Biofilm Formation by Shiga Toxin–Producing Escherichia coli O157:H7 and Non-O157 Strains and Their Tolerance to Sanitizers Commonly Used in the Food Processing Environment3

Shiga toxin–producing Escherichia coli (STEC) strains are important foodborne pathogens. Among these, E. coli O157:H7 is the most frequently isolated STEC serotype responsible for foodborne diseases. However, the non-O157 serotypes have been associated with serious outbreaks and sporadic diseases as well. It has been shown that various STEC serotypes are capable of forming biofilms on different food or food contact surfaces that, when detached, may lead to cross-contamination. Bacterial cells at biofilm stage also are more tolerant to sanitizers compared with their planktonic counterparts, which makes STEC biofilms a serious food safety concern. In the present study, we evaluated the potency of biofilm formation by a variety of STEC strains from serotypes O157:H7, O26:H11, and O111:H8; we also compared biofilm tolerance with two types of common sanitizers, a quaternary ammonium chloride–based sanitizer and chlorine. Our results demonstrated that biofilm formation by various STEC serotypes on a polystyrene surface was highly strain-dependent, whereas the two non-O157 serotypes showed a higher potency of pellicle formation at air-liquid interfaces on a glass surface compared with serotype O157:H7. Significant reductions of viable biofilm cells were achieved with sanitizer treatments. STEC biofilm tolerance to sanitization was strain-dependent regardless of the serotypes. Curli expression appeared to play a critical role in STEC biofilm formation and tolerance to sanitizers. Our data indicated that multiple factors, including bacterial serotype and strain, surface materials, and other environmental conditions, could significantly affect STEC biofilm formation. The high potential for biofilm formation by various STEC serotypes, especially the strong potency of pellicle formation by the curli-positive non-O157 strains with high sanitization tolerance, might contribute to bacterial colonization on food contact surfaces, which may result in downstream product contamination. Shiga toxin–producing Escherichia coli (STEC) strains of various serotypes are important foodborne pathogens that pose a serious public health concern, resulting in significant financial burden. STEC strains have been implicated in numerous outbreaks, with symptoms ranging from bloody diarrhea to other, more severe, diseases such as hemolytic uremic syndrome (HUS), a life-threatening complication that is the major cause of kidney failure for children younger than the age of 5 years (11). Cattle are the principal animal reservoir of these zoonotic pathogens, and foodborne outbreaks of illness have been associated with the consumption of ground beef, dairy products, vegetables, and fruit juices, etc. (20–22). Since it was first identified in 1982, STEC serotype O157:H7 has become the most commonly identified STEC serotype, causing multiple clinical diseases and foodborne outbreaks in North America, Europe, and Asia. Serotype O157:H7 infection can cause bloody diarrhea, HUS, and thrombotic thrombocytopenic purpura, etc. It has been estimated that E. coli O157:H7 is responsible for over 73,000 cases of illness each year in the United States (8). Meanwhile, numerous outbreaks associated with the nonO157 serotypes also have contributed to the public burden of human infections and clinical diseases. Despite increased isolation of non-O157 STEC strains from patients, outbreaks, and environmental sources (32, 33), the public health significance of these pathogens has not been well investigated due to diagnostic limitations and inadequate surveillance. According to studies at the Centers for Disease Control and Prevention (CDC), approximately 70% of the non-O157 STEC infections that emerged from 1983 to 2002 were caused by one of six major serotypes, which are now referred to as ‘‘the big six,’’ including O26, O45, O103, O111, O121, and O145. It was reported that these six STEC serotypes, collectively, caused more human infections in the United States in 2010 than did STEC O157:H7; therefore, the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) has announced the intention to carry out an enforceable program to give these non-O157 * Author for correspondence. Tel: 402-762-4228; Fax: 402-762-4149; E-mail: [email protected]. { The 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 the USDA implies no approval of the product to the exclusion of others that may also be suitable. 