ENHANCING MICROBIAL FOOD SAFETY BY RISK ANALYSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010983
• Multistate No.: S-1056
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Project Director
Nannapaneni, RA.

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

Performing Department
Food Science & Technology

Non Technical Summary
The knowledge of the role of sublethal stress factors on the induction of stable stress resistant phenotypes of L. monocytogenes and Salmonella and their persistence as biofilms in lethal concentration of disinfectants will aid in risk-assessment models for foodborne bacterial pathogens. Influence of strain and temperature on biofilm formation by L. monocytogenes and Salmonella spp. in high and low concentrations offoodresidue on differentfood processing contact surfaces will be known. In food processing environments, L. monocytogenes and Salmonella encounter multiple interventions where they have opportunities to be exposed to sub-lethal stresses that trigger various intracellular stress responses allowing it to be more persistent in the subsequent lethal inactivation. Ourwork is involved in examining the stress adaptive response of high risk L. monocytogenes and Salmonella serotypes commonly occurring in food processingenvironments and determining its correlation to increased persistence/adherence/survival as biofilms and subsequent cross contamination to finished-raw or RTE products. Our recent findings show that high-risk L. monocytogenes serotypes (1/2a) when pre-exposed to mild acid, alkali, heat or oxidative stress can easily survive by 2-4 logs greater than the non-stressed cells. By working with predominant isolates of high-risk L. monocytogenes and Salmonella serotypes from different food processing environments, our objective is to find which of the acid-, alkali-, heat-, or oxidative-stress adapted L. monocytogenes and Salmonella can lead to greater persistence in food processing environments and then lead the development of affordable risk-based strategies for their control.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1020	15%
712	4010	1030	15%
712	4010	1040	15%
712	4010	1060	15%
712	1430	1100	10%
712	1420	1100	10%
712	3260	1100	10%
712	3710	1100	10%

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

Subject Of Investigation
1430 - Greens and leafy vegetables; 3260 - Poultry meat; 4010 - Bacteria; 1420 - Melons; 3710 - Catfish;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1030 - Cellular biology; 1100 - Bacteriology; 1020 - Physiology;

Keywords

listeria monocytogenes

salmonella

biofilms

stress-hardened cells

risk-assessment

Goals / Objectives
Risk Assessment: Assess food safety risks in agriculture systems

Project Methods
This study will determine the growth and survival of L. monocytogenes and Salmonella serotype s and their ability to form biofilm on various food contact surfaces used in food processing environment in high and low concentration of food residue. Representative pool of high-risk L. monocytogenes and Salmonella serotypes isolated from various food processing environments will be screened against mild acid-, alkali-, heat-, and oxidative-stress routinely encountered in processing environments to find stress-resistant phenotypes. Acid-stress:- Highly stable acid-stress resistant sub-populations of L. monocytogenes and Salmonella will be isolated by pre-exposing cells to sublethal acidic pH of 5.0 which can survive post exposure lethal pH concentration of 3.5. Alkali-stress:- Highly stable alkaline-stress resistant sub-populations of L. monocytogenes and Salmonella will be isolated by pre-exposing cells to sublethal pH concentrations of 9.0 which can survive post exposure to lethal pH concentration of 11.5. Oxidative-stress:- Highly stable oxidative-stress resistant sub-populations of L. monocytogenes and Salmonella will be isolated by pre-exposing cells to sublethal H2O2 concentrations of 50 ppm which can survive post exposure to lethal H2O2 concentration of 1000 ppm. The non-stressed cells L. monocytogenes and Salmonella will be prepared using the same generation cycles except that the growth will be at a normal pH of 7.0 in the absence of target stress. All above stress-resistant phenotypes will be screened under high and low food residue conditions commonly encountered during processing environments. The biofilm capacity of L. monocytogenes and Salmonella cells adapted to acid-, alkali- and oxidative stress will be determined: (a) in liquid media containing food residue, and (b) on simulated processing equipment surfaces for differences in persistence of adapted vs non-adapted cells from a pool of diverse L. monocytogenes and Salmonella isolates. Selected high biofilm/persistent and highly stable stress-resistant phenotypes of high-risk Lm serotypes will be tested for their survival on processing equipment mimicking surfaces when exposed to target disinfectants at approved concentrations (used singly or in sequence). Disinfectants to be tested will include chlorine, hydrogen peroxide, quaternary ammonium along with acid, alkali and heating based cleansing treatments typically used in various food processing conditions.

