Shiga toxin-producing Escherichia coli.

CHAPTER THREE

Shiga Toxin-Producing Escherichia coli☆ James L. Smith, Pina M. Fratamico1, Nereus W. Gunther IV USDA, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Diseases Caused by STEC 2.1 Shiga toxin 2.2 Disease course 2.3 Management of patients with bloody diarrhea and HUS 3. Locus of Enterocyte Effacement and Other Virulence Genes 4. Combatting Acidic Conditions 4.1 Acid-resistance mechanisms 4.2 Chaperone-based AR 4.3 Hydrogenase-3-based AR 4.4 Dps-based AR 5. Iron Acquisition 6. Antimicrobial Drug Resistance 7. Ecology 8. Epidemiology 8.1 Incidence 8.2 Transmission 8.3 Geographic distribution 8.4 Age, sex, host factors 9. Prevention of STEC Colonization and Shedding in Cattle 10. Prevention of STEC Contamination of Meat and Dairy Products 11. Prevention of STEC Contamination of Produce 12. Detection, Isolation, and Identification of STEC 13. Comparative Genomics of O157:H7 and Non-O157 STEC 14. Stress Responses 14.1 Cross-protection 14.2 General stress response 14.3 Cold stress 14.4 Heat stress

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☆ Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

Advances in Applied Microbiology, Volume 86 ISSN 0065-2164 http://dx.doi.org/10.1016/B978-0-12-800262-9.00003-2

2014 Published by Elsevier Inc.

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14.5 Acid stress 14.6 Osmotic stress 15. Cell-to-Cell Communication Systems in E. coli 15.1 Intraspecies communication 15.2 Interspecies communication 15.3 Interkingdom communication 15.4 Miscellaneous types of communication 16. Conclusions References

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Abstract In the United States, it is estimated that non-O157 Shiga toxin-producing Escherichia coli (STEC) cause more illnesses than STEC O157:H7, and the majority of cases of non-O157 STEC infections are due to serogroups O26, O45, O103, O111, O121, and O145, referred to as the top six non-O157 STEC. The diseases caused by non-O157 STEC are generally milder than those induced by O157 STEC; nonetheless, non-O157 STEC strains have also been associated with serious illnesses such as hemorrhagic colitis and hemolytic uremic syndrome, as well as death. Ruminants, particularly cattle, are reservoirs for both O157 and non-O157 STEC, which are transmitted to humans by person-to-person or animal contact and by ingestion of food or water contaminated with animal feces. Improved strategies to control STEC colonization and shedding in cattle and contamination of meat and produce are needed. In general, non-O157 STEC respond to stresses such as acid, heat, and other stresses induced during food preparation similar to O157 STEC. Similar to O157:H7, the top six non-O157 STEC are classified as adulterants in beef by the USDA Food Safety and Inspection Service, and regulatory testing for these pathogens began in June 2012. Due to the genetic and phenotypic variability of non-O157 STEC strains, the development of accurate and reliable methods for detection and isolation of these pathogens has been challenging. Since the non-O157 STEC are responsible for a large portion of STEC-related illnesses, more extensive studies on their physiology, genetics, pathogenicity, and evolution are needed in order to develop more effective control strategies.

1. INTRODUCTION Shiga toxin-producing Escherichia coli (STEC), including E. coli serotype O157:H7 and non-O157 serogroups, are major food-borne pathogens worldwide. There is an increasing awareness that several of the non-O157 STEC serogroups are emerging as important pathogens associated with sporadic cases of disease, as well as outbreaks of food-borne illnesses (Johnson, Thorpe, & Sears, 2006; Kaspar, Doyle, & Archer, 2010). The O157: H7/NM serotypes are well-known pathogens that can cause diarrhea,

Shiga Toxin-Producing Escherichia coli

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hemorrhagic colitis, hemolytic uremic syndrome (HUS), and death. Many non-O157 STEC serogroups have been identified; however, not all of them have been shown to cause illness. Hale et al. (2012) estimated that the annual number of illnesses due to STEC in the United States was 231,157 cases. STEC O157 caused 40.3% of the domestically acquired STEC illnesses, whereas the non-O157 serogroups were responsible for 59.7% of the cases. Scallan et al. (2011) estimated that STEC serogroups cause 175,905 food-borne infections per year in the United States. STEC O157 was responsible for 35.9% of these food-borne infections, whereas non-O157 STEC was responsible for 64.1%. Scallan et al. (2011) further estimated that 68% of the domestic O157 infections are food-borne, whereas 82% of nonO157 infections are food-borne. Thus, it is estimated that non-O157 STEC are responsible for a larger portion of total STEC infections in the United States compared to STEC O157. STEC infections represent a notable economic burden due to costs related to medical care, loss of productivity, decrease in the quality of life, and death. The cost of each case of STEC O157 illness has been estimated to be $10,446 (2010 dollars); however, the cost for each case of non-O157 STEC illness is estimated at $1764 (Scharff, 2012). The cost for non-O157 illness is lower because medical care is estimated to be approximately eightfold less than that for illness due to O157, and there is no estimate of deaths due to infection by non-O157 STEC (death accounts for 78% of the cost of an O157 illness). Recently, Marks, Tohamy, and Tsui (2013) have estimated that for the period of 2005 through 2010, the mean annual number of cases of non-O157 STEC infection was 117,712 (range 50,624–239,716) with an estimated cost of $449 (range $231–1007) per illness. Thus, the total annual cost of illnesses due to non-O157 STEC is $51,161,000 (range $19,490–122,156). Marks et al. (2013) estimated one death and 100 hospitalizations per 34,000 non-O157 STEC illnesses. STEC O26, O45, O103, O111, O121, and O145 constitute approximately 75% of the non-O157 STEC serogroups isolated from cases of illness in the United States for the years 1983–2002 (Brooks et al., 2005). These serogroups are considered to be adulterants if present in beef (Anonymous, 2011). In a study of STEC infections in New Mexico (United States) in 2004–2007, non-O157 STEC accounted for 64% of the infections (71 of 111 cases) (Lathrop, Edge, & Bareta, 2009). Serogroups O26 and O111 accounted for 18% and 13%, respectively; the remainder of the non-O157 STEC infections was due to STEC O46, O91, O103, O121, and O177 (Lathrop et al., 2009). For the years 2000–2009, the most

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common non-O157 STEC serogroups (46.4% of the total) isolated from Swiss patients were O26, O103, O121, and O145 (Ka¨ppelli, Ha¨chler, Giezendanner, Beutin, & Stephan, 2011). For the Brussels-Capital region of Belgium, 2008–2010, of 107 non-O157 STEC serogroups isolated from patients, 39.3% belonged to serogroups O26, O63, O111, and O146 (Buvens et al., 2012). Hiroi et al. (2012) determined the serotypes of 138 STEC isolates from Japanese patients from the Shizuoka Prefecture during the period 2003–2008. STEC O157 accounted for 73.2% of the isolates, whereas 20.3% belonged to serogroups O26, O111, and O121 (Hiroi et al., 2012). STEC O157 constituted 18% of 71 isolates from patients in Germany, 1999–2004, whereas 42% of the STEC isolated from German patients consisted of the non-O157 serogroups O26, O91, O103, and O145 (thus, 82% of the isolates were non-O157 STEC) (Werber, Beutin, Pichner, Stark, & Fruth, 2008). STEC strains isolated from patients in Denmark (n ¼ 312) from 2003 to mid-2005 consisted of 50 serogroups and 75 serotypes; 25.7% belonged to serogroup O157 and 41.9% belonged to serogroups O26, O103, O111, O117, O128, O145, and O146 (Nielsen, Scheutz, & Torpdahl, 2006). Thirty-two non-O157 STEC isolated from human stools in Manitoba, Canada consisted of 10 serogroups with 56.3% of the isolates belonging to serogroups O26, O103, and O121 (Thompson, Giercke, Beaudoin, Woodward, & Wylie, 2005). In a survey of non-O157 STEC outbreaks reported in the world literature, the most frequent cause of illness has been associated with serogroup O26 followed by O111 and 0103 (Bettelheim, 2007). Thus, surveys indicate that the nonO157 STEC strains make up a large portion of the STEC isolated from patients in developed countries with the interesting exception of Japan, where O157 is the major STEC serogroup.

2. DISEASES CAUSED BY STEC The individual who ingests food contaminated with STEC will become infected and may have symptoms consisting of watery diarrhea, abdominal pain, fever, and vomiting. Hemorrhagic colitis (bloody diarrhea) is found in 90% of patients, and HUS is seen in 5–15% of patients. In the absence of HUS, there is spontaneous resolution of symptoms in most patients. HUS is characterized by acute kidney failure, microangiopathic hemolytic anemia (damage of small blood vessels with destruction of red blood cells), and thrombocytopenia (decrease of platelets); long-term sequelae may result. The kidneys are frequently attacked but the central


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nervous system, lungs, pancreas, and heart may also be affected. Children under 5 years of age and the elderly are susceptible to the more serious complications induced by HUS (Gyles, 2007; Tarr, Gordon, & Chandler, 2005).

2.1. Shiga toxin STEC produce two bacteriophage-encoded Shiga toxins: Stx1 (virtually identical to the Stx produced by Shigella dysentereriae) and Stx2, which has ca. 60% sequence homology to Stx1. Stx1 and Stx2 are AB5-type toxins. The B-pentamer of the holotoxin binds to globotriaosylceramide (Gb3) present on host microvascular endothelial cell surfaces (kidney, intestine, brain). The expression of Gb3 is high in renal glomerular endothelial cells of humans allowing the binding of Shiga toxins followed by endocytosis of the toxin (Ivarsson, Leroux, & Castagner, 2012). The toxin is transported to the Golgi apparatus and endoplasmic reticulum. The N-terminal A1 domain subunit is cleaved from the C-terminal A2 domain (the domain attached to the B-pentamer) by a protease. A disulfide bond is also reduced leading to full release of the A1 subunit, and the A1 subunit enters the cytosol via chaperone-mediated transfer. The A1 subunit is an N-glycosidase that acts on the 28S RNA of the 60S ribosomal subunit leading to the inhibition of protein synthesis and apoptosis of endothelial cells, particularly those of the kidneys (Ivarsson et al., 2012; Karch, Tarr, & Bielaszewska, 2005). The renal glomerular lesions associated with HUS are due to damage of the endothelial cells. The cells swell and detach from the basement membrane, fibrin thrombi form, and there is narrowing of the capillary lumen. The narrowing leads to a reduced blood supply to glomeruli with loss of kidney function (Gyles et al., 2007). Orth et al. (2009) have demonstrated that purified Stx2 activates the alternative pathway of the complement system and binds to the fluid phase complement regulator, factor H. Activation of the complement cascade and immobilization of the regulator, factor H, results in uncontrolled complement activation leading to complement-induced renal injury. Thus, Stx2 can directly damage the kidney but can also indirectly cause damage through uncontrolled complement activation. A number of variant Shiga toxins are associated with the STEC; Stx1 consists of three variants (stx1a, stx1c, and stx1d), whereas Stx2 has seven variants (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g) (Scheutz et al., 2012). Stx2a is a more potent toxin than Stx1. The LD50 (in mice) of Stx1 is >1000 ng, whereas the LD50 of Stx2a is 6.5 ng, indicating that Stx2a is a more virulent toxin than Stx1 (Fuller, Pellino, Flagler, Strasser, & Weiss,

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2011). A food-borne outbreak caused by E. coli O104:H4 affected over 4000 people in Germany and other countries in 2011, and ca. 23% of cases developed HUS (Beutin & Martin, 2012). The Shiga toxin produced by this strain was Stx2a. STEC O157:H7 was associated with a food-borne outbreak in which 13 individuals were affected, and there were 8 cases of HUS, a surprisingly large number of HUS cases (Soborg et al., 2013). The O157:H7 strain had the gene profile of stx1a and stx2a. Bielaszewska et al. (2013) described 272 strains of STEC O26:H11/H isolated in Europe during the years 1996 through 2012. The stx1a gene was present in 39.3% (107/272), stx2a was present in 51.1% (139/272), and both genes were present in 9.6% (26/272) of the strains. Bielaszewska et al. (2013) further demonstrated that of 107 O26 STEC strains carrying stx1a, only 9 (8.4%) caused HUS and the remainder caused only blood diarrhea. Of 26 strains carrying both sts1a and stx2a, 14 (53.8%) were responsible for HUS and 12 strains caused only bloody diarrhea. Of 139 strains carrying stx2a, 104 (74.8%) strains caused HUS and the remainder caused only bloody diarrhea. The data obtained by Beutin and Martin (2012), Soborg et al. (2013), and Bielaszewska et al. (2013) suggest that Stx2a is strongly associated with the induction of HUS.

