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Proteus spp., Staphylococcus aureus.

2. Inducer bacteria, signal proteins and low nutrient media stimulate bacteriocin production. ARS researchers in Athens, GA together with collaborators in Russia provided specified quantities of three isolated and sequenced signal peptides, which shared a unique signature amino acid sequence, together with specified bacteriocin (BCN) inducer bacteria and, the use of a low nutrient medium to enhance BCN production. They observed that 100% brucella broth provided yields of 25 to 32 mg BCN/L while 10% brucella broth in the presence of inducer bacteria and SP provided yields of 225 to 510 mg BCN/L, an increase of more than 9 fold the quantities of BCN when compared to those levels previously produced. These BCN can now be efficiently produced for application to control bacterial associated problems.

3. Bacteriophages lytic for C. perfringens isolated. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents by ARS researchers in Athens, GA and Russian collaborators. PhiCPV1, one of the more virulent bacteriophages, was classified in the family Podoviridae. Amino acid sequences of the predicted proteins from the phiCPV1 genome ORFs were compared with those from the NCBI database and potential functions of 10 proteins were predicted by sequence homology. One bacteriophage genome encoded lysin was predicted to share homology with N-acetylmuramoyl-L-alanine amidases and a second lytic enzyme was predicted to be a lysozyme-endopeptidase. These enzymes digest peptidoglycan of the bacterial cell wall and could be considered potential therapeutics to control C. perfringens.

4. Impact of Litter Salmonella Status during Feed Withdrawal on the Salmonella Prevalence in the Broiler’s Crop and Ceca following Feed Withdrawal. Salmonella prevalence increases in both the broiler’s crop and ceca following feed withdrawal prior to slaughter which can lead to carcass Salmonella contamination and was thought to be attributed solely to the consumption of contaminated litter. Four pens of market age broilers were challenged with one of two marker Salmonella strains (S. Montevideo or S. Heidelberg) in separate rooms by ARS researchers in Athens, GA. Four days after challenge, one pen of broilers for each marker Salmonella was switched between rooms at the beginning of a 12 hour feed withdrawal period. Broilers were euthanized and crops and ceca aseptically sampled. For the broilers that remained in their marker Salmonella challenge pens during feed withdrawal, from 15% of the crops and 60% of the ceca only S. Montevideo was recovered compared to 100% of the crops and 100% of the ceca from which only S. Heidelberg was recovered. For those broilers that were moved from the S. Montevideo to the S. Heidelberg pen from 20% of the crops S. Heidelberg and from 20% of the crops S. Montevideo was recovered, but only S. Montevideo was recovered from 80% of the ceca (the challenge pen Salmonella). For those broilers moved from the S Heidelberg to the S. Montevideo pen only S. Heidelberg was recovered from 80% of the crops and 100% of the ceca (the challenge pen Salmonella). These results reveal the Salmonella prevalence in the ceca following a 12 hour feed withdrawal period is dependent on the challenge pen Salmonella prior to feed withdrawal and not the Salmonella present in the litter of the feed withdrawal pen. However, the prevalence of crop Salmonella following a 12 hour feed withdrawal period can be the result of both the challenge pen and the feed withdrawal pen Salmonella status. No specific recommendations have been made to regulatory agencies yet.

5. The Prevalence and Number of Salmonella Serogroups Recovered following Carcass Chilling. Variation in carcass Salmonella sampling and chilling methods may result in differences in prevalences and the number of Salmonella serogroups recovered. Commercially processed carcasses were air or immersion chilled to 4°C by ARS researchers at the Russell Research Center pilot plant and the prevalence and number of Salmonella serogroups recovered for matched excised neck-skin and enriched whole carcass samples were evaluated. For air chilled carcasses, neck-skin excision (78%) and whole carcass enrichment (89%) sampling methodologies were comparable for prevalence detection of Salmonella from individual carcasses. From neck-skin samples 3 Salmonella serogroups were identified while from whole carcasses 4 Salmonella serogroups were identified. Immersion chilling resulted in whole carcass enrichment (92%) being superior to neck-skin excision (30%) in prevalence detection of Salmonella from individual carcasses. Neck-skin samples recovered 2 Salmonella serogroups while from whole carcasses 3 serogroup were identified. Prevalence detection of Salmonella from individual carcasses varied greatly between immersion chilled carcasses, without and with 20 ppm free chlorine (respectively), for excised neck-skin (70 vs. 50%), whole carcass rinse (80 vs. 0%), and whole carcass enrichment (100 vs. 90%). Two serogroups were identified from neck-skin and enriched carcasses immersion chilled with and without chlorine. However, only 1 serogroup was detected from whole carcass rinse samples when chlorine was added. Enrichment of neck-skin or the whole carcass increased prevalence and the number of Salmonella serogroups recovered following immersion chilling with and without added chlorine. No specific recommendations have been made to regulatory agencies yet.


