E. Coli

1. ENTEROPATHOGENIC ESCHERICHIA COLI Hin-chung Wong Department of Microbiology Soochow University ___________________________________________________________ 1. INTRODUCTION 2. HEAT-LABILE ENTEROTOXINS 2.1. General Characteristics 2.2. Gene and Regulation 2.3. Mode of Action 3. HEAT-STABLE ENTEROTOXINS 3.1. General Characteristics 3.2. Mode of Action and Regulation 4. ENTEROTOXIN PLASMIDS 5. SHIGA-LIKE TOXINS 5.1. Purification and Structure 5.2. Mode of Action 5.3. Production and Regulation 5.4. Genetics 5.5. Role in Disease 6. HEMOLYSINS 6.1. Production and Purification 6.2. Characteristics 6.3. Genetics 6.4. Role in Virulence and Mode of Action 7. ADHERENCE 7.1. In Enterotoxigenic E. coli 7.2. In Enteropathogenic E. coli 7.3. In Enterohemorrhagic E. coli 1

2. 8. INVASIVENESS 9. DETECTION 9.1. Using glucuronidase assay 9.2. Animal Tissue Culture 9.3. Animal Assays 9.4. Immunological Methods 9.5. Enzymatic bio-nanotransduction 9.6. Nucleic Acid Probes 9.7. Using polymerase chain reaction 10. REFERENCES ___________________________________________________________ 1. INTRODUCTION Escherichia coli is part of the normal large-bowel flora of humans and animals. Although most strains of E. coli are non-pathogenic in the intestine, some can produce diarrhea and by a number of distinct mechanisms. E. coli is usually considered to be an opportunistic pathogen which constitutes a large portion of the normal intestinal flora of humans. This organism can, however, contaminate, colonize, and subsequently cause infection of extraintestinal sites and is a major cause of septicemia, peritonitis, abscesses, meningitis, and urinary tract infections in humans (Cavalieri et al., 1984). E. coli was first incriminated as an enteropathogen in 1945, responsible for an outbreak of infantile diarrhea. Enteropathogenic E. coli (EEC) has been associated with diarrhea in developing countries and localities having poor sanitation. In the developed countries, EEC has been historically associated primarily with infantile diarrhea, but it was later recognized that adults also may suffer from the illness. E. coli are also enteropathogenic in animals (Wasteson et al., 1988; Weinstein et al., 1988b). E. coli O157:H7 causes severe illneses (hemorrhagic) and it does possess distinguishing characteristics, e.g. does not 2

3. ferment sorbitol with 24 h, does not possessα-glucuronidase activity, and does not grow well at all at 44-45.5C (Doyle, 1991). There are several subgroups of EEC: (A) enterotoxigenic (ETEC), (B) enteroinvasive (EIEC), (C) hemorrhagic (EHEC), and (D) enteropathogenic (EPEC) strains. The serogrouping and virulence factors associated with different EEC groups are shown in (Table 1) (39). Some authors classify them into six different pathotypes: ETEC, EIEC, EPEC, enteroaggregative E. coli, diffusely adherent E. coli, and Shiga toxin-producing E. coli (STEC) (Hernandes et al., 2009). In 1995, the enteropathogenic E. coli (EPEC) pathotype is divided into two groups, typical EPEC (tEPEC) and atypical EPEC (aEPEC). The property that distinguishes these two groups is the presence of the EPEC adherence factor plasmid (pEAF), which is only found in tEPEC. aEPEC strains are emerging enteropathogens that have been detected worldwide. The large variety of serotypes and genetic virulence properties of aEPEC strains from nonclassical EPEC serogroups makes it difficult to determine which strains are truly pathogenic (Hernandes et al., 2009). Humans are thought to be the principal if not the only reservoir of toxigenic and invasive strains of E. coli, contaminating foods via contact with food or via contact of processing equipment with water contaminated by human feces. In contrast, animals are reservoirs of the hemorrhagic strain (O157:H7); hence, foods of animal origin may become contaminated via slaughter procedures or post-processing recontamination. However, when E. coli is isolated from foods, pathogenic serotypes are usually absent or represent a very low percentage of the total isolates. 2. HEAT-LABILE ENTEROTOXINS 2.1. General Characteristics Probably the most common type of EEC strains is the enterotoxigenic type. Heat-labile (LT) and heat-stable (ST) enterotoxins are produced. Two partially cross-reacting antigenic variants of plasmid-coded LT, designated LTh and LTp, have been described in E. coli. LTh is associated with 3

4. E. coli isolates from humans, and LTp is associated with E. coli isolates from pigs. The LT family from restricted geographical region exhibited a segregated pattern of dissemination that was revealed by a restriction enzyme site polymorphism (Vinal and Dallas, 1987). Another heat-labile enterotoxin was discovered in extracts of E. coli SA53, a strain isolated from water buffalo. It activated adenylate cyclase. Hyperimmune antisera prepared against LTh and LTp or CT do not neutralize the crude LT-like toxin in Y1 adrenal cell assays. The structural genes for this LT-like toxin are not located on plasmids and are probably located on the bacterial chromosome. The structural gene for LT-like toxin does not have extensive nucleotide sequence homology with the genes encoding the A and B polypeptides of LTp. Subcloning and minicell experiments indicated that the toxin is composed of two polypeptide subunits that are encoded by two genes. The two toxin subunits exhibited mobilities on PAGE gels that are similar to those of CT and LT (Fig. 1) (Pickett et al., 1986). 4

5. It is proposed by Pickett et al. that the LTp and LTh (antigenic variants of LT will both be included in serogroup I and should be designated LTp-I and LTh-I and the LT-like toxin will be the prototype for serogroup II enterotoxins and should be renamed LT-II. Two distinct members of the LT-II family, LT-IIa and LT-IIb, are now known, and both have A and B subunits which are similar in size to those of CT and LT-I (Pickett et al., 1986; Pickett et al., 1987). 2.2. Gene and Regulation The LT gene was cloned into E. coli and two proteins of molecular weights 11,500 (B subunit) and 25,500 (A subunits) were produced (Dallas et al., 1979). The LT A subunit structureal gene (eltA) was sequenced and the amino acid sequence deduced (Spicer and Noble, 1982). It starts with methionine, ends with leucine, and comprises 254 amino acids. The computed molecular weight of LT A is 29,673. The A subunit genes of CT and LT (LT-I) are 78.6% homologous, and the B subunit genes are 78% homologous. The NH2-terminal regions exhibit the highest degree of homology (91%) as compared with CT subunit A, while the COOH-terminal region, containing the sole cystine residue in each toxin is less conserved (52%). Alignment of homologous residues in the COOH-terminal regions of LT A and CT A indicates that a likely site for proteolytic cleavage of LT A is after Arg residue 188 (Spicer and Noble, 1982). The gene of LT-IIa was studied. It is organized in a transcriptional unit similar to those of CT and LT-I. The A subunit gene of LT-IIa was found to be 57% homologous with the A subunit gene of LTh-I and 55% homologous with the A gene of CT. Most of the homology derived from the region of the A gene which encodes the A1 fragment. The B gene of LT-IIa was not homologous with the B gene of LTh-I or CT (Pickett et al., 1987). The LT-IIb gene was also cloned and analysed. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of CT and LT-I. The B subunits of LT-IIa and LT-IIb differ from the LT-I in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT and LT-Is. The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A 5