2.2. Disease course The infectious dose for STEC O157:H7 is low and is considered to be fewer than 100 organisms. After a short incubation period, generally 3–4 days, the patient develops watery diarrhea accompanied by abdominal cramping pain. The watery diarrhea becomes bloody in ca. 90% of cases. Antibiotics or antimotility agents should not be administered due to increased risk of HUS (Boyer & Niaudet, 2011; Karch et al., 2005). Five to 13 days after onset of bloody diarrhea, HUS is seen in 5–15% of patients, which presents with thrombocytopenia and microangiopathic hemolytic anemia with renal insufficiency. A few patients have thrombocytopenia with or without anemia but no renal involvement. Other syndromes occur in a significant portion of patients with HUS including central nervous system involvement (irritability, lethargy, stupor, coma, seizures, and strokes), cardiac dysfunction, intestinal complications (bowel perforation, necrosis, and pancreatitis), and pulmonary problems (fluid overload, pleural effusions (fluid in the lungs)), and adult respiratory distress syndrome (Boyer & Niaudet, 2011; Karch et al., 2005). Patients with HUS caused by STEC O157:H7 infection require dialysis more often than patients suffering from HUS induced by


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non-O157 STEC strains. Deaths have been reported more frequently with O157:H7-induced HUS but also occur with non-O157 STECinduced HUS. Post-HUS sequelae include chronic renal complications, diabetes mellitus, neurological disorders, colonic strictures, hypertension, urinary abnormalities, and biliary stones (Boyer & Niaudet, 2011; Karch et al., 2005).

2.3. Management of patients with bloody diarrhea and HUS In patients with STEC infections, intravenous rehydration and fluid maintenance are necessary to provide protection to the kidneys. Antibiotics should not be given to patients with a suspected STEC infection or with bloody diarrhea since antibiotics are associated with a higher risk of HUS in both children and adults. The effect with administration of antibiotics may be due to bacterial lysis and release of Shiga toxin or may be due to induction of the Stx bacteriophages with subsequent production of toxin. Antimotility agents or narcotics should not be given to STEC-infected patients or to those with bloody diarrhea because these agents are associated with increased HUS or neurological effects induced by HUS. Nonsteroidal anti-inflammatory agents diminish renal blood flow and should not be given to STEC-infected patients (Tarr et al., 2005). Attempts to prevent the progression from bloody diarrhea to HUS have not been successful. Treatment of Stx-induced HUS includes supportive schemes such as management of anemia, bleeding, fluid and electrolyte imbalance, and hypertension. Patients should be monitored for signs of fluid overload due to instability of their renal and vascular status. If hypertension appears, fluid intake should be restricted; vasodilators are the preferred agent for treatment of hypertension. Erythrocyte transfusion is necessary in approximately 80% of patients with HUS. Dialysis in HUS is necessary if potassium or serum urea levels are high and if there is acidosis or hypertension that is not responding to medication (Boyer and Niaudet, 2011; Tarr et al., 2005). Boyer and Niaudet (2011) suggest that Stx-receptor analogs (including Gb3 mimics) and Stx-neutralizing monoclonal antibodies may have potential use in the prevention of HUS.

3. LOCUS OF ENTEROCYTE EFFACEMENT AND OTHER VIRULENCE GENES Ingested STEC remain extracellular in the lumen of the intestinal tract; however, the intracellular environment of the host cell is accessed

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and manipulated by bacterial LEE (locus of enterocyte effacement) and nonLEE effectors injected into the host cell by the type three secretory system (T3SS). The effectors have a variety of functions including hemolysis, phagocytosis inhibition, host lymphocyte response repression, cytotoxicity, inhibition of iron transport to the cell, destruction of microvilli, attaching and effacing (AE) lesions on enterocytes, and action polymerization (Bolton, 2011; Wong et al., 2011). Therefore, injection of the microbial effectors into the host cell enables the bacteria to colonize, multiply, and to cause disease. There are 41 genes located on the LEE pathogenicity island organized in five polycistronic operons, LEE1 through LEE5, two biocistronic operons, and four monocistronic entities (Lara-Ochoa, Oropeza, & Huerta-Saquero, 2010). LEE1, -2, and -3 operons encode the T3SS which translocates bacterial effector proteins into the enterocyte. LEE4 contains esp (EPEC secreted proteins) genes; espADB encodes translocator proteins that form a channel through which the T3SS delivers effector proteins to the host cell. Bacterium-host cell adhesion genes are located in LEE5 and include the eae gene which encodes an outer membrane adhesin (termed intimin). The translocated intimin receptor (i.e., Tir) is encoded by tir, and cesT encodes the Tir chaperone. ler, the first gene in LEE1, encodes Ler, the master regulator of the LEE pathogenicity island (Lara-Ochoa et al., 2010). When the bacterial cell contacts an intestinal cell, the LEE-encoded translocators, EspB and EspD, are inserted into the host cell plasma membrane and form a translocation pore. The LEE-encoded Tir is translocated into the host cell where it integrates into the plasma membrane. A portion of Tir binds to intimin at the surface of the bacterial outer membrane. Once Tir binds intimin, the bacterium is intimately bound to the enterocyte surface. The intimate binding to the intestinal epithelial cells leads to a localized destruction of the microvilli (AE lesions) at the enterocyte brush border and to the polymerization of actin and its accumulation just below the attached bacteria. The polymerized actin forms a cup-like structure, the pedestal, through which the bacterium is intimately attached to the enterocyte (Frankel & Phillips, 2010; Lara-Ochoa et al., 2010). It has been suggested that actin pedestal formation acts as an antiphagocytic mechanism (Campellone, 2010). Therefore, AE lesion formation requires the expression of the LEE pathogenicity island genes and is triggered by Tir. Does LEE play a role in virulence of non-O157 STEC? Mingle et al. (2012) characterized O157:H7 and non-O157 STEC submitted to the New York State Public Health Laboratory during a 6-year period


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(2005–2010). Non-OI57 STEC were identified from 72% of 592 specimens, whereas O157 STEC was only found in 28%. The incidence of the stx1, stx2, hlyA, and eaeA genes for 156 isolates of non-O157 STEC and 132 isolates of O157:H7 STEC is presented in Table 3.1. One hundred percent of the O157:H7 STEC isolates were positive for eaeA and hlyA and 98% were positive for the stx2 gene (Table 3.1). STEC strains isolated from patients with hemorrhagic colitis or HUS are frequently positive for the stx2, eaeA, and hlyA genes (Monaghan et al., 2011). Only 26% of the non-O157 STEC isolates was positive for the stx2 gene (Table 3.1), suggesting that HUS is less likely during an infection with a non-O157 STEC strain. The hlyA and eaeA genes were also found less often in the non-O157 STEC strains (Table 3.1). Since the eae gene is located on the LEE5 cistron, the presence of the gene indicates that the STEC strain has the LEE pathogenicity island. The data shown in Table 3.1 indicate that 76% of 156 strains of non-O157 STEC harbored the LEE pathogenicity island (Mingle et al., 2012). Mingle et al. (2012) determined the incidence of the stx1, stx2, hlyA, and eaeA genes in the six non-O157 STEC serogroups (O26, O45, O103, O111, O121, O145) recently declared as adulterants in beef compared to O157:H7 STEC (Table 3.2). The non-O157 STEC strains were similar to the O157:H7 STEC in that 96–100% of the non-O157 STEC were positive for both hlyA and eaeA genes. The stx2 gene was present in the O121 isolates (stx1 was not present); however, in the other non-O157 strains, the presence of stx2 ranged from 0% to 33%. Thus, the major Shiga toxin gene in the non-O157 serogroups was stx1 (Table 3.2), with a decreased presence of stx2 in the non-O157 strains (Mingle et al., 2012). The study by Mingle et al. (2012) was limited by the small number of non-O157 strains utilized. Other putative virulence factors include the non-LEE encoded effectors (nle genes) that encode translocated substrates of the type III secretion system, proteases, including KatP, EspP, and StcE, fimbrial and non-fimbrial Table 3.1 Incidence of virulence genes in 288 O157 and non-O157 STEC isolates submitted to New York State Public Health Laboratory Percent positive by real-time PCR Serogroup (n)

stx1

stx2

hlyA

eaeA

Non-O157 (156)

81

26

86

76

O157:H7 (132)

33

98

100

100

Modified from Mingle et al. (2012).

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Table 3.2 Incidence of stx1, stx2, hlyA, and eaeA genes in six serogroups of non-O157 STEC strains as compared to O157:H7 STEC Percent positive by real-time PCR Serogroup (n)

stx1

stx2

hlyA

eaeA

O26 (8)

100

12

100

100

O45 (34)

100

0

100

97

O103 (9)

100

11

100

100

O111 (49)

100

12

96

96

O121 (4)

0

100

100

100

O145 (6)

67

33

100

I00

O157:H7 (132)

33

98

100

100

Modified from Mingle et al. (2012).

adhesins, including ToxB, Saa, LpfA, Iha, Lfp, and Sfp, and subtilase cytotoxin (SubAB), as well as others (Bolton, 2011; Coombes et al., 2008; Gyles, 2007; Melton-Celsa, Mohawk, Teel, & O’Brien, 2012).

4. COMBATTING ACIDIC CONDITIONS 4.1. Acid-resistance mechanisms The STEC have three major acid-resistance (AR) mechanisms that protect the cells against exposure to pH 2.0 to 2.5. AR-1 (oxidative system) is induced when STEC strains are grown to the stationary phase in glucosefree Luria-Bertani (LB) broth buffered to pH 5.5. The acid-adapted cells survive exposure to pH 2.5 when diluted into minimal medium, whereas cells grown to the stationary phase in unbuffered LB medium (final pH ca. 8.0) are inactivated when diluted into minimal medium (Foster, 2004). The stationary phase alternative sigma factor, sS (RpoS), and the global regulatory protein, CRP (cAMP receptor protein), are required for induction of AR-1. CRP involvement indicates that AR-1 is repressed by glucose (Foster, 2004). AR-2, the glutamate decarboxylase system, requires glutamate, one of the two glutamate decarboxylase genes (gadA or gadB), and the gadC gene, glutamate/g-aminobutyric acid antiporter, to protect cells against extreme acid environments. Glutamate is transported into the cell via the antiporter GadC and is decarboxylated by GadA or GadB to g-aminobutyric acid with the uptake of a proton. The g-aminobutyric


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acid is transported out of the cell via GadC in exchange for a glutamate entering the cell (Foster, 2004). AR-3, the arginine decarboxylase system, requires arginine, the arginine decarboxylase gene (adiA), the arginine/ agmatine antiporter gene (adiC), and the regulator cysB. Arginine is transported into the cell via the AdiC antiporter and is decarboxylated by AdiA to agmatine with uptake of a proton. Agmatine is transported out of the cell via AdiC in exchange for arginine entering the cell (Foster, 2004). Other AR systems in E. coli include the lysine and ornithine decarboxylase systems, but their role in STEC AR has not been determined (Zhao & Houry, 2010). Thus, the induction of the AR-1, -2, and -3 systems enables E. coli strains to resist the extreme acidic conditions encountered during transit through the mammalian stomach, as well as to the prolonged exposure to more mild acid environments of the host gut. The most efficient AR system is AR-2, the glutamate decarboxylase system. Other than the gadBC operon, the most important genes involved in resistance to acidic conditions are found on the acid fitness island (AFI), a 15 kb, genomic region on the E. coli chromosome which is repressed by histone-like nucleoid-structure protein and controlled by RpoS (stationary phase sS factor) (Tramonti, De Canio, & De Biase, 2008). The 12-gene island is present in non-O157 STEC strains O26: H11, O103:H2, and O111.H; however, the AFI is larger in O157:H7 strains due to the insertion of O-island 140 (Carter et al., 2012). gadA and many of the regulatory genes involved in AR are located on the AFI. The AR-2 system in E. coli is exceedingly complex with over 20 proteins and three small noncoding RNAs regulating the Gad system. This complex system is discussed in detail in the monograph of Zhao and Houry (2010). Bergholz and Whittam (2007) studied the effect of acidity on STEC O157:H7, O26:H11, and O111:H8 strains in a model stomach system. The model stomach system consisted of synthetic gastric fluid mixed with turkey dinner baby food with a final pH of 2.5. Stationary phase bacteria were stored for 24 h at pH 3.5 before inoculation into the model stomach system, and the survival rate (log10 decrease in cell numbers per hour) of the STEC strains was determined after 3 h at 37 C. The mean survival rate of STEC O157 (n ¼ 14) stored at 4 C for 24 h was 3.2-fold higher than cells stored at 22 C. The mean survival rate of the non-O157 STEC (n ¼ 12) was 1.6-fold higher when cells were stored at 4 C before exposure to 2.5 gastric fluid as compared to cells stored at 22 C. The data indicate that the nonO157 STEC were less resistant to gastric acid conditions as compared to E. coli O157 and that prior storage of the STEC strains at 22 C decreased AR (Bergholz & Whittam, 2007). When Bergholz and Whittam (2007)

