**7. Characterization of** *E. coli* **isolates**

Characterization includes detection of bacteria isolates from different sources and typing of bacteria isolates of same species. *E*. *coli* can be characterized by different methods, depending on what attribute is targeted. The methods are categorized as serology, molecular techniques, or cytopathic assays. Molecular characterization includes numerous techniques such as PCR, DNA hybridization, PFGE, restricted fragment length polymorphism (RFLP) and multilocus variable-number tandem repeat analysis (MLVA) to mention a few. These variable methods of bacteria typing have previously been summarized and compared in Ref. [19]. A combination of different methods can be used to complement each other especially when accurate diagnosis is required in a public health threat. A good example of combination of different characterization methods is the work reported by Sabat et al. [23], whereby isolates confirmed to possess somatic antigen O157 by agglutination test were further characterized by PCR subtyping of verotoxigenic (vtx) genes, O:H serotyping, Vero cell assay, sorbitol fermentation, β-glucuronidase activity, dot blot hybridization, and PFGE.

## **7.1. Serotyping**

cultures are stored at 4°C for long time. Low temperature storage of bacteria involves keeping bacteria at low temperatures, ranging from 4 to −80°C. Freezing usually requires addition of glycerol or sugars as cryoprotectants. Deep freezing is the most common preservation method, which maintains both survival and similarity of bacteria population compared with other methods. The choice of the method of preservation depends on several factors, including the nature of bacteria, desired length of time of storage, analysis strategy, and study objectives. Short period preservation, for example, for days or a week, bacteria can be stored under refrigeration temperatures. Pure bacteria culture is grown on agar slants or plates of nondifferential media and stored at 4°C. Screw-capped tubes are recommended when agar slants are used in bacteria preservation. Cultures on Petri dishes should be protected from contamination and rapid drying by sealing the plates with parafilm and stored inverted. Screw-capped tubes with hot sterile media are inclined at an angle to allow the media to solidify into a slant. A loopful of pure bacteria culture is inoculated onto the slant surface and incubated at 37°C

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Freezing is another method used to store bacteria whereby, the degree of coldness corresponds to length of storage period. The colder the storage temperature, the longer the culture will retain viable cells. Freezing temperatures of −20 to −40°C, which is achieved by most laboratory freezers, can be used to preserve bacteria for up to 1 year. Low temperature of −80°C can preserve bacteria for longer than 3 years, whereas cryofreezing at temperatures below

Freezing may damage or kill bacteria cells due to resultant physical and chemical processes taking place. During freezing, water in the bacteria cell is converted to ice and solutes accumulate in the residual free water. Ice crystals formed can damage the cell membrane and the negative solute concentration can denature cell biomolecules. Cryoprotectants such as glycerol lower the freezing point of the bacteria suspension and thus prevent extracellular ice crystal formation and build-up of negative salt concentration. Besides, the lethal intracellular freezing is usually avoided by slow cooling or progressive freezing that allows sufficient water to leave the cell during freezing of extracellular fluid. A slow progressive freezing at a cooling rate of 1°C/min can be achieved by using a rate controlled freezer. Alternatively, similar results can be obtained by "snap freezing." Bacteria cells are snap-frozen by immersing the well-labeled 15% glycerol cell suspension containing cryotubes in dry ice or liquid nitrogen

−130°C, usually in liquid nitrogen, can preserve bacteria for more than 10 years.

before storing them in freezer (−20 to −80°C) or in liquid nitrogen tank (−196°C) [22].

stored at −20, −80, or −196°C.

Bacteria cultures for freeze preservation can be prepared by inoculating a loopful of bacteria culture into nondifferential sterile broth such as nutrient broth followed by 37°C incubation for 24 h. This broth with pure bacteria culture is mixed with glycerol to make it 15–20% glycerol. Pure glycerol is a thick viscous liquid that needs dilution for practical handling. One-to-one dilution of pure glycerol with sterile normal saline is usually required, for example, 100 ml of glycerol is mixed with 100 ml of normal saline. As a result, for any required amount of pure glycerol, the diluted volume should be doubled. For example, if you want to store bacteria in 20% glycerol broth in a cryovial of 2 ml capacity, you need to put 600 μl of culture broth into a cryovial and add 400 μl of diluted glycerol. This 1 ml culture broth of 20% glycerol can be

for 24 h. The slant is then refrigerated for future use of bacteria.

Presence of antigenic components that characterize a specific *E*. *coli* strain can be detected by using specific antibodies, for instance, presence of somatic antigen O, capsular antigen K, and flagella antigen H can be detected by agglutination tests and using specific antisera. The somatic and flagella antigens are tested against each specific antiserum, or they are tested against pools of antisera first and then tested against each of the specific antisera from the positive pools. The number of positive antisera is used in O and H antigen nomenclature, for example, *E*. *coli* O113:H21, O142:H34, and O157:H7. There are more than 180 O somatic antigens and more than 50 H-flagella antigens that are known and used as reference in *E*. *coli* serotyping. [24]. *E*. *coli* antigen serotyping has been described in detail by Ørskov and Ørskov [25].

### **7.2. Polymerase chain reaction (PCR)**

Polymerase chain reaction is performed to characterize *E*. *coli* strains by targeting different virulence genes coding for different virulence factors. Common virulence factors for IPEC include verocytotoxin1, verocytotoxin 2, intimin, heat-stable enterotoxin, human variant, heat-stable enterotoxin, porcine variant, heat labile enterotoxin, and invasive plasmid antigen (**Table 2**). These virulence genes can be detected using multiplex DEC PCR kit as previously described in Ref. [26].

