IMPACT OF ENVIRONMENTAL STRESSORS ON SUSCEPTIBILITY OF SALMONELLA IN PROCESSING PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0229709
• Project No.: ALA018-1-12008
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2017

Project Director
Bilgili, SA.

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Poultry Science

Non Technical Summary
When enteric pathogens are exposed to environmental stresses such as acidic or thermal conditions, some of the exposed cells develop either a tolerance or resistance to these stressors. Various studies have speculated that these stress adapted cells exhibit enhanced resistance to other environmental stressors including those from thermal processing and acid exposure. The magnitude of this increased resistance to other environmental stressors is such that some of the adapted cells could potentially survive the current minimum guidelines for cooking of meat and poultry or the minimum doses of antimicrobials used in processing for control of foodborne pathogens. Current industry practices present a wide scope for foodborne pathogens to be exposed to sub-lethal thermal treatments or antimicrobials which are widely used as microbial interventions during day-to-day meat and poultry processing operations. These treatments are a critical part of a processors food safety Hazard Analysis and Critical Control Point (HACCP) system to ensure safety of meat and poultry products. Preliminary laboratory data suggests that increased resistance as a result of sub-lethal heat exposure or acid exposure could potentially create an unexpected foodborne hazard leading to implications to public health. The limitations of the existing research are that it has been conducted in either laboratory media or in foods that are held in conditions which do not reflect the current practices of storage and distribution of processed meat and poultry. To confirm the laboratory observations and improve our knowledge of stressed bacteria studies need to be conducted in an actual food system for more accurate inferences and reducing the knowledge gaps.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
307	3220	1100	15%
307	3260	1100	15%
307	4010	1100	15%
712	3220	1100	15%
712	3260	1100	20%
712	4010	1100	20%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 307 - Animal Management Systems;

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

Field Of Science
1100 - Bacteriology;

Keywords

salmonella enterica

thermal tolerance

acid adaptation

Goals / Objectives
The overall goal of this study is to determine the pathogenicity and resistance of stressed Salmonella spp. that are previously exposed to other environmental stressors during poultry processing. The proposed study will evaluate prior exposure of Salmonella to environmental stressors such as heat and pH usually found during primary poultry processing as a potential for increased resistance and enhanced pathogenicity. The central hypothesis of for this proposed study is that sub-lethal exposure of Salmonella to environmental stress will result in increased cross-protection of this pathogen against other stressors. To accomplish the overall goal of this project the specific objectives of this study are: 1.Create acid tolerant Salmonella enterica and determine their thermal resistance.2.Create thermal resistant Salmonella enterica and determine their acid tolerance.3.Evaluate thermal death time of acid adapted Salmonella enterica in ground chicken. 4.Inoculate chicken carcasses with heat tolerant Salmonella enterica and evaluate organic acid sprays against control of these bacteria. The outcomes of this proposed study will help processors understand effectiveness of current intervention technologies including heat and acid treatments against atypical/ resistant bacteria that are widely present in the environment and on meat and poultry products. In addition, we believe that generated data will be useful in developing effective intervention methods to control the prevalence and growth of inherently diverse Salmonella that are widely present on meat and poultry products. This in turn will enable accurate and unbiased predictions of pathogen growth to safeguard public health in addition to filling the knowledge gaps in understanding the responses of resistant/ tolerant pathogens present in the environment.

