PRE -AND POST-HARVEST CONTROL OF FOODBORNE PATHOGENS IN POULTRY PROCESSING ENVIRONMENT AND POULTRY PRODUCTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0228506
• Project No.: MIS-326030
• Project Start Date: Mar 1, 2012
• Project End Date: Feb 28, 2017

Project Director
Sharma, C. S.

Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762

Performing Department
Poultry Science

Non Technical Summary
Foodborne illnesses are a regular feature and concern for food safety and public health in the United States. It is estimated that every year foodborne diseases cause 9.4 million illnesses, in the United States, of those about 1 million and 0.8 million illnesses are caused by Salmonella and Campylobacter, respectively (Scallan et al., 2011). Consumption of undercooked poultry products contaminated with Salmonella and Campylobacter have been implicated in foodborne illnesses (Altekruse et al., 1999; CDC 2010a). Poultry is considered an important reservoir of pathogenic bacteria like Salmonella and Campylobacter jejuni and Listeria monocytogenes. Live birds harbor these pathogenic bacteria in their intestines, feathers and skin which lead to contamination of carcasses during slaughtering and processing (Corry, 2007). Keeping in view the public health significance of Salmonella and Campylobacter and frequent contamination of poultry carcasses with these pathogens, USDA's Food Safety and Inspection Service (FSIS) has recently introduced new performance standards for poultry processing plants to reduce Salmonella and Campylobacter contamination in poultry carcasses (USDA FSIS 2010). Listeria monocytogenes is another pathogen of concern to public health. It is estimated to cause approximately 2,500 illnesses, 2,300 hospitalizations, and 500 deaths each year in the United States (Mead et al., 1999). Listeria monocytogenes has been isolated from raw meat and poultry but it is a major concern in ready-to-eat (RTE) meat and poultry products. Because of high case fatality rate and public health significance, USDA FSIS maintains a zero tolerance policy of L. monocytogenes in RTE meat and poultry products. Various outbreaks have been linked to consumption of RTE meat and poultry products contaminated with L. monocytogenes (CDC, 2010b). As poultry and poultry products per capita consumption increase annually, the risk associated with foodborne illnesses from Salmonella, Campylobacter and L. monocytogenes contaminated poultry products will also increase. Many pre-and post-harvest interventions have been applied to restrict the occurrence of foodborne pathogens at grow out farms, processing plants and finished poultry products, but none has resulted in total elimination or removal of foodborne pathogens in poultry products. In order to improve the public health by ensuring food safety, there is great need to decrease the prevalence of these pathogens in poultry products. Therefore, there is critical need for additional interventions to use along with the existing preventive measures to further reduce the contamination of poultry products with foodborne pathogens. The proposed research will be undertaken to reduce or eliminate different foodborne pathogens in raw, processed and finished or RTE poultry products that will be achieved by antimicrobial interventions by using USDA approved antimicrobials at the pre- and post-harvest steps of poultry processing.

Animal Health Component

(N/A)

Research Effort Categories

Basic

40%

Applied

40%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3220	1100	20%
712	3260	1100	45%
712	3280	1100	25%
712	4010	1040	10%

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

Subject Of Investigation
3220 - Meat-type chicken, live animal; 3280 - Other poultry products; 4010 - Bacteria; 3260 - Poultry meat;

Field Of Science
1100 - Bacteriology; 1040 - Molecular biology;

Keywords

poultry processing

poultry products

food safety

food microbiology

salmonella

campylobacter

listeria monocytogenes

ready to eat poultry products

antimicrobials

Goals / Objectives
The long term goals of this proposed research are to develop or improve the methods that will control the occurrence of foodborne pathogens in poultry products. The research will be undertaken with the following objectives to achieve the desired goals. 1. Control of foodborne pathogens such as Salmonella and Campylobacter spp. at the pre-harvest or farm level 2. Control of foodborne pathogens such as Salmonella and Campylobacter spp. during slaughtering and processing of poultry and at post-harvest level 3. Control of foodborne pathogens such as L. monocytogenes in ready-to-eat poultry products 4. To elucidate the antimicrobial mechanism of action of various antimicrobials used in objective 1,2 and 3 against gram-negative and gram-positive foodborne pathogens

