FORMATION AND BIOCIDE TOLERANCE OF SALMONELLA BIOFILMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1022029
• Project Start Date: Jan 10, 2020
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OHIO STATE UNIVERSITY - VET MED
1900 COFFEY ROAD, 127L VMAB
COLUMBUS,OH 43210

Performing Department
Preventive Medicine

Non Technical Summary
Salmonella remains a major contributor to foodborne illness in the United States, however the threat to public and animal health extends beyond food production and consumption. Hospital acquired Salmonella infections are an emerging problem in human and veterinary hospitals, a concerning trend considering the rise of global antimicrobial resistance. Salmonella's ability to form biofilms, a community of connected cells protected by a self-produced "slime", may contribute to its environmental persistence and offer protection against disinfectants. Currently, cleaning and disinfection protocols are not optimized to remove biofilms leaving livestock producers, food processing facilities, and health care environments susceptible to Salmonella outbreaks. The proposed study aims to define biofilm producing capabilities of Salmonella strains of public health importance and determine optimal disinfectants to eradicate biofilms on common surface materials. Our goal is to use this research to guide the design of a cleaning and disinfection protocol applicable to many environments.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4010	1100	50%
712	4010	1100	50%

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

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

salmonella

biofilm

biocide

disinfectant

Goals / Objectives
Nontyphoidal Salmonella (NTS) remains the leading cause of foodborne illness in industrialized countries. However, this threat to public and animal health is not wholly limited to food production and consumption, as nosocomial NTS infections in human and veterinary hospitals are also emerging. Beyond the immediate health threat, this observed rise in nosocomial NTS infections is alarming considering the global emergence, spread, and persistence of multidrug-resistant bacteria. Environmental persistence is consistently cited as a potential risk factor for salmonellosis. NTS has the ability to form biofilms, a collection of adherent cells surrounded by an extracellular polymeric substance (EPS) matrix, which may explain this environmental persistence. Cleaning and disinfection protocols are easy and practical procedures implemented to control infectious disease; however, biofilm formation can result in increased biocide tolerance, allowing pathogens to survive typical sanitation procedures. Further, many biocides have either been tested against planktonic cells only, a limited number of serotypes, or biofilms early in formation (24-48 h), and targeted research on mature biofilms is necessary to mitigate NTS. The goal of this research is to inform and guide the creation of a generic, laboratory-validated cleaning and disinfection protocol that will be applicable across multiple livestock industries, food processing facilities, and health care settings.