6. (Pickett et al., 1989). The carboxy-terminal domain of EtxB (encodes B subunit) mediates A subunit-B subunit interaction. The gene encoding the B subunit of LT was mutated at its 3' end by targeted addition of random nucleotide sequences. The functional and structural properties of the gene products were analysed, that these mutants were defective in their ability to associate stably with A subunits and form holotoxin (Sandkvist et al., 1987). 2.3. Mode of Action LT from E. coli is a protein of approximately 86,000 daltons that consists of one A polypeptide and five B polypeptides held together by noncovalent bonds. LT is closely related to cholera enterotoxin (CT) in structure, antigenicity, and mode of action. Both LT and CT bind to ganglioside GM1 receptors on eukaryotic target cells via their B subunits. The A subunit of LT like CT undergoes a proteolytic cleavage that produces two fragments designated A1 and A2. The A1 fragment catalyzes the NAD-linked ADP ribosylation of a regulatory subunit of adenylate cyclase in the plasma membrane of eucaryotic target cells, resulting in stimulation of adenylate cyclase activity. The activation of adenylate cyclase in mucosal cells in the small intestine causes secretion of fluid and electrolytes into the lumen and produces watery diarrhea. The LT-II, similar to CT and LT-I, increases cAMP by activating adenylate cyclase through the GTP-dependent ADP-ribosylation of specific membrane. Fibroblasts incubated with LT-II had an increased cAMP content (Fig. 2) as well as a fourfold elevation of membrane adenylate cyclase activity (Fig. 3). In membranes, activation of cyclase by toxin was enhanced by NAD, GTP, and dithiothreitol. The effect of LT-II on intact fibroblasts or membranes was increased by trypsin treatment of toxin (Chang et al., 1987). 6

7. 7

8. The B subunit of LTh (Human) is also hemagglutinating. Very strong hemagglutination of both neuraminidase- and pronase-treated human erythrocytes was induced by the B subunit of LTh. Different blood groups reacted differently to such enhancement (Sugii et al., 1988; Sugii and Tsuji, 1989; Sugii and Tsuji, 1990). Combining site of the B subunit may gain access to the receptor exposed on erythrocytes more easily by enzyme treatment. Neuraminidase and pronase are suppose to convert major gangliosides to GM1 and/or expose masked receptors for the B subunit. Both CT and LT strongly react with ganglioside GM1. Separation of ETEC bacteria from target intestinal epithelial monolayers by semipermeable filters prevented activation of adenylate cyclase suggesting that pathogen-host cell contact is required for efficient toxin delivery. Likewise, a non-motile strain bearing a mutation in the flagellar fliD gene was deficient in delivery of LT relative to the ETEC prototype. Although LT secretion via the type II secretion system (T2SS) was responsive to a variety of environmental factors, neither toxin release nor delivery depended on transcriptional activation of genes encoding LT or the T2SS (Dorsey et al., 2006). Enterotoxigenic E. coli LT-induced diarrhea is the leading cause of infant death in developing countries. Ginger significantly blocked the binding of LT to cell-surface receptor G M1 (Fig. 4), resulting in the inhibition of fluid accumulation in the closed ileal Biological-activity-guided searching for active components showed that zingerone (vanillylacetone) was the likely active constituent responsible for the antidiarrheal efficacy of ginger. Further analysis of chemically synthesized zingerone derivatives revealed that compound 31 (2-[(4-methoxybenzyl)oxy] benzoic acid) significantly suppressed LT-induced diarrhea in mice via an excellent surface complementarity with the B subunits of LT(Fig. 6) (Chen et al., 2007b). Similar inhibition effect was found in the extract of Chaenomeles speciosa fruits (寒梅) (Chen et al., 2007a). loops of mice (Fig. 5). 8

9. 9

10. 3. HEAT-STABLE ENTEROTOXINS 3.1. General Characteristics The heat-stable enterotoxins are low-molecular-weight, heat-stable, nonantigenic proteins which do not cause intestinal secretion by activation of adenylate cyclase. At least two types have been described, one with biological activity in suckling mice and piglets (STa, or named as STh, or ST-I) due to stimulation of particulate intestinal guanylate cyclase and a second which induces secretion by an unknown mechanism only in piglets (STb, or known as STp, or ST-II). Generally STs are known to be small peptide toxins consisting of 18 (STb) or 19 (STa) amino acids. The different STa (M.W. about 2,000) from different animal origins are remarkably homogeneous. The amino acid composition of STa of porcine, bovine, and human origins were identical and consisted of 10 of the 18 amino acids usually present in proteins. Six of the 18 amino acids were half-cystines which appear to be present as three disulfide bonds in the native form of the toxin. These disulfide bonds are important for toxic activity. STb is a heat-stable enterotoxin which does not cause intestinal fluid secretion in the suckling mouse as STa does, but does cause intestinal fluid secretion in pig intestinal loop assays. It is insoluble in methanol, while STa is methanol-soluble. Since STs are small peptides and are nonantigenic, fusion proteins (e.g. STa with outer membrane protein C, etc.) (Saarilahti et al., 1989) or synthetic ST 10

11. peptide conjugated with ovalbumin (Frantz et al., 1987) could be use as the immunoprophylactic agents against diarrhea caused by STs. Nontoxic fusion protein of LT was also contructed (Clements, 1990). In swine, the most common and severe enterotoxigenic E. coli (ETEC) infections are caused by strains that express K88 F4+ fimbriae, heat-labile enterotoxin (LT), heat-stable enterotoxin b (STb), and enteroaggregative E. coli heat-stable toxin 1. Previous studies based on a design that involved enterotoxin genes cloned into a nontoxigenic fimbriated strain have suggested that LT but not STb plays an important role in dehydrating diarrheal disease in piglets <1 week old and also enhances bacterial colonization of the intestine. In a study, these two toxins were examined in terms of importance for piglets >1 week old with the construction of isogenic single- and double-deletion mutants and inoculation of 9-day-old F4ac receptor-positive gnotobiotic piglets. Based on the postinoculation percent weight change per h and serum bicarbonate concentrations, the virulence of the STb- mutant (Delta estB) did not significantly differ from that of the parent. However, deletion of the LT genes (Delta eltAB) in the STb(-) mutant resulted in a complete abrogation of weight loss, dehydration, and metabolic acidosis in inoculated pigs, and LT complementation restored the virulence of this strain. These results support the hypothesis that LT is a more significant contributor than STb to the virulence of F4(+) ETEC infections in young F4ac receptor-positive pigs less than 2 weeks old (Erume et al., 2008). 3.2. Mode of Action and Regulation The enteropathogenic strains (O8:KX105) which colonized the small intestine of piglets produced STb and LT enterotoxins, wheras the nonenteropathogenic strains produced the STb. In another report, a hypertoxigenic E. coli strain which produced 17- to 60-fold more ST than laboratory ETEC. This strain rapidly induced severe form of scours in calves and piglets (Saeed et al., 1986). The action of STa stimulated the cGMP level in epithelial cell. Accumulation of cGMP by STa was synergistically enhanced by phorbol esters which are direct activators of protein kinase but not guanylate cyclase (Weikel et al., 1990). 11

12. The cysteine residues were substituted in vivo by oligonucleotide-directed site-specific mutangenesis to dissociate each disulfide bond and examined the biological activitites of the resulting mutants (Fig.7, 8). All three disulfide bonds formed at fixed positions are required for full expression of the biological activity of STb. It has some fexibilities in its conformation to exert toxic activity and that the role of each disulfide bond in exerting toxic activity is not quite the same (Okamoto et al., 1987). The STs share biologically active sequences which reside in the C-terminal 13 amino acid residues. Substitution of the asparagines residue at position 11 of STb by other amino acids resulted in significant decrease in enterotoxic activities, although the conformation was not changed (Okamoto et al., 1988). The amino acid sequences and disulfide bonds of the heat-stable enterotoxins of E. coli, Yersinia enterocolitica, and Vibrio cholerae non-O1 are shown in Fig. 9 (Okamoto et al., 1988; Okamoto et al., 1987). 12

13. Analogs of ST were made, including the native 18-amino-acid ST, the 14-amino-acid carboxy terminus of this native peptide with a proline at position 12, and the 14-amino-acid carboxy terminus of in which the proline at position 12 was substituted with glycine (Table 1). Each analog bound to the receptor in a dose-dependent fashion, native ST with the highest adherence (Fig. 10). Similarly, these peptides maximally activated particulate guanylate cyclase and stimulate intestinal secretion in suckling mice, and native ST with the highest potency (Fig. 11, Table 2). It demonstrats that the four amino-terminal residues contribute significantly to the potency of these peptides. In addition, the turn imposed by the proline residue at position 12 is not absolutely required for receptor occupancy or activation of the biochemical cascade that results in intestinal secretion. However, it significantly increases the potency of the toxin (Waldman and O'Hanley, 1989). 13

