Mixed Biofilm Formation by Shiga Toxinâ•fiProducing Escherichia coli and Salmonella enterica Serovar Typhimurium Enhanced Bacterial Resistance to Sanitization due to Extracellular Polymeric Substances

Shiga toxin–producing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium are important foodborne pathogens capable of forming single-species biofilms or coexisting in multispecies biofilm communities. Bacterial biofilm cells are usually more resistant to sanitization than their planktonic counterparts, so these foodborne pathogens in biofilms pose a serious food safety concern. We investigated how the coexistence of E. coli O157:H7 and Salmonella Typhimurium strains would affect bacterial planktonic growth competition and mixed biofilm composition. Furthermore, we also investigated how mixed biofilm formation would affect bacterial resistance to common sanitizers. Salmonella Typhimurium strains were able to outcompete E. coli strains in the planktonic growth phase; however, mixed biofilm development was highly dependent upon companion strain properties in terms of the expression of bacterial extracellular polymeric substances (EPS), including curli fimbriae and exopolysaccharide cellulose. The EPS-producing strains with higher biofilm-forming abilities were able to establish themselves in mixed biofilms more efficiently. In comparison to single-strain biofilms, Salmonella or E. coli strains with negative EPS expression obtained significantly enhanced resistance to sanitization by forming mixed biofilms with an EPS-producing companion strain of the other species. These observations indicate that the bacterial EPS components not only enhance the sanitizer resistance of the EPS-producing strains but also render protections to their companion strains, regardless of species, in mixed biofilms. Our study highlights the potential risk of cross-contamination by multispecies biofilms in food safety and the need for increased attention to proper sanitization practices in food processing facilities. Shiga toxin–producing Escherichia coli strains are important foodborne pathogens which pose a serious public health concern with a significant financial burden. O157:H7 is the most-commonly identified Shiga toxin–producing E. coli serotype associated with foodborne outbreaks and clinical diseases. It was estimated that E. coli O157:H7 was responsible for over 73,000 illnesses each year in the United States (6). The symptoms caused by E. coli O157:H7 infections range from bloody diarrhea to other, more-severe diseases, such as hemolytic uremic syndrome, a lifethreatening complication which is the major cause of kidney failure for children younger than 5 years. Meanwhile, Salmonella infections have been reported as the second leading cause of bacterial foodborne illness in the United States, responsible for approximately 11% of all infections caused by foodborne pathogens (30). Over 95% of human salmonellosis cases have been associated with the consumption of contaminated foods, which have included red meat and poultry. Among the many Salmonella serotypes, Salmonella enterica serovar Typhimurium has been reported as the major and frequent cause of human gastroenteritis. It has been estimated that human infections by S. enterica were responsible for approximately 1.4 million clinical cases each year in the United States, resulting in over 17,000 hospitalizations and a financial burden of approximately $2.3 to $3.6 billion on the United States’ economy (10). These serious public health concerns and financial consequences highlight the critical need to prevent food contamination by these foodborne pathogens. Biofilm formation is one of the major strategies that support bacterial survival under adverse circumstances. In the food industry, the attachment of foodborne pathogens on food products and contact surfaces can be enhanced by biofilm formation. Bacterial cells in biofilms are usually more resistant to sanitizing agents than planktonic cells of the same species, and the strong attachment of the biofilm cells on food surfaces also may affect the efficiency of * Author for correspondence. Tel: 402-762-4228; Fax: 402-762-4149; E-mail: [email protected]. { U.S. Department of Agriculture is an equal opportunity provider and employer. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. 1513 Journal of Food Protection, Vol. 76, No. 9, 2013, Pages 1513–1522 doi:10.4315/0362-028X.JFP-13-077