Non-O157 Shiga Toxin-Producing Escherichia coli: Prevalence Associated with Meat Animals and Controlling Interventions 1

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1 FOOD SAFETY Non-O57 Shiga Toxin-Producing Escherichia coli: Prevalence Associated with Meat Animals and Controlling Interventions Norasak Kalchayanand* a, Terrance M. Arthur a, Joseph M. Bosilevac a, and Tommy L. Wheeler a INTRODUCTION Escherichia coli are a large and diverse group of bacteria, with most strains of the species considered harmless. However, some kinds of E. coli cause disease by producing any of a variety of toxigenic agents. One such group of pathogenic E. coli produce a class of potent cytotoxins called Shiga toxins (stx). Shiga toxins are also referred to as verotoxins (Konowalchuk et al., 977); hence, the designations of Shiga toxin-producing E. coli (STEC) and verocytotoxigenic E. coli are used interchangeably. Members of this subset of E. coli are also referred to as enterohemorrhagic E. coli (EHEC; Nataro and Kaper, 998). The EHEC designation has been used for those STEC that harbor the additional virulence factors intimin and the large EHEC plasmid. The common feature and main virulence factor of STEC is the production of Stx or Stx2 proteins. Other factors thought to enhance the virulence of STEC strains are intimin (eae), a protein essential for the attachment and the formation of attaching and effacing lesions on gastrointestinal epithelial cells, and E. coli hemolysin (ehxa; Paton and Paton, 998) which is carried on the EHEC plasmid. The most commonly identified STEC in North America is E. coli O57:H7, which causes approximately 3,704 confirmed cases of foodborne illness each year in the United States (Scallan et al., 20). Norasak Kalchayanand *Corresponding author. a US Department of Agriculture, Agricultural Research Service, Roman L. Hruska US Meat Animal Research Center, PO Box 66, State Spur 8D, Clay Center, NE norasak.kalchayanand@ars.usda.gov Names are necessary to report factually on available data; however, the US Department of Agriculture neither guarantees nor warrants the standard of the product, and the use of the name by the US Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable. In addition to E. coli O57:H7, other kinds of STEC, called non-o57 STEC, cause human diseases. The Centers for Disease Control and Prevention estimate that non- O57 STEC are responsible for about,579 confirmed cases of illness annually (Scallan et al., 20) but cause relatively fewer cases of hemolytic uremic syndrome (HUS) than E. coli O57:H7. The true number of illnesses caused by non-o57 STEC may be underestimated because detection and isolation of non-o57 STEC in stools and foodstuffs is laborious and time-consuming, with only about 4% of clinical laboratories routinely screening for these pathogens. In addition, non-o57 STEC detection is complicated by the lack of common biochemical characteristics in distinguishing non-o57 STEC from other E. coli strains. More than 200 virulent non-o57 serotypes have been isolated from outbreaks, sporadic cases of HUS, and severe diarrhea in the United States and other countries (Brooks et al., 2005). In the United States, six O groups (comprising 3 serotypes) have been described by the Centers for Disease Control and Prevention to be the cause of 7% of non-o57 STEC disease (Brooks et al., 2005). These serotypes have been identified as O26:H or nonmotile (NM); O45:H2 or NM; O03:H2, H, H25, or NM; O:H8 or NM; O2:H9 or H7; and O45:NM. Similar to E. coli O57:H7, non-o57 STEC serotypes are often associated with cattle and ruminants. The presence of non-o57 serotypes from beef carcasses, retail beef, and raw milk has been reported previously (Arthur et al., 2002; Barkocy-Gallagher et al., 2003; Hussein and Bollinger 2005a; Hussein and Sakuma T. 2005). Although cattle often carry multiple serotypes of STEC, they should not be viewed as equally virulent. Most of the STEC serotypes that have been isolated from cattle or beef appear to be a minimal or insignificant health risk to humans because they lack accessory virulence factors. A virulence classification scheme proposed by Karmali et al. (2003) identifies STEC seropathotypes based on associations of the serotype and severity of disease caused. This classification system is divided into 5 groups from A to E, with American Meat Science Association

