**3. Bacterial survival in the human digestive tract**

include fimbrial and fimbrillar structures. Among them, seven are generally more prevalent than others: CFA/I (colonization factor antigen) and CS1 to CS6 (*coli* surface antigen) [30]. Most CF receptors have not been yet identified, but CFs are thought to bind to glycopro‐ tein conjugates in mucus fraction from the small intestine and on the surface of host cells. Non‐fimbrial adhesins such as TibA, a glycosylated autotransporter; Tia, an outer membrane protein; and EtpA, which acts as a molecular bridge binding host cell receptors to the tips of

EHEC colonization involves attaching and effacing (A/E) lesions on the enterocytes, which are characterized by ultra‐structural changes, including loss of microvilli and intimate attach‐ ment of the bacterium to the cell surface [32]. Genes encoding A/E lesion formation are local‐ ized on a pathogenicity island, the locus for enterocyte effacement (LEE), which encodes a bacterial type III secretion system (T3SS). Colonization is mainly mediated by the primary adhesin, namely intimin (encoded by *eae* gene), but other putative adherence factors have been described, such as long polar fimbriae—Lpf—or curli [33]. A number of other non‐fim‐ brial EHEC adhesins have been implicated in adhesion including the plasmid‐encoded *toxB*,

Mucin‐degrading enzymes, which allow temporary access to intestinal cell membrane and promote bacterial adhesion have been recently identified in both ETEC and EHEC. In ETEC, YghJ, a mucin‐binding metalloprotease [34] and EatA, a member of serine protease autotrans‐ porters of the Enterobacteriaceae (SPATE) family [35] have been described. In EHEC, one protein has been shown to have mucinase activity: StcE, an extracellular zinc metalloprotease

Toxins are considered as the main virulence factor for both ETEC and EHEC as they are responsible for the main clinical symptoms and/or systemic complications. In ETEC, secre‐ tory diarrheas are mediated through the action of heat‐stable (ST) and/or heat‐labile (LT)

ETEC strains are able to secrete either one or two toxins (LT and/or ST), but it has been shown that LT toxin is less likely to cause disease than ST or LT/ST ETEC toxins [7]. LT toxins encoded by the *eltAB* gene are similar in structure and function to Cholera toxin by sharing 80% homol‐

ring of LTB subunits responsible for binding and internalization [37]. LT are mainly secreted associated with outer membrane vesicles (OMVs) and bind irreversibly to monoganglioside (GM1) on the host cell. LT leads to an increase in cAMP that induces cystic fibrosis transmem‐ brane regulator (CFTR) phosphorylation, eliciting massive fluid loss and watery diarrhea. In addition to causing diarrhea, LT plays multiple roles in modulating host cell function and providing a competitive advantage for ETEC adherence to cultured intestinal epithelial cells. ST toxins encoded by the *estAB* gene are small cysteine‐rich peptides which mimic the human hormone guanylin. They are divided into two structural and antigenically distinct groups: STa and STb which reversibly bind to guanylyl cyclase C (GC‐C) and sulphatide, respectively

configuration with a catalytically active LTA subunit and a pentameric

ETEC flagella, have also been implicated in the pathogenesis [31].

8 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

the chromosomally encoded adhesins Iha, Cah, and OmpA [32, 33].

which specifically recognizes α‐O‐glycan‐containing substrates [36].

*2.4.3. Secretion of toxins*

ogy. LT shows an AB5

[37], leading to CFTR activation and diarrhea.

enterotoxins.

Bacterial survival in the human GI tract is a key parameter in ETEC and EHEC physiopathol‐ ogy. Nevertheless, how pathogens can survive in the human digestive environment remains largely unknown as studies in humans are impossible. For regulatory, ethical, technical, and cost reasons, artificial digestive systems are increasingly used as an alternative to *in vivo* stud‐ ies in humans. Until now, almost no data are available for ETEC under human digestive simulated conditions while a number of studies have assessed the survival of EHEC during human *in vitro* digestion.

Masters et al. [41] have shown that after exposure to pH 2, ETEC became undetectable by plate counting after 2 hours. A recent study using flow cytometry analysis indicated that there was no significant difference in the percentage of live bacteria when ETEC were subjected to pH 5 or pH 7 [42]. Only one study has investigated the impact of 30 g/L bile on the survival of ETEC *in vitro*. Despite the known bactericidal effect of bile in the intestine, growth curves for ETEC in Luria Bertani (LB) media and LB‐bile showed similar slopes during the exponential growth phase [43].

