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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR CHARACTERIZATION AND GASTROINTESTINAL TRACT ECOLOGY OF COMMENSAL HUMAN FOOD-BORNE BACTERIAL PATHOGENS IN THE CHICKEN

Location: Poultry Microbiological Safety Research

2011 Annual Report


1a.Objectives (from AD-416)
1. Complete the molecular characterization of selected isolates of the pathogens Campylobacter spp., Salmonella spp. and Clostridium perfringens from poultry, utilizing repetitive-sequence polymerase chain reaction (PCR). Make a comparison regarding cost and efficiency of identification with other methods of differentiating bacteria, such as, multi-locus sequence analysis or pulsed-field gel.

2. Identify host and pathogen genes important to colonization and/or toxin formation by Campylobacter jejuni and Clostridium perfringens in poultry, and monitor host and pathogen gene expression by RNA microarray analysis. Complete comparative genomic analyses between robust and poor colonizers to identify gene targets that could be disrupted to decrease pathogen presence in the gut environment.

3. Qualitatively and quantitatively identify and compare selected microbial populations in the chicken gastrointestinal and reproductive tracts and in the internal organs of healthy and of diseased birds. Use biophotonics models where appropriate, and develop approaches for processing biofilms to provide for quantitative measurement of bacterial populations in situ.


1b.Approach (from AD-416)
In an effort to further characterize food-borne bacteria, we will:.
1)perform repetitive-sequence-polymerase chain reaction (PCR) analysis of Campylobacter spp., Salmonella spp, and Clostridium perfringens isolates, formally recovered from various stages of well-defined poultry production and processing operations;.
2)determine if repetitive-sequence-PCR technology can speciate Campylobacter spp.;.
3)perform comparisons regarding cost, technical difficulty and efficiency of repetitive-sequence-PCR technology relative to previously established subtyping methods (DNA sequence analysis including multi-locus sequence typing [MLST] and pulse field gel electrophoresis [PFGE]) for each pathogen; and.
4)develop an internet accessible database for each poultry associated pathogen assayed by repetitive-sequence-PCR.

In an effort to identify factors responsible for colonization of poultry by Campylobacter jejuni:.
1)pathogen gene content; and.
2)differential gene expression will be monitored by suppressive subtractive hybridization and RNA microarray analysis, respectively. Genes and gene products identified during these experiments will be further characterized and investigated for the development of possible intervention strategies.

In an effort to further our understanding of how environmental changes affect microbial populations associated with poultry production and processing environments we will:.
1)quantitatively characterize chicken intestinal tract populations in the presence or absence of antimicrobial growth promoters (AGP’s) provided in poultry feed; and (2) develop biophotonic methods and techniques to investigate microbial populations in the chicken intestinal tract. Environmental factors (such as AGP in feed) determined to produce changes in microbial populations will be identified and further investigated for the development of possible alternative intervention strategies such as bacteriocins in chicken feed (Stern et al., 2005; 2006).

In an effort to further characterize the role of the fertilized egg in the transmission of Campylobacter spp. in chickens, we will introduce a genetically characterized strain of Campylobacter jejuni (Hiett, et al., 2002), to day-of hatch chicks and test for this particular isolate in the inoculated chickens for a 70 week period. Detection of bacterial pathogens in novel locations in the chicken will allow for the development of more targeted intervention strategies.

In an effort to further our understanding of biofilm formation and persistence in poultry operations, we will:.
1)develop techniques to label pathogens present in biofilms, especially Listeria monocytogenes; and (2) develop methods for the production of biofilms. Environmental factors contributing to biofilm formation will be identified and further investigated for the development of possible intervention strategies.


3.Progress Report
Epidemiology and Molecular Characterization: Lipoologosaccharide (LOS) polymerase chain reaction and Multilocus Sequence Typing (MLST) analyses were performed on an epidemiologically characterized subset of 51 C. jejuni isolates. C. jejuni isolates within specific LOS classes invaded Caco-2 cells at significantly higher rates than isolates in other LOS classes. Also, LOS class correlates with specific MLST clonal complexes. Isolates that segregate into particular LOS classes and specific MLST clonal complexes will be chosen for further detailed genomic pyrosequence analyses to.
1)facilitate the development of specific interventions against C. jejuni, and.
2)develop testable biomarkers of virulence.