2 varying degrees of severity of disease and propensity to cause outbreaks. Group A includes E. coli O57:H7 and NM, which are common causes of outbreaks and are frequently associated with HUS in most countries. Groups B and C are of moderate frequency and severity in causing disease, and seropathotypes group D and E belong to the lowest frequency and severity, usually producing mild disease, if any (Karmali et al., 2003; Gyles, 2007). Based on this classification, the six O groups described by the Centers for Disease Control and Prevention fall into groups B and C, suggesting that non-o57 STEC are emerging organisms as sources of foodborne illness. It is clear that non-o57 STEC pose a human health risk. Understanding which serotypes are commonly found in foodstuffs and are the most virulent when people are exposed to these pathogens will help to identify which existing interventions are effective in reducing pathogenic non-o57 STEC to minimize human infections. THE STEC It is likely that not all STEC strains found in animal reservoirs are pathogenic to humans (Karmali et al., 2003). However, several STEC strains are responsible for human gastrointestinal illnesses, from mild diarrhea to bloody diarrhea to hemorrhagic colitis and HUS. Most outbreaks and sporadic cases of bloody diarrhea and HUS have been attributed to strains of STEC serotype O57:H7. In Europe, and recently in the United States, however, the role of non-o57 STEC strains such as O26:H/ NM, O45:H2/NM, O9:H2/NM, O03:H2, O:NM, O3:H2, O2:H9, O28:H2/NM, and O45:H28/ NM as the cause of HUS, bloody diarrhea, and other gastrointestinal illnesses are increasingly being recognized (European Food Safety Authority, 200a). The STEC have been characterized by a variety of methods. Serotyping is used extensively to categorize strains of E. coli (Blanco et al., 2004a,b; Prager et al., 2005). The serotyping is based on the O (Ohne) antigen determined by the polysaccharide portion of cell wall lipopolysaccharide and the H (Hauch) antigen attributable to flagella protein. There are 74 O antigens and 53 H antigens in the international serotyping scheme, with E. coli isolates having various combinations of O and H antigens (Scheutz et al., 2004). SURVEILLANCE AND PATHOGENICITY OF NON-O57 STEC Non-O57 STEC strains are not new types of E. coli organisms, and infections caused by non-o57 STEC are widespread and have been reported from many parts of the world (Kasper and Doyle, 2009). Serotype O03 was the first non-o57 STEC strain identified as a suspected cause of sporadic cases of HUS, in 975 in France (Karmali et al., 985). The earliest reported outbreak caused by serotype O45:NM was in Japan in 984, but the vehicle of infection was not determined (Johnson et al., 996. To date, the first and only US non-o57 STEC outbreak associated with beef occurred in 200 and was caused by O26. Non-O57 STEC outbreaks were reported with increasing frequency worldwide from 984 to 2008 (Table ). Outbreaks of non-o57 STEC increased from 29 cases in to,770 cases in , suggesting that the rate of non-o57 STEC infections was increasing or, more likely, that detection of these organisms has improved. According to a 200 Foodborne Diseases Active Surveillance Network (FoodNet) report, which included preliminary data from 2009, the rate of infection with O57 STEC was at least 25% lower than it was a decade ago (Centers for Disease Control and Prevention, 200b). The opposite directions between a decreasing infection rate of O57 STEC and an increasing incidence of foodborne disease caused by non-o57 STEC suggest that clinical surveillance for E. coli O57:H7 is no longer sufficient to identify foodborne STEC infections. From 984 to 2009, the most common non-o57 STEC serotypes identified worldwide were 37% O26, 3% O, 6% O03, 5% O2, 5% O45, and % O45 (Table 2), whereas in the United States from 983 to 2002, the breakdown in proportions of non-o57 STEC serotypes was 22% O26, 6% O, 2% O03, 8% O2, 7% O45, and 5% O45 (Brooks et al., 2005). The cases of HUS and outbreaks of bloody diarrhea associated with non-o57 STEC serotypes O26 and O appeared to be