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stored the STEC strains for 24 h at pH 3.5 or 7.0, they found that acidadapted STEC were more resistant to gastric acid conditions than cells adapted to pH 7.0. Acid-adapted O157 strains (n ¼ 4) had a mean survival rate 6.1-fold higher as compared to cells adapted pH 7.0. Non-O157 STEC (four strains) adapted to pH 3.5 had a survival rate 2.3-fold higher than cells adapted to pH 7.0. Both O157 and non-O157 STEC showed better survival in the low pH model stomach system if they were adapted to low pH prior to exposure to gastric acid (Bergholz & Whittam, 2007). In addition, Bergholz and Whittam (2007) showed that the transcription levels of gadA and gadB were ca. three to fourfold higher in the O157 STEC as compared to nonO157 STEC. Thus, the greater activity of the glutamate decarboxylase system may explain the ability of E. coli O157 to withstand gastric acidity better than the non-O157 STEC strains examined. However, it is apparent that studies concerning the effect of gastric acid on non-O157 STEC are needed. Utilizing 30 E. coli O157:H7, 18 O26:H1, 4 O111:H8, and 14 O121: H19 STEC strains, Large, Walk, and Whittam (2005) studied the effects of the oxidative AR-1, glutamate decarboxylase (AR-2), and arginine decarboxylase (AR-3) systems on these STEC when they were exposed to low pH. The data obtained by Large and her coworkers are presented in Table 3.3. The AR-1was less effective in protecting STEC against acidic conditions as compared to the AR-2 or AR-3 systems, and the AR-2 system provided the most protection to STEC under extreme acidic conditions when compared to the AR-1 or AR-3 systems. With all three systems, the percent killing/h was always higher with the O157:H7 STEC as compared to the O26/O111 (these two STEC belong to the same clonal group based on multilocus sequence analysis) or O121 STEC strains, suggesting that O157:H7 are not exceptionally resistant to extreme acidic conditions in comparison to non-O157 STEC strains when utilizing the AR-1, -2, Table 3.3 The effect of the oxidative, glutamate decarboxylase, arginine decarboxylase systems on the killing of STEC serotypes on exposure to low pH STEC Oxidative system Glutamate decarboxylase Arginine decarboxylase serogroup/ (AR-1), pH 2.5 system (AR-2), pH 2.0 system (AR-3), pH 2.5 serotype (percent kill/h) (percent kill/h) (percent kill/h)

O157:H7

66.9

18.7

36.9

O26/O111 47.5

4.5

27.6

10.9

24.1

O121

54.3

Modified from Large et al. (2005).


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or -3 systems (Large et al., 2005). From the results obtained by Bergholz and Whittam (2007) and Large et al. (2005), it is not clear that O157:H7 is more acid resistant than non-O157 strains. However, their studies included very few non-O157 strains, and a larger study is necessary. Another potential AR mechanism in E. coli is the deamidation of glutamine to glutamate by glutaminase (YbaS) with the release of NH3. Lu et al. (2013) have demonstrated that glutamine is plentiful in many food products. Only glutamate and glutamine greatly enhanced the survival of E. coli (K-12 strain MG1655) during acid shock at pH 2.5; deletion of the ybaS gene (encodes for glutaminase) abolished glutamine potentiation of survival of E. coli during acid shock. The NH3 released by the action of the YbaS enzyme on the transformation of glutamine to glutamate neutralizes protons by forming NH4 þ and raises the intracellular pH. Lu et al. (2013) further demonstrated that the amino acid antiporter, GadC, is responsible for the uptake of glutamine from the environment and its transport into the cell. The glutamate produced from glutamine is transformed to g-aminobutyric acid (with uptake of a proton) by the action of GadA/GadB and transported out of the cell via GadC. The YbaS-GadC system may work in tandem with the decarboxylation of glutamate via the GadA/GadB system with GadC acting as an amino acid transporter specific for glutamine, glutamate, and g-aminobutyric acid (Lu et al., 2013). The ybaS gene is present in STEC O157:H7 strain EDL933 (Dong & Schellhorn, 2009), and it is probable that the gene is found in other STEC strains, as well.

4.2. Chaperone-based AR Proteins that function in the cytoplasm are protected against acid stress by the AR-2 and AR-3 pathways (Zhao and Houry, 2010). Periplasmic proteins (those proteins that are functional in the space between the cytoplasmic membrane and the outer cell membrane) are protected against acid stress by a different AR system. Nonspecific transporters such as porins are present in the outer membrane, which allow small molecules, including protons, to freely diffuse into the periplasmic space. Therefore, during acid stress, the periplasmic proteins are directly exposed to a pH that is more acidic than the cytoplasm, thus making the periplasmic proteins more susceptible to acid-induced denaturation and aggregation as compared to cytoplasmic proteins (Hong, Wu, Fu, & Chang, 2012; Zhao and Houry, 2010). Two periplasmic chaperones encoded by the genes hdeA and hdeB are key factors in AR in the periplasmic proteins of STEC. hdeA and hdeB are expressed

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under normal conditions but are strongly induced at low pH, and both genes are found on the AFI. HdeA and HdeB function at a low pH by dissociating into monomers that bind periplasmic proteins and prevent their aggregation into insoluble proteins (Carter et al., 2012; Hong et al., 2012). Carter et al. (2012) studied hdeA/hdeB mutants of STEC O157:H7 (two strains) and O145:NM (one strain), as well as E. coli K12 (two strains) and O55:H7 (two strains) strains when exposed to low pH. E. coli strains were grown in LB broth plus 1% NaCl (LBFS) at 37 C to the stationary phase and then diluted into LB at pH 2 (HCl) for 2 h. STEC O157:H7 showed ca. twofold decrease in survival; O55:H7, ca. 1.5-fold decrease; K12, ca. 10-fold decrease; and O145:NM showed ca. 150-fold decrease in survival after 2 h exposure to pH 2 (Carter et al., 2012). When the strains were grown to the stationary phase in LB lacking NaCl at 28 C, O157:H7 demonstrated ca. onefold decrease in survival at pH 2; O55:H7, ca. twofold; K12, ca. 12-fold; and O145:NM showed a ca. 2200-fold decrease in survival with exposure for 2 h at pH 2 (Carter et al., 2012). Thus, the data obtained by Carter et al. (2012) indicated that the genes hdeA/B were not utilized or were played only a minor role in the survival of O157:H7 or O55:H7; the genes were moderately necessary for survival of K12; and hdeA/B played a very important role in the survival of O145:NM under extreme acid conditions. The HdeA and HdeB proteins were detected in E. coli K-12, in serotype O55:H7, and in STEC O145:NM; however, only HdeA was detected in STEC O157:H7. An investigation of single nucleotide polymorphism demonstrated that there was a G!A transition in the O157:H7 hdeB gene; this transition in the putative start codon of the gene indicates that the STEC O157:H7 strain could not produce the HdeB protein (Carter et al., 2012). The G!A shift was found in 20 strains of STEC O157:H7 but not in 12 non-O157 E. coli, including serotype O55:H7 and STEC serotypes O26:H11, O45:H2, O111:H8, O111:H11, O111:HN, and O145: NM. hdeB silencing in STEC O157:H7 suggests that that serotype evolved mechanisms for survival under extreme acid conditions that do not depend on HdeA/B chaperones (Carter et al., 2012).

4.3. Hydrogenase-3-based AR Hydrogenase-3 (hyd-3) along with formate dehydrogenase combine to form the formate hydrogen lyase complex, which under anaerobic (fermentative) conditions breaks down formate to CO2 and 2Hþ. The two protons are converted to H2 (Bagramyan & Trchounian, 2003). Using a △hycE mutant


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(lacks hyd-3) of K-12 W3110, Noguchi, Riggins, Eldahan, Kitko, and Slonczewski (2010) demonstrated ca. 85% loss of survival of the organism when subjected to pH 2 for 2 h at 37 C anaerobically, whereas under the same conditions, the wild-type strain showed only ca. 40% loss of survival. Noguchi et al. (2010) concluded that proton conversion to H2 by Hyd-3 under anaerobic conditions is required for AR. It is probable that STEC strains utilize the Hyd-3 mechanism to combat acidic conditions anaerobically.

4.4. Dps-based AR Jeong, Hung, Baumler, Byrd, and Kaspar (2008) demonstrated that Dps (DNA-binding protein in starved cells) has a role in acid tolerance of STEC O157:H7. Dps has several roles in E. coli: DNA binding, iron sequestration, and ferroxidase activity. Thus, Dps is involved in iron storage, forms stable complexes with DNA, and diminishes iron-mediated oxidative stress (Calhoun & Kwon, 2010). Exposure of stationary phase cells of O157:H7 to pH 2.0-adjusted LB broth at 37 C for 3 h showed that the survival of a dps mutant was 1000-fold less than that of the wild type (Jeong et al., 2008). They also showed that Dps binds plasmid DNA at pH 2.0 and protected that DNA from acid-induced strand breaks. Thus, STEC have several mechanisms by which they combat acid conditions. The ability to survive and grow in acidic environments ensures that the organisms will be viable in foods deliberately acidified, in fermented foods, and in the host during passage through the gastrointestinal tract.

5. IRON ACQUISITION In most bacteria, iron is an essential macronutrient and acts as a cofactor in a number of important physiological reactions including virulence. In the iron-limited environment of the vertebrate host, bacteria such as the STEC have mechanisms to acquire needed iron from the host’s tightly bound iron reserves (Saha, Saha, Donofrio, & Besterveit, 2012). Examining a number of STEC strains (155 strains of O157:H7/H; 141 strains of nonO157), Kresse et al. (2007) found that all of the strains carried the catechol siderophore enterobactin (also known as enterochelin), an iron-chelator, which binds iron and transports it into the cell. The hydroxamate siderophore aerobactin was absent in STEC O157:H7 (155 strains) and O26: H/H11 (31 strains); however, aerobactin was present in a few nonO157/H strains (24/110) (Kresse et al., 2007). Various aspects of microbial siderophores have been reviewed recently (Saha et al., 2012).

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Law and Kelly (1995) found that 20 STEC O157:H7 strains produced enterohemolysin and the growth of all strains was stimulated by heme and hemoglobin. Of 16 human non-O157 STEC, 11 produced enterohemolysin, and the growth of 5 was stimulated by heme and 3 by hemoglobin (Law & Kelly, 1995). Kresse et al. (2007) found that 145/155 strains of O157:H7/H produced enterohemolysin, and 142 strains utilized heme for growth; 29/31 strains of STEC O26:H/H11 produced enterohemolysin, and 28 utilized heme for growth; 26/28 strains of O103:H2 produced enterohemolysin but none utilized heme for growth; and 42/82 of other non-O157 produced enterohemolysin and 53 strains utilized heme for growth (Kresse et al., 2007). Some E. coli strains carry the high pathogenicity island (HPI) of Yersinia. The irp (encodes for the iron repressible protein) loci on the island encode the siderophore yersiniabactin (Koczura & Kaznowski, 2003). Karch et al. (1999) studied 206 STEC strains isolated from patients for the presence of the HPI and found that the island was present in the genome of 31/31 eae-positive O26:H11/H and 7/12 eae-positive O128:H2/H STEC. The HPI was absent from eae-positive strains of the following STEC: 37 O157:H7/H, 14 O111:H, 13 O103:H2, and 13 O145:H. The data obtained by Kresse et al. (2007), Law and Kelly (1995), and Karch et al. (1999) indicate that all O157 strains use enterobactin to chelate host iron but do not use aerobactin or yersiniabactin, and most O157 strains produce enterohemolysin and are able to use heme and hemoglobin as iron sources (Kresse et al., 2007). While all of the non-O157 strains utilized the siderophore enterobactin (Kresse et al., 2007), a few non-O157 strains also utilized aerobactin (24/141) (Kresse et al., 2007) or yersiniabactin (56/169) (Karch et al., 1999). Two studies indicated that enterohemolysin was produced by 97/141 non-O157 STEC (Kresse et al., 2007) and 122/169 non-O157 STEC (Karch et al., 1999). However, the only mention of the utilization of enterohemolysin products by non-O157 STEC was by Kresse et al. (2007) who indicated that heme was utilized by 61/141 non-O157 STEC. Thus, the number of strains of non-O157 STEC, which can produce enterohemolysin or utilize heme and hemoglobin, appears to be limited in comparison to O157 STEC.

