**5. Convergence of** *E. coli* **isolates**

*E*. *coli* form a large diverse species of bacteria that is difficult to handle when targeting a specific strain. Working on any *E*. *coli*-suspect colony from less discriminatory procedures prolong the time interval to isolate confirmation and utilizes more resources. The use of selective media helps to achieve this goal, but only when target *E*. *coli* strain is well defined. Otherwise, other approaches should be employed. Selection of *E*. *coli* isolates with some common features may be used to narrow down the population size. Converging similar *E*. *coli* isolates can be done by employing different procedures that utilize antimicrobial resistance, enzymatic, and immunogenic reactions and genetic characteristics of specific *E*. *coli* to mention a few. The choice of the method of converging *E*. *coli* isolates depends on different factors including study objectives, bacteria characteristics, skill, and resource availability.

Resistance to a single antimicrobial agent or combined resistance to more than one antimicrobial agent can be used to get *E*. *coli* isolates with common features. Resistance to common antimicrobial agents used in an area can be used to screen *E*. *coli* isolates before further analyses. For example, in a study to assess genetic similarities between *E*. *coli* isolates from 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 their genetic relatedness.

sequencing. For example, Turabelidze and colleagues [16] sequenced pathogenic *E*. *coli* that was congregated by PFGE. It was reported that these isolates had identical PFGE band, but their differences were revealed by sequencing. Likewise, Trees et al. [17] sequenced 240 isolates related to outbreaks from different sources by PFGE fingerprinting. As a result, whole genome sequencing of 228 isolates showed that they were Shiga toxin-producing *E*. *coli*, whereas other

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Moreover, *E*. *coli* isolates can be brought together by making use of common antigenic features they possess. Antibodies specific to the bacteria antigen are used to trap the bacteria in enrichment broth. Magnetic beads are coated with specific antibodies for specific bacteria antigen. When beads are applied to the culture broth, antigen will attract antibody resulting in bacteria-bead complex. The complexes are brought together by a magnetic field and concentrate at the bottom of the tube. After decantation, concentrated bacteria-bead complexes are inoculated on a solid media and incubated at 37°C for 24 h. The culture is then analyzed by other methods such as PCR or sequencing. Immunomagnetic separation (IMS) can be used to isolate different bacterial and fungal species. Different strains of Shiga toxin-producing *E*. *coli* can be isolated by this procedure. These include all Shiga toxin-producing *E*. *coli* with

Chromogenic media can also be used to concentrate bacteria possessing some enzymes whose action on sugars brings changes that are detected and depicted by indicating color change. *E*. *coli* are distinguished from other coliforms by the presence of β-glucuronidase activity on glucuronide. Examples of chromogenic media for coliform discrimination are Brilliance *E*. *coli* agar and Petrifilm Select *E*. *coli* count plate. Apart from differentiating the coliforms, these media can be used to sort out between β-glucuronidase positive and negative *E*. *coli* since there are a few *E*. *coli* strains that are β-glucuronidase negative, for example, *E*. *coli* O157:H7 [19]. Beta-glucuronidase positive isolates will appear purple on Brilliance *E*. *coli* agar or dark-green on Petrifilm Select *E*. *coli* count plate. The use of chromogenic media is usually followed by analyses by other techniques, for example, PCR, PFGE, or sequencing [20].

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

12 isolates were non-Shiga toxin-producing diarrheagenic *E*. *coli*.

somatic antigen O157, O26, O45, O103, O111, O113, O121, and O145 [18].

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

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 sequencing. For example, Turabelidze and colleagues [16] sequenced pathogenic *E*. *coli* that was congregated by PFGE. It was reported that these isolates had identical PFGE band, but their differences were revealed by sequencing. Likewise, Trees et al. [17] sequenced 240 isolates related to outbreaks from different sources by PFGE fingerprinting. As a result, whole genome sequencing of 228 isolates showed that they were Shiga toxin-producing *E*. *coli*, whereas other 12 isolates were non-Shiga toxin-producing diarrheagenic *E*. *coli*.

Moreover, *E*. *coli* isolates can be brought together by making use of common antigenic features they possess. Antibodies specific to the bacteria antigen are used to trap the bacteria in enrichment broth. Magnetic beads are coated with specific antibodies for specific bacteria antigen. When beads are applied to the culture broth, antigen will attract antibody resulting in bacteria-bead complex. The complexes are brought together by a magnetic field and concentrate at the bottom of the tube. After decantation, concentrated bacteria-bead complexes are inoculated on a solid media and incubated at 37°C for 24 h. The culture is then analyzed by other methods such as PCR or sequencing. Immunomagnetic separation (IMS) can be used to isolate different bacterial and fungal species. Different strains of Shiga toxin-producing *E*. *coli* can be isolated by this procedure. These include all Shiga toxin-producing *E*. *coli* with somatic antigen O157, O26, O45, O103, O111, O113, O121, and O145 [18].

Chromogenic media can also be used to concentrate bacteria possessing some enzymes whose action on sugars brings changes that are detected and depicted by indicating color change. *E*. *coli* are distinguished from other coliforms by the presence of β-glucuronidase activity on glucuronide. Examples of chromogenic media for coliform discrimination are Brilliance *E*. *coli* agar and Petrifilm Select *E*. *coli* count plate. Apart from differentiating the coliforms, these media can be used to sort out between β-glucuronidase positive and negative *E*. *coli* since there are a few *E*. *coli* strains that are β-glucuronidase negative, for example, *E*. *coli* O157:H7 [19]. Beta-glucuronidase positive isolates will appear purple on Brilliance *E*. *coli* agar or dark-green on Petrifilm Select *E*. *coli* count plate. The use of chromogenic media is usually followed by analyses by other techniques, for example, PCR, PFGE, or sequencing [20].