Project Methods
Experimental PlanSpecific Aim 1: Characterize biofilm densities of clinically relevant Salmonella serovars (S. Typhimurium, Heidelberg, Montevideo, Agona, Newport, Dublin, I 4,[5],12:i:-, Enteriditis) at 48 h and 168 h.Research Approach: We will conduct crystal violet microtiter dish biofilm formation assays for 12 isolates per serotype (n=96) at 48 h and 168 h post-inoculation. These timepoints were chosen to reflect common sanitation practices of non-product surfaces in food processing facilities (every other day) and intermittent cleaning practices seen in farm settings (e.g. once a week). Salmonella isolates will be bovine, avian, and equine in origin. Serotypes were selected based on reported prevalence in culture-confirmed human Salmonella infections[3] (S. Enteriditis, Newport, Typhimurium, I 4,[5],12:i:-, Montevideo, Heidelberg, Agona) and serotype profiles (S.Enteriditis, Montevideo, Typhimurium, Dublin, Heidelberg, Newport, Agona) isolated from meat and poultry by the United States Department of Agriculture Food Safety and Inspection Service [8]. Each serotype will be tested in triplicate in two separate trials. Scanning electron microscopy will be utilized to illustrate morphological differences between 48 h and 168 h biofilms and between Salmonella serotypes. Live/dead staining will be conducted to understand viability of cells within the biofilm.Specific Aim 2: Determine minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), and minimum biofilm eradication concentrations (MBEC) of commonly used disinfectants for planktonic Salmonella, 48 h Salmonella biofilms, and 168 h Salmonella biofilms.Research Approach: MIC and MBC data will be determined for planktonic Salmonella cells via 96 well microtiter plate susceptibility assay. Biofilms will be matured to 48 h and 168 h and analyzed using MBEC Assay® system. Six common disinfectants encompassing chlorine and peroxy based oxidizing agents, quaternary ammonium compounds, and bisbiguanides will be tested. Neutralizer, a chemical solution formulated to block the germicidal action of biocides, will be tested against each serotype and biocide. This will ensure the neutralizer has no bactericidal effects and fully negates biocidal action when applied, allowing the results to appropriately reflect biocide efficacy after a specified contact time. Serotypes will be tested in triplicate and repeated in two separate trialsSpecific Aim 3: Using MBEC data generated in specific aim 2, biocides will be tested against biofilms adhered to different surface materials (concrete, stainless steel, plastic, rubber, and aluminum) common in livestock production, food processing facilities, and human and veterinary health care settings.Research Approach: Biofilms will be grown in a CDC Biofilm Bioreactor® on coupons to emulate dynamic conditions found in nature. MBEC data generated in specific aim 2 will be tested against all serotypes and optimized for each surface material used. Scanning electron microscopy and live/dead staining will be used to visualize biofilm morphology before and after disinfectant exposure and confirm biofilm eradication. The agar candle dip method, adapted from Moley et al. (2019), will be utilized to confirm long-term biofilm eradication. The agar candle dip method was pioneered by Dr. Paul Stoodley's laboratory at The Ohio State University College of Medicine Department of Microbial Infection and Immunity. Dr. Stoodley is a Professor in the Department of Microbial Infection and Immunity. The Stoodley Laboratory has strong expertise in biofilm dynamics and their removal from medical devices. Dr. Stoodley is supportive of the proposed project and the potential for further collaboration in this line of work.Pitfalls and Limitations: This area of research is critical to reducing foodborne and nosocomial Salmonella infections across many industries. However, laboratory results do not always accurately mimic "real world" environments, and the efficacy of these results must be evaluated beyond the scope of this study, prior to their adoption. Ultimately, we believe our comprehensive approach, including validating neutralizer toxicity and efficacy, testing biocide efficacy on multiple materials, and expanding the subset of serotypes analyzed will allow for a wholistic, yet practical approach towards optimizing cleaning and disinfection protocols capable of removing Salmonella biofilms.

Progress 01/10/20 to 09/30/21

Outputs
Target Audience:The target audience of this research includes livestock producers and food processors, as well as individuals involved with infection control in hospital environments. This research will also be useful to other investigators within this area to expand lines of inquiry into other Salmonella serovars or other bacteria of interest. Changes/Problems:The COVID-19 pandemic significantly delayed this project, since students were not allowed in labs for a period of time. Additionally, there was difficult with acquisition of laboratory supplies and equipment. Nontheless, we will complete all of the project goals, albeit much later than anticipated. What opportunities for training and professional development has the project provided?The research team includes a doctoral student, post-doctoral student, and undergraduate students. The doctoral student has become proficient in laboratory techniques including biofilm formation assays, bacterial quantification via plating, and biocide susceptibility assays. Both the doctoral and post-doctoral student have gained considerable experience in troubleshooting inevitable problems. Additionally, this project has provided undergraduates experience in research and, in some cases, their first experience practicing microbiological techniques How have the results been disseminated to communities of interest?Data from the biofilm formation assays was presented at the 2021 Conference of Research Workers in Animal Disease. Several manuscripts are currently in preparation. This research will inform the basic parameters of an enhanced cleaning and disinfection protocol to be tested in a commercial environment. What do you plan to do during the next reporting period to accomplish the goals?Additional replicates from Specific Aims 2 and 3 are currently underway to reconfirm observations. Subsequently, data analysis and manuscripts will be finalized for publication this calendar year. Although this is the final report for the project, data generation, analysis, and preparation of outputs will continue for these projects.