6. ANTIMICROBIAL DRUG RESISTANCE Due to the use (and misuse) of antimicrobial drugs, bacteria have become resistant to these drugs, and indeed, resistance has shown a steady increase over time. Antibiotic-resistant bacteria create health problems in


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clinical situations and have increased significance in farm and companion animals since these animals become reservoirs of drug-resistant bacteria and can pass the organisms to humans through direct contact or via the food chain. In addition, resistant bacteria can be spread from place to place due to global or domestic travel and through the global food markets (Da Silva & Mendonça, 2012). Tadesse et al. (2012) demonstrated that in the United States, antibiotic resistance in E. coli during the period of 1950–2002 for drugs introduced in1936 through 1961 increased, ranging from 24.1% to 40.9%. However, drugs introduced during the period of 1984 through 1987 showed only a smaller increase in resistance ranging from 0.4% to 5.6%. Schroeder, Zhao, et al. (2002) studied antimicrobial drug resistance in E. coli O157 strains isolated from humans, cattle, swine, and food for the years 1985–2000. The isolates were tested against a battery of cephalosporins, penicillins, sulfonamides, and quinolones, as well as against chloramphenicol, gentamicin, and tetracycline. Most of the STEC O157 strains (n ¼ 182) were susceptible to all the antimicrobial drugs; however, 4 were resistant to 1 drug, 16 were resistant to 2 drugs, and 8 strains were resistant to 3 drugs. All STEC O157:H7 isolates were susceptible to cefoxitin, ceftriaxone, ceftiofur, gentamicin, nalidixic acid, ciprofloxacin, and trimethoprim-suflamethoxazole (Schroeder, Zhao, et al., 2002). In addition, Schroeder, Meng, et al. (2002) isolated 196 STEC strains belonging to serogroups, O26, O103, O111, O128, and O145 from humans and animals and determined their resistance to antimicrobial drugs. These serogroups, similar to STEC O157, were resistant to several antimicrobials and some strains showed multiple resistance. Recent studies indicate that an increasing number of STEC serotypes are showing multiresistance to a number of drugs belonging to various antimicrobial drug classes including the penicillins, aminoglycosides, tetracyclines, sulfonamides, and fluoroquinolones (Buvens, Bogaerts, Glupczynski, Lauwers, & Pie´rard, 2010; Ennis, McDowell, & Bolton, 2012; Hiroi et al., 2012; Lee, 2009; Scott et al., 2009; von Mu¨ffling et al., 2007). It is probable that antimicrobial drug resistance will increase in the six important non-O157 STEC serogroups (O26, O45, O103, O111, 0121, O145), as well as in other non-O157 STEC. Antimicrobial drug resistance can be acquired by spontaneous chromosomal mutation or by horizontal gene transfer via uptake of foreign DNA containing drug resistance genes. These genes may be present on plasmids, transposons, integrons, or bacteriophages (Giedraitiene˙, Vitkauskiene˙, Naginiene˙, & Pavilonis, 2011). There are several biochemical mechanisms

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by which an organism becomes resistant to antimicrobial drugs. These mechanisms include drug inactivation by enzymatic modification, target modification, altered outer membrane permeability, activation of efflux pumps, and bypassing antibiotic inhibition by using a different metabolic pathway (Giedraitiene˙ et al., 2011).

7. ECOLOGY STEC are zoonotic food-borne and water-borne pathogens associated with numerous animal species. Ruminants are the major reservoir for STEC O157, as well as the non-O157 STEC (Kaspar et al., 2010). Since STEC are present in the intestinal tract, the feces of animals containing STEC will contaminate any environment with which the animals come in contact. Kaspar et al. (2010) stated that cattle are probably the most important reservoir of non-O157 STEC that cause human illness. Therefore, ingestion of beef, milk, cheese, and other dairy products may lead to outbreaks and sporadic cases of illnesses due to STEC. Oporto, Esteban, Aduriz, Juste, and Hurtado (2008) studied STEC prevalence in 345 herds (17 swine, 122 dairy sheep, 124 beef, and 82 dairy cattle) by rectal fecal sampling during October 2003–May 2005 in Northern Spain. The prevalence rates of non-O157 STEC for the sheep dairy herds were 50.8%, 46.0% for beef cattle herds, 20.7% for dairy cattle herds, and 0.0% for swine herds (Oporto et al., 2008). Worldwide, fecal analyses indicate that the prevalence rates of STEC O157 in beef cattle range from 0.2% to 27.8%, whereas the prevalence rates for non-O157 STEC range from 2.1% to 70.1% (Hussein & Bollinger, 2005b). The number of STEC serotypes present in beef cattle was 261; 44 serotypes had been associated with HUS and 37 other serotypes were known to cause diarrhea and hemorrhagic colitis (Hussein & Bollinger, 2005b). Monaghan et al. (2011) isolated non-O157 STEC from soil and bovine fecal samples from 10 farms in Ireland for a 1-year period (July 2007–July 2008). They identified 107 non-O157 STEC comprising 17 serogroups including O2 (12.1%), O26 (13.1%), O113 (29%), and O168 (9.3%). Masana et al. (2011) isolated 293 non-O157 STEC from carcasses and bovine feces in nine beef abattoirs in Argentina over a 17-month period. Non-O157 STEC serotypes reported as human pathogens throughout the world made up 45.7% of the Argentinian isolates. These serotypes included O8:H19, O15:H27, O22:H8, O22:H16, O79: H19, O82:H8, O91:H21, O103:[H2], O111:NM, O113:H21, O116: H21, O130:H11, O145:NM, O153:H25, O163:H19, O165:NM, O174:


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H21, O178:H19, and O179:H8. The data on the presence of non-O157 STEC in Irish and Argentinian bovine samples indicated a great variety in the STEC serogroups isolated. In a survey involving a large number of studies, Hussein and Bollinger (2005a) found that 155 STEC serotypes were present in beef products, 31 serotypes were associated with HUS and 25 were associated with diarrhea and hemorrhagic colitis. In products such as ground beef, unspecified retail cuts, beef sausage, and whole beef carcasses, the prevalence rates of STEC O157 ranged from 0.01% to 54.2%, and the rates for non-O157 STEC ranged from 1.7% to 62.5% (Hussein and Bollinger, 2005a). In a 24-month survey of ground beef involving 4133 samples from 18 commercial ground beef producers in the United States, Bosilevac and Koohmaraie (2011) demonstrated Shiga toxin genes in 24.3% (1006/4133) of the samples. Three hundred (7.3% of 4133) samples yielded 338 unique nonO157 STEC strains consisting of 99 serotypes. Ten of the isolates (0.24% of 4133 samples) were considered to be potential pathogens based on the presence of specific virulence genes, including eae, subA, and nle genes (Bosilevac & Koohmaraie, 2011). The source of contamination of beef carcasses and beef products is likely the transfer of STEC from cattle hides and the intestinal tract to carcass surfaces during slaughter. The prevalence rate for O157:H7 on cattle hides from the Midwestern United States for a 1-year period was 60.6% and that of non-O157 STEC was 56.3% (BarkocyGallagher et al., 2003). Monaghan et al. (2012) tested 450 beef cattle hides and 450 beef carcasses from three Irish abattoirs during a 12-month period and found that 67% of the hides and 27% of the carcasses were non-O157 STEC-positive. Since the prevalence of STEC in bovine feces can be high, it is not surprising that beef carcasses and meat can become contaminated with STEC from feces and hides. Among 170 STEC isolated from dairy cattle from five dairy farms in Argentina, the most frequently isolated STEC serogroups were O113 (20 isolates), O130 (38 isolates), and O178 (31 isolates) (Fernańdez, Irino, Sanz, Padola, & Parma, 2010). These three serogroups comprised 52.4% of the total STEC isolates. In a survey of the published literature from a number of countries, Hussein and Sakuma (2005) determined that dairy cattle feces demonstrated a prevalence rate for STEC O157 strains ranging from 0.2% to 48.8% and the rate for non-O157 STEC ranged from 0.4% to 74.0%. There were 193 STEC serotypes, and 24 of these serotypes have been isolated from HUS patients. Non-O157 STEC, including pathogenic serotypes, have been isolated from raw cow’s, ewe’s, and goat’s milk, as well

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as from cheeses made from those raw milks (Baylis, 2009). Obviously, the non-O157 STEC can survive the cheese-making process. Hussein and Sakuma (2005) reported that a large number of human infections (396 cases) in various countries were due to ingestion of STEC O157 in products from dairy cattle, including raw milk, ground beef, cheese, yogurt, cream, butter, and contact with dairy animals or manure. However, only a few cases (17 cases) of non-O157 STEC infections were cited by Hussein and Sakuma (2005) that were due to ingestion of raw milk and cheese, or contact with dairy animals or manure. The low numbers of cases related to non-O157 STEC infections are probably due to the failure to isolate and identify non-O157 STEC. Frank, Kapfhammer, Werber, Stark, and Held (2008) demonstrated that there was an association between reported German O157 and non-O157 STEC cases and cattle density. There was a 68% increase in STEC gastroenteritis per 100 additional cattle/km2. This reported relationship between cattle density and incidence of STEC infections may be true for other countries; however, data are lacking. Wild ruminants, red deer, roe deer, chamois, and ibex, carry non-O157 STEC, including serogroups O26, O45, O91, O103, O111, O113, O121, and O145 (Hofer, Cernela, & Stephen, 2012). A total of 52 STEC strains were isolated from the feces of wild ruminants. The stx1d gene variant was present in 21 strains and stx2b was present in 24; the eae and ehxA genes were found in 2 strains and 24 strains, respectively (Hofer et al., 2012). The authors concluded that the STEC strains isolated from wild ruminants did not show gene patterns typical of highly pathogenic strains. Data from outbreaks caused by non-O157 STEC indicate that ingestion of both meat and non-meat products contaminated by non-O157 STEC can induce disease (Kaspar et al., 2010; Mathusa, Chen, Enache, & Hontz, 2010). Any food product derived from wild or domestic ruminants may contain STEC. Water sources that have been contaminated with ruminant feces can also be vehicles of STEC infection. Therefore, STEC-contaminated recreational waters can be a source of infection to swimmers and boaters; contaminated irrigation waters can be a source of STEC in fruits, vegetables, and other produce. It is possible that almost any raw food will contain STEC, and during meal preparation, crosscontamination of a food by a raw food containing STEC may occur if careful hygiene is not practiced (Kaspar et al., 2010; Mathusa et al., 2010). Due to the worldwide dispersal of ruminant animals, it is not surprising that the environment, soil, and water may be contaminated with STEC present in ruminant feces.


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8. EPIDEMIOLOGY 8.1. Incidence Investigations on the incidence of non-O157 STEC as disease agents indicate that these pathogens are a significant cause of STEC-induced illnesses. For example, in Manitoba, Canada, 63% of the total STEC infections were caused by non-O157 STEC (Thompson et al., 2005), 74% in Denmark (Nielsen et al., 2006), 82% in Germany (Werber et al., 2008), 80% in the Netherlands (van Duynhoven et al., 2008), and 59.7% in the United States (Hale et al., 2012). However, non-O157 STEC caused only 27% of the total STEC-induced infections in Ireland in 2008 (Garvey, McKeown, Carroll, & McNamara, 2009). Thus, a limited number of studies suggest that nonO157 STEC are a major cause of Shiga toxin-induced illnesses.

8.2. Transmission The non-O157 STEC are oral-fecal organisms and infections may be acquired by direct contact with an infected person, wild or domestic animals harboring STEC, or animal environments. Visits to farms or petting zoos, as well as contact with household pets have been associated with non-O157 STEC-induced diseases (Kaspar et al., 2010). Hale et al. (2012) have estimated that in the United Sates, 8% (range 4–15%) of the non-O157 STEC infections and 6% (range 3–11%) of O157 are attributable to contact with animals. In institutions such as day-care centers, schools, and senior care facilities, person-to-person spread is a major means of infecting fellow inmates and visitors with the pathogens (Kaspar et al., 2010). Water and food contaminated with animal feces have been shown to transmit non-O157 STEC to humans (Kaspar et al., 2010; Mathusa et al., 2010). In the United States in the years 1999 through 2008, the most common food vehicle associated with non-O157 illness was beef (40%) followed by dairy products (20%) (Batz, Hoffmann, & Morris, 2012).

8.3. Geographic distribution Kaspar et al. (2010) indicate that non-O157 STEC outbreaks (n ¼ 80) have occurred in the United States, Australia, Japan, Ireland, and other countries in Europe. Other countries that have reported non-O157 STEC infections include New Zealand, Chile, and Argentina, and infections have likely

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occurred in other countries, as well (Johnson et al., 2006). Thus, illnesses associated with non-O157 STEC occur in many developed countries.

8.4. Age, sex, host factors All age groups may be infected with STEC. In a German report of 85 cases of non-O157 STEC infections, the individuals ranged from 3 months to 72 years of age (Beutin, Zimmermann, & Gleier, 1998). In a food-borne outbreak due to STEC O111 in Oklahoma, United States, which involved at least 341 persons, the ages of the infected individuals ranged from 3 months to 89 years (median age, 51 years; Calderon et al., 2010). Beutin et al. (1998) reported that of 84 patients infected with non-O157 STEC in Germany, 60.7% were female. In a discussion of 940 non-O157 STEC infections in the United States during 1983–2002, the sex of 676 patients was known. Women made up 55.0% of the individuals infected with non-O157 STEC (Brooks et al., 2005). Hadler et al. (2011) reported that of 229 patients infected with non-O157 STEC during 2000–2009 in Connecticut, United States, 134 patients were women (58.5%). In a food-borne outbreak due to STEC O111 in Oklahoma, 225 patients out of 341 were women (66.0%) (Calderon et al., 2010). The data suggest that it is possible that women may be more susceptible to non-O157 STEC infections. Other explanations are that women may be infected during food preparation, thus accounting for their higher level of infection. It is probable that the very young, the elderly, and the immunocompromised are more susceptible to STECinduced gastroenteritis. It is also probable that individuals whose occupations involve contact with animals will be more likely to be infected with STEC.