Project Methods
Objective 1: At the pre-harvest level various dietary modifications and management aspects (for example competitive exclusion, modifications in broiler feed and water, feed withdrawal) related to broiler production will be undertaken to assess their role in pre-harvest food safety. Different commercial bacteriophage products targeting Salmonella will be tested on poultry feathers and carcasses to reduce the level of Salmonella contamination. Bacteriophage dose and contact time to kill common poultry-borne Salmonella serotypes will be determined in vitro. Preliminary studies will be carried on live birds to determine the appropriate number of birds per treatment and number of replications for this study in order to have least variability in results/findings. Objective 2: For the control of foodborne pathogens like Salmonella and Campylobacter spp. during poultry processing and at post-harvesting, novel USDA approved antimicrobials will be tested for their efficacy at different steps in poultry processing such as scalding, post-pick, chilling and pre-chill and post-chill carcass dips/sprays. Novel antimicrobials such as lauric arginate (LAE), will be tested for its efficacy against Salmonella and Campylobacter in ground chicken, ground turkey and chicken breast fillets. As an example, the approach to using LAE for control of Salmonella in poultry products: ground chicken procured locally will be inoculated with different Salmonella serotypes. The biocide effect of LAE against these Salmonella serotypes will also be determined in vitro. Inoculated ground chicken will be placed in sterile bags and treated with different concentrations of LAE. Each bag of treated ground chicken will be stored at 4C for 3 days. Duplicate samples per treatment will be analyzed after 0, 1, 2 and 3 days for Salmonella and total psychrotrophic bacteria, pH and objective color. This study will be repeated three times and recovery of the surviving Salmonella populations will be made on selective media. Randomized block design will be used to determine differences between trials, among treatments and storage days, and treatment vs day interaction. Objective 3: Novel USDA approved antimicrobials like listeria-lytic bacteriophages and different lactic acid bacterial strains will be tested for their efficacy against L. monocytogenes under vacuum or modified atmosphere packaging in various RTE poultry products. Briefly, RTE poultry products such as turkey ham procured locally or produced in meat processing lab will be inoculated with cocktail mixture of L. monocytogenes strains and treated with viable LAB strains. Objective 4: Studies will also be carried out to elucidate the mode of action of these antimicrobials for better understanding and application to control foodborne pathogens in poultry products. The methods used to assess the mechanism of action will depend on the type of antimicrobial selected. Various techniques such as transmission electron microscopy, and nucleic acid staining combined with fluorescent microscopy and flow cytometry will be utilized to elucidate the mode of action of antimicrobials.

Progress 03/01/12 to 02/28/17

Outputs
Target Audience:Poultry Processors in the broiler chicken and turkey industry and food safety profession in industry and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities were provided to graduate as well as undergraduate students through their participation in laboratory procedures for conducting research in food microbiology and safety of poultry products, data analysis, and the dissemination of information by means of oral presentations and assistance in the writing and publication of abstracts and manuscripts. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest such as poultry and food processors, food safety professionals through their attendance at the annual meetings in which the results were presented in the form of oral and/or poster presentations. The concise information from these projects was also published in meeting abstracts. What do you plan to do during the next reporting period to accomplish the goals?This will be the final report for this project.