common across Europe, South America, and Australia (Bettelheim, 2007). However, in an outbreak study in Minnesota from 2000 to 2006, 29 isolates of O26 and 20 isolates of O did not cause HUS in any of the patients (Hedican et al., 2009). Further analysis indicated that those O26 and O isolates carried either stx alone or at least stx2, suggesting that severity among STEC strains cannot be explained by Shiga toxins and is likely due to STEC carrying a combination of virulence factors. The main virulence factor contributing to pathogenicity is the production of Stx, Stx2, or both, each including several variants (Scheutz and Strockbine, 2005). Strains producing Stx2 alone or both Stx and Stx2 cause more serious illness than those producing only Stx (Ostroff et al., 989). The mode of toxic action starts with the toxin molecule binding to host cell receptors, facilitating transfer of the toxin into the cells. Once inside the host cell, the A-subunit of the toxin, which possesses enzyme activity, Table. Worldwide outbreaks caused by non-o57 Shiga toxin-producing Escherichia coli Years Cases HUS Deaths , HUS = hemolytic uremic syndrome. Adapted from Kasper and Doyle (2009) th Annual Reciprocal Meat Conference

3 Table 2. Most frequent serotypes distribution among worldwide outbreaks Years O26 O45 O03 O O2 O45 Others Total Adapted from Kasper and Doyle (2009). interferes with host cell protein synthesis and induces an inflammatory response (Nataro and Kaper, 998). In addition to Shiga toxin, virulent STEC strains isolated from human clinical cases often contain intimin (eae), a protein essential for the attaching and effacing lesions on gastrointestinal epithelial cells, and enterohemolysin (ehxa). Several reviews discuss these virulence factors in more depth (Nataro and Kaper, 998; Caprioli et al., 2005; Gyles, 2007). Some strains of STEC not harboring intimin and enterohemolysin genes have been shown to be capable of causing human illnesses (Neill, 997). It was therefore suggested that each STEC strain be considered a potential EHEC (Bürk et al., 2002). Clinical manifestations of non- O57 STEC infection range from mild, watery diarrhea to hemorrhagic colitis, HUS, and death. A 2-yr prospective study of 5,45 cases of sporadic diarrhea in Canada revealed that there were significant differences between the clinical syndromes associated with non-o57 STEC and O57 infection (Pai et al., 988). Patients with non-o57 infection had a longer duration of diarrhea (9. vs. 5.7 d), but less frequent bloody diarrhea (42 vs. 97%) compared with patients with O57 infection, suggesting that bloody diarrhea associated with STEC infection cannot be presumed to be due to O57 infection. RESERVOIRS OF NON-O57 STEC Ruminants have been identified as the major reservoir of STEC (Beutin et al., 993; Bettelheim, 2003; Blanco et al., 2004b; Karmali et al., 200). Fecal contamination of the farm or feedlot environment causes cyclic colonization of ruminant animals and aids in the persistence of the pathogens in the farm or feedlot. In North America, cattle are the major reservoir of STEC, but in countries such as Australia (Djordjevic et al., 2004), sheep are of greater significance. Non-O57 STEC have been isolated from several animals (Fratamico et al., 2004; Vu-Khac and Comick, 2008; Rivas et al., 2008), including domestic pets, such as cats (Busch et al., 2007) and dogs (De Paula and Marin, 2008). Worldwide, the prevalence rates of non-o57 STEC ranged from 4.7 to 44.8% in grazing cattle and 4.6 to 55.9% in feedlot cattle (Hussein and Bollinger, 2005b). Studies of US dairy cattle have reported a non-o57 STEC prevalence of 0 to 9% (Wachsmuth et al., 99; Wells et al., 99; Cray et al., 996; Thran et al., 200). The prevalence of non-o57 STEC in US beef cattle feces has been shown to range from 9 to 30% (Barkocy-Gallagher et al., 2003; Renter et al., 2005) and the prevalence on hides was 56.3% (Barkocy-Gallagher et al., 2003; Table 3). Prevalence of the pathogen on hides may reflect several sources of contamination, such as soil, feces from other animals, and the environment (Barkocy-Gallagher et al., 2003). The STEC strains O26, O, and O57 were reported to be able to survive in feces for several weeks (Fukushima et al., 999). Cattle