9. PREVENTION OF STEC COLONIZATION AND SHEDDING IN CATTLE STEC (both O157 and non-O157) intestinal colonization and fecal shedding is common in cattle (Menrath et al., 2010), and Bolton et al. (2011) demonstrated that non-O157 STEC (14 serotypes) persisted in farm soils for several months. Proper composting of cattle manure leads to the destruction of non-O157 STEC and renders the product suitable as a natural fertilizer (Fremaux, Delignette-Muller, Prigent-Combaret, Gleizal, & Vernozy-Rozand, 2007; Gonçalves & Marin, 2007). Reduction in STEC shedding on farms would reduce the exposure of humans to STECcontaminated water sources (both drinking and irrigation water) and to


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infection by direct animal contact. In a study of cattle from Midwestern U.S. beef processing plants during the years 2001 and 2002, the mean prevalence of non-O157 STEC from approximately 300 cattle was 14.4% (range 13.9–27.1) from feces collected from the distal portion of the colon. Non-O157 STEC were more prevalent in the spring and fall months (Barkocy-Gallagher et al., 2003). The mean prevalence of O157:H7 from these cattle was 6.0% (range 0.3–12.9) with the highest prevalence found in the summer months. Thus, the data indicate that non-O157 STEC may be more common in cattle than O157:H7 (Barkocy-Gallagher et al., 2003). A variety of feeding regimens have been tested in attempts to reduce or prevent STEC colonization and shedding in cattle. Callaway, Carr, Edrington, Anderson, and Nisbet (2009) and Jacob, Callaway, and Nagaraja (2009) have reviewed dietary interventions on the colonization and shedding of E. coli O157:H7 in cattle, but studies on the effect of diet on non-O157 STEC in cattle have not been done. Feeding studies have included the effect of grain types, grain processing methods, forage, and distiller’s grains on shedding of O157 strains by cattle. Frequently, the results of these studies were conflicting and inconsistent, and often many studies could not be reproduced. It is probable that the host–organism relationship involved in STEC colonization and shedding is complex, and dietary influences are only a part of that complexity (Callaway et al., 2009; Jacob et al., 2009). A U.S. Government Accountability Office report (GAO, 2012) suggested a number of preslaughter interventions with potential use in the reduction of STEC colonization and shedding in cattle. These include antimicrobial compounds, bacteriophages, colicins, natural product extracts, prebiotics, probiotics, sodium chlorate, and vaccines. However, these interventions have limitations. STEC may develop resistance to antimicrobials, bacteriophages, and colicins. It may be difficult to produce bacteriophages, colicins, probiotics, and natural product extracts (e.g., essential oils from citrus peel) in large enough quantities at reasonable cost for use in the large cattle population of the United States. The production of an anti-STEC vaccine would be costly to produce and administer. Snedeker, Campbell, and Sargeant (2012) and Varela, Dick, and Wilson (2013) reviewed vaccination studies involved in the reduction of fecal shedding of O157:H7 during the period of 2004 through 2009. The vaccines were based on type III-secreted proteins or on siderophore receptor/porin proteins; both vaccines significantly reduced fecal shedding of O157:H7 in cattle. Preharvest control of fecal shedding by vaccination diminished the level of O157:H7 entering

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slaughter; however, vaccination did not completely eliminate the organism (Snedeker et al., 2012; Varela et al., 2013). A large number of studies indicate that preharvest treatment of cattle with lactic acid bacteria used as probiotics (Sargeant, Amezcua, Rajic, & Waddell, 2007), anti-O157 vaccines (Sargeant et al., 2007; Snedeker et al., 2012; Varela et al., 2013), or chlorate (Anderson et al., 2005; Sargeant et al., 2007) can reduce fecal shedding of E. coli O157; however, the results were not consistent. It would appear that these practices are unreliable as preharvest interventions to reduce serogroup O157 colonization and shedding in cattle and are of limited use.

10. PREVENTION OF STEC CONTAMINATION OF MEAT AND DAIRY PRODUCTS Since preharvest interventions cannot be relied on to prevent STEC colonization or shedding in cattle, other interventions are necessary during slaughter, meat processing, and at the retail/consumer level to prevent contamination of meat with STEC. STEC O157:H7 inoculated onto cattle hides at105 CFU/cm2 were reduced by ca. 102–3 CFU/cm2 when the hides were sprayed with water, 4% sodium metasilicate, 3% sodium hydroxide, 10% acetic acid, or 10% lactic acid (Carlson et al., 2008). Spraying with sodium hydroxide (1.5%) plus sodium hypochlorite (0.2%) eliminated almost all of the E. coli O157 from the hides. Dehairing of the hides with 2.4% potassium cyanate or 6.2% sodium sulfide was also effective in removal of almost all of the O157 organisms (Carlson et al., 2008). Bosilevac, Nou, Barkocy-Gallagher, Arthus, and Koohmaraie (2006) demonstrated that a hot water spray (74 C) was more effective than a 2% lactic acid spray in reducing the level of O157 on pre-evisceration beef carcasses. Kalchayanand et al. (2012) inoculated STEC serotypes O26:H11, O45:H2, O103:H2, O111: NM, O121:H19, O121:H7, O145:NM, and O157:H7 onto the surfaces of pre-rigor beef flanks at microbial levels of ca. log 104 CFU/cm2 and subjected the meat surfaces to 15-s spraying with acidified sodium chlorite (1000 ppm), peroxyacetic acid (200 ppm), lactic acid (4%), or hot water (85 C). The non-O157 and O157 STEC behaved similarly with these treatments. Hot water spraying was the most effective of the treatments and reduced the STEC levels by ca. 10-fold from a mean of log 4.2 CFU/cm2 (range 3.6–4.6) to a mean of log 0.39 (range 0.2–0.9) (Kalchayanand et al., 2012). The data obtained by Kalchayanand et al. (2012), using a model for beef carcasses, indicated that STEC present on the surface of meat will not be completely removed by these intervention


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techniques. Thus, it appears that ensuring that a beef carcass is completely free of O157 and non-O157 STEC is difficult to achieve. Rigorous in-plant hygiene involving equipment and personnel will do much in keeping meat products free of STEC contamination. Rigorous personal hygiene must be practiced by consumers to prevent cross-contamination of meat by raw products that may contain STEC. Consumers must store meat at proper temperatures and cook meat thoroughly to ensure a safe product. Proper sanitation is critical during the milking process. If the dairy cow is shedding STEC, organisms on the udder and teats will contaminate the milk. The udder and teats should be washed with a cleaning solution and thoroughly dried before milking. After milk is collected, it should be cooled and pasteurized. Pasteurized milk and dairy products produced from pasteurized milk should be handled in such a way to prevent cross-contamination with STEC or other pathogens in the processing environment (Hussein & Sakuma, 2005).

11. PREVENTION OF STEC CONTAMINATION OF PRODUCE Outbreaks linked to produce caused by STEC O157:H7 and nonO157 STEC have been reported (http://bites.ksu.edu/leafy-greensrelated-outbreaks). There are a number of ways in which produce may become contaminated with pathogens, including STEC. Fecal material from cattle or other ruminants or animals can contaminate the environment and be washed into water sources resulting in contaminated drinking water, irrigation water, and produce. In 2010, there was an outbreak caused by STEC O145 associated with contaminated shredded Romaine lettuce harvested from a farm in Arizona. There were 27 confirmed and 4 probable cases of illness in five states (Taylor et al., 2013). Forty-five percent of the affected individuals required hospitalization, 10% of the cases developed HUS, and there were no deaths. In 2011–2012, there was an outbreak associated with STEC O26-contaminated clover sprouts sold at a gourmet sandwich shop chain (www.cdc.gov/ecoli/2012/O26-02-12/index.html). There were 29 cases of illness, 25% of the cases required hospitalization, and there were no deaths or HUS. A very large outbreak occurred in Germany in 2011 associated with a non-typical STEC strain (Beutin & Martin, 2012). There were over 4000 cases of illness, ca. 900 cases of HUS (ca. 23% of the cases), and there were 54 deaths. The outbreak was associated with consumption of sprouts from fenugreek seeds. The E. coli

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serotype was O104:H4. It did not carry stx1, eae, or ehxA, but it carried the stx2a variant. The outbreak strain was sequenced and shown to be very similar to an enteroaggregative E. coli (EAEC), and it contained the EAEC virulence plasmid, which carries genes associated with adherence. The E. coli O104:H4 strain was resistant to several antibiotics. Therefore, the strain associated with this large outbreak was likely an EAEC that acquired the stx2a gene. This strain has also been referred to as an enteroaggregative hemorrhagic E. coli (Brzuszkiewicz et al., 2011). Potential sources of contamination of produce are soil amendments such as raw animal manure, contaminated water, infected workers, unsanitary conditions at the field or packing house, and the presence of animals in the fields. Therefore, control of STEC includes controlling animals entering the fields, ensuring the quality of irrigation water, and adequate training of workers. Currently, testing for STEC on many produce farms is being conducted, and when samples are positive, the plots are not harvested. Once produce is contaminated, removing or inactivating pathogens is difficult. Conventional postharvest washing and sanitizing treatments may achieve at most 2–3 log reductions of surface bacteria. Chlorine-based sanitizers are the most widely used, but other sanitizers using citric, lactic, acetic, and peroxyacetic acid, as well as acidified electrolyzed water and ozonated water are also being evaluated. Saldanã, Sańchez, Xicohtencatl-Cortes, Puente, and Giroń (2011) showed that STEC can enter the stomata, roots, and cut edges of produce and become internalized. They found that spinach became colonized with E. coli O157: H7 through the coordinated expression of curli, pili, and the type III secretion system. The adhesion and internalization of pathogens limits the usefulness of conventional processing and chemical sanitizing methods for inactivation of the bacteria. Physical interventions for inactivation of pathogens include irradiation and UV light. Irradiation up to 4 kGy was approved for lettuce and spinach to improve safety and enhance shelf life. Modified atmosphere packaging to reduce oxygen and prevent bacterial growth is used to increase shelf life but its effect on pathogens, including STEC, needs further study. High pressure, pulsed electric field, and a technology known as cold plasma are also being investigated. Cold plasma is a nonthermal process that employs an ionized gas containing reactive chemical species that inactivate bacteria. Poorly cleaned and maintained equipment can harbor microorganisms, including pathogens, and provide a reservoir of contamination. The Food and Drug Administration (FDA) recommends cleaning and sanitizing


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procedures for food contact surfaces, including equipment and processing and storage areas using hot water, quaternary ammonium compounds, or other sanitizers. Developing multiple-hurdle or sequential intervention treatments will likely be the most effective approach to minimizing the transmission of STEC or other enteric pathogens through produce. The FDA has released various guidance documents addressing microbiological hazards of produce and recommended control measures. These guidance documents can be found at the FDA website (http://www.fda.gov/Food/ GuidanceRegulation/default.htm). An European Food Safety Authority scientific report provides an assessment of the exposure of consumers to STEC through consumption of raw vegetables and possible control measures (http://www.efsa.europa.eu/en/efsajournal/doc/2274.pdf). The implementation of effective interventions from farm-to-table will help to minimize illnesses due to fruits and vegetables. These include the use of Good Agricultural Practices during growing and harvesting of produce and adherence to the FDA guidelines for fresh cut fruits and vegetables.

12. DETECTION, ISOLATION, AND IDENTIFICATION OF STEC A comprehensive review by Wang, Yang, Kase, and Meng (2013) describes a variety of methods, including nucleic acid-based, immunological, and other types of methodologies recently developed for detection on non-O157 STEC, as well as the associated challenges with non-O157 STEC method development. E. coli O157:H7 was declared an adulterant in 1994 by the USDA Food Safety and Inspection Service (FSIS) establishing a zero tolerance policy for this pathogen. In 1996, FSIS established HACCP (Hazard Analysis and Critical Control Points) system requirements for processing plants. In 1999, irradiation of red meat was approved by the USDA. In 2000, non-O157 STEC infections became nationally reportable, and because it became evident that non-O157 STEC are carried by cattle and can cause serious human illness, a public meeting was held by the FSIS, FDA, and the Centers for Disease Control and Prevention in 2007 to solicit input from consumers, academia, industry, and other government agencies on whether certain non-O157 STEC serogroups should be declared adulterants. The rationale for this decision is described in a FSIS document that describes the risk profile for pathogenic non-O157 STEC (Pihkala et al., 2012). At the time, there was no detection capability for these pathogens.