Impacts
What was accomplished under these goals? Over the past 40 months, our lab has discovered and developed strategies for understanding administering new antimicrobials for reducing Salmonella, Campylobacter, and Listeria from either pre or post-harvest facilities. In addressing these issues morphological and adaptive aspects of each pathogenic organism was identified and explored further to discover how to best eliminate them from the abusive area. These activities have led to 16 published manuscripts with 2 more still under review. A total of 19 abstracts have been published and presented as oral or poster presentations. Along with the scientific presentations presented at national and international meeting, the research topic has provided interest to the poultry community which has led to 3 invited talks on this topic. The research projects have supported the mentorship of 3 undergraduate students, 5 Master's students and 1 Ph.D. student who have all successfully defended their degree. These actions and accomplishments support the overall goal of this project.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Saha, S., N. Dhowlaghar, A. Lawrence, R. Nannapaneni, and C. S. Sharma. 2015. Transmission electron microscopy study of Listeria Monocytogenes serotype 1/2a cells exposed to sublethal heat stress and carvacrol. 79th Mississippi Academy of Sciences Annual Meeting, Hattiesburg, MS.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Abeysundara, P.D.A., N. Dhowlaghar, R. Nannapaneni, C.S. Sharma and B. Mahmoud. 2016. Influence of cold stress at 4�C for 24 hon the survival of Listeria monocytogenes (Bug600) and ScottA in lethal alkali, acid, and oxidative stress. J. Food Prot. (Submitted).
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Abeysundara, P.D.A., R. Nannapaneni, K.A. Soni, C.S. Sharma and B. Mahmoud. 2016. Induction and stability of oxidative adaptation in Listeria monocytogenes EGD (Bug600) and F1057 in sublethal concentrations of H202 and NaOH. Int. J. of Food Micro. 238: 288-294.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Shen, Q., P. Pandare, K.A. Soni, R. Nannapaneni, B. Mahmoud and C.S. Sharma. 2016. Influence of temperature on alkali stress adaptation in Listeria monocytogenes. Food Control 62: 74-80.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Sukumaran, A.T., A.S. Kiess, R. Nannapaneni, and C.S. Sharma. 2016. Reduction of Salmonella on chicken breast fillets stored under aerobic and modified atmosphere packaging by the application oflytic bacteriophage preparation. Poult. Sci. 95: 688-675.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhaka, J., R. Nannapaneni, and C.S. Sharma. 2016. Comparison of thermal resistance of rugose phenotype of Salmonella Typhimurium with normal smooth morphology at different growth phases. Poult. Sci. Vol 95 (E-Suppl. 1) 21.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Obe, T., R. Nannapaneni, and C.S. Sharma. 2016. Development ofrugose morphotype of Salmonella Typhimurium following exposure to sub-inhibitory chlorine concentrations that exhibit chlorine resistance and strong biofilm forming ability. Poult. Sci. Vol 95 (E-Suppl. 1) 35.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Moore, A. and C.S. Sharma. 2016. Antimicrobial efficacy of acidified lactic acid, propionic acid, and lauric arginate against Salmonella in ground chicken frames. Poult. Sci. Vol 95 (E� Suppl. 1) 256.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Dhowlaghar, N., P. Abeysundara, R. Nannapaneni, C.S. Sharma, B. Mahmoud, Z. Haque, W.H. Cheng. 2016. Growth and biofilm formation by Listeria monocytogenes in high and low concentrations of catfish mucus extract. IFT Annual meeting, July 16-19, 2016 Chicago, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Abeysundara, P., N. Dhowlaghar, R. Nannapaneni, C.S. Sharma, B. Mahmoud, and W. Schilling. 2016. Growth and biofilm formation by Listeria monocytogenes in high and low concentrations of cantaloupe extract. IFT Annual meeting, July 16-19, 2016 Chicago, IL.
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Bansal, M., R. Nannapaneni, C. S. Sharma and A. S. Kiess*. 2017. Listeria monocytogenes Response to Sub-lethal Chlorine Induced Oxidative Stress on Homologous and Heterologous Stress Adaptation. Frontiers of Micro. (Submitted).
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Obe, T., R. Nannapaneni, C. S. Sharma, and A. S. Kiess*. 2017. Rugose Morphotype of Salmonella enterica serovar Typhimurium ATCC 14028 Exhibits Chlorine Resistance and Strong Biofilm Forming Ability. Int. J. of Poult. Sci. (Submitted)
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Obe, T., R. Nannapaneni, C. S. Sharma, and A. S. Kiess*. 2017. Homologous Stress Adaptation, Antibiotic Resistance and Biofilm Forming Ability of Salmonella enterica serovar Heidelberg ATCC 8326 on Different Food-Contact Surfaces Following Exposure to Sublethal Chlorine Concentrations. Poult. Sci. (Accepted) Doi: 10:3382/PS/pex346.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Moore, A., R. Nannapaneni, A. Kiess, and C.S. Sharma. 2016. Evaluation of USDA approved antimicrobials on the reduction of Salmonella and Campylobacter in ground chicken frames and their effect on meat quality. Poult. Sci. 96: 2385-2392.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Obe, T., R. Nannapaneni, and C.S. Sharma. 2017. Homologous stress adaptation, antibiotic resistance, and biofilm formation of Salmonella Heidelberg on different food-contact surfaces following exposure to sub-lethal chlorine concentrations. Poult. Sci. Vol 96 (E-Suppl. 1) 252.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bansal, M., R. Nannapaneni, and C.S. Sharma. 2017. Influence of chlorine induced sub-lethal oxidative stress on homologous stress adaptation and biofilm formation in Listeria monocytogenes. Poult. Sci. Vol 96 (E-Suppl. 1) 256.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target audiences include poultry processors in the broiler chicken and turkey industry and food safety professionals in industry and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities were provided to graduate as well as undergraduate students through their participation in laboratory procedures for conducting research in food microbiology and safety of poultry products, data analysis, and the dissemination of information by means of oral presentations and assistance in the writing and publication of abstracts and manuscripts. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest such as poultry and food processors, food safety professionals through their attendance at the annual meetings in which the results were presented in the form of oral and/or poster presentations. The concise information from these projects was also published in meeting abstracts. What do you plan to do during the next reporting period to accomplish the goals?The plan for the next reporting period will be to complete the projects currently under progress and to initiate new projects to control Salmonella, Campylobacter and Listeria monocytogenes in poultry products by novel intervention technologies. The findings from these projects will be disseminated to public and scientific communities in meetings and through publications in peer-reviewed journals.

