**8. Common** *E. coli* **pathotypes**

Intestinal pathogenic *E*. *coli* form a large proportion of pathogenic *E*. *coli*. They include VTEC, EPEC, ETEC, EAEC, DAEC, and EIEC.

**Verocytotoxigenic** *E*. *coli* (**VTEC**) produces verocytotoxins also known as Shiga toxins. The most common VTEC is O157:H7 strain. VTEC are characterized by possession of genes encoding for *vtx1* and *vtx2*, although they carry other virulence genes such as *eae* and *ehxA*. Animals are principal reservoirs of VTEC, and the main route of transmission is fecal-oral. In humans, especially children and elderly, VTEC cause abdominal cramps associated with diarrhea or dysentery. Complicated cases of VTEC infection may lead to HUS. VTEC can be isolated from different sources by different approaches, but the choice will depend on the objectives. Reliance on sugar fermentation ability, for example, sorbitol or presence of specific enzymes, for example, beta-glucuronidase, may lead to focus on specific fraction of the pathogen. On the other hand, targeting verocytotoxin-producing genes will give the overall burden of VTEC from a target source. In this scenario, the use of IMS technique may be recommended [28]. Characterization of isolates for VTEC detection may include immunological methods by using specific antibodies against target VTEC strain or PCR by targeting specific genes. VTEC isolates typing can be done by serology, using specific antisera, PFGE, DNA hybridization, and sequencing.

**Enteropathogenic** *E*. *coli* (**EPEC**) possess *eae* just as do some VTEC strains. As a result they cause attaching and effacing lesion and hence diarrhea. Classical EPEC differs from atypical EPEC (A/EEC) by possession of *bfpA* gene. However, atypical EPEC is a more prevalent cause of diarrhea [38]. Human EPEC infection follows fecal-oral route and isolation can be done from different sources such as water, food, animal, and environment. However, characterization emphasize should be put on distinguishing EPEC from VTEC by presence of *eae* gene and absence of vtx genes. Also, classical EPEC and atypical EPEC should be differentiated by assessing the presence of *bfpA* gene that encode for bundle-forming pili. These features can be determined by characterization procedures such as PCR and DNA hybridization [14]. PFGE typing can be applied to compare strains during outbreaks.

sequencing. Besides, the fast advancing technology is taking the investigative life science from a few DNA fragments analysis into another level of whole genome sequencing. Next Generation Sequencing analyses the entire genome in a short time of single sequencing run. As a result, analysis and comparison of whole genome of isolates lead to correct diagnostic inference. Principally, next generation sequencing is similar to conventional Sanger method, but the former, through sequencing by synthesis, allows detection of single bases as they are incorporated into a growing DNA strand until the whole genome is read. Moreover, millions

Sequencing is superior to other methods in characterization of genetic material. For example, whole genome sequencing can detect false positive and false negative clonal relationship of isolates from PFGE fingerprinting [34]. Regardless of the approach to the genome as a whole, the actual process of DNA sequencing is the same. Guidelines and protocols for sequencing are described in detail by a number of researchers in Refs. [35, 36], such that it is possible for

The genetic expression of *E*. *coli*, especially pathogenic *E*. *coli*, can be evaluated by applying the toxin extract from the bacteria to the monolayer Vero cell culture. Cytopathic effects to the cells will indicate virulence activities of the genes. Details of cytotoxic effect assay on Vero cell have been documented in Ref. [37]. Mouse inoculation can also be done to assess virulence of genes.

Intestinal pathogenic *E*. *coli* form a large proportion of pathogenic *E*. *coli*. They include VTEC,