Progress 10/01/16 to 09/30/18

Outputs
Target Audience:Results of this work has been disseminated to various Food Industry clients and for the USDA, FDA and University Scientists at the International Association for Food Protection Annual Meetings, and Institute of Food Technologists Annual Meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students received extensive training in food safety and food microbiology research techniques by undertaking their thesis project focusing in this focused area of biofilms of foodborne bacterial pathogens. How have the results been disseminated to communities of interest?Results of this work has been disseminated to various Food Industry clients and Scientists at the International Association for Food Protection Annual Meetings and Institute of Food Technologists Annual Meetings. Results of this work have been published as four journal articles in an international food microbiology journals for reaching wider audience. What do you plan to do during the next reporting period to accomplish the goals?Understanding the role of cold stress on the survival of L. monocytogenes cells when subsequently exposed to acid, alkali and oxidative stress is important with respect to eliminating this foodborne pathogen. This work will be included in the next report.

Impacts
What was accomplished under these goals? Salmonella enterica ssp. enterica serovar Hadar outbreak with 10 cases that was reported from a New Jersey restaurant was linked to catfish. Due to this outbreak, Centers for Disease Control and Prevention (CDC) has listed catfish as a vehicle for the spread of Salmonella. Salmonella was isolated from freshwater catfish, catfish ponds and different organs of catfish such as skin, gills, faces, gut and intestine. During catfish processing, residues such as water runoff, muscle extract and mucus present on the skin surface may promote Salmonella for its growth and subsequent biofilm formation. In this study, we determined the effect of strain and temperature on the growth and biofilm formation of Salmonella spp. in high and low concentrations of catfish mucus extract on different food-contact surfaces at 22°C and 10°C. The second objective of this study was to evaluate the efficacy of disinfectants at recommended concentrations and contact times for removing Salmonella biofilms cells on a stainless steel surface containing catfish mucus extract. Growth and biofilm formation of all Salmonella strains increased with higher concentrations of catfish mucus extract at both 10°C and 22°C. In 15 µg/ml of catfish mucus extract inoculated with 3 log CFU/ml, the biofilm levels of Salmonella on stainless steel surface reached to 3.5 log CFU/cm2 at 10°C or 5.5 log CFU/cm2 at 22°C in 7 days. In 375 µg/ml of catfish mucus extract inoculated with 3 log CFU/ml, the biofilm levels of Salmonella on the stainless steel surface reached 4.5 log CFU/cm2 at 10°C and 6.5 log CFU/cm2 at 22°C in 7 days. No differences were observed between Salmonella strains tested for biofilm formation in catfish mucus extract on the stainless steel surface. The biofilm formation by Salmonella Blockley (7175) in catfish mucus extract was less (P < 0.05) on buna-N rubber when compared to stainless steel, polyethylene and polyurethane surfaces. Salmonella biofilm cells were not detectable on the stainless steel surface after treatment with a mixture of disinfectants but were still present when single compound disinfectants were used. For example, disinfectants containing a mix of QAC with H2O2 or PAA with H2O2 and octanoic acid were effective at reducing of biofilm cells on the stainless steel surface such that they were non-detectable. In another part of biofilm study, we determined the growth and survival of Salmonella enterica in the presence of high and low concentrations (375 µg/ml and 15 µg/ml) of catfish mucus extract at 10 °C and 22 °C for 63 days. The second objective of this study was to investigate the biofilm formation of Salmonella enterica serovar Blockley (7175) in catfish mucus extract for 48 h at 22 °C on four food-contact surfaces and to observe the biofilm populations using Scanning Electron Microscopy (SEM). The surface properties, surface roughness and surface energies were determined using contact angle measurement and atomic force microscopy. In 375 µg/ml of catfish mucus extract that was inoculated with 3 log CFU/ml, the growth of Salmonella counts were increased to a maximum of 6-7 log CFU/ml at 10 °C and 7-8 log CFU/ml at 22 °C in 7-14 d and decreased by 1-2 log CFU/ml from these peak levels at both 10 °C and 22 °C from 21-63 d. In 15 µg/ml of catfish mucus extract, Salmonella counts were in the range of 4-5 log CFU/ml at 10 °C and 5-6 log CFU/ml at 22 °C over 7-63 d of storage. By contrast, Salmonella counts were non-detectable in the absence of catfish mucus by 21-28 d of storage at 10 °C or 22 °C. The biofilm counts of S. Blockley (7175) on a stainless steel surface were 4 log CFU/cm2 and 5.5 log CFU/cm2 in 15 µg/ml and 375 µg/ml of catfish mucus extract respectively after 48 h incubation at 22 °C. SEM revelead that biofilm formation by S. Blockley (7175) was less in 15 µg/ml than 375 µg/ml of catfish mucus extract on stainless steel. In addition, SEM indicated that the visible biofilms were least on buna-N rubber as compared to stainless steel, polyethylene and polyurethane surfaces. Contact angle and atomic force microscopy confirmed that buna-N rubber was highly hydrophobic with low surface energy and low roughness when compared to other three surfaces. These findings indicate that Salmonella can utilize catfish