** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** THE HIGH-MORTALITY PATHOGEN LISTERIA MONOCYTOGENES IS ENDEMIC AND CAN EASILY BE INTRODUCED INTO MEAT PROCESSING FACILITIES, BUT THE CONDITIONS THAT ALLOW IT TO PERSIST IN THESE LOCATIONS ARE NOT FULLY KNOWN. LISTERIA MONOCYTOGENES IS A BACTERIAL PATHOGEN ASSOCIATED WITH READY-TO-EAT MEATS, SOFT CHEESES, ICE CREAM, AND MANY OTHER FOOD PRODUCTS. IT IS OF PARTICULAR CONCERN DUE TO A 30% MORTALITY RATE AND ELEVATED RISKS OF PREGNANCY LOSS AND LONG-TERM DISABILITY. DESPITE MONUMENTAL EFFORTS BY THE FOOD INDUSTRY TO PREVENT CONTAMINATION, THERE ARE REGULAR OUTBREAKS OF LISTERIOSIS, ESPECIALLY AMONG THE YOUNG, OLD, AND IMMUNOCOMPROMISED. IN JUST THE LAST FIVE YEARS IT HAS BEEN THE CAUSATIVE AGENT FOR AT LEAST 15 OUTBREAKS, RESULTING IN 194 ILLNESSES, 176 HOSPITALIZATIONS, AND 18 DEATHS. THESE OUTBREAKS ARE NOT ONLY DEVASTATING IN TERMS OF PUBLIC HEALTH, BUT THEY ALSO LEAD TO LARGE FOOD PRODUCT RECALLS, WHICH HAVE A HIGH ECONOMIC COST AND CONTINUETO EXASPERATE THE GLOBAL FOOD WASTE PROBLEM. THE RISK THIS PATHOGEN POSES TO OUR MOST VULNERABLE POPULATIONS MAKES IT WORTH THE CONTINUED SCIENTIFIC PURSUIT OF NEW METHODS TO IDENTIFY AND PREVENT POTENTIAL OUTBREAKS BEFORE THEY BEGIN.UNDERSTANDING WHY LISTERIA PERSISTS IN SOME MEAT PROCESSING FACILITIES IS CRITICAL TO ADDRESS THIS PUBLIC HEALTH ISSUE. THESE FACILITIES UNDERGO RIGOROUS DAILY CLEANING AND SANITATION PROTOCOLS, AND THIS HOSTILE ENVIRONMENT HAS SELECTED FOR ORGANISMS THAT HAVE SURVIVAL ADAPTATIONS; ONE SUCH ADAPTATION IS THE ABILITY TO FORM BIOFILMS, A STRUCTURED NETWORK OF PROTEIN AND SUGARS THAT PROTECTS THE ORGANISMS LIVING WITHIN IT. THESE BIOFILMS CAN PROVIDE A PERFECT ENVIRONMENTAL NICHE FOR LISTERIA PERSISTENCE. HOWEVER, WE STILL KNOW VERY LITTLE ABOUT THE ROLE OF SPECIFIC ORGANISMS IN A BIOFILM MICROBIOME AND THEIR POTENTIAL IMPACTS ON FOOD PRODUCTS. THEREFORE, IN THIS PROJECT, WE WILL DETERMINE WHETHER THERE ARE MEAT PROCESSING FACILITY-ASSOCIATED BIOFILM MICROBIAL COMMUNITIES THAT SUPPORT OR INHIBIT THE GROWTH AND SURVIVAL OF LISTERIA MONOCYTOGENES.TO ADDRESS THIS CRITICAL RESEARCH QUESTION, WE WILL EMPLOY DNA SEQUENCING AND 'OMICS TECHNOLOGIES TO FULLY REVEAL THE POPULATIONS AND ACTIVITIES OF THE MICROBES INVOLVED IN FACILITY BIOFILMS. WE WILL WORK WITH THE MEAT PROCESSING COMMUNITY TO COLLECT BIOFILM SAMPLES FROM SMALL MEAT FACILITIES LOCATED IN ALABAMA AND THE