Impacts
What was accomplished under these goals? The antimicrobial efficacy of oregano oil nanoemulsion against 5 Salmonella enterica serotypes (S. Enteritidis, S. Heidelberg, S. Newport, S. Reading, and S. Typhimurium) and 2 Listeria monocytogenes serotypes (1/2a and 4b) was evaluated in vitro by colorimetric broth microdilution assay. The MIC values of oregano oil nanoemulsion against Salmonella and L. monocytogenes were much lower as compared with oregano essential oil. Specifically, the MIC values for nanoemulsion ranged from 0.0195% (195 µL/L) to 0.039% (390 µL/L) as compared with MIC values for oregano essential oil that ranged from 0.156% (1560 µL/L) to 0.312% (3120 µL/L). Oregano oil nanoemulsion was most effective against S. Typhimurium (ATCC 14028) and S. Reading (MIC = 195 µL/L). For all other Salmonella and L. moncytogenes serotypes, the MIC value remained same (MIC = 390 µL/L). These findings suggest that nanoemuslion is more effective than essential oil in inhibiting the growth of Salmonella and L. moncytogenes. Moreover, for post-harvest applications, nanoemulsions might be more useful practically than essential oils. Mechanically separated chicken (MSC) has been associated with high levels of Salmonella contamination. This study was conducted to examine the efficacy of peracetic acid (PAA), cetylpyridinium chloride (CPC) and sodium hypochlorite (NaOCl) in reducing Salmonella in ground chicken frames simulating a product similar to MSC. Fresh chicken frames obtained from a commercial poultry processor were treated with PAA (0.1%), CPC (0.4%), and NaOCl (0.005%) by dipping the individual frames in treatment solutions for 10 s. Each treatment was replicated on 10 different frames. All chicken frame samples were ground in blender to obtain a product similar to MSC. Each ground sample weighing (325 ± 32.5 g) was analyzed for the presence of Salmonella. Two out of 10 (20%) control samples were found to be positive for Salmonella. None of the samples treated with PAA were positive for Salmonella whereas 10% (1/10) and 16.7% (1/6) of the samples treated with CPC and chlorine were positive for Salmonella, respectively. There was no significant difference for Salmonella prevalence in control or treated samples (P > 0.05). We are also investigating the effectiveness of commonly used antimicrobials during poultry processing in reducing Salmonella contamination in MSC with Salmonella challenge studies. The findings from this work could provide useful information to the poultry processing industry for reducing Salmonella in MSC. The efficacy of Salmonella lytic bacteriophage preparation (SalmoFresh) in reducing Salmonella Heidelberg on turkey breast cutlets and ground turkey was evaluated. In a broth model assay, the phage preparation completely inhibited the growth of four S. enterica serotypes (S. Enteritidis, S. Heidelberg, S. Kentucky, and S. Typhimurium) at 37°C at a multiplicity of infection of 10,000 PFU/CFU. At 4°C in 0.1% peptone water (PW), phage treatment at a multiplicity of infection of 10,000 resulted in ca. 4.0-log CFU/ml reductions of Salmonella Enteritidis, Salmonella Heidelberg, and Salmonella Typhimurium. When raw turkey breast cutlets inoculated with Salmonella Heidelberg (∼103CFU/g) were treated with phage preparation (107PFU/g) and stored at 4°C, the phage treatment caused reductions of 0.8, 0.6, and 1.3 log CFU/g (P ≤ 0.05) of Salmonella Heidelberg on day 0, 1, and 7, respectively, compared with the counts in the control. However, no significant reduction of Salmonella Heidelberg (P > 0.05) was observed in ground turkey when turkey meat pieces inoculated with Salmonella Heidelberg were surface treated with phage preparation (107PFU/g) before grinding. These findings indicate that the bacteriophage preparation was effective in reducing Salmonella on turkey breast cutlets as a surface treatment but did not cause any reduction of Salmonella Heidelberg in ground turkey. The effectiveness of SalmoFresh™ in reducing Salmonella on chicken breast fillets was examined in combination with lauric arginate (LAE) or cetylpyridinium chloride (CPC). In another experiment, a sequential spray application of this bacteriophage (phage) solution on Salmonella inoculated chicken skin after a 20s dip in chemical antimicrobials (LAE, CPC, peracetic acid, or chlorine) was also examined in reducing Salmonella counts on chicken skin. The application of phage in combination with CPC or LAE reduced S. Typhimurium, S. Heidelberg, and S. Enteritidis up to 5 log units in vitro at 4°C. On chicken breast fillets, phage in combination with CPC or LAE resulted in significant (p<0.05) reductions of Salmonella ranging from 0.5 to 1.3logCFU/g as compared to control up to 7days of refrigerated storage. When phage was applied sequentially with chemical antimicrobials, all the treatments resulted in significant reductions of Salmonella. The application of chlorine (30ppm) and PAA (400ppm) followed by phage spray (109PFU/ml) resulted in highest Salmonella reductions of 1.6-1.7 and 2.2-2.5logCFU/cm2, respectively. In conclusion, the surface applications of phage in combination with LAE or CPC significantly reduced Salmonella counts on chicken breast fillets. However, higher reductions in Salmonella counts were achieved on chicken skin by the sequential application of chemical