hides and feces have been shown to be major sources of pathogens at slaughter facilities (Barkocy-Gallagher et al., 200; Elder et al., 2000). Barkocy-Gallagher et al., (2003) reported that the prevalence of non-o57 STEC on preevisceration carcasses (before antimicrobial interventions) was 58% and was reduced to 9% after all antimicrobial interventions (Table 3). Similarly, Arthur et al. (2002) reported that 53.9% of beef carcasses in large processing plants carried at least one type of non-o57 STEC prior to evisceration, but that the prevalence was reduced to 8.3% with various intervention strategies. The prevalence of non-o57 STEC on hides was lowest in winter, spring, and summer (averaging 49.2%) and highest (77%) in fall (Barkocy-Gallagher et al., 2003). Serotypes O26 and O were detected in Korean cattle from May to October (Jeon et al., 2006). Transfer of STEC to carcasses usually occurs during hide removal (Elder et al, 2000; McEvoy et al., 2003). Non-O57 STEC serotypes are typically more prevalent in beef products than is E. coli O57:H7 (Hussein and Bollinger, 2005a). This stands to reason because E. coli O57:H7 represents only one STEC serotype, as opposed to the hundreds of non-o57 Table 3. Prevalence rates (%) of non-o57 Shiga toxinproducing Escherichia coli (STEC) from cattle, sheep, and swine at various stages of processing Species Surface 2 Pre 3 Post 4 Cattle Sheep Pork Prevalence rates are based on the presence of stx and/or stx2 gene(s) in samples as determined by polymerase chain reaction. Not all instances of stx genes were confirmed through isolation of a STEC. 2 Surface refers to cattle hides, sheep pelts, and pork skin present at slaughter and before removal, washing, scalding, or flaming. 3 Preevisceration carcasses have had the hide or pelt removed or the hair singed off. No or very few interventions have been applied to the carcass at this time before evisceration. 4 Post refers to the final carcasses in the chiller after all multiple-hurdle interventions have been applied. 3 Adapted from Barkocy-Gallagher et al. (2003). 4 Adapted from Kalchayanand et al. (2007). 5 J. M. Bosilevac (US Department of Agriculture, Agricultural Research Service, Roman L. Hruska US Meat Animal Research Center, Clay Center, NE), personal communication. American Meat Science Association 3

4 STEC serotypes that have been associated with cattle and beef products. The prevalence rates of non-o57 STEC ranged from.7 to 58.0% in packing plants, from 3.0 to 63.5% in retail stores, and averaged 3.0% in fast food restaurants (Hussein and Bollinger, 2005a,b). The prevalence of non-o57 STEC in imported and domestic boneless beef trim used for ground beef in the United States has recently been reported to be as high as 0 to 30% (Bosilevac et al., 2007). Bosilevac and Koohmaraie (20) also screened 4,33 samples of US commercial ground beef, and 99 different serotypes were identified. The most frequent O serotypes, in order of frequency, were O3, O8, O22, O7, O63, O74, O6, and O20. However, only 0.24% of these samples were identified as pathogenic STEC, of which serotype O03:H2 was the most frequently isolated. The O57 and non-o57 STEC have been detected in lambs from several countries (Blanco et al., 2003; Djordjevic et al., 2004; Zweifel et al., 2004; Cookson et al., 2006; Kalchayanand et al., 2007). However, E. coli O57:H7 was a minor component of the STEC identified in these studies. In addition, several serotypes detected in sheep in different countries were similar, indicating that some serotypes may be adapted to colonizing sheep. Samples from various steps during processing from 3 commercial lamb processing plants in the United States revealed that the prevalence rates of non-o57 STEC on pelts, preevisceration carcasses, and postintervention carcasses were 86.2, 78.6, and 8.6%, respectively (Table 3). The high prevalence of non-o57 STEC on postintervention carcasses was probably due to the lack of an effective antimicrobial intervention, such as hot water treatment, at all the processing plants studied. A total of 69 different non-o57 serotypes were identified, and approximately 4% of these serotypes have previously been associated with severe human illness, but none of them were among the top six