14. 14

15. The action of STb in the intestine of pig is still not consistant. It is susceptible to trypsin degradation and that variable amounts of trypsin-like activity in swine jejuna are responsible for inconsistent responses to STb in jejunal loops of swine (Whipp, 1987). When endogenous protease activity was blocked with trypsin inhibitor, STb evoked a dose-dependent secretory response in infant mice and jejunal loops of rats and pig (Whipp, 1990). The gene determined the nucleotide sequence for STb has been cloned. The only open reading frame with STb activity encoded for a 71-amino acid protein. It began with methionine-lysine-lysine followed by 8 to 20 hydrophobic amino acids and had a calculated molecular weight of 5,090. The STb structural gene (estA) was cloned into high-expression vector pKC30 downstream from the strong PL promoter and the expression was studied (Lawrence et al., 1990). 10-20-fold increase in mRNA was produced by the recombinant strain. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. Pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin 15

16. molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell (Fig. 12). These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Therefore, the MacAB-TolC system may capture the periplasmic STII molecules and exports the toxin molecules to the exterior (Yamanaka et al., 2008). 4. ENTEROTOXIN PLASMIDS Enterotoxin plasmids from classical strains (frequently associated with diarrhea, e.g. O6, O25, O27, O128, and O159) did not transfer by conjugation from clinical isolates, whereas those from rare strains (rarely associated with diarrhea, e.g. O7, O17, O80, O98, O139, and O153) transferred almost always from the clinical isolates by conjugation. Analyses of enterotoxin plasmids by restriction endonucleases and hybridization with the enterotoxin probes revealed that the strains with the same O serotype and toxigenicity carry closely related enterotoxin plasmids. These results suggest that classical strains resulted from the dissemination of ancestral clones which received enterotoxin plasmids long ago, while the rare strains acquired the enterotoxin plasmids recently by conjugation and have not yet been spread to the same degree as the ancestral 16

17. clones. 5. SHIGA-LIKE TOXINS Enteropathogenic E. coli (EPEC, e.g. O26:H11, O128:B12, O138:K81) and enterohemorrhagic E. coli (EHEC, e.g. O157:H7) are not enteroinvasive and do not produce the classical heat-stable and heat-labile enterotoxins. A cytotoxin known as Shiga-like toxin was demonstrated in EPEC and EHEC. A cytotoxin which could be neutralized by anti-shiga toxin is known as Shiga-like toxin I (SLT-I), and the non-neutralizable one is known as shiga-like toxin II (SLT-II) (Downes et al., 1988; O'Brien and Holmes, 1987). A total of 9% of LT-II-producing isolates from cows and buffalo hybridized with DNA probes for genes coding for SLT-II (Seriwatana et al., 1988b). In Thailand, SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle (Suthienkul et al., 1990). Vero cell cytotoxin E. coli O157:H7 was isolated from 3.7% of beef, 1.5% of port, 1.5% of poultry, and 2% of lamb samples in Canada (Doyle and Schoeni, 1987). The SLT-I is also known as verotoxin 1 and the SLT-II is also known as verotoxin 2. E. coli producing large among of SLTs are also named as VTEC (Verotoxin producing E. coli)(Smith and Scotland, 1988). Iron is known to depress Shiga toxin production by Shigella dysenteriae 1, and temperature has been shown to regulate several genes required for Shigella invasiveness and also expression of virulence plasmid in Yersinia. Iron also suppressed SLT-I synthesis in E. coli lysogenized with phage 933J but did not demonstrably repress toxin synthesis in E. coli strains carrying the cloned slt-I genes (Table 3). Temperature had no effect on SLT-I synthesis (Weinstein et al., 1988a). 17

18. 5.1. Purification and Structure The SLT-I and SLT-II have been purified to homogeneity. The bacteria grown in iron-depleted medium were disrupted by French press and the toxins purifed by anti-shiga toxin affinity chromatography or by conventional biochemical methods. The SLT-I A subunit has a MW 32,200 and the SLT-I B subunit has a MW of 7,700 (O'Brien and Holmes, 1987). The SLT-II was purified from E. coli strain containing the cloned toxin genes on recombinant plasmid. Purification was accomplished by a series of column chromatography techniques including chromatography (against SLT-II). The SLT-II consisted of A and B subunits with apparent molecular weights of 32,000 and 10,200, respectively. Also an A subunit of M.W. 25,000 was also observed and identified by immunoblot analysis (Downes et al., 1988). A variant of SLT-II was purified and bands of molecular weights of 33,000, 27,500, and 7,500 were identified to be A, Aa, and B subunits, respectively. Electrophoresis under nonreducing conditions resulted in disappearance of the 27,000 band (MacLeod and Gyles, 1990). By analogy with Shiga toxin, the most likely A-to-B subunit ratio for SLT-I is 1:5. Then the MW of holotoxin is about 70,000 (O'Brien and Holmes, 1987). monoclonal antibody affinity 18

19. 5.2. Mode of Action The purified SLTs have the same biological activities as and comparable specific activities to purified Shiga toxin. The molecular basis is probably the catalytic inactivation of 60S ribosomes in toxin-sensitive (receptor-expressing) cells (Fig. 14) (O'Brien and Holmes, 1987). The receptor for SLT-I and SLT-II has been identified and is the same as for Shiga toxin; it is a globotriosyl ceramide containing a galactose-à-(1->4)-galactose-á-(1->4)-glucose ceramide (Smith and Scotland, 1988). 19

20. But experiment in gnotobiotic piglets showed that SLTs is not essential for expression of virulence by EHEC strains O157:H7 (Tzipori et al., 1987). 5.3. Production and Regulation Production of SLT is activated in iron-limited media. By a gene fusion experiment, the sltA and sltB genes are regulated by a fur locus. A gene fusion between the promoter and proximal portion of the SLT gene with gene for bacterial alkaline phosphatase was made. Growth in low-iron conditions resulted in a 13- to 16-fold increase in alkaline phosphatase activity. In the presence of a null mutation in the fur locus, however, alkaline phosphatase activity was constitutively high regardless of the iron concentration. These data indicate negative regulation of the slt operon by the fur gene product (Calderwood and Mekalanos, 1987). 5.4. Genetics Phage conversion is responsible for controlling the production of several important bacterial toxins, including diphtheria toxin, streptococcal erythrogenic toxin, botulinum toxin, and staphylococcal enterotoxin A. Production of SLTs was also shown to be determined by specific phages in selected strains of both EPEC and EHEC serotypes isolated from humans (Fig. 15) (Smith and Scotland, 1988). In contrast, production of SLT in E. coli strains isolated from pigs was not found to be controlled by phages. The organization and expression of the operon for SLT-I are shown schematically in (Fig. 16). The structural genes that encode SLT-I were cloned on a 2.5-kb DNA fragment from a phage from strain H19, on a 3.1-kb DNA fragment from phage 933J, and on a 1.7-kb DNA fragment from phage H19B (Huang et al., 1986; Newland et al., 1985). 20

21. The recombinant plasmid containing the 1.7-kb fragment from phage H19B confers the ability to produce high levels of SLTs on transformed E. coli cells. By an in vitro transcription/translation system that the cloned fragment specifies the two subunit peptides which have masses of 31 and 5.5 kDa which different from 70,000 estimated (Huang et al., 1986). 21