Project Methods
Salmonella spp. will be grown in laboratory media (Brain Heart Infusion; BHI) supplemented with 1 percent glucose (BHI plus 1 percent Glucose) to a stationary phase and held for 12-18 hours. The inclusion of glucose allows for the production of organic acids, which lowers the final pH of the culture medium significantly without acid-shocking the bacterial cultures. Thermal tolerance of acid adapted cells will be determined by decimal reduction values (D-values) and compared to those of non-adapted cells. D-values will be determined in BHI and BHI plus 1 percent glucose broths.Heat tolerant cultures of Salmonella spp. will be developed by growing multiple strains in BHI medium maintained at sub-lethal temperatures for 18-24 hours in the stationary phase. The purpose of growing cultures in sub-lethal temperatures is to select for heat tolerant strains that can be tested for resistance to other stressors such as organic acids. Heat tolerant cells will then be enumerated by growing on laboratory media supplemented with glucose and compared to those grown in regular media. A comparison of this will illustrate level of cross protection obtained as a result of heat shock/ adaptation. Poultry carcasses will be inoculated with stress adapted (acid and heat) bacteria while non-adapted strains will be separately inoculated to serve as controls.Inoculated chicken carcasses will then be treated by spraying organic acids in varying concentrations and held at 4 C for up to 24 hours to allow maximum attachment of bacteria. As an additional set of controls, adapted and non-adapted bacteria will be inoculated onto the chicken carcasses, held at 4 C for up to 24 hours without treatment with organic acid rinses. The thermal tolerance of Salmonella spp. will be determined in laboratory media and in the inoculated ground meat (chicken) by comparing decimal reduction values (D-values) at different temperatures. Briefly, 5 g of inoculated ground chicken will be placed in a vacuum bag, pressed to obtain as thin a thickness as possible, and sealed. These bags will be immersed in water baths set at appropriate temperatures (52, 58, and 62 C) and the timing will start as soon as the set point is reached. Control bags with thermocouples will serve as temperature monitors. Similarly D-values of the bacteria will be measured in the inoculated laboratory media and the D-values will be compared to that of the bacteria inoculated into ground chicken. Because of the high probability of injured bacteria as a result of exposure to stressors such as heat and acid, the bacterial populations will be enumerated on selective and non-selective media (Buchanan and Edelson 1999 noted as much as a 5 log10 difference between injured and un-injured populations). Initial experiments will be conducted to determine optimal incubation conditions (for example 25 C for 72 h compared to 37 C for 48 h) for maximum recovery of the injured bacteria.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research was performed by a Graduate Student with the result of a PhD. How have the results been disseminated to communities of interest?International and National Meetings and Peer reviewed journal articles What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We determined the thermal death time of acid adapted Salmonella to see if there were differences as compared to non thermally adapted cells.

Publications

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

Outputs
Target Audience:Academia Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Yes, at International Professional Meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Presentation ofresults at International Meetings.

Publications

• Type: Theses/Dissertations Status: Accepted Year Published: 2014 Citation: Assessment of the Effects of Acid-Adaptation on the Thermal Tolerance of Listeria monocytogenes and Salmonella Heidelberg. Smith,Jacob.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2014 Citation: J.M. Smith and M. Singh. Effect of acid adaptation on heat treatment of Salmonella Heidelberg in ground turkey. Poultry Science Meeting
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: J.M. Smith, S.F. Bilgili, and M. Singh. Temperature effect on the growth of non-adapted and acid adapted Salmonella Heidelberg and Listeria monocytogenes. Poultry Science Meeting

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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Research was concluded in regards to objective 3 this past calendar year. Results of the in vivo experimentation indicated that there are no significant differences between acid-adapted and non acid-adpated Salmonella.

Publications

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

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research is being conducted to insure that current cooking guidelines for poultry products are not impacted by current industry practices,specifically the use of acid sprays to deter the growth of pathogenic and spoilage bacteria. This research is important and can potentilally impact all households that prepare and consume poultry products. We have performed the in vitro portion of this experiment specific to research objectives 1 and 2. Currently, we are performing the second trial for research objective 3, with plans to move forward and complete all objectives by the end of this calander year. Thus far, data has shown that the acid adapted cells are for the most part exhibit the same heat tolerance as non-adapted cells.

Publications

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

Outputs
OUTPUTS: Current food safety trends focus on achieving best practices in pathogen control. Many interventions strategies have been investigated to eliminate Campylobacter both ante-mortem and post-mortem. The bactericidal activity of intervention treatments relies heavily on the disruption of an organism's physiological processes and treatments in experimental research are validated based on the log10 scale. These intervention processes can be categorized as: thermal, irradiation-based, and chemical based. Application of approved chemical treatments during processing is another method to reduce pathogens on poultry carcasses. In order for antimicrobials to be effective and relevant to the industry, they must be approved and have validated efficacy against microorganisms. In addition, the concentration and contact time needs to be appropriate for a particular processing step. Finally, they must be cost effective and without negative impacts on product quality. Chemical antimicrobials are evaluated for many different factors and their use on meat, poultry, and other food products should be approved by the appropriate regulatory agencies. While worker safety and production of harmful effluents should be monitored and minimized for environmental concerns, another important concern is that a chemical antimicrobial should not have any organoleptic effects on the products. In commercial poultry chilling process, chemical intervention strategies used have evolved over time. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have been ables to perform surveys and tabulate practices that are being followed in the poultry processing plants and better understand high risk areas within a plant and the interventions being used against foodborne pathogens. By understanding high risk areas and testing for pathogens throughout the plants, suggestions are being developed for implementation in plants in order to reduce the risk of contamination of poultry.

Publications

• No publications reported this period