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On September 2011, FSIS announced that the top six STEC serogroups would be declared as adulterants, since like O157:H7, they are carried by cattle, can survive ordinary cooking, have a low infectious dose, and can cause serious human illness. To develop methodologies for detection of non-O157 STEC, an understanding of the diversity of this group of pathogens is necessary, as well as the genes that are important for causing severe illness. The Shiga toxin genes are the most critical; however, strains that cause severe illness also carry the LEEencoded eae gene that is important for attachment to intestinal cells. The method used by the FSIS for regulatory testing for the top six non-O157 STEC can be found at the FSIS website for the Microbiology Laboratory Guidebook (http://www.fsis.usda.gov/wps/portal/fsis/topics/inspection/! ut/p/a0/04_Sj9CPykssy0xPLMnMz0vMAfGjzOINAg3MDC2dDbz8LQ3 dDDz9wgL9vZ2dDSwcTfQLsh0VAZcBLLc!/?1dmy¤t¼true&urile¼ wcm%3Apath%3A/fsis-content/internet/main/topics/science/laboratoriesand-procedures/guidebooks-and-methods/microbiology-laboratory-guidebook/ microbiology-laboratory-guidebook), and it involves enrichment of the beef sample, followed by extraction of genomic DNA, which is used to perform TaqMan-based multiplex PCR assays targeting the stx1, stx2, and eae genes (also includes an internal control). Samples that are positive for both stx and eae are subjected to multiplex PCR assays targeting genes specific to the top six STEC O groups. If samples are positive for one of the top six O groups, the enrichment is subjected to immunomagnetic separation (IMS) followed by plating onto a selective and differential agar medium (modified Rainbow Agar O157), and presumptive isolates are confirmed by latex agglutination, the same multiplex PCR assays used for screening, and biochemical tests. Many test kit manufacturers have also adopted a stepwise approach of detecting the virulence factors, stx and eae, and subsequently testing for the presence of the specific O groups. Many challenges related to non-O157 STEC detection still remain, since these comprise a heterogeneous group of pathogens with different phenotypic features. Although the top six serogroups cause 70–80% of non-O157 STEC-associated illnesses, should we also be concerned about the other serogroups causing the remaining 20–30% of illnesses? Should we target the top six O groups, or should we detect all STEC that cause serious illness that belong to any STEC serogroup. In addition, the sensitivity of nonO157 strains to selective agents commonly used in enrichment media varies; therefore, determining which enrichment medium to use that allows growth


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of all non-O157 STEC strains has been a challenge (Vimont, DelignetteMuller, & Vernozy-Rozand, 2007). It is important to differentiate between pathogenic STEC and other STEC that do not have a high potential to cause illness. There are hundreds of STEC serotypes that likely do not have the potential to cause serious illness. There are several questions to address. Should we target the stx and eae genes in screening assays for the non-O157 STEC or should the assays also target other genes that are found in highly pathogenic STEC? Should assays target all of the stx and eae subtypes, even though primarily only stx2a, stx2c, and stx2d are found in highly pathogenic STEC, and the top six STEC serogroups are each primarily associated with specific eae variants? Furthermore, enteropathogenic E. coli and other bacteria may carry the eae gene. Therefore, if multiplex PCR screening for eae and stx is performed, it is difficult to determine if the target genes are found in one or more than one bacterial source. Thus, the presence of both target genes in a sample may not necessarily be a cause for concern. One other issue to keep in mind is that the STEC O104:H4 German outbreak strain and STEC O91, O113, O128 and other serogroups that cause serious illness lack the eae gene. Thus, screening assays targeting stx and eae would not detect eae-negative serogroups that may cause illness. Although IMS and latex agglutination reagents are becoming more readily available for the top six STEC, the quality of these reagents requires further investigation. High-quality antibodies are useful in the preparation of IMS and latex reagents, as well as enzyme-linked immunosorbent assays and other types of immunological test systems. Another important concern is that even if IMS is used, non-O157 STEC cannot easily be isolated from the selective and differential agars that are currently commercially available, since they are difficult to distinguish from nonpathogenic E. coli. Furthermore, there are selective agents used in agar media, which may inhibit some STEC strains. The use of Rainbow Agar O157 for isolation of non-O157 STEC has been described (Fratamico et al., 2011), and the typical color of the non-O157 STEC colonies on Rainbow Agar O157 was purple, magenta/mauve, blue-violet, gray, pink with dark pink center, and violet/light purple, for E. coli O26, O45, O103, O111, O121, and O145, respectively. However, colonies formed by other bacteria may often be of the same color as those from the non-O157 STEC, and colonies formed by different strains of the same STEC serogroup may not always be of the same color.

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13. COMPARATIVE GENOMICS OF O157:H7 AND NON-O157 STEC The ability to determine the genomic content of a bacterial organism and compare that to the genomic make up of other bacteria is an essential research technique. The genetic complement of bacteria defines the abilities of the organism. In the case of a pathogenic organism, these abilities include the virulence factors that determine how the pathogen causes disease, under what conditions, and to which host organisms. Therefore, genomic and comparative genomic research provides the ability to understand how specific bacteria function as pathogens and how it is possible to circumvent the pathogens’ abilities and protect humans from the diseases they cause. STEC represent a relatively diverse group of bacteria which possess a range of disease causing potential from strains causing life-threatening disease to those with little or no disease-causing capacity. One of the best researched subgroups of STECs is serotype O157:H7 STEC, which has historically demonstrated considerable potential to cause human disease. Four O157:H7 STEC have been carefully sequenced to produce complete, well reviewed, closed chromosomal genomic sequences (Eppinger, Mammel, Leclerc, Ravel, and Cebula, 2011; Hayashi et al., 2001; Kulasekara et al., 2009; Perna et al., 2001). These genome sequences often serve as reference databases for ongoing comparative genomic projects of STEC strains (Song et al., 2012). More recently, non-O157 STEC strains have emerged as significant human pathogens and have become the subject of genomic-based research. The number of non-O157 STEC causing human disease cases has risen 60.5% globally between 2000 and 2005 (Bugarel, Beutin, & Fach, 2010). The application of comparative genomics to STEC research has been utilized in assessing the disease risk that specific strains possess, determining the evolutionary process by which certain STEC strains have become dangerous pathogens and for epidemiological purposes, to track STEC outbreak strains. Comparative genomic-based risk assessment of STEC strains has focused predominately on the identification of virulence genes in STEC responsible for causing serious disease. The goal is to differentiate dangerous STEC from less dangerous STEC. The STEC classified as enterohemorrhagic E. coli (EHEC) cause serious human disease including the life-threatening HUS. When the genomes of O157 and non-O157 STEC strains classified as EHEC strains were sequenced, compared to nonpathogenic E. coli these


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virulent strains were shown to have roughly 1 Mb of additional sequence in their genomes producing at least 1000 additional genes of which greater than 100 were identified as virulence genes (Hayashi et al., 2001; Kulasekara et al., 2009; Ogura et al., 2007; Perna et al., 2001). Despite the similarities in genome size between O157 and non-O157 disease-causing STEC, there was little correlation between the additional genes present in O157 and non-O157 STEC with only 20% of genes conserved between the two groups (Ogura et al., 2007). Most of the conserved genes were virulence factors found on prophages or plasmids. Efforts to identify which of these virulence genes are essential have relied on comparative genomic techniques. The genomic sequences of STEC strains isolated from cases of human disease or from food animals were compared to identify virulence genes that were essential to all strains causing serious human disease (Beutin, Krause, Zimmermann, Kaulfuss, & Gleier, 2004; Bugarel et al., 2010; Islam et al., 2008; Slanec, Fruth, Creuzburg, & Schmidt, 2009). The results of such studies demonstrated that although there were greater than 400 different STEC serotypes, 69% of disease-causing STEC belonged to just 11 serogroups (Beutin et al., 2004). Additionally, the virulence genes eae (intimin) and iha (adhesion) were found to play important roles in STEC virulence (Beutin et al., 2004; Slanec et al., 2009). The eae gene was found to be associated more often with both O157 and non-O157 STEC belonging to the EHEC classification (Slanec et al., 2009). Also, different eae variants were shown to cause different levels of disease severity. However, it should be noted that eae-negative STEC can also cause disease (Beutin et al., 2004; Slanec et al., 2009). The iha gene proved to be the most commonly present virulence gene in a comparison of the genomes of 75 STEC strains (Slanec et al., 2009). An additional study identified genes located on O-island 122 (OI-122) that were strongly associated with severe disease outcomes in STEC strains (Bugarel, Martin, Fach, & Beutin, 2011). The goal of identifying virulence genes associated with STEC carries the practical application of developing a testing method to identify high-risk STEC strains. A macroarray developed to determine the presence of stx (Shiga toxin), eae, ehxA (enterohemolysin), and a series of nle (nonlocus of enterocyte effacement encoded effecter) genes produced gene detection signatures that have identified STEC strains highly virulent to humans (Bugarel et al., 2010). In addition to identifying the genes that make STEC dangerous pathogens, comparative genomic studies have sought to describe the evolution by which the E. coli strains have become pathogens. The early whole genome

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sequencing of O157 STEC strains demonstrated that the virulence genes unique to the O157 strains were clustered together in genetic islands suggesting their acquisition was the result of horizontal transfers from other organisms (Hayashi et al., 2001; Perna et al., 2001). Further observations of virulence genes associated with prophage sequences from bacteriophage supported the theory of horizontal transfer being integral to pathogenic O157 evolution. Comparative genomic studies of O157 and non-O157 STEC also demonstrated that virulence factors common between these two groups are again associated with prophages and plasmids, implicating horizontal transfer in the development of disease-causing non-O157 STEC (Ogura et al., 2007, 2009). Sequence comparisons between STEC strains demonstrated that those belonging to the EHEC classification clustered more closely together with EHEC strains of different serogroups than with other strains belonging to the same serogroup (Didelot, Meric, Falush, & Darling, 2012; Sims & Kim, 2011). The genomic results suggest that through horizontal transfer of a specific set of virulence genes, STEC from diverse serotypes have co-evolved converging from separate backgrounds into similar pathogens (Didelot et al., 2012). Comparing STEC genomes also has suggested that the presence of the same H antigen more accurately predicts common ancestry between STEC strains than does the O antigen (Ju et al., 2012). The evolutionary study of emerging subgroups of STEC, such as LEE-negative STEC, has benefited from comparative genomic investigations. A whole genome comparative analysis showed LEE-negative STEC to be a very diverse group evolutionally divergent from LEE-positive STEC clustering more closely with non-STEC strains (Steyert et al., 2012). Epidemiological studies of STEC strains have also began to utilize comparative genomics. As new outbreak strains are isolated, the ability to determine where they came from is essential. High-density oligo arrays have been used to compare the genetic content of independent isolates collected during an outbreak caused by STEC (Jackson, Patel, Barnaba, LeClerc, & Cebula, 2011). This type of technique differentiates closely related strains; however, direct whole genome sequence comparison provides the most complete evaluation. This technique was used to compare an STEC strain from a Norwegian outbreak to a German outbreak 4 years later showing the two strains to be closely related particularly with regards to their stx containing phage sequences (L’Abee-Lund et al., 2012). The Norwegian strain was identified as an EHEC type STEC strain as was the German strain originally. However, through comparative genomics, the German strain was shown to be more properly classified as an EAEC-STEC (Rasko et al., 2011).


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Furthermore, a comparative genetic study of the stx2 bacteriophage sequence of the German strain suggested that the strain had recently acquired the Shiga toxin gene from an O111:H-like strain (Perna et al., 2001). With the advent of high-throughput DNA sequencing techniques, the ability to rapidly and cost effectively sequence whole bacterial genomes is readily available. This has permitted the utilization of comparative genomics in a wider range of research settings. For STEC research, it has allowed the identification of virulence genes that permit certain STEC to become dangerous disease-causing strains. It has identified mechanisms by which STEC strains acquire and share these essential virulence genes. Additionally, comparative genomics can classify and group STEC strains describing from where new outbreak strains may be originating.

14. STRESS RESPONSES Bacteria are continually buffeted by potentially stressful environmental conditions. In order to survive and grow in the presence of stressful conditions, bacteria invoke mechanisms that allow them to adapt to the new environment. Food-borne pathogens, such as STEC, are stressed during food processing, food storage, and food preparation; the most common stresses are heat, cold, osmotic, and acid stress (Chung, Bang, & Drake, 2006; Jones, 2012). Recently, Smith and Fratamico (2012) reviewed the effect of various stresses on non-O157 STEC.

14.1. Cross-protection An interesting phenomenon that can occur when bacteria face stressful conditions is cross-protection. Cross-protection is the ability of a stress to induce protection against a different stress (or stresses) (Chung et al., 2006). For example, when strains of O157:H7 STEC were heat-shocked at 48 C for 10 min and then subjected to pH 2.5 (HCl) for 6 h at 37 C, the heat-shocked STEC were 10–100 times more resistant to the acidic conditions as compared to cells that were not heat-shocked (Wang & Doyle, 1998). Leenanon and Drake (2001) produced acid-adapted STEC O157: H7 (grown in 1% glucose medium at 37 C for 18 h; final pH 4.8–4.9) and found that they showed increased resistance to heat when exposed to 56 C for 50 min as compared to cells that were not acid-adapted. Similarly, they starved O157:H7 cells (washed cells suspended in saline at pH 6.6 for 48 h at 37 C) and demonstrated that the starved cells were more resistant to heat exposure (56 C for 50 min) as compared to unstarved cells

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(Leenanon & Drake, 2001). Chung et al. (2006) have a table listing a number of publications in which stress-induced cross-protection in E. coli are discussed.