non-o57 O groups listed by the Centers for Disease Control and Prevention (Kalchayanand et al., 2007). The STEC serotypes isolated from swine often come from animals suffering from postweaning diarrhea or edema disease (Aarestrup et al., 997; Blanco et al., 2006). Serogroups O38, O39, O4, O49, and O57 are normally associated with edema disease. However, when these serogroups were compared with the same serogroups isolated from humans, they were shown to differ in ) their virulence patterns, and therefore are unlikely human pathogens, and 2) their interactions with intestinal epithelial cells (Sonntag et al., 2005). Otherwise, studies of STEC prevalence in healthy pigs reported that some strains showed potential to be human pathogens (Rios et al., 999; Fratamico et al., 2004). Unpublished observations of O57 and non-o57 STEC have identified many EHEC and pathogenic STEC serotypes present on pork carcasses during the initial stages of processing (Table 3; unpublished data; [(US Department of Agriculture, Agricultural Research Service, Roman L. Hruska US Meat Animal Research Center, Clay Center, NE), personal communication. J. M. Bosilevac, personal communication). INTERVENTIONS FOR CONTROL OF NON-O57 STEC One of the greatest challenges in the meat industry is to keep the product safe and free from pathogen contamination. It is clear that non-o57 STEC threaten the health of consumers and cause economic losses. At the current time, the US Food Safety and Inspection Service is considering designating selected non-o57 STEC strains for regulation. In light of such a designation, there is a need to understand how non-o57 STEC strains are affected by current antimicrobial interventions, most of which have been validated against E. coli O57:H7. Several preslaughter interventions are currently under development, whereas numerous postslaughter interventions targeting E. coli O57:H7 have been validated and implemented to decontaminate meat and meat products during the slaughtering process. The susceptibility of non-o57 STEC to these intervention technologies may be similar to other E. coli even though there are known differences among strains in acid tolerance and sensitivity to some other compounds (Molina et al., 2005). PRESLAUGHTER INTERVENTIONS The farm or feedlot is the origin of microorganisms introduced onto carcasses during slaughter and dressing. It appears that changes in diet and management practices could precipitate increased shedding of pathogens. Initial studies on high-grain diets, as opposed to high-fiber diets, reported increased numbers of E. coli; however, further research on this topic has not provided confirmation of the previous results (Diez-Gonzales et al., 998; Keen et al., 999; Scott et al., 2000; McSweeney et al., 2002; Krause et al., 2003). Feeding cattle barley-based diets (Berg et al., 2004) or wet distillers grains (Wells et al., 2009) increased shedding of E. coli O57:H7 compared with feeding standard corn diets. There also is significant research on the feeding of probiotics ( good bacteria ) to livestock to competitively exclude the pathogens. Some organisms have shown promise in reducing the incidence of E. coli O57:H7 in calves (Zhao et al., 998). A lactobacillus-based direct-fed microbial has shown promise in decreasing the shedding of E. coli O57:H7 (Brashears et al., 2003). In contrast, a direct-fed microbial consisting of Bacillus subtilis strain 66 fed to cattle under feedlot conditions for 84 days did not reduce shedding of E. coli O57:H7 (Arthur et al., 200). Sodium chlorate treatment has been shown to reduce populations of Salmonella Typhimurium and E. coli O57:H7 in the intestinal content of swine and cattle (Anderson et al., 200; Callaway et al., 2002), and work is underway to see if this can be used in the field. No regulatory approval has been granted to date in the United States, the European Union, or Australia for the use of this compound. Water troughs have been shown as 4 64 th Annual Reciprocal Meat Conference