Impacts
What was accomplished under these goals? Specific Aim 1: The first Aim of the project is to characterize the biofilm densities of clinically relevant Salmonella at 48 and 168 h post inoculation. We hypothesized that biofilm density would vary between serovars and increase from 48 to 168 h. Salmonella isolates were curated from veterinary diagnostic laboratories and augmented with isolates recovered from previous research projects. Eighty-five isolates were characterized from eight serovars (S. 4,[5],12:i:-, Agona, Dublin, Enteriditis, Heidelberg, Montevideo, Newport, and Typhimurium). Both hypotheses were accepted. It was found that there was substantial variation between Salmonella serovars, with sers. Montevideo and Dublin forming weak biofilms by 48 h. and Salmonella sers. Heidelberg and 4,[5],12:i:- consistently forming strong biofilms by 48 h. At 168 h., the majority of isolates tested formed strong biofilms, suggesting that 'weak' biofilm formers may more aptly be described as 'slow' biofilm formers. These results also suggest that intermittent or inconsistent cleaning in livestock housing environments may allow for the establishment of mature biofilms that require higher concentrations of biocides or additional cleaning steps to ensure eradication. A manuscript is currently in preparation for submission to the Journal of Applied Microbiology. Specific Aim 2: The second Aim of the project was to determine inhibitory and bactericidal concentrations of disinfectants against planktonic Salmonella and Salmonella biofilms. As contact time is a critical component of cleaning and disinfection protocols, we included and evaluated the use of a neutralizer in these experiments. Interestingly, Salmonella Montevideo was found to be sensitive to neutralizers. Additionally, disinfectant susceptibility assays utilizing a neutralizer were found to be less effective compared to susceptibility results where a neutralizer was not introduced. Bleach, although commonly used in on- farm cleaning and disinfection protocols, was not able to appreciably reduce or eliminate Salmonella and may be inappropriate for use in such situations. This work is ongoing, and we expected to have completed SA2 activities by the end of Spring 2022. Specific Aim 3: The third Aim of the project is to assess the impact different surface material adherence will have on disinfectant efficacy against mature Salmonella biofilms. We will used a bioreactor to measure the disinfectant efficacy. This work is projected to be completed by the end of Summer 2022. Further funding was acquired to probe genomic determinants that drive associations between biofilm production and biocide tolerance. Additional lines of investigation into associations between antimicrobial resistance and biofilm forming capabilities were generated by this research.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Biofilm forming abilities of Salmonella serovars isolated from clinically ill livestock at 48 and 168 hrs. S.R. Locke, P. Vinayamohan, G Habing. 2021. Conference of Research Workers in Animal Disease.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Biofilm forming abilities of Salmonella serovars isolated from clinically ill livestock at 48 and 168 hrs. S.R. Locke, P. Vinayamohan, G Habing. 2022. College of Veterinary Medicine Research Day.

Progress 10/01/20 to 09/30/21

Outputs
Target Audience:The target audience of this research includes livestock producers and food processors, as well as individuals involved with infection control in hospital environments. This research will also be useful to other investigators within this area to expand lines of inquiry into other Salmonella serovars or other bacteria of interest. Changes/Problems:The COVID-19 pandemic significantly delayed this project, since students were not allowed in labs for a period of time. Additionally, there was difficult withacquisition of laboratory supplies and equipment. Nontheless, we will complete all of the project goals, albeit much later than anticipated. What opportunities for training and professional development has the project provided?The research team includes a doctoral student, post-doctoral student, and undergraduate students. The doctoral student has become proficient in laboratory techniques including biofilm formation assays, bacterial quantification via plating, and biocide susceptibility assays. Both the doctoral and post-doctoral student have gained considerable experience in troubleshooting inevitable problems. Additionally, this project has provided undergraduates experience in research and, in some cases, their first experience practicing microbiological techniques. How have the results been disseminated to communities of interest?Data from the biofilm formation assays was presented at the 2021 Conference of Research Workers in Animal Disease. Several manuscripts are currently in preparation.This research will inform the basic parameters of an enhanced cleaning and disinfection protocol to be tested in a commercial environment. What do you plan to do during the next reporting period to accomplish the goals?Additional replicates from Specific Aims 2 and 3 are currently underway to reconfirm observations. Subsequently, data analysis and manuscripts will be finalized for publication this calendar year. Although this is the final report for the project, data generation, analysis, and preparation of outputs will continue for these projects.