1418 Journal of Food Protection, Vol. 75, No. 8, 2012, Pages 1418–1428 doi:10.4315/0362-028X.JFP-11-427 STECs the same regulatory scrutiny as STEC O157:H7 in the national’s beef supply (45). Among these six non-O157 serotypes, O26 is the most common non-O157 STEC isolated from specimens submitted to CDC for serotyping. O26 was found to be responsible for 40% of all non-O157 and 14% of total STEC-related cases of HUS in Austria and Germany between 1996 and 2003 (1). This serotype also was found to play an important role in the etiology of acute diarrhea in young children (13, 34). Recovery of serotype O26 from cattle and beef products has been reported in different countries (1, 29, 47). On the other hand, serotype O111 is the second most common non-O157 STEC strain isolated from specimens submitted to the CDC. This serotype has been associated with sporadic cases and outbreaks of bloody diarrhea. The first case of community O111 outbreak was reported in Texas in 1999, and further analysis of samples from patients confirmed that STEC O111:H8 was the causative agent of the infection. The largest foodborne outbreak due to STEC O111 in the United States was reported in Oklahoma in 2008; it caused over 340 cases of human infections and severe diarrhea illness (6). In addition, one study reported that O111 was responsible for the majority of HUS cases in the United States (5). O111:H8 and O111:nonmotile strains are the two most frequently isolated O111 serotypes, and these pathogens have been detected in cattle fecal samples and in ground beef products (42). It has been shown that various STEC serotypes have the ability to attach, colonize, and form biofilms on a wide variety of food contact surfaces commonly used in meatprocessing plants as well as on vegetables and meat products (25, 39). Food processing equipment design and surface materials also could affect bacterial attachment and biofilm formation. In addition, bacterial biofilms are usually much more tolerant to sanitizing agents than free-flowing cells of the same species, so it is difficult to completely inactivate biofilms formed on the equipment and in the environment, and the surviving biofilm cells may detach from the surface and contaminate food products. Strong attachment of the STEC biofilms on food surfaces may also affect the efficiency of antimicrobial interventions applied to food products for reducing contamination. Thus, STEC biofilm formation is a serious potential hazard in food hygiene and may become a source of cross-contamination in the food processing environment. Prevention, removal, and inactivation of STEC biofilms, therefore, are critical for improving hygiene, controlling contamination, and enhancing food safety. Considerable research has been directed at evaluating the impact of STEC biofilms on food safety, as well as understanding the mechanisms and genetic basis for biofilm formation by these pathogens (3, 11, 25, 27, 38, 39). Studies also have investigated the tolerance of STEC cells in biofilms to decontamination reagents (18, 19, 26, 40). However, most of these studies have focused on serotype O157:H7 since it is the serotype most commonly associated with foodborne outbreaks and clinical diseases. In contrast, there are relatively few reports on the ability of non-O157 serotypes to form biofilms (2, 9, 28, 41) and to tolerate sanitizers. In the present study, we compared various strains of STEC serotypes O157:H7, O26:H11, and O111:H8 for their ability to form biofilms under different conditions and for the tolerance of their biofilms to sanitizing regents commonly used in the food processing environment. MATERIALS AND METHODS Bacterial strains, culture conditions, and curli expression. Ten strains each of STEC O157:H7, STEC O26:H11, and STEC O111:H8 that varied epidemiologically by sources were used for biofilm formation and sanitization study (Table 1). Each isolate was characterized by enzyme-linked immunosorbent assay, using antiO157, O26, O111, and H7 monoclonal antibodies and multiplex PCR for stx1, stx2, eae, hlyA, rfbO157, fliCH7 rfbO26, fliCH11 rfbO111, and fliCH8 (11, 12, 14, 16, 31, 36, 48). Additionally, each strain was genotyped for a polymorphism residing within the translocated intimin receptor gene (tir 255 T . A) (3). Curli expression of these STEC strains was screened using Congo red indicator (CRI) plates as previously described (15). All strains were stored at 270uC in Lennox broth (LB; Acumedia Manufacturers, Baltimore, MD) containing 15% glycerol. Prior to use, each strain was streaked from the glycerol stock onto LB agar plates and grown overnight at 37uC. To prepare bacterial broth cultures at stationary phase for biofilm formation and sanitization study, bacteria from a single colony on LB agar plates were inoculated into LB–low salt (LB-LS) broth and incubated overnight (16 to 18 h) at 37uC with orbital shaking at