EXPEC commonly carry virulence factor causing urinary tract or nervous tissue infection characterized by syndromes such as urosepsis, pyelonephritis, prostatitis, cystitis, and meningitis. More than 30 virulence factors carried by EXPEC have been reported in Refs. [27, 28].


**Table 2.** Gene target, primer sequence, and amplicon size for common intestinal pathogenic *E*. *coli* virulence factors (Adapted from Persson et al. [26]). These include *papA*, *papC*, *papEF*, *papG*, *papG* II (±III), *papG* III (±II), *papG* II + III, *sfa*, *focDE*, *sfaS*, *focG*, *afa*/*draBC*, *iha*, *bmaE*, *gafD*, *fimH*, *hlyD*, *cnf1*, *cdtB*, *fyuA*, *iutA*, *iroN*, *ireA*, *kpsM* II, K1 *kpsM*, K2 *kpsM*, *kpsMT* III, *rfc*, *cvaC*, *traT*, *iss*, *ibeA*, *ompT*, H7 *fliC*, *malX*, and *ibeA*. Commercial multi-

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Verocytotoxin (*vtx*) genes form the most variable group of IPEC virulence factors that can further be characterized by PCR into *vtx1* and *vtx2*. Within *vtx1* and *vtx2* groups further subtyping can be done as previously described in Ref. [29]. As a result, 10 subtypes have been identified, three for *vtx1* (*vtx1a*, *vtx1c* and *vtx1d*) and seven for *vtx2* (*vtx2a*, *vtx2b*, *vtx2c*, *vtx2d*, *vtx2e*, *vtx2f* and *vtx2g*). This subtyping is important because the subtype differ in virulence and disease syndrome they cause. Moreover, these details are needed when comparison of

Detection of virulence factors and genetic relatedness of *E*. *coli* isolates can also be assessed by DNA hybridization. This a phenomenon whereby a single strand of DNA anneals to a complementary single-stranded DNA fragment (probe) to form a hybrid. Since the probe is labeled, formation of a hybrid molecule is detected and hence showing presence of its complementary (target) nucleic acid strand. Apart from detection of conventional virulence genes, DNA hybridization can be used as a complementary to PCR to check for additional virulence factors [14, 30]. Analyses of additional virulence factors by hybridization can assist in differentiation of closely related isolates. For instance, EPEC pathotypes possess *eae* gene, and they can be differentiate into classical EPEC and A/EEC through DNA hybridization. Classical EPEC possesses *bfp* that codes for bundle-forming pili (BFP) [14, 31]. Different DNA probes can be used in hybridization such as *vtx1*, *vtx2*, *eae*, enterohaemolysin (*ehxA*), EPEC adherence factor (*EAF*), bundle-forming pilus (*bfpA*), *saa*, *astA*, *and vtx2f*. The protocols for DNA hybrid-

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

plex PCR kits are available for detection different virulence genes for EXPEC.

isolates from different cases/outbreaks is desired.

ization have previously explained in Refs. [30, 32, 33].

**7.3. DNA sequencing**

These include *papA*, *papC*, *papEF*, *papG*, *papG* II (±III), *papG* III (±II), *papG* II + III, *sfa*, *focDE*, *sfaS*, *focG*, *afa*/*draBC*, *iha*, *bmaE*, *gafD*, *fimH*, *hlyD*, *cnf1*, *cdtB*, *fyuA*, *iutA*, *iroN*, *ireA*, *kpsM* II, K1 *kpsM*, K2 *kpsM*, *kpsMT* III, *rfc*, *cvaC*, *traT*, *iss*, *ibeA*, *ompT*, H7 *fliC*, *malX*, and *ibeA*. Commercial multiplex PCR kits are available for detection different virulence genes for EXPEC.

Verocytotoxin (*vtx*) genes form the most variable group of IPEC virulence factors that can further be characterized by PCR into *vtx1* and *vtx2*. Within *vtx1* and *vtx2* groups further subtyping can be done as previously described in Ref. [29]. As a result, 10 subtypes have been identified, three for *vtx1* (*vtx1a*, *vtx1c* and *vtx1d*) and seven for *vtx2* (*vtx2a*, *vtx2b*, *vtx2c*, *vtx2d*, *vtx2e*, *vtx2f* and *vtx2g*). This subtyping is important because the subtype differ in virulence and disease syndrome they cause. Moreover, these details are needed when comparison of isolates from different cases/outbreaks is desired.

Detection of virulence factors and genetic relatedness of *E*. *coli* isolates can also be assessed by DNA hybridization. This a phenomenon whereby a single strand of DNA anneals to a complementary single-stranded DNA fragment (probe) to form a hybrid. Since the probe is labeled, formation of a hybrid molecule is detected and hence showing presence of its complementary (target) nucleic acid strand. Apart from detection of conventional virulence genes, DNA hybridization can be used as a complementary to PCR to check for additional virulence factors [14, 30]. Analyses of additional virulence factors by hybridization can assist in differentiation of closely related isolates. For instance, EPEC pathotypes possess *eae* gene, and they can be differentiate into classical EPEC and A/EEC through DNA hybridization. Classical EPEC possesses *bfp* that codes for bundle-forming pili (BFP) [14, 31]. Different DNA probes can be used in hybridization such as *vtx1*, *vtx2*, *eae*, enterohaemolysin (*ehxA*), EPEC adherence factor (*EAF*), bundle-forming pilus (*bfpA*), *saa*, *astA*, *and vtx2f*. The protocols for DNA hybridization have previously explained in Refs. [30, 32, 33].