mucus as a nutrient source to survive for longer periods and promote biofilm formation for its persistence on different food-contact surfaces. In summary, the results from the above study are highly informative for catfish industry in controlling Salmonella contamination in farm and the processing environment. These findings will aid in developing proper cleaning and sanitation procedures in the catfish processing environment to reduce the contamination and persistence of Salmonella biofilms. The nationwide listeriosis outbreak that occurred in the United States during 2011 highlighted the importance of preventing cantaloupe contamination with Listeria monocytogenes (Lm) within farm and processing environments. The objectives of this study were to determine the effects of strain and temperature on growth and biofilm formation of Lm in cantaloupe flesh and peel extracts on different food-contact surfaces. Growth of Lm strains was markedly greater at high concentration of cantaloupe extracts and temperature in comparison to low concentration and temperature. For 50 mg/ml of cantaloupe extract inoculated with 3 log CFU/ml, the growth of Lm was 8.5 log CFU/ml in 32 h at 22°C and 6-7 log CFU/ml in 72 h at 10°C. For 2 mg/ml of cantaloupe extract that was inoculated with Lm, the growth was 7-7.5 log CFU/ml in 72 h at 22°C and 3.5 log CFU/ml in 72 h at 10°C. There were no differences (P ? 0.05) among Lm strains for biofilm formation in cantaloupe extracts, but biofilm formation was greater at high temperature and high concentration. For 50 mg/ml cantaloupe extracts inoculated with 3 log CFU/ml, the biofilm formation of Lm on stainless steel surface was approximately 7 log CFU/coupon at 22°C in 4-7 days and 5-6 log CFU/coupon at 10°C in 7 days. For 2 mg/ml cantaloupe extracts, the biofilm formation of Lm on the stainless-steel surface was approximately 5-6 log CFU/coupon at 22°C and 4-4.5 log CFU/coupon at 10°C in 7 days. The biofilm formation by cantaloupe outbreak strain Lm 2011L-2625 in cantaloupe extracts was least on buna-n rubber when compared to stainless steel, polyethylene and polyurethane surfaces (P < 0.05). These findings show that a very low concentration of nutrients from cantaloupe flesh or peel can induce Lm growth and subsequent biofilm formation on different food-contact processing surfaces.In summary, these findings illustrate the critical importance of appropriate cleaning and sanitizing of cantaloupe processing surfaces because even the presence of small sediments of cantaloupe residue on the food-contact surfaces can promote growth and subsequent biofilm formation in the processing environments, which creates serious food safety risks.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Dhowlaghar, N., Abeysundara, P. D., Nannapaneni, R., Schilling, M. W., Chang, K.-C., Cheng, W.-H., Sharma, C. S. (2018). Growth and biofilm formation by Salmonella spp. in catfish mucus extract on four food-contact surfaces at 22�C and 10�C and their reduction. Food Microbiology, 70, 172-180.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Dhowlaghar, N., Bansal, M., Schilling, W., Nannapaneni, R. (2018). Scanning electron microscopy of Salmonella biofilms on various food-contact surfaces in catfish mucus. Food Microbiology, 74, 143-150.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Dhowlaghar, N. (Presenter), Bansal, M. (Author), Schilling, M. W. (Author), Nannapaneni, R., (2017). "Scanning electron microscopy of Salmonella biofilms on various food-contact surfaces in catfish mucus." Poster presented at the American Association of Microbiology-South Central Branch Meeting (ASM-SCB), October 19, 2017, American Association of Microbiology-South Central Branch, Little Rock, AR.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Abeysundara, P., Dhowlaghar, N., Nannapaneni, R., Sharma, C., Mahmoud, B., Schilling, M. W., (2016). "Growth and biofilm formation of Listeria monocytogenes at high and low concentrations of cantaloupe extract." Poster presented at the Institute of Food Technologists Annual Meeting, Chicago, IL.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Abeysundara, P. D., Dhowlaghar, N., Nannapaneni, R., Schilling, M. W., Chang, S., Mahmoud, B. S. M., Sharma, C. S., Ma, D. P. (2017). Growth and biofilm formation by Listeria monocytogenes in cantaloupe flesh and peel extracts on four food-contact surfaces at 22 �C and 10 �C. Food Control, 80, 131-142.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Results of this work has been disseminated to various Food Industry clients and Scientists at the International Association for Food Protection Annual Meetings, andInstitute of Food Technologists Annual Meetings heldduring 2016-17. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students received extensive training in food safety and food microbiology research techniques by undertakingtheir thesis project focusing in this area. How have the results been disseminated to communities of interest?Results of this work has been disseminated to various Food Industry clients and Scientists at the International Association for Food Protection Annual Meetings andInstitute of Food Technologists Annual Meetings. Results of this work have been published as twojournal articles in an international food microbiology journals forreachingwider audience. What do you plan to do during the next reporting period to accomplish the goals?Anotherjournal article will be published on this work after the completion of remaining experiments by scanning electron microscopy of the biofilms of Salmonella on different processing surfaces.