5 another source of contamination. Chlorinating water appears to be the treatment of choice for controlling pathogen populations in water, but animal water troughs often contain large amounts of organic material, which reduces the effectiveness of these treatments. Sanitation of drinking water using lactic acid and acidic calcium sulfate in combination with benzoate, caprylic acid, butyric acid, or chlorine dioxide resulted in more than 3 log reductions of E. coli O57:H7, O26:H, and O:NM in contaminated trough water. However, cattle consumed much less water when these chemicals were present, suggesting that these compounds should be applied periodically rather than be added continuously (Zhao et al., 2006). Research on preslaughter interventions is increasing, and agents such as vaccines and bacteriophages are being evaluated (Loneragan and Brashears, 2005; LeJeune and Wetzel, 2007; Johnson et al., 2008). Although research indicates promising outcomes for the use of phages (Waddell et al., 2000) or vaccines (Potter et al., 2004) for the control of E. coli O57:H7 in live animals, several large vaccine trials under commercial conditions have just been completed or are currently underway, and results should be available in the near future. One of the challenges of preslaughter interventions is recontamination of animals during holding in lairage at the processing plant before they enter the slaughtering process (Arthur et al., 2008). Washing the hide with water or other chemical compounds has been proven to be effective in reducing the pathogen load on hides before hide removal and results in less carcass contamination (Arthur et al., 2007; Bosilevac et al., 2005). Therefore, hide washing should be considered a useful intervention to reduce non-o57 STEC as well. POSTSLAUGHTER INTERVENTIONS Meat carcasses are difficult to decontaminate because of their shape and structure. Most treatments require physical contact with the carcass surface and an even coverage of the surface. Because carcasses have irregular shapes and surfaces, the possibility exists that one part of the carcass will be overexposed to the treatment, whereas another part may be underexposed to the treatment. In the United States, beef processors have invested in a multiplehurdle system of sequential interventions at various processing steps, including hide washing, steam-vacuuming, trimming, carcass washing, and subprimal treatments with various compounds, to ensure the safety of their products. Studies that have evaluated the effectiveness of sequential, multiple-hurdle intervention systems to improve meat safety have confirmed their efficacy (Bacon et al., 2000; Arthur et al., 2004). The use of 2 or more food safety technologies in sequence may achieve a synergistic effect, or at least an additive effect. Arthur et al. (2004) demonstrated that by minimizing the deposition of bacteria onto the carcass and using subsequent effective food safety technologies, processors can maintain E. coli O57 populations below detectable levels on all the carcasses tested after chilling. Many interventions that are effective in reducing of E. coli O57:H7 are also likely to be effective for non-o57 STEC. The use of hot water, lactic acid, bromine compound washes, and steam to clean carcasses effectively reduces contamination with E. coli O57:H7 (Koohmaraie et al., 2005; Bosilevac et al., 2006; Kalchayanand et al., 2009). Some of these intervention techniques were found to be similarly effective against O26:H and O:H8. (Cutter and Rivera-Betancourt, 2000). Additional compounds are currently being evaluated. Solutions of lactic acid (2 to 4%) are the most frequently used interventions in commercial processing plants for both beef and lamb dressing. Other compounds, such as peroxyacetic acid and acidified sodium chlorite, are being evaluated for their effectiveness in reducing non- O57 STEC inoculated on fresh beef. These compounds reduce populations of serotypes O26 and O similar to the effects on E. coli O57:H7 (Table 4). However, the effectiveness in reducing pathogens depended on the antimicrobial compounds used, and it increased in the following order: lactic acid > peroxyacetic acid > acidified sodium chlorite (Table 4). Acid decontamination of beef carcasses may lead to the development of acid-resistant STEC strains. There is the potential for sublethal inactivation of pathogens, which would allow the organism to colonize a niche within the plant environment during treatment (Samelis et al., 