antimicrobials followed by phage spray. The sequential application of chlorine, PAA, and phage can provide additional hurdles to reduce Salmonella on fresh poultry carcasses or cut up parts. In another study, the combined efficacy of carvacrol and modified atmosphere packaging (MAP) was examined in reducing Salmonella on turkey breast cutlets stored at 4°C. Turkey breast cutlets were dip treated with carvacrol (0.25, 0.5, and 1% v/v) for 30s and stored under MAP (95% carbon dioxide and 5% oxygen) to evaluate the efficacy against Salmonella, Campylobacter jejuni and lactic acid bacteria on turkey breast cutlets. The combined application of carvacrol and MAP resulted in 1.0-2.0logCFU/g reduction (P≤0.05) of both Salmonella and Campylobacter on turkey breast cutlets for 7d storage at 4°C. MAP alone and in combination with carvacrol reduced lactic acid bacteria (P≤0.05) on cutlets stored at 4°C for 21d period. There was no difference (P≤0.05) in meat color among treatments and controls except for an increased paleness of meat (P≤0.05) observed for the 1% carvacrol treated cutlets stored under MAP after 21d of storage. The high concentration of carbon dioxide and carvacrol treatments did not cause any alteration in meat pH (P≤0.05). In conclusion, carvacrol was effective at a low concentration of 0.25% (v/v) in reducing Salmonella and C.jejuni by ~1.0logCFU/g when stored under MAP. In a study to determine the prevalence of antibiotic resistant Salmonella strians in retail poultry products, atotal of 240 retail poultry meat samples were collected from various stores in Mississippi. Thirty three Salmonella enterica isolates were recovered from ground turkey (n=20) and chicken breast fillets (n=13) samples. Clonal relatedness among allthe isolates was assessed by pulsed-field gel electrophoresis (PFGE). The predominant serotypes isolated from ground turkey were S. Heidelberg (35%) and S. Saintpaul (35%) followed by S. Newport (10%) whereas S. Typhimurium var.5- was the most prevalent serotype (77%) in chicken meat isolates. Multidrug resistance (≥3 antimicrobial classes) was observed in 100% of S. Heidelberg (n=7/7) and 29% of S. Saintpaul (n=2/7) isolates. Resistance to ciprofloxacin, nalidixic acid, sulfamethoxazole/ trimethoprim, and chloramphenicolwas not observed in any isolate. The PFGE profiles indicated that Salmonella isolates were grouped mostly based on their serotypes.The results indicated that the existence of antibiotic resistant and MDR Salmonella in retail poultry products can be a significant food safety concern.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Divek V.T. Nair, R. Nannapaneni, A. Kiess, and C.S. Sharma. 2015. The combined efficacy of carvacrol and modified atmosphere packaging on the survival of Salmonella, Campylobacter jejuni and lactic acid bacteria on turkey breast cutlets. Food Microbiology. 49: 134141
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Sukumaran, A. T., A. Kiess, R. Nannapaneni and C.S. Sharma. 2015. Reduction of Salmonella on chicken meat and chicken skin by combined or sequential application of lytic bacteriophage with chemical antimicrobials. International Journal of Food Microbiology 207:8-15
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Sharma, C.S. J. Dhakal, and R. Nannapaneni. 2015. Efficacy of Salmonella lytic bacteriophage preparation in reducing Salmonella in vitro, on turkey breast cutlets and ground turkey. Journal of Food Protection 78:13571362.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Sukumaran, A. T., A. Kiess, R. Nannapaneni and C.S. Sharma. 2015. Reduction of Salmonella on Chicken Breast Fillets Stored Under Aerobic or Modified Atmosphere Packaging by the Application of Lytic Bacteriophage Preparation. Poultry Science.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: C.S. Sharma, K. Bhargava, and A. Ates. 2015. Antimicrobial activity of oregano oil nanoemulsion against Salmonella enterica and Listeria monocytogenes. 2015 Annual Meeting of Poultry Science Association at Louisville, KY on July 27-30, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Moore A., J. Dhakal, A. Sukumaran, A. Ates, and C.S. Sharma. 2015. Efficacy of commercial antimicrobials in reduction of Salmonella in ground chicken frames. 2015 Annual Meeting of Poultry Science Association at Louisville, KY on July 27-30, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Sharma C.S., R. Nayak, A. Ates, J. Deck, S. Foley, A. Kiess, R. Nannapaneni. 2015. Molecular Characterization of Multidrug Resistant Salmonella enterica Strains Isolated from Retail Poultry Products. Presented at 4th ASM Conference on Antimicrobial Resistance in Zoonotic Bacteria and Foodborne Pathogens, Washington, DC, May 8-11, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Dhakal J. and C.S. Sharma. 2015. Effect of sub-lethal oxidative stress of chlorine on biofilm forming ability of various Salmonella strains. Presented at 2015 International Poultry Scientific Forum held at Atlanta, January 26-27, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Sukumaran, A.T. and C.S. Sharma. 2015. Reduction of Salmonella on chicken skin by the sequential application of chemical treatments and Salmonella lytic bacteriophage preparation. Presented at 2015 International Poultry Scientific Forum held at Atlanta, January 26-27, 2015