Impacts
What was accomplished under these goals? The objective of this study was to determine the effect of strain and temperature on the growth and biofilm formation of Salmonella spp. in high and low concentrations of catfish mucus extract on different food-contact surfaces at 22°C and 10°C. The second objective of this study was to evaluate the efficacy of disinfectants at recommended concentrations and contact times for removing Salmonella biofilms cells on a stainless steel surface containing catfish mucus extract. Growth and biofilm formation of all Salmonella strains increased with higher concentrations of catfish mucus extract at both 10°C and 22°C. In 15 µg/ml of catfish mucus extract inoculated with 3 log CFU/ml, the biofilm levels of Salmonella on stainless steel surface reached to 3.5 log CFU/cm2 at 10°C or 5.5 log CFU/cm2 at 22°C in 7 days. In 375 µg/ml of catfish mucus extract inoculated with 3 log CFU/ml, the biofilm levels of Salmonella on the stainless steel surface reached 4.5 log CFU/cm2 at 10°C and 6.5 log CFU/cm2 at 22°C in 7 days. No differences were observed between Salmonella strains tested for biofilm formation in catfish mucus extract on the stainless steel surface. The biofilm formation by Salmonella Blockley (7175) in catfish mucus extract was less (P < 0.05) on buna-N rubber when compared to stainless steel, polyethylene and polyurethane surfaces. Salmonella biofilm cells were not detectable on the stainless steel surface after treatment with a mixture of disinfectants but were still present when single compound disinfectants were used. The nationwide listeriosis outbreak that occurred in the United States during 2011 highlighted the importance of preventing cantaloupe contamination with Listeria monocytogenes (Lm) within farm and processing environments. The objectives of this study were to determine the effects of strain and temperature on growth and biofilm formation of Lm in cantaloupe flesh and peel extracts on different food-contact surfaces. Growth of Lm strains was markedly greater at high concentration of cantaloupe extracts and temperature in comparison to low concentration and temperature. For 50 mg/ml of cantaloupe extract inoculated with 3 log CFU/ml, the growth of Lm was 8.5 log CFU/ml in 32 h at 22°C and 6-7 log CFU/ml in 72 h at 10°C. For 2 mg/ml of cantaloupe extract that was inoculated with Lm, the growth was 7-7.5 log CFU/ml in 72 h at 22°C and 3.5 log CFU/ml in 72 h at 10°C. There were no differences (P ? 0.05) among Lm strains for biofilm formation in cantaloupe extracts, but biofilm formation was greater at high temperature and high concentration. For 50 mg/ml cantaloupe extracts inoculated with 3 log CFU/ml, the biofilm formation of Lm on stainless steel surface was approximately 7 log CFU/coupon at 22°C in 4-7 days and 5-6 log CFU/coupon at 10°C in 7 days. For 2 mg/ml cantaloupe extracts, the biofilm formation of Lm on the stainless-steel surface was approximately 5-6 log CFU/coupon at 22°C and 4-4.5 log CFU/coupon at 10°C in 7 days. The biofilm formation by cantaloupe outbreak strain Lm 2011L-2625 in cantaloupe extracts was least on buna-n rubber when compared to stainless steel, polyethylene and polyurethane surfaces (P < 0.05). These findings show that a very low concentration of nutrients from cantaloupe flesh or peel can induce Lm growth and subsequent biofilm formation on different food-contact processing surfaces.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Dhowlaghar, N., Abeysundara, P. D. A., Nannapaneni, R., Schilling, M. W., Chang, S. C., Cheng, W.-H., Sharma, C. S. (2018). Biofilm formation by Salmonella spp. in catfish mucus extract under industrial conditions. Food Microbiology, 70, 172-180.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Abeysundara, P. D., Dhowlaghar, N., Nannapaneni, R., Schilling, M. W., Chang, S., Mahmoud, B. S. M., Sharma, C. S., Ma, D. P. (2017). Growth and biofilm formation by Listeria monocytogenes in cantaloupe flesh and peel extracts on four food-contact surfaces at 22 �C and 10 �C. Food Control, 80, 131-142.