22. A 0.75 kb probe of the SLT gene was used to hybridize clinical strains. Some SLT-positive strains only hybridized at low stringency and these results indicate that there are differences in the SLT genes of EEC (Willshaw et al., 1985). Genes controlling production of SLT-II were cloned from the phage 933W. Subcloning analysis identified a region within the 4.9-kb EcoRI fragment was associated with SLT-II production by minicell experiment. These experiments showed the genetic organization of the SLT-II genes to be the same as that of the SLT-I genes, with the coding sequence for the large A subunit adjacent to that for the smaller B subunit. The mobilities of the SLT-II subunits in SDS-PAGE gels were slightly greater than those determined for the SLT-I subunits. Although apparent processing of the SLT-I subunits was observed with polymyxin B treatment of the labeled minicells, no processing of the SLT-II subunits was detected. Southern blot hybridization studies suggested that the DNA fragment carrying the SLT-II structural genes shares approximately 50 to 60% homology with the DNA of the SLT-I structural genes (Newland et al., 1987). The molecular weight of the A and B subunits of SLT-II, deduced from the translated nucleotide sequences, were 33,135 and 7187, respectively (Smith and Scotland, 1988). The structural gene of a SLT-II variant (SLT-IIv) produced by a strain of E. coli was cloned and analysed. The A subunit genes for this SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit were less homologous (79%). The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-α-1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for shiga-toxin and SLTs. SLT-IIv may bind to a different cellular receptor than do other members of the shiga toxin family (Weinstein et al., 1988b). 5.5. Role in Disease Epidemiological Evidence As is the case with Shiga toxin, there is no direct proof that E. coli SLTs plays a role in disease. Some of the strongest circumstantial evidence comes from epidemiological studies of E. coli strains isolated from humans and animals. 22

23. Most of the high level cytotoxin producers were associated with diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome (HUS) (Table 4) (Smith and Scotland, 1988). It appears that food is the primary source of infection in man. E. coli O157 has been isolated from hamburger meat and unpasteurised milk which was also associated with hemorrhagic colitis and HUS. The O157:H7 were isolated from 1.5-3.7% of samples of beef, pork, poultry and lamb (Smith and Scotland, 1988). Animal Models A number of animal models, including chicks, mice, rabbits, calves, and pigs, has been used to study the pathogenesis of SLT producing E. coli. 6. HEMOLYSINS E. coli produce cell-free and cell-bound hemolysins, designated as theα- (AH) 23

24. andβ-hemolysin, respectively. This is an unfortunate designation and has caused some confusion since both the α- and β-hemolysins cause β-hemolysis (clear zone of lysis) around colonies on blood agar plates. Anγ-hemolysin was also produced by mutants resistant to nalidixic acid and this hemolysin does not hemolyze human or rabbit RBC but does hemolyze RBC of other species (Cavalieri et al., 1984). The AH has been studied intensively and discussed as follows. 6.1. Production and Purification Hemolysin-producing E. coli isolates are found primarily but not exclusively in serogroups O4, O6, O18, and O75. Ah is produced by growing hemolytic isolates in an alkaline meat extract broth, casein hydrolysate, or a chemical defined medium at 37C, aerobic, anaerobic condition and also in CO2. Aerobic growth enhances AH production. Both AH and β-hemolysin are produced during the log phase of growth (Cavalieri et al., 1984). Iron concentration above 100μM represses hemolysin production. It is suggested that a major function of AH in vivo may be to provide iron for growth under iron-limiting conditions. AH has been purified by various biochemical methods. However, electrophoresis of AH preparations in PAGE gels under nondenaturing conditions was unsuccessful, probably due to the high molecular weight of the molecule. Electrophoresis after denaturing with SDS and 2-mercaptoethanol and boiling revealed one well-defined protein band. AH was also purified by affinity chromatography. An immunoadsorbent was prepared by coupling Affi-Gel and the monoclonal antibody (Bohach and Snyder, 1986). 6.2. Characteristics AH, purified by affinity column chromatography, contains several proteins and lipopolysaccharides. Thus, AH exists as a macromolecular complex and may be exported from E. coli cells by outer membrane fragmentation (Bohach and Snyder, 1986). Complexing of AH proteins to LPS could account for discrepancies between measurements of the size of active AH (150,000 to 24

25. 300,000 daltons) and the 106,000 to 110,000-dal predicted by the size of its structural gene (Pollard et al., 1990). Treatment of AH with DNase, RNase, lecithinase, or lysozyme has no effect on AH activity, indicating that nucleotides, lecithin, or peptidoglycan do not comprise the active site. However, enzymatic treatment with lipases destroys hemolytic activity, suggesting that a lipid component may be necessary for AH activity (Cavalieri et al., 1984). Divalent cation calcium, strontium, or barium was required to demonstrate hemolytic activity in cultures of E. coli. The requirement of Calcium is not completely understood. Boehm et al. (Boehm et al., 1990) showed that calcium is required for binding of AH to erythrocyte membranes. Calcium autoradiography of the recombinant hemolysins separated by SDS-PAGE and transferred to nitrocellulose showed that full-length, active hemolysin bound calcium (Fig.17). AH is not a heat-stable protein, and it is inactivated by heating at 56C for as little as 10 min. However, some species of AH are relatively more stable to heat. Stability to heat is depending on the medium of treatment. Also, AH is inactivated by formalin and urea. 25

26. 6.3. Genetics The AH gene locates on various incompatible plasmids. It was shown that Insertion element (IS) occur in these plasmids that is the possible explanation for the finding of hemolysin determinants on various types of plasmids (Cavalieri et al., 1984). Strains harboring these plasmids produce a cell-free extracellular pool of hemolysin as well as an intracellular pool. Two types of nonhemolytic mutants were obtained by chemical and transposon mutagenesis of the plasmids. Some mutants produce intracellular AH but cannot export it into the medium. Cloning and the following analysis of AH gene have been done. At least three cistrons, designated as hlyA, hlyB, and hlyC, clustered in the AH determinant were found to be involved in synthesis and secretion of AH (Fig. 18) (Cavalieri et al., 1984). hlyA is responsible for synthesis of precursor, hlyC is responsible for the processing, and hlyB is responsible for export of AH. The gene product of hlyA was found to be a 106,000- to 107,000-dal nonsecreted cytoplasmic protein which is probably the inactive hemolysin precursor. hlyC codes for a 18,000-dal protein that appears to be involved in the conversion of the precursor hemolysin to active hemolysin with a proposed molecular weight of 58,000. The hlyC gene product is believed to have dual functions of (i) activation and (ii) transport of hemolysin through the cytoplasmic membrane to the periplasm. The hemolysin not treated with HlyC could not bind to RBC (Boehm et al., 1990). hlyB is not involved in synthesis of hemolysin but is required for transport of hemolysin from the periplam to the exterior of the cell. The hlyBa cistron codes for a 46,000-dal protein located in the outer membrane that binds the hemolysin and transports it through the outer membrane. hlyBb codes for a protein of 62,000-dal, most of which is found in the outer membrane, and presumably functions in release of hemolysin from the outer membrane (Cavalieri et al., 1984). 26

27. Hemolysin determinant on chromosome has also been cloned and studied. As with AH plasmid, at least three cistrons (A, B, and C) are present on the chromosomal AH determinant. Cistron hlyA seems to be most variable, whereas hlyB and hlyC are highly conserved. The primary structure of E. coli hemolysin (HlyA) contains a 9-amino-acid sequence which is tandemly repeated 13 times near the C terminus and which is essential for hemolytic activity. The domain involved in binding calcium was identified as the tandemly repeated sequences, since the deletion derivative missing 11 of the 13 repeats did not bind calcium (Boehm et al., 1990). 6.4. Role in Virulence and Mode of Action AH is toxic and lethal when intravenously injected to animal. It also shows cytotoxicity and the toxicity can be neutralized by antiserum treatment. The AH had a rather low activity in membranes formed of pure lipids, such as phosphatidylcholine or phosphatidylserine. In membranes from asolectin, a crude lipid mixture from soybean, hemolysin was able to increase the conductance by many order of magnitude in a steep concentration-dependent fashion. The asolectin may contain a receptor needed for membrane activity of 27

28. the toxin. The results of single-channel records showed that the membrane activity of hemolysin is due to the formation of ion-permeable channels with a single-channel conductance of about 500 pS in 0.15 M KCl (Benz et al., 1989). 7. ADHERENCE 7.1. In Enterotoxigenic E. coli Adhesion of ETEC to the small intestinal mucosa is now recognized as an important early event in colonization and the development of diarrheal disease. Special classes of protein fimbriae which promote mucosal adhesion of ETEC have been identified in some ETEC strains. The colonization factor antigens (CFAs) now include CFA/I, CFA/II, CFA/III, and CFA/IV (formerly PCF8775). A putative human ETEC colonization factor (PCF0159:H4) has been described in ETEC serotype O159:H4. The CFA/I, CFA/III, and PCF0159 are probably homogenous rodlike fimbrial antigens (Fig. 19). CFA/II is composed of three surface-associated antigenic components termed coli surface antigens (CS), CS1, CS2, and CS3. Strains of serotype O6:H16 produce either CS1 or CS2 in associated with CS3, while CS3 alone is found in most other CFA/II serotypes. CFA/IV also exhibits heterogeneity and currently consists of three distinct CS antigens, CS4, CS5, and CS6; CS4 and CS5 are rodlike fimbriae, where a structure has not been reported for CS6. The adhesion mediated by the fimbriae is shown in Fig. 20 (Knutton et al., 1989b). 28