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

Outputs
Target Audience: Target audiences include poultry processors in the broiler chicken and turkey industry and food safety professionals in industry and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training opportunities were provided to graduate as well as undergraduate students through their participation in laboratory procedures for conducting research in food microbiology and safety of poultry products, data analysis, and the dissemination of information by means of oral presentations and assistance in the writing and publication of abstracts and manuscripts. How have the results been disseminated to communities of interest? Results have been disseminated to communities of interest such as poultry and food processors, food safety professionals through their attendance at the annual meetings in which the results were presented in the form of oral and/or poster presentations. The research findings have been published in peer-reviewed journal articles and the concise information from these projects was also published in meeting abstracts What do you plan to do during the next reporting period to accomplish the goals? The plan for the next reporting period will be to complete the projects currently under progress and to initiate new projects to control Salmonella, Campylobacter and Listeria monocytogenes in poultry products by novel intervention technologies. The findings from these projects will be disseminated to public and scientific communities in meetings and through publications in peer-reviewed journals.

Impacts
What was accomplished under these goals? In this study, the combined efficacy of carvacrol and modified atmosphere packaging (MAP) was evaluated in reducing Salmonella, and Campylobacter on turkey breast cutlets stored at 4?C. Turkey breast cutlets were inoculated with a three strain cocktail of Salmonella (S. Enteritidis, S. Heidelberg and S. Typhimurium) and two strain cocktail of Campylobacter jejuni followed by dipping in 0.25, 0.5 and 1% carvacrol for 30s and stored under MAP (~ 95% CO2 and 5% O2) at 4?C. Positive (inoculated, not treated with carvacrol and stored under aerobic and modified atmosphere) controls were also included in the study. Samples were analyzed on d1, 3, and 7 of storage for the survivability of Salmonella and C. jejuni. The combined application of carvacrol and MAP caused significant (P < 0.05) reductions (1.0 to 2.0 log CFU/g) of Salmonella and Campylobacter on turkey breast cutlets on each day of analysis. There was no difference in the meat color among treatments and controls (P > 0.05). In conclusion, carvacrol was effective in reducing Salmonella, C. jejuni on turkey breast cutlets with concentrations as low as 0.25% when used in combination with high CO2 packaging. In another study the efficacy of Salmonella lytic bacteriophages cocktail on the inhibition of growth of various Salmonella serotypes (Enteritidis, Hadar, Heidelberg, Kentucky, and Typhimurium) was determined using a 96-well plate assay by measuring the optical density at 630 nm. Diluted overnight cultures of Salmonella (104CFU/ml) were treated with bacteriophage at a multiplicity of infection (MOI) of 1, 100, 1000, 10000 PFU/CFU and incubated at 37°C for 48h. The efficacy of phage on reducing Salmonella at 4°C in TSB was determined by treating Salmonella cultures (106 CFU/ml) with phage (MOI of 100, 1000, 10000) for 24h. Chicken breast fillets inoculated with a three-strain cocktail of Salmonella (Enteritidis, Heidelberg and Typhimurium ~103 CFU/g) were treated with phage concentrations of 107 PFU/g and stored up to 10d at 4°C. Bacteriophage preparation at 10000 MOI (completely inhibited growth of Salmonella at 37°C for 48 hours. At 4°C, bacteriophage preparation caused ca. 4.5 log reduction of Salmonella up to 24h. On raw chicken breast fillets, phage application caused 1.2, 1.3, 1.8, and 1.7 log CFU/g reductions of Salmonella as compared to control on d 0, 1, 7, and 10 of storage at 4°C, respectively. All reductions were statistically significant (P≤ 0.05). These findings indicate that the bacteriophage preparation is effective in reducing Salmonella on chicken breast fillets. This phage cocktail could be an effective tool for reducing Salmonella on chicken parts during processing. In continuation of the previous study on the efficacy of phage against Salmonella, another study was conducted to determine the survivability of Salmonella bacteriophage preparation in antimicrobials commonly used during poultry processing and to determine the efficacy of bacteriophage in combination with compatible antimicrobials in reducing Salmonella in vitro and on chicken breast fillets. Bacteriophage preparation (109 PFU/ml) was mixed with cetylpyridnium chloride (CPC 0.4, 0.6 and 1%), lauric arginate ester (LAE 200 and 5000ppm) and peracetic acid (PAA 30, 40, 50, 100, 200 and 700ppm) and stored at 4?C for 24h. The number of surviving bacteriophages was determined by soft agar overlay technique. There was no effect (P > 0.05) of all tested concentrations of CPC and LAE on the survival of bacteriophage as compared to the control (bacteriophage + DI water) whereas bacteriophage was not able to survive in PAA solutions (100, 200 and 700ppm). However, phage counts up to 6.5 log PFU/ml were recovered in low strength PAA solutions (30, 40 and 50ppm) as compared to control (9.3 log PFU/ml). The application of phage (102 and 103 multiplicity of infection (MOI)) in combination with 0.6% CPC and 200ppm LAE was examined for reduction of Salmonella (S.Typhimurium, S.Heidelberg and S.Enteritidis) in 0.1% peptone water at 40C for 24 hand on chicken breast fillets inoculated with cocktail of the three serotypes stored up to 7 d. Irrespective of the serotypes, antimicrobial, storage time and MOI of phage, all treatments resulted in complete inactivation of Salmonella in vitro compared to the control (6 log CFU/ml). On chicken breast fillets, all the combinations of phage and antimicrobials resulted in significant (P<0.05) reductions of Salmonella throughout d7 of storage with reductions ranging from 0.85 log CFU/g to 2.15 log CFU/g as compared to positive controls (3.2-3.6 log CFU/g). Among all the treatments, the combination of phage (103MOI) and CPC showed the highest reductions of 1.1, 2.15 and 1.4 log CFU/g on d0, 1 and 7, respectively. In conclusion, phage preparation was able to survive in CPC and LAE and their combination was effective in reducing Salmonella on chicken breast fillets. This study was conducted to determine the prevalence ofSalmonellaand antibiotic resistant Salmonellaphenotypes in retail poultry products. Ground turkey (n=120) and skinless, boneless chicken breast fillet (n=117) samples were randomly collected from 3 different local grocery stores. All samples were analyzed for the presence ofSalmonellausing selective enrichment procedure of the USDA FSIS.Salmonellaisolates were confirmed by serology and biochemical characterization. All the isolates were serotyped. Antibiotic resistant phenotypes ofSalmonellaisolates were determined as per the Clinical and Laboratory Standards Institute method.Salmonellawas detected in 16.7% (20/120) and 11.1% (13/117) of ground turkey and chicken breast fillet samples, respectively. The predominant serotypes isolated from ground turkey wereS.Heidelberg andS.Saintpaul (7 isolates each), whereasS.Typhimurium var 5- (10 isolates) was the primary serotype isolated from chicken breast samples. All except one isolates ofS.Typimurium var 5- were resistance to sulfisoxazole and tetracycline. Among turkey isolates, four isolates were pan-susceptible, five isolates displayed resistance to one single antibiotic, one isolate to two drugs, three isolates to three, 4 isolates to four, 2 isolates to seven agents and one isolate to 10 antimicrobials. Multidrug resistance (MDR) was primarily observed inS.Heidelberg isolates. All the sevenS.Heidelberg isolates were resistant to at least three antibiotics. All the strains were confirmed by PCR assay by targeting invA gene. The isolated were also tested for the presence of blaCMY gene for extended spectrum beta lactamases were The prevalence of MDR Salmonellain retail poultry products can be a food safety concern and more effective measures are needed to eliminate the prevalence of MDRSalmonellastrains in retail poultry products.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Divek V.T. Nair, R. Nannapaneni, A. Kiess and B. Mahmoud and C.S. Sharma. 2014. Antimicrobial efficacy of lauric arginate against Campylobacter jejuni and spoilage organisms on chicken breast fillets. Poultry Science 93: 2636-2640
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Divek V.T. Nair, R. Nannapaneni, A. Kiess, W. Schilling and C.S. Sharma. 2014. Reduction of Salmonella on turkey breast cutlets by plant derived compounds. Foodborne Pathogens and Disease (In Press)
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Divek V.T. Nair, R. Nannapaneni, A. Kiess, and C.S. Sharma. 2014. The combined efficacy of carvacrol and modified atmosphere packaging on the survival of Salmonella, Campylobacter jejuni and lactic acid bacteria on turkey breast cutlets. Food Microbiology (Under Review)
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sharma C.S. and A. Ates. 2014. Prevalence, serotypes and antibiotic resistant phenotypes of Salmonella enterica isolates in retail poultry products. Presented at 2014 Annual Meeting of Poultry Science Association (PSA), July 14-17, 2014 at Corpus Christi, TX
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Nair V.T. Divek and C.S. Sharma. 2014. Antimicrobial efficacy of carvacrol against Salmonella, Campylobacter and lactic acid bacteria on turkey breast cutlets under modified atmosphere packaging. Presented at 2014 Annual Meeting of Poultry Science Association (PSA), July 14-17, 2014 at Corpus Christi, TX
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sukumaran, A.T. and C.S. Sharma. 2014. Evaluation of compatibility of Salmonella lytic bacteriophage with different antimicrobials and their combinations on reduction of Salmonella in vitro and on chicken breast fillets. Presented at 2014 Annual Meeting of Poultry Science Association (PSA), July 14-17, 2014 at Corpus Christi, TX
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Janak Dhakal, Anuraj T. Sukumaran, Divek Nair, R. Nannapaneni and C.S. Sharma. 2014. Reduction of Salmonella in vitro and on the surface of chicken breast fillets by bacteriophage preparation. Presented at 2014 Annual Meeting of International Association of Food Protection (IAFP), August 3-6, 2014 at Indianapolis, IN
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Nair V.T. Divek and C.S. Sharma. 2014. Antimicrobial activity of carvacrol against Salmonella on turkey breast cutlets packaged under modified atmosphere. Presented at 2014 International Poultry Scientific Forum held at Atlanta, January 27-28, 2014