Impacts
What was accomplished under these goals? Specific Aim 1: The first Aim of the project is to characterize the biofilm densities of clinically relevant Salmonella at 48 and 168 h post inoculation. We hypothesized that biofilm density would vary between serovars and increase from 48 to 168 h. Salmonella isolates were curated from veterinary diagnostic laboratories and augmented with isolates recovered from previous research projects. Eighty-five isolates were characterized from eight serovars (S. 4,[5],12:i:-, Agona, Dublin, Enteriditis, Heidelberg, Montevideo, Newport, and Typhimurium). Both hypotheses were accepted. It was found that there was substantial variation between Salmonella serovars, with sers. Montevideo and Dublin forming weak biofilms by 48 h. and Salmonella sers. Heidelberg and 4,[5],12:i:- consistently forming strong biofilms by 48 h. At 168 h., the majority of isolates tested formed strong biofilms, suggesting that 'weak' biofilm formers may more aptly be described as 'slow' biofilm formers. These results also suggest that intermittent or inconsistent cleaning in livestock housing environments may allow for the establishment of mature biofilms that require higher concentrations of biocides or additional cleaning steps to ensure eradication. A manuscript is currently in preparation for submission to the Journal of Applied Microbiology. Specific Aim 2: The second Aim of the project was to determine inhibitory and bactericidal concentrations of disinfectants against planktonic Salmonella and Salmonella biofilms. As contact time is a critical component of cleaning and disinfection protocols, we included and evaluated the use of a neutralizer in these experiments. Interestingly, Salmonella Montevideo was found to be sensitive to neutralizers. Additionally, disinfectant susceptibility assays utilizing a neutralizer were found to be less effective compared to susceptibility results where a neutralizer was not introduced. Bleach, although commonly used in on-farm cleaning and disinfection protocols, was not able to appreciably reduce or eliminate Salmonella and may be inappropriate for use in such situations. This work is ongoing, and we expected to have completed SA2 activities by the end of Spring 2022. Specific Aim 3: The third Aim of the project is to assess the impact different surface material adherence will have on disinfectant efficacy against mature Salmonella biofilms. We will used a bioreactor to measure the disinfectant efficacy. This work is projected to be completed by the end of Summer 2022. Further funding was acquired to probe genomic determinants that drive associations between biofilm production and biocide tolerance. Additional lines of investigation into associations between antimicrobial resistance and biofilm forming capabilities were generated by this research.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Biofilm forming abilities of Salmonella serovars isolated from clinically ill livestock at 48 and 168 hrs. S.R. Locke, P. Vinayamohan, G Habing. 2021. Conference of Research Workers in Animal Disease.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Biofilm forming abilities of Salmonella serovars isolated from clinically ill livestock at 48 and 168 hrs. S.R. Locke, P. Vinayamohan, G Habing. 2022. College of Veterinary Medicine Research Day.

Progress 01/10/20 to 09/30/20

Outputs
Target Audience:The project seeks to understand how biofilms contribute to survival ofSalmonellain the environment, and further the design of evidence-based protocols for the appropriate and effective cleaning and disinfection. Therfore, the target audience for the project are all individuals involved with the control and prevention ofSalmonellaintroduction into farms and/or food production sites. Changes/Problems:The project was substantially delayed by the COVID-19 pandemic; nonetheless, we are proceeding quickly and making the necessary plans to complete all the activities described in the project. Additionally, contamination has been a substantial challenge in the project, and interfered with characterization of the biofilms. We've identified the source of the contamination, and this phase of the project is nearly complete. What opportunities for training and professional development has the project provided?The activities in this projec have served as dissertation research chapters within the doctoral program for a graduate student. How have the results been disseminated to communities of interest?The results have been substantially delayed by the pandemic; however, the graduate student will be presenting these results to to researchers at the Conference of Research Workers in Animal Disease in December 2021. Additionally, a manuscript detailing the results is in preparation. What do you plan to do during the next reporting period to accomplish the goals?Over the next reporting period, we will complete characterization of biofilm growth, and move into the next phases of the project. In the next phases of the project, we will determine the minimum biofilm eradication concentration of common biodcides against young and mature biofilms, and assess difference in disinfectant efficacy on several surface materials (e.g. concrete, stainless steel, plastic). These data are essential to design evidence-based cleaning and disinfection protocols.

Impacts
What was accomplished under these goals? We 've thus far measured the biofilm formation on approximately 150 strains ofSalmonellaat 2 and 7 days of growth. This is labor intensive process requiring non-gene-based growth techniques. These data will be a springboard towards further characterization of genomic determinants of biofilm formation, and identifying in-vitro efficacy of biocides againstSalmonella.?

Publications