29. A new nonfimbrial adhesive factor (antigen 8786), with mol.Wt. 16,300 Da, was found on the bacterial surface of enterotoxigenic E. coli O117:H4 (Aubel et al., 1991). A plasmid was also demonstrated coding for CS5, CS6, heat-stable enterotoxin, and colicin in O167 (Thomas et al., 1987). Infact, the structural genes of colonization factors are located on high-molecular-weight plasmids, except CS1 and CS2, which are chromosomal (Aubel et al., 1991). Synthesis and Action The synthesis of fimbriae by E. coli 469-3 (O21:H-) which was isolated from an infantile enteritis was studied at different temperature. It synthesized fimbriae at 37C, but not at 18C. No transcripts were detected, indicating that environmental temperature affects expression by regulating transcription (Williams and Hinson, 1987). Colonizing factor occurs in ETEC, however, its role in causing diarrhea remains unclear. Small bowel colonization by colonizing, nontoxigenic E. coli impairs water and electrolyte absorption and sucrase activity in the absence or recognized enterotoxin, cytotoxin, invasion, or effacement traits (Schlager et al., 1990). 29

30. Toxicity of heat-labile enterotoxin secreted by an ETEC was 40-fold enhanced in mixtures containing organisms capable of adhering (Streptococcus pyogenes) to the cell (Y-1 adrenal mouse cells) compared with monolayers exposed to organisms whose adherence was inhibited by mannoside (Ofek et al., 1990). Toxin produced by bacteria adherent to cells are targeted more efficiently and become relatively inaccessible to neutralization by toxin inhibitors (Table 5) (VL645 abd VL647 are isogenic strains Fim+ and Fim- strains of E. coli, each harboring LT+ plasmid, H-10407-p is an enterogenic strain lacking CFA/I but expressing type 1 fimbriae) (Ofek et al., 1990). The function of the coli surface antigens of CFA/IV (PCF8775) was studied. Mutants of PCF8775-positive enterotoxin producing E. coli were obtained. Mutants carrying CS6 alone colonized the intestine equally as well as strains carrying CS4-CS6 or CS5-CS6 did, whereas CS-negative mutants were excreted in the stool for a significantly shorter period. Rabbits previously infected with mutants carrying CS6 alone or CS6 in combination with CS4 or CS5 developed diarrhea with a significantly lower frequency after reinfection with a normally highly diaarheagenic dose. These results suggest that the CS6 component is a colonization factor in rabbits and that it is also capable of inducing protective immunity (Svennerholm et al., 1988). Molecular Biology of CFA The CFA/I gene has been isolated and sequenced. The amino acid sequence deduced from the nucleotide sequence is composed of 170 amino acids. The first 23 amino acids are considered to be the signal peptide of the CFA/I protein since they are not present in the protein sequence. Among the remaining amino acids, only two are different from the protein sequence. The CFA/I gene has a typical Shine-Dalgarno sequence located 10 bp upstream from the initiation 30

31. codon. The sequence TACAAT located 48 bp upstream from the initiation codon was tentatively designated the -10 sequence of the CFA/I gene promoter. No sequence homologous to the consensus -35 promoter sequence was found. A pair of inverted repeat sequences followed by a stretch of eight A's are located 45 bp downstream from the termination codon of the CFA/I gene; this region may be a p-independent transcriptional terminator (Karjalainen et al., 1989). The genetic determinant encoding the biosynthesis of 987P fimbriae in the E. coli strains isolated from neonatal pigs has been cloned into cosmid vector pHC79, and also cloned into pBR322. Analysis revealed that the 987P gene cluster contains a transposon that encodes the synthesis of ST and is flanked by inverted repeats of IS1. Hybridization experiments with the ST- and 987P-specific probes demonstrated that a variety of ST+ 987P+ wild-type E. coli strains contained contiguous ST-987P DNA, most likely on their chromosome (Klaasen et al., 1990). 7.2. In Enteropathogenic E. coli It has been shown that many EPEC strains adhere to cells (e.g. HEp-2, HeLa) in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA) (Benz and Schmidt, 1989). It was demonstrated that hemagglutination (pattern termed HAIII) factor of EPEC is very similar to the type 1-fimbriae antigenically. Type 1-fimbriae have been shown to mediate adherence to intestinal mucosa. But not all the EPEC strains carry type 1-fimbriae, so other structures are likely to be involved in the adhesion process. Electron microscopy failed to show fimbriae or pilus-like structures on the bacteria which exhibited adherence to HEp-2 and HeLa cells (Pal and Ghose, 1990). Adherence Factor on Plasmids EPEC are capable of adhering to tissue culture cells. EPEC of classic serotypes usually form distinctive microcolonies on the surface of epithelial tissue culture cells. This so called localized adherence (LA) is associated with the presence of a plasmid of 50 to 70 MDa. Such plasmids encode the so-called EPEC adherence factor (EAF) (Fletcher et al., 1990). The fragment A from 31

32. pMAR2 was used as probe in Southern blot analysis, and the result showed high degree of sequence conservation among these plasmids. Adherence genes from pMAR2 were cloned as two distinct plasmid regions which confer the adherence phenotype only when complementing each other in trans (Nataro et al., 1987). A DNA probe has been constructed from one of the adherence plasmids (pMAR-2) and has been used in field trials to detect EPEC (Fletcher et al., 1990). By comparing the restriction maps, other plasmids associated with cell adhesion are not similar to pMAR-2 (Fletcher et al., 1990). Another plasmid, pYR111 from serotype O111:NM, was also associated with localized adherence (LA) with HeLa cells. Curing of this plasmid yielded strains which lost the ability to exhibited LA, resistance to the antibiotics, and expression of lipopolysaccharide (LPS) O-antigenic polysaccharide (Riley et al., 1987). By conjugation experiment, both the mannose-resistant hemagglutinin and cell adherence factors were shown to be encoded by the same plasmid in two diarrheagenic E. coli strains belonging to an EPEC (O86) (Table 6) (Pal and Ghose, 1990). The diffuse adherence (DA) was also demonstrated to be associated with a 32

33. large plasmid in EPEC. EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) DA to HeLa cells, (ii) carried two large (about 100-kb) plasmids and one small plasmid (3-kb), and (iii) no fimbriae. A 6.0-kb fragment from the largest plasmid was cloned to E. coli and expressed DA phenotype. This insert encoded the production of a 100,000-dal protein mediating adhesion (Benz and Schmidt, 1989). The cellular adherence factors were associated with cell surface structures of bacteria that were proteinaceous in nature. So, cellular adherence properties could be substantially reduced by pronase treatment and by heat treatment (100C for 5 min) of bacteria (Pal and Ghose, 1990). Adherence Factor on Chromosome However, adherence factor may also exist in chromosome. TnphoA insertion mutants of EPEC with various adherence and pathogenic activity were obtained. By Southern hybridization of plasmid and total DNA of each strain was performed to determine the location of each TnphoA insert, and each TnphoA insert along with flanking EPEC sequences was also cloned. These studies resulted in the grouping of the mutants into five main categories: (A) strains with plasmid and chromosomal insertions that alter adherence, (B) chromosomal insertions that alter the ability to induce actin polymerization, (C) chromosomal insertions that do not affect adherence or actin polymerization (Table 7). It indicates that genes affecting EPEC adherence may be located on both the plasmid and chromosome, that several genes are involved in the induction of actin polymerization in epithelial cells, and that EPEC invasion is a complex process involving multiple genetic loci (Donnenberg et al., 1990). The chromosomally encoded gene products are required for full expression of enteropathogenicity (Knutton et al., 1987b; Knutton et al., 1987a). 33