Colonization Factors: Campylobacter spp., non-carbohydrate consuming bacteria, have unique nutritional and environmental requirements. C. jejuni is most often associated with poultry, while C. coli are more frequently associated with swine. Temperature-dependent genes of the bacteria are important to colonization and temperature-dependent colonization factors are in part responsible for the species-specific colonization characteristics of C. coli. We determined the differential utilization of 190 different sole carbon substrates by C. coli at 37oC and 42oC using phenotype microarray (PM) technology. The amino acids L-asparagine and L-serine induced more respiration by C. coli at the lower temperature of 37°C (human) as compared to 42°C. Conversely, L-glutamine was utilized to a significantly greater extent at the higher temperature of 42°C (chicken). Further investigations are needed to determine the basis for the temperature-dependent utilization of substrates by Campylobacter spp. and their role in species-specific colonization.

Microbial Ecology: Various protocols were evaluated for sampling Salmonella Enteritidis and Salmonella Typhimurium, from eggshells of hatching broiler eggs. Comparisons of rinse and crush sampling methods using high bacterial challenge numbers demonstrated equal levels of recovery; however, intermediate challenge levels (~102 cells/eggshell) revealed that the crush sampling method resulted in 20% more Salmonella positive eggshells when compared to the rinse methodology. Low challenge levels (~101 cells/eggshell) were ineffective for bacterial recovery.

The use of antibiotic growth promoters during poultry production is a public health concern due to antibiotic resistance development in bacteria. Lupulone, a beta-acid from the hops plant Humulus lupulus, is a potential feed additive growth promoter and antibacterial. Lupulone significantly decreased numbers of Clostridium perfringens, a pathogenic bacterium causing human food-borne disease among humans, in chicken gastrointestinal tracts. No significant changes were noted in the overall microbiota for the cecum or the midgut indicating that only pathogenic clostridia were reduced in the chicken gastrointestinal tract.

This project has reached its term date and been replaced by project 6612-32000-059-00D.


4.Accomplishments
1. Delineating Campylobacter spp. epidemiology. Several investigations focused on delineating Campylobacter spp. epidemiology have been conducted; however, a complete understanding of the critical sources for Campylobacter spp. transmission through integrated poultry operations continues to remain elusive. ARS investigators in Athens, GA, sampled three vertically integrated broiler flocks, during production and processing, using 5 media, 2 temperatures, and 2 atmospheres. Additionally, processed birds were placed at 4°C whereupon exudate was sampled at day 2. Recovered Campylobacter spp. isolates were subtyped using both flaA short variable region and multi-locus sequence typing. Preliminary analyses demonstrated that cultural recovery of Campylobacter spp., relative to sample type, was similar among 4 media, independent of temperature or atmosphere conditions. Levels of Campylobacter spp. recovered were approximately 106 cfu/ml for fecal samples and 102 cfu/ml for both rinse and exudate samples. Initial subtype analyses of isolates from 1 flock recovered with these 4 media and from the 3 sample types, revealed a predominant sequence type (ST-939) belonging to the ST-353 clonal complex. These results will provide insight into the comparability of investigations using various recovery media. Additionally, comparison with results obtained from 16S rRNA gene pyrosequencing investigations will assist in refinement of a more efficacious recovery media for Campylobacter spp. Identification of the predominant Campylobacter spp. subtypes involved in the colonization of agricultural animals and human clinical cases is critical for the development of targeted intervention strategies such that a safer food product can be delivered to consumers.

2. Distinct sampling techniques (crush vs rinse sampling) for the recovery of Salmonella Enteritidis and Salmonella Typhimurium. ARS scientists in Athens, GA, compared distinct sampling techniques (crush vs rinse sampling) for the recovery of Salmonella Enteritidis and Salmonella Typhimurium from the eggshells of hatching broiler eggs. When high inoculation levels of pathogens were employed, both methods resulted in similar recovery rates. However, when Salmonella challenge levels of ~100 cfu/eggshell were employed, the crush sampling method resulted in 20% more Salmonella positive eggshells (69% vs. 49%) compared to the rinse method. Furthermore, low challenge levels (~101 cfu/eggshell) revealed that both methods were ineffective for recovery. Accurate determination of the most efficacious bacterial sampling methodology is critical for the development of targeted intervention strategies such that a safer food product can be delivered to consumers.

