**6. Storage of** *E. coli* **isolates**

humans, cattle, and the environment, Lupindu and his colleagues [12] chose to isolate *E*. *coli* with resistance to tetracycline and ampicillin to concentrate *E*. *coli* from the general population. *E*. *coli* isolated from MacConkey agar was subjected to ampicillin-tetracycline solution using Petrifilm Select *E*. *coli* count (SEC) plate. Out of 1046 *E*. *coli* isolated from MacConkey agar, 118 isolates were resistant to ampicillin-tetracycline drug combination. Antimicrobial stock solution and bacteria inoculation were executed as previously described in Ref. [13]. One milliliter of antimicrobial stock solution containing 0.32 mg ampicillin and 0.64 mg was placed on the bottom lid. After 2 h of absorption of the antimicrobial solution, 2 μl of standardized sample suspension was spot-inoculated onto the antimicrobial embedded lower lid of Petrifilm SEC plate. The upper lid was closed after 10 min, and the plate incubated at 42°C for 24 h. Round, medium-sized *E*. *coli* colonies appear dark-green due to the presence of β-glucuronidase activity on glucuronide substrate in indicator embedded medium. This procedure was also used to confirm the *E*. *coli* isolates that were further analyzed by PFGE for

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

Ability of some *E*. *coli* strains to ferment different sugars can be used to concentrate strains of interest. All *E*. *coli* are lactose fermenters, but only some can ferment sorbitol. Sorbitol (instead of lactose) is mixed with MacConkey agar to form sorbitol MacConkey agar. This media can be used to discriminate sorbitol fermenting *E*. *coli* from nonsorbitol fermenters (NSF) and hence narrow down the *E*. *coli* population to a group of interest. The most common pathogenic *E*. *coli* that can be targeted by this procedure is *E*. *coli* O157:H7. Majority of *E*. *coli* O157:H7 and a few other diarrheagenic *E*. *coli* strains do not ferment sorbitol. Many studies to isolate *E*. *coli* O157:H7 have used sorbitol MacConkey agar. For example, Lupindu and friends [14], instead of focusing on every brown-colored, medium-sized round colony grown on MacConkey agar, they went for nonsorbitol fermenters in search for O157:H7. Sorbitol MacConkey agar was supplemented with antimicrobials cefexime and tellurite to inhibit growth of other bacteria such as *Aeromonas* and *Proteus* species and thus improving the recognition of nonsorbitol fermenting *E*. *coli*. The plates were inoculated with sample suspension and incubated at 37°C for 24 h. Nonsorbitol fermenting bacteria appeared colorless. NSF *E*. *coli* were confirmed by biochemical method. In this procedure, where one isolate was selected from each sample, the authors managed to recover 143 NSF *E*. *coli* isolates from the total of 1046 samples analyzed. The NSF *E*. *coli* isolates were further analyzed by molecular techniques, for example, PCR and DNA hybridization and serology to determine their virulence genes and pathotypes. Of 95

NSF *E*. *coli* isolates from cattle, 4 (4.2%) were *E*. *coli* O157:H7, carrying *vtx2c* genes.

Concentration of *E*. *coli* isolates can be achieved by molecular techniques where a specific part of the DNA is compared for different isolates. PFGE is one of the commonly used methods to bring together *E*. *coli* isolates with similar attribute prior to further analyses. Specific base pair sites of the DNA are cut by special enzymes, amplified, and electrophoresed by applying electric voltage in three directions periodically. It is suitable even for comparison of large DNA fragments up to 20 kb. PFGE can be reliably used as final analyses in the outbreak investigation in Ref. [12], but sequencing is becoming an adjunct to PFGE whereby isolates with identical PFGE bands are further subtyped by sequencing to give a more detailed discrimination in Ref. [15]. In outbreak situations, isolates are fingerprinted by PFGE, but detailed discrimination among isolates especially from different outbreak in different locations is obtained by

their genetic relatedness.

Preservation of bacteria aims at slowing the rate of harmful reactions in bacteria cultures so as to maintain viability and genetic attributes for future use. When imminent analyses require intact live cell, the storage method becomes very important. Different methods can be used to store pure *E*. *coli* and other bacteria isolates for future analyses [21]. Removal of water from the bacteria culture (drying) can be one option in preserving bacteria cells, while low temperature storage can also reduce the rate of chemical reaction in the cell culture and hence prolong bacteria viability. Drying of the bacteria cells may involve freeze and vacuum drying. In freeze drying, also called lyophilization or cryodesiccation, bacteria are suspended in a medium which maintain their viability through freezing, water removal, and storage. Principally, the bacteria in 15% glycerol suspension are frozen on dry ice or liquid nitrogen and subjected to high vacuum line that allows bacteria to dry through water sublimation. In vacuum drying, the bacteria are dried over calcium chloride in vacuum. Both freeze and vacuum-dried bacteria 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.

All *E*. *coli* strains can be revived by inoculation on blood agar, nutrient agar, or any nonselective media. A loopful of culture is inoculated onto the agar and incubated at 37°C for 18–24 h. Do not allow to thaw whenever frozen cultures intended for further storage are in use.

Isolation and Characterization of *Escherichia coli* from Animals, Humans, and Environment

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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,

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].

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

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].

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

β-glucuronidase activity, dot blot hybridization, and PFGE.

**7.1. Serotyping**

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

described in Ref. [26].

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

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

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

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 stored at −20, −80, or −196°C.

All *E*. *coli* strains can be revived by inoculation on blood agar, nutrient agar, or any nonselective media. A loopful of culture is inoculated onto the agar and incubated at 37°C for 18–24 h. Do not allow to thaw whenever frozen cultures intended for further storage are in use.