34. Studying the adhesion by using electron microscope, a two-stage model of EPEC adhesion is proposed (Fig. 21, 22) (Knutton et al., 1987b): (i) initial attachment of bacteria promoted by plasmid-encoded adhesion, and (ii) effacement of brush border microvilli and intimate EPEC attachment. The second stage can occur in the absence of the first atage, but the presence of plasmid-encoded adhesions appears to greatly enhance the ability of EPEC to colonize the mucosa. 34

35. 35

36. Adherence and Pathogenesis Although EPEC strains possess toxic and adhesive capabilities which are likely to be involved in the disease process, it has been proposed that the intimate attachment of EPEC strains to intestinal mucosa could disturb the function of the microvillous border and bring about diarrhea. Evidence that adhesive non-toxigenic organisms can cause disease has been demonstrated in a distinct class of strains of E. coli. These strains of E. coli do not belong to the EPEC serotypes and are designated enteroadherent E. coli (EAEC) (Knutton et al., 1987b). Methodology Fluorescence Actin Staining (FAS) Method When bacteria are attached, microvilli are lost. The underlying cytoskeleton of the epithelial cell is disorganized, with a proliferation of filamentous actin. The polymerization of actin at the sites of the attaching and effacing lesion forms the basis of a recently described diagnostic test for EPEC. Fluorescein isothiocyanate (FITC)-phalloidin, the fluorescein conjugate of a phallotoxin, binds specifically to polymerized actin (Knutton et al., 1989a; Knutton et al., 1991). 7.3. In Enterohemorrhagic E. coli A plasmid of 60 MDa was also found in EHEC (e.g. O157:H7) (Toth et al., 1990; Tzipori et al., 1987). But experiment in gnotobiotic piglets showed that the presence of such plasmid is not essential for expression of virulence (Tzipori et al., 1987). Such plasmid appears to modify the eukaryotic cell adherence of E. coli O157:H7 and to confer that adherence on E. coli HB101 through surface structures other than pili. By electron microscopy, the wildtype strain and the plasmid cured strain which showed reduced adherence had pili (Fig. 23) (Toth et al., 1990). 36

37. 8. INVASIVENESS Enteroinvasive E. coli (EIEC) are strains with pathogenicity close to Shigella. Epithelial-cell invasiveness as detected by the ability of the organism to cause keratoconjunctivitis in the guinea-pig eye (Sereny test) is absent in enteropathogenic E. coli. Strains of Salmonella and Shigella were internalized after attachment to animal cells. Such process also occurs after the adherence of the E. coli to HEp-2 cells or Henle 407 cells and multiplication has been seen to take place. The E. coli invasion of human intestinal tissue in vivo has not been demonstrated (Law, 1988; Miliotis et al., 1989). 9. DETECTION 9.1. Using glucuronidase assay It was reported in 1976 thatβ-glucuronidase is limited to E. coli, Shigella species, and Salmonella species in the family Enterobacteriaceae. Then, β-glucuronidase substrates have been incorporated in diverse media to detect E. coli in samples from a variety of sources, such as environmental, food, seawater, and clinical sources. Constitutive enzyme test demonstrate that 87 to 97% of E. 37

38. coli isolates are positive, and inducible procedures show 91 to 100% positivity. A method designated as Colilert system is described as follows. The sample is plated on MacConkey agar, and the suspected colonies are picked and resuspended in Colilert tube (Access Analytical Systems, Branford, Conn., U.S.A.) (medium containing 4-methylumbelliferyl-β-glucuronide, MUG, as the fluorogenic indicator) rehydrated with 10 ml of destilled water. Tubes are read for fluorescence after 24, 28, and 120 h of incubation at 35C. The tube becomes yellow if total coliforms were present and fluorescent (under long UV light source) if E. coli is present (Rice et al., 1990). Using the Colilert system, 95.5% of E. coli isolates from human and animals were β-glucuronidase-positive in 24 h and 99.5% positive after 28 h of incubation (Rice et al., 1990). However, in another report 34% of E. coli from volunteers was β-glucuronidase-negative assayed in lauryl sulfate tryptose broth containing MUG (LST-MUG, Difco) (Chang et al., 1989). 9.2. Animal Tissue Culture Some of the bioassays for detecting EEC are listed in Table 8 (O'Brien and Holmes, 1987). Some cell lines such as Chinese Hamster Ovary (CHO) and Y1 Adrenal cells response to the LT. Cells could be cultivated in petri dishes or microplates. Bacterial cultures plus animal tissue culture medium are added to cells and incubate for 1 to several hours and replaced with fresh culture medium. The rounding of cells could be observed at 18 to 24 h (Sack and Sack, 1975). The EPEC and EHEC adhere to the intestinal mucosa and produce and 38

39. attaching and effacing lesion in the brush border microvillous membrane, this phenomenon also appears in tissue culture cells exposed to these bacteria or toxins. Dense concentrations of microfilaments are present in the apical cytoplasm beneath attached bacteria. Such polymerization of actin can by detected by Fluorescein-labeled phallotoxin (FAS). So FAS can be a simple, highly sensitive diagnostic test for EPEC and EHEC (Knutton et al., 1989a). 9.3. Animal Assays Rabbit ligated ileal loop assay (RIL) is usually employed. Test using infant mice is a convenient assay for STa. Supernatants of cultures (0.1 ml) could be injected with a no. 30 hypodermic needle into the milk-filled stomachs of infant mice (1-4 day old) and fluid accumulation in the intestine was measured after 4 h by determining the ration of intestine to whole body weight. Usually two drops of a 2% solution of pontamine sky blue 6BX (DuPont) are added to each 1 ml of inoculum. Results from mice with no dye in the intestine tract at autopsy are discarded. CT did not dilate infant-mouse intestine significantly, even in high concentrations. The younger mice and/or mice held at lower temperatures (e.g. 25C) tended to accumulate intestinal fluid, higher gut weight/body weight ratios (Moon et al., 1978). 9.4. Immunological Methods Membrane Filter-Enzyme-Labeled Antibody Procedure A hydrophobic grid membrane filter (HGMF)-enzyme-labeled antibody method was developed for the rapid detection of hemorrhagic O157 in food. An O-antigen-specific monoclonal antibody was labeled by horseradish peroxidase-protein A. The blended sample (e.g. meat) was pipetted through disposable prefilters (pore size 100-æm), dispensed in 10 ml of peptone water, and filtered through the HGMFs. The membranes were incubated at 43C for 16 to 20 h on HC agar. Antiserum, horseradish peroxidase-protein A conjugate were added in 1 ml of Tris-buffered saline (TBS, 20 mM Tris-500 mM NaCl, pH7.5) containing 1% gelatin. Filtered blocked in 3% gelatin-TBS and colored developed in coloring reagent containing 4-chloro-1-naphthol, hydrogen peroxide, etc. This method yielded presumptive identification within 24 h and 39

40. recovered, on average, 95% of the E. coli O157:H7 artifically inoculated into the meats (Todd et al., 1988). Enzyme-linked Immunosorbent Assay (ELISA) A competitive ELISA for STa is commercially available (COLI ST EIA, Denka Seiken). This use a peroxidase-conjugated monoclonal antibody to STa together with STa prepared by peptide synthesis (Scotland et al., 1989). Monoclonal anti-LT and anti-ST antibodies were also used in GM1-ganglioside ELISA which does not require advanced equipments(Scotland et al., 1989). Monoclonal antibodies against different epitopes on colonization factors of ETEC were produced (Lopez-Vidal et al., 1988) and these antibodies can be used to detect these virulence factors. Shiga-like toxin (Stx) can be detected by enzyme immunoassay (EIA) (ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar (Chromagar O157; BD BBL, Sparks, MD) (Grys et al., 2009). Antibody array constructed on solid supports using nitrocellulose membrane and poly-l-lysine (PLL) glass slide was developed for the detection of E. coli (Karoonuthaisiri et al., 2009). Different types of immunosensors (biosensors) proposed for rapid identification of E. coli O157:H7 were evaluated (Tokarskyy and Marshall, 2008). Latex Agglutination Test A simple latex agglutination test (E. coli O157 latex test, DR620, Oxoid) was developed for rapid presumptive detection of E. coli serotype O157:H7 during outbreak of hemorrhagic colitis. It is highly efficient and reliable test with 100% sensitivity and specificity. The latex test includes two reagents: test latex, consisting of latex particles sensitized with specific rabbit antibody reactive with the E. coli O157 antigen, and control latex, consisting of latex particles sensitized with preimmune rabbit globulins. It is a slide agglutination format, best used in conjunction with Sorbitol-MacConkey medium (SMAC). The SMAC medium is not very suitable for screening food samples (March and Ratnam, 1989). A V. cholerae enterotoxin Reverse Phase Latex Agglutination kit is commercialized by Seiken and this kit is also can be used to detect LT of E. coli. 40