14.2. General stress response When E. coli enters the stationary phase or undergoes nutrient deprivation or stress, there is an increase in the accumulation of RpoS (sS). RpoSdependent gene expression induces the general stress response. The general stress response allows the induction of cellular genes leading to resistance to the encountered stress and in addition, resistance to that specific stress allows resistance to other stressful events (Battesti, Majdalani, & Gottesman, 2011; Jones, 2012).

14.3. Cold stress The effect of a temperature downshift on bacteria results in decreased membrane fluidity, stabilization of the structure of nucleic acids with a reduction in the efficiency of mRNA translation and transcription, obstruction of ribosome function, and inefficient protein folding. Cold shock proteins are induced to cope with the deleterious effects of the downshift in temperature (Phadtare & Severinov, 2010). Cold shock proteins induce increased formation of unsaturated fatty acids and their incorporation into the membrane with reestablishment of membrane fluidity, as well as mRNA translation and transcription, restoration of ribosome function, and proper folding of cellular proteins (Phadtare and Severinov, 2010; Jones, 2012).

14.4. Heat stress Heat, at a lethal level, is an effective means of inactivating bacteria if applied for a sufficient length of time. However, a nonlethal heat treatment may lead to heat tolerance through the induction of the heat shock response. A major component of the heat shock response is the upregulation of heat shock proteins (HSPs). The HSPs are regulated by the s32 transcription factor (encoded by rpoH). Translation of s32 increases at high temperatures; s32 directs transcription of RNA polymerase from heat shock promoters leading to induction of HSPs. s32 functions relate to cytoplasmic protein damage, whereas sE serves to protect periplasmic proteins during heat stress. The HSPs act as molecular chaperones affecting protein folding and repair or as ATP-dependent proteases that degrade abnormally folded proteins that were generated by the stress (Chung et al., 2006; Guisbert, Yura,


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Rhodius, & Gross, 2008; Jones, 2012). Thus, HSPs provide the cell with functional proteins allowing survival and/or growth during heat stress.

14.5. Acid stress Acid stress in foods is due to the combined effects of Hþ ions and organic acids due to fermentation or when organic acids are added as food preservatives. Undissociated organic acids enter the bacterial cell and upon dissociation release protons, which increases intracellular acidity leading to eventual cell inactivation (Chung et al., 2006; Jones, 2012). Mechanisms involved in acid tolerance, which allow survival and growth of STEC under acidic conditions are discussed in Section 4.

14.6. Osmotic stress Increasing the osmotic pressure of a food through drying or by the addition of sugars or salts leads to the reduction of water available to the bacterial cell. The major reaction toward an osmotic upshift is the efflux of water from the microorganisms into the external environment. Increased osmolarity of the external environment is associated with inhibition of DNA replication, nutrient uptake, and growth by the bacterial cell, the internal osmotic pressure must be higher than that of the external environment in order to maintain bacterial viability and growth (Chung et al., 2006; Jones, 2012). When there is an increase in the osmotic pressure of the external environment, cellular osmoregulation mechanisms such as the uptake of charged solutes or synthesis and concentration of specific organic solutes (i.e., compatible solutes) allow the equilibrium of intracellular osmotic pressure with that of the external osmotic pressure (Capozzi, Fiocco, Amodio, Gallone, & Spano, 2009; Chung et al., 2006). Peng, Tasara, Hummerjohann, and Stephan (2011) provide an interesting review on how STEC respond to stresses encountered during cheese making.

15. CELL-TO-CELL COMMUNICATION SYSTEMS IN E. COLI Communication is mediated in bacteria through chemical signals synthesized and secreted by the organisms themselves. These signals allow communication between cells of the same species, different species, or different kingdoms. The communication process is used by microorganisms to sense specific changes in the environment, which then allow them to adapt to the new conditions (Bandara, Lam, Jin, & Samaranayake, 2012;

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Skandamis & Nychas, 2012). As the bacterial population increases, the synthesis of the signaling molecules increases, and they are then secreted into the extracellular environment. When the signaling molecules reach a threshold level, they re-enter the bacterial cell and alter the expression of target genes. The communication process is referred to as quorum sensing (Bandara et al., 2012; Skandamis & Nychas, 2012).

15.1. Intraspecies communication Intraspecies communication in gram-negative bacteria is mediated through N-acyl homoserine lactones (AHLs). The homoserine lactone ring is N-acylated at the C-1 position with a fatty acyl group ranging from 4 to 18 carbons; the acyl group can be a straight chain or is modified at the acyl C-3 position by a double bond, an oxo group, or by a hydroxyl group. The AHLs are synthesized by AHL synthases (LuxI). The AHLs are sensed by a response transcriptional regulator protein (LuxR), producing a LuxR/AHL complex which regulates the up- or downexpression of target genes (Bandara et al., 2012; Skandamis & Nychas, 2012). The gram-negative species Escherichia, Klebsiella, Salmonella, and Shigella lack a LuxI homolog and therefore do not synthesize AHLs. These organisms do produce a LuxR homolog known as SdiA (suppressor of cell division inhibitor), which can accept AHLs produced by other microorganisms (Smith, Fratamico, & Yan, 2011). Several studies on the role of SdiA in STEC utilized overexpression of sdiA on a plasmid; however, overexpression of the gene does not give a true picture of the physiological role of SdiA (Smith et al., 2011). There have been a few studies concerning the role of the chromosomal sdiA gene in STEC O157:H7 strains. Sharma, Bearson, and Bearson (2010) demonstrated that deletion of the sdiA gene in STEC O157:H7 (strain 86-24 △stx2 △lac) led to enhanced adherence of bacteria to HEp-2 cells (human laryngeal epithelial) as well as enhanced the expression of fliC (encodes flagellin) and csgA (encodes curlin of the curli fimbriae), but had little effect on the expression of LEE genes. The data indicate that SdiA acts as a repressor of genes encoding flagella and curli leading to decreased motility and decreased adherence to host cells (Sharma et al., 2010). Sharma and coworkers did not study the effect of AHL addition on the wild-type STEC O157. Utilizing STEC O157:H7 strain 700927, Dyszel et al. (2010) found that the expression of the fliE gene (involved in synthesis of flagella) was almost completely repressed in the presence of oxo-C6-homoserine lactone, whereas the expression of genes associated


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with AR (gadE, yhiD, hdeA) was upregulated (>2-fold) in the presence of the AHL. However, the AR phenotype of the organism was AHLindependent, suggesting that the basal level of SdiA was high enough to induce AR (Dyszel et al., 2010). Using weaned calves, Sharma and Bearson (2013) demonstrated that deletion of sdiA reduced the level of fecal shedding of STEC 86-24 from 106 cfu to 102 cfu/g feces (102 cfu was the limit of detection) over a period of 27 days in contrast to the wild type, which was still shed at ca 104 cfu/g feces at 27 days. Thus, the deletion of the sdiA gene reduced the extent and the duration of fecal shedding of the O157 strain in calves. The LEE locus is a pathogenicity island present in STEC O157:H7 strains. The genes located in LEE encode an array of effector proteins that enter host cells and manipulate their cellular physiology to facilitate bacterial colonization and pathogenicity (Schmidt, 2010). In the presence of oxo-C6-homoserine lactone, the transcription of LEE genes by wild-type STEC O157:H7 (strain 86-24) was decreased several-fold as compared to the △sdiA strain (with or without AHL) or the wild type in the absence of AHL (Hughes et al., 2010). These results suggest that AHL represses the transcription of genes in LEE through SdiA. The ler gene encodes a positive transcriptional regulator of LEE, and in the presence of AHL, SdiA represses ler and consequently, LEE (Sharma & Bearson, 2013). Gramnegative bacteria in the rumen produced AHLs (Hughes et al., 2010); however, AHLs were not present in the bovine intestinal tract (Hughes et al., 2010; Swearingen, Sabag-Daigle, and Ahmer (2013). Lim et al. (2007) found that STEC O157:H7 colonization in cattle occurred only at the rectoanal junction and not at any other bovine intestinal site. SdiA þ AHL inhibit transcription of the LEE genes in the rumen; however, in the absence of AHL in the intestine, the transcription of the LEE genes is derepressed, and colonization of the rectoanal junction by STEC occurs. The expression of STEC O157:H7 gad AR genes (the glutamate decarboxylase pathway for AR) increased approximately twofold in the presence of oxo-C8-homoserine lactone in the wild-type bacterium, and gad transcription was absent in △sdiA with or without AHL, indicating that the AHL/SdiA complex was responsible for the increased expression (Hughes et al., 2010); however, SdiA activated the gad genes (at a lower rate) even in the absence of AHL. Thus, the regulation of the gad genes by SdiA is only partially dependent on the presence of AHL. The increase in bacterial AR in the rumen prepares the bacterial cells to resist the acid condition of the stomach. The work of Hughes and coworkers (2010) indicates that rumen AHLs

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in combination with SdiA downregulate the expression of the LEE in STEC O157:H7 and upregulate the expression of the gad AR system in the bovine rumen.

15.2. Interspecies communication Gram-positive and -negative bacteria have a common quorum sensing system that mediates interspecies communication, the autoinducer 2 (AI-2) system. The enzyme LuxS (encoded by luxS) synthesizes 4,5dihydroxy-2,3-pentanedione (DPD) from S-ribosyl homocysteine; DPD spontaneously cyclizes to form AI-2 (Bandara et al., 2012; Skandamis & Nychas, 2012). AI-2 is a furanosyl-borate diester in Vibrio harveyi; however, the AI-2 molecule in Salmonella and E. coli does not contain boron (Walters & Sperandio, 2006b). Studies have appeared in which investigators have used “conditioned medium” (CM) as a source of AI-2. CM consists of a cell-free filtered medium in which an AI-2-producing organism was grown. For example, Soni, Lu, Jesudhasan, Hume, and Pillai (2008) studied the effect of CM on survival and gene expression of a STEC O157:H7 △luxS mutant. STEC O157:H7 ATCC strain 43895 was the source of the CM. The mutant did not produce AI-2 but could respond to it. In the presence of CM, the mutant showed ca. 60% survival at 20 days at 4 C, but with autoclaved CM, the survival was ca. 20%. The addition of beef extract (from fresh ground beef ) completely inhibited O157:H7 survival with both CM preparations (Soni et al., 2008). In the presence of CM, the expression of the haa gene (involved with negative regulation of a-hemolysis) was increased ca. twofold and that of the yadK (fimbrial gene) was increased ca. threefold as compared to autoclaved CM. However, beef extract had no effect on gene expression with either CM preparation (Soni et al., 2008). Since LB broth (plus 0.5% glucose) was used to prepare the CM, the components that may have caused the stimulatory effects of the CM on survival and gene expression are difficult to determine due to the complexity of the medium. Inoculation of STEC O157:H7 strain 86-24 (produces AI-2) or strain VS-94 (does not produce AI-2; isogenic mutant of 86-24) in LB broth containing stainless steel coupons at 25 C for 72 h indicated that both strains produced a similar level of biofilm on the coupons (Yoon & Sofos, 2008). Therefore, AI-2 does not appear to have a role in biofilm formation in these E. coli O157 strains. Using STEC O157:H7 strain 86-24 and its isogenic △luxS mutant (VS-94), Kendall, Rasko, and Sperandio (2007) found


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there was no significant difference in the expression of LEE1 (escU) and LEE2 (escC) genes between the wild type and the luxS mutant, whereas the expression of a LEE3 (escV) gene was increased in the mutant, and the expression of LEE5 (eaeA) and LEE4 (espA) was decreased in the mutant strain. The genes escU, escC, escV, and espA are involved in the type III secretion apparatus, and the eaeA gene encodes intimin. The results obtained by Kendall et al. (2007) suggest that AI-2 is not necessary for the transcription of escU or escC but is involved in transcription of eaeA and espA. The increased expression escV in the luxS mutant suggests that AI-2 downregulates the expression of the gene in the wild-type strain. The luxS mutation has no effect on the expression of the stx2A gene (encodes the A subunit of Stx2) as compared to the wild type (Kendall et al., 2007), indicating that AI-2 is not involved. In STEC O157:H7 strain VS-94 (△luxS), Lee, Zhang, Hegde, Bentley, and Jayaraman (2008) demonstrated that there was increased transcription of espA and eae at 37 C when DPD was added; however, there was no significant change in expression at 30 C. Lee et al. (2008) suggested that AI-2 signaling occurs primarily at 37 C. Kendall et al. (2007) also studied the expression of non-LEE genes such as the nleA gene (involved in virulence) and fimbrial genes, as well as etrA and eivF (negative regulators of LEE) in the wild type and luxS mutant. The expression of nleA in the wild type and mutant was similar, indicating that AI-2 is not involved with expression of that gene. However, the expression of etrA and elvF was decreased in the luxS mutant, indicating that AI-2 is necessary for the expression of these genes (Kendall et al., 2007). There was decreased transcription of fimbrial genes in the luxS mutant as compared to the wild type; thus, AI-2 is involved in the expression of certain fimbrial genes. Theoretically, the addition of DPD (the precursor to AI-2) to the luxS mutant strain should make the mutant behave like the wild type, which contains endogenous AI-2. The addition of DPD to the luxS mutant (VS-94) did not lead to the wild-type level of expression of the genes studied by Kendall et al. (2007), suggesting that DPD does not fully replace AI-2 or that the DPD (100 mM) level was too low. Also, it may be possible that the DPD preparation acts as a chemical inhibitor of the expression of the genes. Kendall et al. (2007) did not add DPD to the wild type; addition of DPD to the wild type should prove or disprove chemical inhibition. Using strains 86-24 and VS-94, Soni et al. (2007) demonstrated the upregulation of fliC in the wild type as compared to the mutant strain. Using the agar motility assay, Lee et al. (2008) found that the wild-type strain

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produced a larger halo than the mutant strain. The results of Soni et al. (2007) and Lee et al. (2008) suggest that AI-2 is necessary for motility. However, the addition of enzymatically produced AI-2 (25 mM) to the mutant strain had no effect on the size of the motility halo (Lee et al., 2008).

