**7.3. DNA sequencing**

**Virulence factor**

Verocytotoxin 1

Verocytotoxin 2

Intimin Heat-stable enterotoxin-human

Heat-stable enterotoxin-porcine

Heat-labile enterotoxin

Invasive plasmid antigen

**Table 2.**

*ipaH*

*eltA*

*estA*-porcine

*estA*-human

*Eae*

*vtx2*

**Gene target**

*vtx1*

**Primer sequence (5′-)**

GTTTGCAGTTGATGTCAGAGGGA

CAACGAATGGCGATTTATCTGC

GCCTGTCGCCAGTTATCTGACA

GGAATGCAAATCAGTCGTCACTC

GGYCAGCGTTTTTTCCTTCCTG

TCGTCACCARAGGAATCGGAG

TTTCGCTCAGGATGCTAAACCAG

CAGGATTACAACACAATTCACAGCAGTA

CTTTCCCCTCTTTTAGTCAGTCAACTG

CAGGATTACAACAAAGTTCACAGCAG

AAACCGGCTTTGTCAGATATGATGA

TGTGCTCAGATTCTGGGTCTCCT

TTGACCGCCTTTCCGATACC

ATCCGCATCACCGCTCAGAC

Gene target, primer sequence, and amplicon size for common intestinal pathogenic *E*. *coli* virulence factors (Adapted from Persson et al. [26]).

647

479

160

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

151

377

420

260

**Amplicon size (bp)**

This is the determination of precise order of bases in the nucleotides that make a specific segment of a DNA. Apart from characterization of genetic material for the purpose of identification of *E*. *coli* strain, DNA sequencing assist in comparison of genetic makeup from different sources, for example, in assessment of the association of different disease outbreak. Generally, sequencing use electrophoresis to separate pieces of DNA into bands. DNA molecules move through the gel when an electric current is applied and molecules are separated according to size, small molecules move faster. During sequencing, bases are tagged with fluorescence dyes, each base type producing a different color, for example, thymine = blue, cytosine = green, adenine = red, and guanine = yellow. Artificial modified bases are added to the DNA mixture. DNA molecules will undergo copying many times. When one of the modified bases is incorporated into the DNA molecule, elongation of the chain stops and all DNA pieces in that batch will have an ending with that particular modified base. The next batch of DNA copy will have a different artificial base at the end and so on. As a result, different DNA batches will end with different base T, A, G, and C, each with a specific color. So the base sequence in the assembled DNA material will be determined by a color pattern of the last (modified) base. The information is stored in computer memory and used for interpretation. This is a traditional Sanger 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 of reactions take place in parallel and many samples can be analyzed at once.

**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

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**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

**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

**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

**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.

genes can be easily detected by serological assays [39] and multiplex DEC PCR.

typing can be applied to compare strains during outbreaks.

typing by PFGE are possible [41].

Afa/Dr genes [42].

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 many laboratories to manage the procedure.

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

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.