200). The sublethal injured organisms can be controlled by using another hurdle system, such as novel processing technologies. Novel technologies are those technologies that use little heat to preserve the product while minimizing the quality and nutrient losses. Examples include high hydrostatic pressure processing, pulsed electric field, high-intensity light, electrolyzed water treatment, ultrasonics, and irradiation. High hydrostatic pressure processing and ultraviolet light are the most promising technologies because there Table 4. Populations (log colony-forming units/cm 2 of serotype) of non-o57 Shiga toxin-producing Escherichia coli (STEC) and E. coli O57:H7 following antimicrobial interventions Treatment O26 O O57 Control LA Control ASC Control POA LA = lactic acid (adapted from Cutter and Rivera-Betancourt, 2000); ASC = acidified sodium chlorite (,000 ppm, unpublished data); N. Kalchayanand (US Department of Agriculture, Agricultural Research Service, Roman L. Hruska US Meat Animal Research Center, Clay Center, NE); POA = peroxyacetic acid (200 ppm, unpublished data). N. Kalchayanand (US Department of Agriculture, Agricultural Research Service, Roman L. Hruska US Meat Animal Research Center, Clay Center, NE). American Meat Science Association 5

6 are few barriers to approval by regulatory authorities, no special labeling requirements because no chemicals are used, and, if used appropriately, no changes to texture or flavor of the product. Ultraviolet light causes permanent cross-links to form in the microbial DNA, preventing the cell from carrying out its normal functions (Sastry et al., 2000). Ultraviolet light reduced Salmonella inoculated on chicken breasts by.2 log (Chun et al., 200). Pulsed ultraviolet light has been used to inactivate E. coli O57:H7 and Listeria monocytogenes on salmon fillets (Ozer and Demirci, 2006). About a -log reduction was achieved without a detrimental effect on product quality. High hydrostatic pressure processing works by submerging packaged food in a liquid medium (usually water) in an enclosed vessel. The pressure within the vessel is increased by pumping more liquid into the pressure vessel. Pressures of 00 to 600 MPa have been explored as potential food safety treatments for meat. The effects of extreme pressure on microorganisms are not fully understood, but substantial reductions of E. coli O57:H7 and Salmonella (>5 log cycles) have been demonstrated (Alpas et al., 999). Microbial reductions on fresh meat are enhanced when high-pressure treatment is combined with mild heating or chilling, but color changes were observed after 0 min of treatment. The use of pulsed high pressure can be more effective than a continuous single application, so treatment times can be reduced (Hayakawa et al., 994). Recently, Cargill adopted high hydrostatic pressure processing as an intervention hurdle in its raw ground beef patty production. In summary, non-o57 STEC are commonly associated with ruminants and are found throughout the processing steps during slaughter. Although some non-o57 STEC may not be virulent, several strains have been found with the same serotypes and virulence genotypes as those determined to cause human disease. Many studies have demonstrated the effect of various intervention technologies on E. coli O57:H7, but there is a limited dataset to confirm similar effects on non-o57 STEC. Although it is likely that non-o57 STEC will respond similarly to antimicrobial interventions, non-o57 STEC are composed of many serotypes. Thus, they have the potential to present some currently unappreciated resistance to the interventions and processes in place to control E. coli O57:H7 in commercial beef processing plants. Therefore, further evaluation of non-o57 STEC responses to interventions is needed. REFERENCES Aarestrup, F. M., S. E. Jorsal, P. Ahrens, N. E. Jensen, and A. Meyling Molecular characterization of Escherichia coli strains isolated from pigs with edema disease. J. Clin. Microbiol. 35: Alpas, H., N. Kalchayanand, F. Bozoglu, A. Sikes, C. P. Dunne, and B. Ray Variation in resistance to hydrostatic pressure among strains of food-borne pathogens. Appl. Environ. Microbiol. 65: Anderson, R. C., M. 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