SOUTHEASTERN UNITED STATES. WITHIN EACH FACILITY, WE WILL PLACE STAINLESS STEEL COUPONS IN SELECT DRAINS THROUGHOUT THE PROCESSING SPACES AND ALLOW BIOFILMS TO GROW OVER A PERIOD OF THREE MONTHS, WHICH WILL ALLOW US TO FORM A BIO-MAP OF BIOFILM MICROBES REPRESENTATIVE OF ALL SMALL PROCESSORS. ONCE WE HAVE COLLECTED THE SAMPLES, WE WILL DIVIDE THE COUPON INTO THREE PORTIONS. THE FIRST WILL UNDERGO DNA SEQUENCING, WHICH WILL ALLOW US TO IDENTIFY THE SPECIFIC MICROORGANISMS PRESENT IN THE SAMPLE, THE RELATIVE ABUNDANCE OF EACH ORGANISM IN THE COMMUNITY, AND THEIR OVERALL MICROBIAL DIVERSITY. THE SECOND PORTION WILL UNDERGO RNA SE,QUENCING, WHICH WILL ALLOW US TO DETERMINE THE BIOLOGICAL FUNCTIONS THESE ORGANISMS ARE GENERATING (E.G., BIOFILM FORMATION, COMPETITION). THE FINAL PORTION WILL BE CULTURED WITH LISTERIA MONOCYTOGENES, AND THEN ALSO UNDERGO RNA SEQUENCING, SO WE CAN DETERMINE WHAT MICROBIAL ACTIVITIES CHANGE IN RESPONSE TO THE ADDITION OF THE PATHOGEN. WE HYPOTHESIZE THAT THE ADDITION OF LISTERIA TO THESE COMMUNITIES WILL DRIVE A RE-ORGANIZATION OF THE COMMUNITY, WHICH WILL BE EVIDENT BY THESE FUNCTIONAL CHANGES. DIFFERENT COMMUNITIES WILL LIKELY HAVE DIFFERENT RESPONSES, AND WITH THIS INFORMATION WE CAN IDENTIFY BIOFILM COMMUNITIES MORE LIKELY TO RESPOND WITH COMPETITIVE EXCLUSION TO LISTERIA, THEREFORE PREVENTING ITS COLONIZATION.THE GOAL OF THIS RESEARCH PROJECT IS TO BETTER UNDERSTAND THE DYNAMICS OF MICROBES WITHIN MEAT PROCESSING FACILITY BIOFILMS. OUR METHODS WILL ALLOW US TO DIRECTLY ADDRESS THE DIVERSITY OF MICROBIAL COMMUNITIES IN THESE ENVIRONMENTS ACROSS THE REGION AND TO GAIN INSIGHT INTO WHY THEY FORM AND WHAT ROLES EACH MEMBER PERFORMS. WITH THE INTRODUCTION OF AN IMPORTANT PATHOGEN, LISTERIA MONOCYTOGENES, WE WILL ALSO LEARN HOW THESE COMMUNITIES RESPOND TO THE INTRODUCTION OF NEW ORGANISMS, AND WHETHER THIS INCREASES THE AMOUNT OF COMPETITION FOR NUTRIENTS WITHIN THE LIMITED SPACE. ULTIMATELY, WE BELIEVE THIS WILL LEAD TO THE CREATION OF AN EARLY-WARNING SYSTEM FOR DETECTING THE GROWTH POTENTIAL OF LISTERIA IN A HIGH-RISK SPACE SUCH AS MEAT PROCESSING FACILITIES BEFORE IT IS ACTUALLY COLONIZED BY THE PATHOGEN. THROUGH THIS, WE WILL AIM TO DECREASE THE RISK OF PATHOGEN CONTAMINATION FOR THE SMALL PROCESSOR, DECREASE THE ECONOMIC BURDEN ASSOCIATED WITH FOOD RECALLS AND ILLNESS, AND IMPROVE THE SAFETY OF FOOD PRODUCTS LOCALLY, REGIONALLY, AND, ULTIMATELY, NATIONWIDE.