Review Publications
Buhr, R.J., Bourassa, D.V., Cox Jr, N.A., Richardson, L.J., Phillips, R.W., Kelley, L.C. 2009. Evaluation of a quail embryo model for the detection of botulinum toxin type A activity. 2010. The Botulium Journal. 1(3) p281-296.

Behling, R.G., Eifert, J., Erickson, M.C., Gurtler, J., Kornacki, J.L., Line, J.E., Radcliff, R., Ryser, E.T., Stawick, B., Yan, Z. 2010. Selected pathogens of concern to industrial food processors: infectious, toxigenic, toxico-infectious, selected emerging pathogenic bacteria. In: Kornack, J.L. Editor. Principles of Microbiological Troubleshooting in the Industrial Food Processing Environment. New York, NY: Springer. 140 p.

Yoon, S.C., Lawrence, K.C., Line, J.E., Siragusa, G.R., Feldner, P.W., Park, B., Windham, W.R. 2010. Detection of Campylobacter Colonies using Hyperspectral Imaging. Sensing and Instrumentation for Food Quality and Safety. Vol. 4, Issue 1. pg. 35-49.

Line, J.E., Hiett, K.L., Guard, J.Y., Seal, B.S. 2010. Differential carbon source utilization by Campylobacter jejuni strain 11168 in response to growth temperature variation. Journal of Microbiological Methods. 80:198-202.

Hinton Jr, A., Cason Jr, J.A., Buhr, R.J., Liljebjelke, K.A. 2009. Bacteria recovered from whole-carcass rinsates of broiler carcasses washed in a spray cabinet with lauric acid-potassium hydroxide. International Journal of Poultry Science. 8:1022-1027.

Stern, N.J., Svetoch, E.A. 2010. Applications and consequences of bacteriocins to control Campylobacter spp. in poultry production. Joint Meeting of the ADSA, AMSA, ASAS and PSA. Volume 64 page 217.

Stern, N.J., Svetoch, E.A., Eruslanov, B.V., Perelygin, V.V., Levchuk, V.P., Urakovq, N., Volodina, L.I., Kovalev, Y.N., Kudryavtesava, T.V., Pokhilenko, V.D., Borzenkov, V.N., Svetoch, O.E., Mitsevich, E.V., Mitsevich, I.P. 2010. Bacteriocins and novel bacterial strains. Journal of Food Protection. Vol 68,p11-17.

Svetoch, E.A., Eruslanov, B.V., Kovalev, Y.N., Mitsevich, E.V., Levchuk, V.P., Fursova, N.K., Perelygin, V.V., Stepanshin, Y.G., Seal, B.S., Stern, N.J. 2009. Antimicrobial activities of bacteriocins E 50-52 and B 602 against antobiotic resistant strains involved in nosocomial infections. Probiotics and Antimicrobial Proteins. Volume 1 Pages 136-142.

Svetoch, E.A., Eruslanov, B.V., Perelygin, V.V., Levchuk, V.P., Seal, B.S., Stern, N.J. 2010. Inducer bacteria, unique signal peptides and low nutrient media stimulate in-vitro bacteriocin production by Lactobacillus spp. and Enterococcus spp. strains. Journal of Agricultural and Food Chemistry. Volume 58 pages 6033-6038.

Last Modified: 10/25/2014
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