41. Commercial latex agglutination kit for the detection of LT-producing E. coli is also available (Oxoid, TD920) (Notermans et al., 1991). VTEC can be detected by VTEC-Screen 'Seiken'. In a modified method, only parts of the commercial kit were used, including the Polymyxin B sulution, diluent, sensitized latex, control latex and the positive control. The basis for the test was the coagglutination test for strains of E. coli which produce the heat-labile enterotoxin (LT). Growth from each culture was emulsified in 50 μl polymyxin solution in the well of a U-bottomed microtitre tray. For each strain, 25 μl diluent were dispensed into one well of a V-bottomed microtitre tray. Using a multichannel pipette, 25 μl of supernantant fluid were carefully removed from the U-bottomed tray, avoiding contact with the precipitated bacterial cells, and added to the diluent. Following thorough mixing, 25 μl were placed in an adjacent well. To one of this pair of wells was added 25 μl sensitized latex and to the second well, 25 μl control latex. In addition, 25 μl positive control were placed into two wells and treated with the two latex preparations. The tray was sealed and left in the dark at room temperature for 2 h. It was then centrifuged 500 g for 15 min at 22°C and set at an angle of about 45° for up to 1 h at room temperature. The tray was regularly examined for bleeding of the control latex while the positive control with the sensitized latex remained intact. Where bleeding occurred in the test wells, these were considered negative; where they remained intact, these were considered positive (Bettelheim, 2001) E. coli in water samples were assayed by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis (r = 0.62 for E. coli) (Fig. 24). Simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results (Bushon et al., 2009). In the ATP method, ATP of E. coli was released into solution to which luciferin-luciferase was added to produce light. 41

42. 9.5. Enzymatic bio-nanotransduction Enzymatic bio-nanotransduction is based on the production and measurement of biological nano-signals (nucleic acid sequences) in response to the biological recognition of the targeted organism or toxin. Specifically, biological recognition molecules (such as antibodies) are linked to DNA templates that code for a T7 RNA polymerase promoter and a given nucleotide sequence. The specific capture and concentration of a target organism or toxin that is bound to a recognition molecule is followed by an in vitro transcription reaction of the bound DNA template. Detection of the RNA nano-signals on a detection platform is correlated with the presence or absence of the target in the original sample. By this approach, it is possible to detect multiple targets and target types (e.g., DNA, RNA, protein, whole cells) in a single sample by changing the recognition element (e.g., antibody, peptidomimetic) linked to the DNA template. In addition, it is possible to link this flexible detection system to nucleic acid detection platforms such as biosensors (Branen et al., 2007). 9.6. Nucleic Acid Probes nucleic acid probe, aptamer, 42

43. There are a number of reports on the detection of various toxin producers of EEC using various nucleic acid probes in different formats. Nucleic acid probes based on different virulence factors have been developed for the detection of pathogenic E. coli. Detection of Heat-labile and Heat-stable Enterotoxins LT probe isolated from pEWD299 after cleavage with HindIII and EcoRI (Sommerfelt et al., 1988a), or a 850-bp HincII digestion were used (Echeverria et al., 1989). Oligonucleotide probes were used used for the detection of LT, e.g. 5'-A CGT TCC GGA GGT CTT ATG CCC AGA GGG CAT AAT-3' (Sommerfelt et al., 1988a). PCR were used to amplify LT. A conservative sequence of LT subunit A was amplified by PCR and detected by radio- or enzyme-labeled oligonucleotide probes. 10μl of saline suspension of bacteria was used and combined in a total volume of 10 μl with a 10X PCR buffer; a 10 μl of 8 mM deoxynucleoside triphosphate stock (2 mM each of dATP, dGTP, dTTP, and dCTP); and 4 μl of each primer (10μM each), and distilled water to final volume of 99 μl. The reaction mixtures were overlaied with mineral oil and heated initially to 95C for 10 min to lyse the bacteria and denature the DNA (Olive, 1989; Victor et al., 1991). 215-bp HapII fragment from the STa-II gene of pSLM004 into the AccI site of pUC8 and later cleaved with BamHI and PstI and used as probe (Sommerfelt et al., 1988a). Aslo, a 157-bp HinfI fragment from the STa-I gene cloned into the SmaI site of pUC8 and cleaved by EcoRI and BamHI and used as probe. Oligonucleotide probes were also used, e.g. STa-I, 5'-GAA CTT TGT AAT CCT GCC TGT GCT GGA TGT-3'; STa-II, 5'-GAATTG TGT AAT CCT GCT TGT ACC GGG TGC-3' (Sommerfelt et al., 1988b; Sommerfelt et al., 1988a). A number of gene probes for various types of ST were used to detect ST genes in E. coli strains isolated in Brazil (Maas et al., 1985). One to four oligonucleotide probes labeled by alkaline phosphatase are used to detect each of the enterotoxin gene types and these probes are provided by Bresatec Ltd., Adelaide, South Australia, Australia (Medon et al., 1988). A single RNA probe was synthesized and used to detect simultaneously the LT and ST in E. coli strains. 911-bp of DNA where the regulatory elements and the signal peptide of eltB (for LT) were replaced by estA2 DNA (for ST) was 43

44. cloned into pSP plasmids containing SP6 promoter. Plasmid pYK159 (estA-eltB) was first linearized with HindIII and then transcribed with SP6 RNA polymerase with biotin-labeled synthetic UTP. The results with the biotinulated or radioactive probe correlated 100% with the biological assay results for both toxins. This probe could facilitate large epidemiological studies (S:aez-Llorens et al., 1989). Detection of Invasion An EIEC probe, 17-kb EcoRI digestion fragment of pRM17, was studied (Echeverria et al., 1989). Detection Shiga-like Toxin Toxin gene probes consisting of the 1,142-bp TaqI-HincII fragment of parts of the SLT-IA and SLT-IB subunits and the 842-bp SmaI-PstI fragment of the SLT-IIA subunit were excised, purified, and labeled with 32P. Colony blot from cultures on Modified Trypticase soy agar (BBL Microbiol.) and dot-blot with cultures from enrichment broth (Modified Trypticase soy broth, BBL) were hybridize with these probes. The dot-blot technique yielded results within 48 h and can be used as a fast and sensitive method of detection for SLT-producers (Samadpour et al., 1990). In Thailand, these probes were used in determination of SLT-producers in children with diarrhea (Brown et al., 1989a; Seriwatana et al., 1988a), also in meats (Suthienkul et al., 1990). Synthetic oligonucleotide probes labeled with 32P were also used for detecting E. coli producing SLT-I, SLT-II, and a variant of SLT-II (Brown et al., 1989b; Karch and Meyer, 1989a; Meyer et al., 1989). The results were affected by the hybridization temperatures (Brown et al., 1989b). Oligonucleotide probes for the detection of LTI, ST-Ia, ST-Ib, inv, EAF, SLT-I, and SLT-II were described (Olsvik et al., 1991). Unique restriction fragments present in plasmids of serogroup O157 E. coli strains were used as DNA probe for the detection of enterohemorrhagic E. coli (Huck et al., 1995). Detection of adherence factor 44