3. Campylobacter spp. are small bacteria incapable of metabolizing glucose. C. jejuni is most often recovered from poultry, while C. coli are more frequently recovered from swine. Temperature has been suggested to trigger potential colonization or virulence factors in C. jejuni; recent studies support this suggestion as it was demonstrated that temperature-dependent genes are important to colonization. It is possible that temperature-dependent colonization factors are in part responsible for the species-specific colonization characteristics of C. coli as well. ARS scientists in Athens, GA, determined the utilization of 190 different sole carbon substrates by C. coli ATCC 49941 at 37°C and 42°C using phenotype microarray (PM) technology. Temperature was shown to significantly affect utilization of specific amino acids. Knowledge of temperature-dependent substrate utilization by different Campylobacter spp. will provide information regarding species-specific colonization. This information could lead to improvements in culture media for detection and isolation of the pathogen as well as to future intervention methods that ultimately reduce human exposure. Identification, at the phenotype level, of factors involved in the colonization of agricultural animals by zoonotic pathogens is critical for the development of targeted intervention strategies such that a safer food product can be delivered to consumers.

4. Antimicrobial resistance genes and the subsequent antibiotic resistance in bacteria. The use of antibiotic growth promoters during poultry production is a public health concern due to the harborage of antimicrobial resistance genes and the subsequent antibiotic resistance in bacteria. Lupulone, a hop ß-acid from the hops plant Humulus lupulus, has been considered a potential feed additive growth promoter and antibacterial. ARS scientists in Athens, GA, demonstrated that Lupulone significantly decreased the chicken gastrointestinal numbers of Clostridium perfringens. Metagenomic analyses revealed no significant changes in the overall microbiota for the cecum or the midgut indicating that only the pathogenic clostridia were reduced in the chicken gastrointestinal system. The identification of changes, upon administration of pre-harvest interventions, in the microbial ecology of the broiler chicken gastrointestinal tract is critical for the scientifically sound development of targeted intervention strategies such that a safer food product can be delivered to consumers.


Review Publications
Hiett, K.L., Line, J.E., Conlan, A.J., Coward, C., Van Diemen, P.M., Stevens, M.P., Jones, M.A. 2010. Transmission and dose–response experiments for social animals: a reappraisal of the colonization biology of Campylobacter jejuni in chickens. Journal of the Royal Society Interface. DOI: 10.1098/rsif.2011.0125.

Tillman, G.E., Oakley, B., Haas, G.J., Wise, M.G., Smith, M.A., Siragusa, G.R. 2011. Quantitative Profiling of the Chicken Intestine Microbiome Following Administration of Lupulone, a Plant-Based Antimicrobial.. FEMS Microbiology Ecology. 77(2):395-403. DOI:10.1111/j.1574-6941.2011.01119.x.

Hannah, J.F., Wilson, J.L., Cox Jr, N.A., Cason Jr, J.A., Bourassa, D.V., Richardson, L.J., Musgrove, M.T., Rigsby, L.L., Buhr, R.J. 2011. Comparison of shell bacteria from unwashed and washed table eggs harvested from caged laying hens and cage-free floor-housed laying hens. Poultry Science. 90(7):1586-1593.

Mead, G.C., Lammerding, A., Cox Jr, N.A., Doyle, M.P., Humbert, F., Kulikovskiy, A., Panin, A., Nascimento, V., Wierup, M., Biggs, R., Buhr, R.J., Cahill, S., Cason Jr, J.A., Chalermchaikit, T., Filho, R., Forshell, L., Hidalgo, H., Hofacre, C., Hupkes, H., Landinez, M., Madsen, M., Mulder, R., Richardson, L.J., Shi, Z., Smith, D., Toyofuku, H., Tuominen, P., Uyttendaele, M., Zwietering, M. 2010. Scientific and technical factors affecting the setting of salmonella criteria for raw poultry: a global perspective. Journal of Food Protection. 73(8):1566-1590.

Cox Jr, N.A., Cason Jr, J.A., Richarson, J. 2010. Minimization of Salmonella Contamination on Raw Poultry. Annual Review of Food Science & Technology. 2:75-95.

Spickler, J.L., Buhr, R.J., Cox Jr, N.A., Bourassa, D.V., Rigsby, L.L. 2011. Comparison between rinse and crush-and-rub sampling for aerobic bacteria recovery from broiler hatching eggs after sanitization. Poultry Science. 90:(7)1609-1615.

Last Modified: 8/27/2014
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