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Target audiences include poultry processors in the broiler chicken and turkey industry and food safety professionals in industry and academia. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training opportunities were provided to graduate as well as undergraduate students through their participation in laboratory procedures for conducting research in food microbiology and safety of poultry products, data analysis, and the dissemination of information by means of oral presentations and assistance in the writing and publication of abstracts and manuscripts. How have the results been disseminated to communities of interest? Results have been disseminated to communities of interest such as poultry and food processors, food safety professionals through their attendance at the annual meetings in which the results were presented in the form of oral and/or poster presentations. The concise information from these projects was also published in meeting abstracts. What do you plan to do during the next reporting period to accomplish the goals? The plan for the next reporting period will be to complete the projects currently under progress and to initiate new projects to control Salmonella, Campylobacter and Listeria monocytogenes in poultry products by novel intervention technologies. The findings from these projects will be disseminated to public and scientific communities in meetings and through publications in peer-reviewed journals.

Impacts
What was accomplished under these goals? In the current study the antimicrobial efficacy of LAE against Campylobacter jejuni was determined in vitro and on chicken breast fillets. In addition, the effect of LAE treatments on growth of spoilage microorganisms on chicken breast fillets was also determined. Chicken breast fillets were inoculated with C. jejuni (ATCC 33291 & 33560) and treated with 0, 200 and 400 ppm of LAE, packaged and stored at 4?C for 7 days. non-inoculated skinless chicken breast fillets were treated with 0, 200 and 400 ppm of LAE, packaged and stored at 4?C for analysis after 0, 3, 9, and 14 days for growth of mesophilic and psychrotrophic organisms. In another study, the antimicrobial efficacy of plant-derived compounds: carvacrol, eugenol, trans-cinnamaldehyde and thyme essential oil was evaluated against Salmonella in vitro and on fresh turkey breast cutlets. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of natural antimicrobials were tested against different Salmonella serotypes microdilution method. Turkey breast cutlets were inoculated with a three-strain cocktail of Salmonella (S. Heidelberg, S. Typhimurium and S. Enteritidis) and treated with 0.5, 1, 2 and 5% (v/v) of these natural antimicrobials as a 2 min. dip treatment at 4°C. Carvacrol was further tested as 30 s and 60s dip treatment at 4°C to reduce Salmonella on turkey breast cutlets. Lauric arginate caused significant reduction (P < 0.05) of Campylobacter on chicken breast fillets at 200 and 400 ppm levels. But except on day 0, there was no significant difference between 200 and 400ppm treatments. Treating chicken breast fillets with 400 ppm of LAE caused 2.3-log reduction of psychrotrophs (P < 0.05) as compared to control on day 0 of storage. However, no difference existed (P > 0.05) in the growth of mesophiles and psychrotrophs on chicken breast fillets after treatment with 200 and 400 ppm LAE as compared to control after 3 days. The results of study indicated that LAE is effective in inactivating C. jejuni but has no effect against spoilage micro flora at the currently approved level of 200 ppm in poultry products. Higher concentrations of LAE may be needed to increase the shelf life of poultry products. In study on the efficacy of plant derived antimicrobial compounds against salmonella, The MIC and MBC values of the antimicrobial against Salmonella were in the following order: carvacrol < red thyme oil < white thyme oil < trans-cinnamaldehyde < eugenol. Results of dip treatments for 2min showed that all the tested antimicrobials at 1, 2 and 5 % concentrations resulted in significant (P < 0.05) reductions of Salmonella on turkey breast cutlets. Carvacrol showed maximum activity with 1, 1.7 and 2.6 log cfu/g reductions of Salmonella on turkey breast cutlets at 1, 2 and 5 %( v/v) concentrations respectively. Trans – cinnamaldehyde and red thyme oil at concentrations of 1, 2 and 5% resulted in 0.9, 0.9 and 2.3 log cfu/ and 0.4, 0.5 and 1 log cfu/g reductions of Salmonella, respectively. Eugenol treatments led to 0.7, 1.1 and 1 log cfu/g reductions of Salmonella with 1, 2 and 5% concentrations. Dipping treatments of turkey breast cutlets with 5% carvacrol for 30s and 60s resulted in 1.0 and 1.8 log cfu/g reductions (P < 0.05) of Salmonella, respectively. None of the natural antimicrobial agents caused any alterations in the pH of meat (P> 0.05). The sensory evaluation showed that the panelist were able to detect the difference (P ≤ 0.05) in aroma of turkey breast cutlets dipped in carvacrol as compared to cutlets dipped in sterile distilled water for both cooked and raw samples. This study revealed that plant derived compounds could control Salmonella on turkey breast when applied dip treatments. Further studies are being carried out on reducing the effective concentration of these plants derived compounds in meat system to get more consumer acceptance.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: D. V. T. Nair and C. S. Sharma. 2013. Reduction of Salmonella on turkey breast cutlets by plant derived molecules - carvacrol, eugenol and trans-cinnamalde�hyde. Poultry Science Vol. 92 (E-suppl. 1) 24.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Divek Nair, Chander Shekhar Sharma, Ramakrishna Nannapaneni, Barakat Mahmoud. 2013. Antimicrobial effects of lauric arginate against Campylobacter jejuni and spoilage organisms on chicken breast fillets. Journal of Food Protection. Supplement A, Vol. 76: 176

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Currently, the poultry industry in the United States is faced with a problem of high prevalence of Salmonella in ground and comminuted poultry products such as ground chicken, ground turkey, and mechanically separated poultry. Recently ground turkey has been involved in a large multistate foodborne outbreak caused by Salmonella. Thus, it is critical to apply additional novel interventions to reduce the prevalence of Salmonella in ground poultry products. In the current study, the antimicrobial efficacy of lauric arginate (LAE), a USDA approved novel antimicrobial was tested against Salmonella survivability in ground chicken under refrigerated storage. The effect of LAE treatments on total aerobes, pH and objective color were also assessed in ground chicken. Ground chicken samples were inoculated with a Salmonella cocktail consisting of four different serotypes (S. Enteritidis , S. Heidelberg, S. Kentucky and S. Typhimurium) and then treated with 200 and 400 ppm of LAE. The samples were stored at 4 C and analyzed on d 0, 1, 3, 5, and 7 for Salmonella, total aerobes, color and pH. PARTICIPANTS: Chander S. Sharma, Amanda Ates, P. Joseph, K. Soni, M.W. Schilling, and Aaron Kiess TARGET AUDIENCES: Poultry processors, USDA FSIS, USDA NIFA PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
During this study, Salmonella counts in untreated control ranged from 4.3 to 5.0 log cfu/g d 0 through 7 d of storage. No antimicrobial effects were observed for any LAE treatments on Salmonella growth in ground chicken, as evidenced by similar (P > 0.05) Salmonella counts among the positive control and treatment with 200 and 400ppm of LAE from d 0 through d 7. The findings from this study suggested that LAE is ineffective in reducing Salmonella in ground chicken when used at the currently approved levels of 200 ppm in ground poultry and even using the LAE up to 400 ppm didn't exhibit any effect on growth of Salmonella in ground chicken. In an earlier study we found that LAE was effective in reducing Salmonella on chicken breast fillets unlike the lack of antimicrobial effect observed in this study. The high fat concentration of ground chicken might be responsible for lack of antimicrobial efficacy of LAE against Salmonella and higher concentrations of LAE may be needed to achieve the reduction of Salmonella in ground chicken. Total aerobic counts increased with simultaneous increase in storage time for all treatments and control and there was no treatment effect on growth of total aerobes in ground chicken. Lauric arginate treatments had no significant effect on the pH and color of ground chicken.

Publications

• Sharma C.S., Ates, A., Joseph, P., Soni, K.A., Schilling, M.W., and Kiess, A. (2013). Evaluation of antimicrobial effects of lauric arginate on reduction of Salmonella in ground chicken. Int. J. Food Sci. Technol. (In Press)