**Verocytotoxigenic** *E*. *coli* (**VTEC**) produces verocytotoxins also known as Shiga toxins. The most common VTEC is O157:H7 strain. VTEC are characterized by possession of genes encoding for *vtx1* and *vtx2*, although they carry other virulence genes such as *eae* and *ehxA*. Animals are principal reservoirs of VTEC, and the main route of transmission is fecal-oral. In humans, especially children and elderly, VTEC cause abdominal cramps associated with diarrhea or dysentery. Complicated cases of VTEC infection may lead to HUS. VTEC can be isolated from different sources by different approaches, but the choice will depend on the objectives. Reliance on sugar fermentation ability, for example, sorbitol or presence of specific enzymes, for example, beta-glucuronidase, may lead to focus on specific fraction of the pathogen. On the other hand, targeting verocytotoxin-producing genes will give the overall burden of VTEC from a target source. In this scenario, the use of IMS technique may be recommended [28]. Characterization of isolates for VTEC detection may include immunological methods by using specific antibodies against target VTEC strain or PCR by targeting specific genes. VTEC isolates typing can be done by serology, using specific antisera, PFGE, DNA hybridization,

of reactions take place in parallel and many samples can be analyzed at once.

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

many laboratories to manage the procedure.

**7.4. Phenotypic characterization of** *E. coli*

**8. Common** *E. coli* **pathotypes**

EPEC, ETEC, EAEC, DAEC, and EIEC.

and sequencing.

**Enterotoxigenic** *E*. *coli* (**ETEC**) are responsible for watery diarrhea in humans due to impaired sodium absorption and enhanced chloride secretion caused by enterotoxins. Fecal-oral contamination is responsible for transmission through food and water, and the syndrome is common to travellers and children. A simple procedure for detection of ETEC from stool has been described earlier in Ref. [38]. Heat-stable and heat-labile enterotoxins encoded by heat-stable enterotoxin (*estA*) and heat-labile enterotoxin (*eltA*) genes, respectively, are responsible. These genes can be easily detected by serological assays [39] and multiplex DEC PCR.

**Enteroaggregative** *E*. *coli* (**EAEC**) causes acute and persistent diarrhea in humans. This group has diverse strains differing in many aspects but have a common feature of forming a "stacked brick" pattern of adhesion to the human epithelial cell line HEp-2. This feature is used in HeLa cell adherence method to detect EAEC strains [40]. They often produce heatstable toxin EAST1, Shigella enterotoxin (ShET1), and Haemolysin E, which cause host cell damage and induce inflammation leading to diarrhea especially in travellers, children, and immunocompromised patients. The EAEC strains are found in mixed infections whereby isolation by MacConkey ager, detection by conventional biochemical methods, and PCR and typing by PFGE are possible [41].

**Diffusely adherent** *E*. *coli* (**DAEC**) are responsible for acute diarrhea in humans. DAEC are characterized by the ability to adhere to Hep-2 cells in a diffuse fashion as confirmed by HeLa cells assays. Isolation is done conventionally and detection by PCR can be done by targeting Afa/Dr genes [42].

**Enteroinvasive** *E*. *coli* (**EIEC**) cause profuse diarrhea or dysentery in human through mechanical damage of host epithelial cell by using adhesin protein for binding and invading/entering intestinal cells. They do not produce toxin. EIEC resembles Shigella species biochemically and genetically. Most of them do not ferment lactose. Following conventional isolation methods, EIEC are detected by invasion plasmid antigens (*ipaH*) gene-targeted PCR [43]. The invasiveness of EIEC can be assessed by plaque formation on HeLa cell or guinea pig conjunctivitis assays.

**Extra**-**intestinal pathogenic** *E*. *coli* (**EXPEC**) cause a wide range of bacteraemia-associated disease syndromes. EXPEC have been isolated in patients with cystitis, pyelonephritis, or prostatitis [28]. Other syndromes associated with EXPEC include septic arthritis or pyomyositis, nontraumatic meningitis, or hematogenous osteomyelitis and pneumonia [44]. This group is comprised of UPEC, NMEC, and SEPEC [1]. Infection normally follows fecal-oral route. Samples to collect will depend on infected system; urine samples can be collected for urinary tract infection-related syndromes, such as cystitis, Pyelonephritis, or Prostatitis [28], whereas blood, joint fluid, psoas fluid, or sputum are target samples when nonurinary syndromes are concerned [44]. Isolation of *E*. *coli* for EXPEC detection can follow methods that have been mentioned previously for other pathotypes. Detection of EXPEC can be done by multiplex PCR targeting different genes some of which have been previously described and dot blot hybridization [1, 20, 27, 28]. Typing of isolates from different sources can be done by different procedures including PFGE [20].

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