15.3. Interkingdom communication Bacteria–host communication is mediated through the AI-3/epinephrine (EPI)/norepinephrine (NE) signaling system (Walters & Sperandio, 2006b). Two classes of signals are sensed by STEC to activate virulence genes: AI-3, which is produced by the normal flora of the gastrointestinal tract and the catecholamine hormones, EPI and NE, produced by the host and are found in the gastrointestinal tract. The LEE genes and the flagella regulon are activated through the AI-3/EPI/NE signaling system; these signals are sensed by sensor kinases that lead to a regulatory cascade activating the flagella regulon and LEE genes (Walters & Sperandio, 2006b). In the STEC O157:H7 luxS mutant (VS-94), there is decreased production of AI-3 due to an alteration in cellular metabolism, therefore the luxS gene is needed for efficient synthesis of AI-3 (Walters, Sircili, & Sperandio, 2006). Walters and Sperandio (2006a) studied the kinetics of LEE gene transcription, protein expression, and function of the type III secretion apparatus in strains 86-24 or VS-94. The luxS mutant (VS-94) showed decreased transcription from LEE promoters, decreased levels of proteins involved in type III secretion (EscJ, Tir, EspA), and decreased secretion of type III secretory proteins, EspA and EspB. In addition, the luxS mutant showed a delay in formation of AE lesions on HeLa cells. The addition of 50 mM of EPI to the wild type and luxS mutant led to a significant increase in expression of LEE genes; however, the increase was greater in the wild type, indicating that a possible synergistic relationship between AI-3 and EPI exists in STEC O157:H7 (Walters & Sperandio, 2006a). The AI-3/EPI/NE signals are sensed by the adrenergic receptor, QseC, a histidine sensor kinase present in the bacterial membrane (Clarke, Hughes, Zhu, Boedeker, & Sperandio, 2006). QseC senses and binds to AI-3, EPI, and NE. QseC initiates autophosphorylation in the presence of EPI and transfers that phosphate to QseB (the response regulator for QseC). Thus, QseB/QseC is a functional two-component system. Phosphorylated QseB binds to and activates the transcription of the flhDC promoter (encodes the FlhDC master regulators of the flagella regulon). Addition of 5 mM EPI or 100 nM AI-3 to the luxS mutant (VS-94) increased flhDC transcription to


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wild-type levels. Addition of AI-3 or EPI to a qseC mutant had no effect (Clarke et al., 2006). Since there is no animal model for STEC infection, Clarke et al. (2006) inoculated rabbits with a rabbit enteropathogenic E. coli qseC mutant VS243 and wild-type strain E22. This RPEC wild type induced similar intestinal lesions as occurs with STEC infections. The investigators demonstrated that the qseC mutant was attenuated for virulence in rabbits, thus indicating that QseB/QseC is likely associated with pathogenicity in STEC. The results obtained by Clarke et al. (2006) indicate that the AI-3/EPI/NE signaling system has an important role in virulence and motility of STEC strains. STEC O157:H7 produces AE lesions on intestinal epithelial cells; the microvilli are destroyed and there is induction of actin arrangement to form pedestals that cup each bacterium. The AI-3/EPI/NE signaling system activates the transcription of the genes involved in the formation of the AE lesions in strain 86-24. The signaling system is sensed by the twocomponent QseEF system (QseE is the sensor kinase and QseF is the response regulator). The qseEF genes are cotranscribed and gene transcription is activated by EPI through the QseC sensor. AE lesions are not formed in a qseF mutant (Reading et al., 2007). QseF activates transcription of the espFu gene. EspFu is an effector protein translocated to the host cell by the bacteria, which induces actin polymerization during pedestal formation. A plasmid with the espFu gene restored the formation of AE lesions in the qseF mutant. Regulation of AE formation is mediated through the QseEF two-component system (Reading et al., 2007). A review of interkingdom communication between bacteria and the mammalian has been recently published (Karavolos, Winzer, Williams, & Khan, 2012).

15.4. Miscellaneous types of communication 15.4.1 Indole Indole is an intercellular signal in both gram-positive and gram-negative bacteria and has been shown to control a number of bacterial processes such as spore formation, plasmid stability, drug resistance, biofilm formation, and virulence (Lee & Lee, 2010). Hirakawa, Kodama, Takumi-Kobayashi, Honda, and Yamaguchi (2009) demonstrated that indole is a signal for expression of type III secretion system translocators in the Sakai strain of STEC O157:H7. In a tnaA (encodes tryptophanase, catalyzes synthesis of indole from tryptophan) deletion mutant, there was decreased secretion of EspA and EspB via the secretion system leading to reduced formation of A/E lesions in HeLa cells. Addition of indole to the tnaA mutant

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enhanced secretion of EspA and EspB and restored A/E lesion formation (Hirakawa et al., 2009). Thus, indole has a positive role in regulating genes of the LEE locus. Bansal et al. (2007) studied the effect of indole, EPI, and NE on chemotaxis, motility, biofilm formation, cell attachment, and gene expression in E. coli O157:H7 (strain EDL933). In a chemotaxis assay, E. coli was attracted to EPI and NE; however, there was migration of the organism away from indole. Using 50 mM EPI and NE and 500 mM indole, there was no significant effect on the bacterial growth rate. Indole decreased motility by ca. 2.8-fold, whereas EPI and NE increased motility by ca. 1.4-fold as compared to untreated controls. Addition of EPI and NE resulted in a 1.7- and 1.5-fold increase in biofilm production, respectively, on polystyrene plates, whereas indole decreased the formation of biofilms by 2.4-fold. Adherence to HeLa cells was increased 3.4-fold by EPI and 5.l-fold by NE, but was decreased 3.1-fold by indole when compared to untreated cells (Bansal et al., 2007). In E. coli biofilms, DNA microarrays indicated that EPI/NE upregulated expression of genes involved in surface colonization and virulence, whereas indole repressed the expression of those genes. The results obtained by Bansal et al. (2007) indicated that EPI/NE and indole affect STEC chemotaxis, motility, biofilm formation, and adherence to HeLa cells, suggesting that these signaling molecules impact STEC O157:H7 colonization of the large intestine. Lee et al. (2007) demonstrated that STEC strain EDL933 formed strong biofilms under static conditions (on polystyrene) and in a continuous flow system. The hydroxyindoles, 5- and 7-hydroxyindole, inhibited biofilm formation similarly to indole. However, isatin (1Hindole-2,3-dione) stimulated biofilm formation due to its repression of the transcription of tnaA, which led to a decrease in indole production (Lee et al., 2007). The levels of indole and the hydroxyindoles that inhibited biofilm formation had no effect on bacterial growth. 15.4.2 Ethanolamine Ethanolamine (EA), a breakdown product of cell membrane phospholipid phosphatidyl EA, is found in the gastrointestinal tract and can serve as a nitrogen source to STEC O157:H7, providing the organisms a competitive advantage in the gut. In addition, EA is a signal that relays to the organisms that they are present in an intestinal environment, and EA also acts as a trigger for the expression of virulence genes (Garsin, 2012). Using STEC O157: H7 strain 86-24, Kendall, Gruber, Parker, and Sperandio (2012) demonstrated that growth in a minimal medium containing EA as the nitrogen


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source led to the increased expression of the stx2a gene, sensor kinase genes (qseC, qseE), and LEE genes (ler, espA, eae). Levels of EA that cannot support growth are also capable of activating expression of STEC virulence genes (Kendall et al., 2012). A survey of the literature indicated that studies on the roles of SdiA, AI-2, indole, EA, and the AI-3/EPI/NE signaling on gene regulation in the nonO157 STEC have either not been done or not reported. It is probable that these systems are available to the non-O157 STEC strains, and studies to determine this are warranted.

16. CONCLUSIONS Non-O157 STEC are a diverse group of pathogens, and in the United States and many other countries a few serogroups are responsible for the majority of human infections. However, not all strains belonging to these specific serogroups and not all non-O157 STEC serotypes that have been identified are capable of causing severe disease. A better understanding of the ecology and virulence gene profiles of STEC that cause severe disease, as well the prevalence of highly virulent non-O157 STEC serogroups and serotypes in animals, foods, and in the environment is needed to develop effective control strategies. Several studies that have been conducted demonstrate that non-O157 STEC respond similarly to stresses as O157:H7 and are inactivated with the same treatments that have been evaluated or are currently being used for inactivation of E. coli O157:H7 during food processing. However, since STEC comprise a diverse group of pathogens, additional studies in this area using a wide range of strains are needed. Effective control measures from farm-to-table include reducing STEC carriage in cattle, preventing contamination during slaughter, and preventing contamination of produce both pre- and postharvest. There is great interest from both the food industry and regulatory agencies to enhance food-handling practices and develop improved interventions for fresh produce. Since many outbreaks linked to produce contaminated with non-O157 STEC have occurred (Beutin & Martin, 2012; Pihkala et al., 2012; Taylor et al., 2013), many efforts worldwide are underway to address this. Improvements in methodologies for rapid detection, identification, and isolation of non-O157 STEC, as well as subtyping methods that can be used for outbreak investigations are essential. Better-formulated enrichment media and selective and differential agar media for isolation of non-O157 STEC should be carefully designed and tested to ensure applicability across

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relevant non-O157 serogroups. Furthermore, procedures for detecting nonO157 STEC should be integrated as much as possible with those in place for detection of E. coli O157:H7 and should be adaptable to accommodate detection of emerging serogroups of public health concern. Finally, there needs to be rapid and vigorous detection and investigation of outbreaks and increased testing by clinical laboratories to monitor for emerging non-O157 STEC.

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Detection, Characterization, and Typing of Shiga Toxin-Producing Escherichia coli.

Six Novel O Genotypes from Shiga Toxin-Producing Escherichia coli.

Neuropsychological outcome after complicated Shiga toxin-producing Escherichia coli infection.

Oxidative Stress in Shiga Toxin Production by Enterohemorrhagic Escherichia coli.

Characterization of Shiga Toxin Subtypes and Virulence Genes in Porcine Shiga Toxin-Producing Escherichia coli.

A Poly-N-acetylglucosamine-Shiga toxin broad-spectrum conjugate vaccine for Shiga toxin-producing Escherichia coli.

Thermal inactivation of Escherichia coli O157:H7 and non-O157 shiga toxin-producing Escherichia coli cells in mechanically tenderized veal.

Probiotic Escherichia coli Nissle 1917 reduces growth, Shiga toxin expression, release and thus cytotoxicity of enterohemorrhagic Escherichia coli.

Colonization of Enteroaggregative Escherichia coli and Shiga toxin-producing Escherichia coli in chickens and humans in southern Vietnam.

Comparative genomic analysis of Shiga toxin-producing and non-Shiga toxin-producing Escherichia coli O157 isolated from outbreaks in Korea.

Transcription of the Shiga-like toxin type II and Shiga-like toxin type II variant operons of Escherichia coli.

In silico analysis of Shiga toxins (Stxs) to identify new potential vaccine targets for Shiga toxin-producing Escherichia coli.

Cinnamon Oil Inhibits Shiga Toxin Type 2 Phage Induction and Shiga Toxin Type 2 Production in Escherichia coli O157:H7.

Prevalence of Salmonella enterica and Shiga toxin-producing Escherichia coli in zoo animals from Chile.

Isolation of atypical enteropathogenic and shiga toxin encoding Escherichia coli strains from poultry in Tehran, Iran.

Hemolytic uremic syndrome associated with Escherichia coli O8:H19 and Shiga toxin 2f gene.

Geographic divergence of bovine and human Shiga toxin–producing Escherichia coli O157:H7 genotypes, New Zealand.

Novel sequence types of non-O157 Shiga toxin-producing Escherichia coli isolated from cattle.

Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing.

Comparison of Enrichment Broths for Supporting Growth of Shiga Toxin-Producing Escherichia coli.

Agitation down-regulates immunoglobulin binding protein EibG expression in Shiga toxin-producing Escherichia coli (STEC).

Advances in detection methods for Shiga toxin-producing Escherichia coli (STEC).

Non-O157 Shiga toxin-producing Escherichia coli in U. S. retail ground beef.

Thermal inactivation kinetics of Shiga toxin-producing Escherichia coli in buffalo Mozzarella curd.