With regard to EHEC, most of the studies have been carried out, like for ETEC, using simpli‐ fied *in vitro* approaches integrating a limited number of digestive parameters, such as acid pH or bile salts [44, 45]. Even if the pathogen is considered as acid resistant, large variations in survival rates have been obtained for *E. coli* O157:H7 in acidified culture media or in simu‐ lated gastric fluid [45, 46]. This wide range of response may be explained by differences in culture conditions, bacterial strains, and pH values used to simulate the gastric phase. Other more recent studies have evaluated the survival of EHEC strains by using dynamic multi‐ compartmental *in vitro* models that closely mimic the gastric, small intestinal, and colonic human digestive conditions. In the TNO GastroIntestinal model (TIM), which simulates the stomach and the three segments of the human small intestine, it has been shown that EHEC survival was affected in the stomach and duodenum (when ingested within a food matrix but not with a glass of water), while bacterial growth was observed at the end of digestion in the jejunum and ileum [47–49]. This growth renewal in the distal parts of the small intes‐ tine was probably linked to the occurrence of less stringent conditions, such as neutral pH and lower concentrations of bile salts due to their reabsorption (as occurred *in vivo*). EHEC survival in the TIM model was found to be strain/serotype dependent [48] and influenced by food matrices [47–49] and age conditions [48]. In particular, thanks to the potential of the TIM model, Roussel et al. [49] have shown that differences in digestive physicochemi‐ cal parameters related to age conditions may partly explain the higher susceptibility of chil‐ dren (compared to adults) to EHEC infections and HUS. Additional studies performed under human‐simulated colonic conditions (including colonic microbiota) have shown that EHEC strains were not able to colonize [50], probably due to the barrier effect of gut microbiota or to the high short‐chain fatty acid (SCFAs) concentrations found in the colon and known to inhibit EHEC growth [51, 52]. Taken together, these data suggest that the ability of EHEC to colonize the human gut would be rather linked to growth renewal of the pathogen in the distal parts of the small intestine than the ability to maintain in the colon.

the available studies have been performed in simple *in vitro* digestive conditions, recent data have been obtained for EHEC in more physiological conditions simulated by dynamic multi‐

Enterotoxigenic and Enterohemorrhagic *Escherichia coli*: Survival and Modulation of Virulence in the Human...

http://dx.doi.org/10.5772/intechopen.68309

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Once ingested, pathogens are exposed to the host digestive tract characterized by acid con‐ ditions in the stomach where pH gradually decreases during digestion from around 6 to 2,

For ETEC, the release of ST seems to be not pH‐dependent [54], while it is acknowledged that extracellular pH has an influence on the release of LT toxin which increases with alkalinity [55, 56]. ETEC seems to use the pH gradient in the GI tract to modulate LT toxin production and secretion: when bacteria reach the small intestine, alkaline pH induces both transcription

For EHEC, House et al. [57] have examined, using DNA microarrays, the gene expression profiles of EHEC O157 that had been acid stressed and then neutralized relative to the same unstressed strain. Virulence factors associated with adhesion, motility, and type III secretion were significantly modulated leading to enhancement of motility and host cell adhesion. The T3SS genes encoding proteins that mediate colonization and infection in the large intestine were downregulated following acid stress [33, 57]. Impact of low pH on Stx gene expression and production is not yet fully understood: House et al. [57] have shown no change whereas other studies have revealed that acid pH decreases Stx production [58, 59]. In the gastric and small intestinal TIM model, Roussel et al. [49] have shown that *stx1* and *stx2* genes were upregulated in the gastric compartment even if Stx‐mediated cytotoxicity is generally associ‐ ated with distal parts of the small intestine or large intestine. Higher expression levels were observed under child digestive conditions compared to adult ones where less acidic condi‐ tions are found, which is in accordance with the results of Yuk et al. [58] and Huang et al. [59].

Once the small intestine is reached, bile salts form a major challenge to pathogens, with bile

Chatterjee and Chowdhury [60] have shown *in vitro* that 2 g/L crude bile can prevent the binding of LT toxin to GM1 and that this effect was associated to arachidonic, linoleic, and oleic unsaturated fatty acids detected in crude bile. The same authors demonstrated *in vivo* in rabbit ileal loops that linoleic acid prevented LT‐mediated fluid accumulation in a dose‐ dependent manner [60]. In another study by Nicklasson et al. [61], 1.5 g/L crude bile and 2 g/L bile salts sodium deoxycholate and sodium glycocholate‐induced *in vitro* the expression of CS5‐encoding gene *csfD*. A global transcriptional analysis of two ETEC strains showed that bile salts at a concentration of 30 g/L in LB medium upregulated *estA*, *eltA,* or *etpA* (encoding for STa, LTa enterotoxins, and EtpA, respectively) while *csoA* and *cstA* (encoding for CS1 and

concentrations sequentially decreasing from duodenum to colon due to reabsorption.

**4.1. Regulation by physicochemical parameters of the human gut**

followed by pH close to neutrality in the small intestine.

compartmental models.

and maximal release of LT [42].

*4.1.1. pH*

*4.1.2. Bile*