45. A 21-base oligonucleotide probe was constructed on the basis of a sequence from within the 1-kb E. coli adherence factor (EAF) probe and was shown to have greater sensitivity and specificity than the EAF fragment probe in detecting localized adherent E. coli. These isolates of enteropathogenic E. coli that exhibit localized adherence to HEp-2 cells (Jerse et al., 1990). 9.7. Using polymerase chain reaction Polymerase chain reaction (PCR) methods have been developed. Nonradioactive reporting material, digoxigenin-11-dUTP, was incorporated (Jackson, 1991). PCR has been used for amplifying segment of distinct SLT genes (Karch and Meyer, 1989b). The primers deduced from a conserved region among SLT genes are so-called degenerate-sequence primers; e.g., they contain intentionally introduced sequence ambiguities to overcome minor sequence variations within different SLT genes. In direct gel hybridization with genomic DNA, both primers recognized SLT-I and SLT-II sequences The PCR was used as a tool for the detection of heat-labile enterotoxin (LT) gene. The nucleotide sequences of the left- and right-hand amplimers were 5'-CCTCTCTATATGCACACGGAGCTCCCCAG-3' and 5'-CTATATGTTGAC TGCCCGGACTTCGACC-3', respectively. For identification of the amplified PCR product, an internal 20-mer probe with the sequence 5'-ATACGGAATC GATGGCAGGC-3' was used. When 3 CFU of E. coli LT was added to the 25-g samples of minced meat prior to enrichment culturing, the PCR assay yielded positive results (Wernars et al., 1991). Inosine-containing oligonucleotide primers were used for enzymatic amplification of different alleles of the heat-stable toxin type I gene. Inosine residues have no destabilizing effect on DNA duplexes regardless of the opposing bases (Candrian et al., 1991). PCR and gene probe detection of target lacZ (β-galactosidase) and uidA (β-glucuronidase) genes were used to detect total coliform bacteria and E. coli, respectively, for determining water quality (Bej et al., 1991a; Bej et al., 1991b). On the basis of virulence markers and clinical data, test strains could be split into different pathogroups, such as uropathogenic E. coli, enteropathogenic E. coli, Shiga toxin-producing E. coli, and enteroaggregative E. coli. H serotyping 45

46. and genotyping of the flagellin (fliC) gene revealed 11 different H types and a close association between certain H types, virulence markers, and pathogroups was found. Nucleotide sequence analysis of the O-antigen gene cluster revealed putative genes for biosynthesis of O15 antigen. PCR assays were developed for sensitive and specific detection of the O15-antigen-specific genes wzx and wzy (Fig. 25) (Beutin et al., 2005). A novel AB5 subtilase cytotoxin is produced by certain Shiga toxigenic E. coli (STEC) strains. This potentially lethal toxin may contribute to severe gastrointestinal and systemic disease in humans. A multiplex PCR assay for the detection of the novel toxin A subunit gene subA, as well as stx1 and stx2. The three primer pairs used in the assay do not interfere with each other and generate amplification products of 556, 180, and 255 bp, respectively (Fig. 26) (Paton and Paton, 2005). 46

47. Real-time PCR methods were also developed. PCR protocol for rapid identification of enterohemorrhagic E. coli on a LightCycler instrument. In a multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are detected in a single reaction capillary. A complete analysis of up to 32 samples takes about 45 min (Bellin et al., 2001). Real-time PCR method targeting on the fliC gene was developed. The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan minor groove binder probe specific for fliC-H21 were designed and used in a 5'-nuclease PCR assay. The primers fliCH21-F (5’-ACAGGAT AAAGATGGCAAACAAGTT) and fliCH21-R (5’-GCAGCCACTGCAAGC TTAGTT) and the TaqMan minor groove binder (MGB) probe (5’-FAM- ACCAGCAGTGATACTCMGB) were selected using Primer Express software (Applied Biosystems). This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC-H21 from one cell per reaction mixture was possible, and an initial 47

48. inoculum of 10 STEC H21 colony-forming units per 25 g of ground beef was detected after overnight enrichment (Fig. 27) (Auvray et al., 2008). Samples from Australia were also tested using real-time PCR (with SYBR Green I dye) for the presence of potential pathogenic microorganisms. Results showed that in the 27 rainwater samples tested, 17 (63%), 21 (78%), 13 (48%), and 24 (89%) were positive for E. coli, enterococci, C. perfringens, and 48

49. Bacteroides spp., respectively. 11 (41%), 7 (26%), 4 (15%), 3 (11%), and 1 (4%) were PCR positive for the Campylobacter coli ceuE gene, the Legionella pneumophila mip gene, the Aeromonas hydrophila lip gene, the Salmonella invA gene, and the Campylobacter jejuni mapA gene (Ahmed et al., 2008). 10. REFERENCES Ahmed,W., Huygens,F., Goonetilleke,A., Gardner,T. 2008. Real-time PCR detection of pathogenic microorganisms in roof-harvested rainwater in Southeast Queensland, Australia. Applied & Environmental Microbiology 74, 5490-5496. Aubel,D., Darfeuille-Michaud,A., Joly,B. 1991. New adhesive factor (antigen 8786) on a human enterotoxigenic Escherichia coli O117:H4 strain isolated in Africa. Infection & Immunity 59, 1290-1299. Auvray,F., Lecureuil,C., Tache,J., Perelle,S., Fach,P. 2008. Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment. Journal of Applied Microbiology 104, 899-905. Bej,A.K., DiCesare,J.L., Haff,L., Atlas,R.M. 1991a. Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Applied & Environmental Microbiology 57, 1013-1017. Bej,A.K., McCarty,S.C., Atlas,R.M. 1991b. Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring. Applied & Environmental Microbiology 57, 2429-2432. Bellin,T., Pulz,M., Matussek,A., Hempen,H.G., Gunzer,F. 2001. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. Journal of Clinical Microbiology 39, 370-374. Benz,I.,Schmidt,M.A. 1989. Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli. Infection & Immunity 57, 1506-1511. Benz,R., Schmid,A., Wagner,W., Goebel,W. 1989. Pore formation by the 49

50. Escherichia coli hemolysin: evidence for an association-dissociation equilibrium of the pore-forming aggregates. Infection & Immunity 57, 887-895. Bettelheim,K.A. 2001. Development of a rapid method for the detection of verocytotoxin- producing Escherichia coli (VTEC). Letters in Applied Microbiology 33, 31-35. Beutin,L., Tao,J., Feng,L., Krause,G., Zimmermann,S., Gleier,K., Xia,Q., Wang,L. 2005. Sequence analysis of the Escherichia coli O15 antigen gene cluster and development of a PCR assay for rapid detection of intestinal and extraintestinal pathogenic E. coli O15 strains. Journal of Clinical Microbiology 43, 703-710. Boehm,D.F., Welch,R.A., Snyder,I.S. 1990. Domains of Escherichia coli hemolysin (HlyA) involved in binding of calcium and erythrocyte membranes. Infection & Immunity 58, 1959-1964. Bohach,G.A.,Snyder,I.S. 1986. Composition of affinity-purified alpha- hemolysin of Escherichia coli. Infection & Immunity 53, 435-437. Branen,J.R., Hass,M.J., Douthit,E.R., Maki,W.C., Branen,A.L. 2007. Detection of Escherichia coli O157, Salmonella enterica serovar Typhimurium, and staphylococcal enterotoxin B in a single sample using enzymatic bio-nanotransduction. Journal of Food Protection 70, 841-850. Brown,J.E., Lexomboon,U., Neill,R.N., Newland,J.W. 1989a. Determination by DNA hybridization of Shiga-like-toxin-producing Escherichia coli in children with diarrhea in Thailand. Journal of Clinical Microbiology 27, 291-294. Echeverria,P., Taylor,D.N., Seriwatana,J., Vanapruks,V., Brown,J.E., Sethabutr,O., Jackson,M.P., Lolekha,S., Echeverria,P. 1989b. Hybridization of Escherichia coli producing Shiga-like toxin I, Shiga-like toxin II, and a variant of Shiga-like toxin II with synthetic oligonucleotide probes. Infection & Immunity 57, 2811-2814. Bushon,R.N., Brady,A.M., Likirdopulos,C.A., Cireddu,J.V. 2009. Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique. Journal of Applied Microbiology 106, 432-441. 50