**4. Confirmation of** *E. coli* **isolates**

Confirmation of *E*. *coli* isolates can be done by biochemical, enzymatic, or molecular methods. The choice of the method depends on many factors including availability of resources. The confirmation methods include biochemical methods, such as IMViC and Analytical Profile Index 20E (API 20E) systems, enzymatic methods, for example, use of brilliance *E*. *coli* agar or Petrifilm Select *E*. *coli* count plate, and molecular techniques such as MALD-TOF.

### **4.1. IMViC tests**

**3. Isolation of** *E. coli* **and quality control**

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

ture media at 45°C selects for thermophilic *E*. *coli* strains.

Different options are available for the isolation of *E*. *coli*. The choice depends on target strain and objective of isolation. The ability to ferment lactose gives an option to use MacConkey agar to discriminate *E*. *coli* from other nonlactose fermenting coliforms from fecal, stool, food, water, and soil samples. Sample suspension (for solid samples) is made at any concentration, for example, 5% in normal saline or phosphate buffer solution and inoculated onto MacConkey agar followed by 18–24 h incubation at 37°C. Pink, round medium-sized colonies are picked as *E*. *coli* suspect colonies. All *E*. *coli* strains can be captured on MacConkey agar, and this approach gives a wide spectrum of strains to work on. Incubation of inoculated cul-

The concentration of sample suspension may be set at different levels such as 1 g of solid sample in 19 ml of normal saline or phosphate buffer solution (5%), 1 g in 9 ml (10%) or 1 g in 4 ml of diluent (20%). However, the concentration of sample suspension will affect the number of colonies on the culture plate. This is well evidenced in bacteria count procedures

Sample suspension can be enriched by 24 h incubation at 37°C in nondifferential broth such as Muller-Hinton or nutrient broth. This procedure will allow multiplication of *E*. *coli* and hence increase the chance of *E*. *coli* isolation especially when infrequent strains, such as pathogens, are the target. The generation (doubling) time for *E*. *coli* at 37°C incubation is 17–18 min [8], therefore, in 18–24 h incubation there will be 60–80 *E*. *coli* cell generations. However, clonal variability will decrease when samples are enriched because same bacteria increase in number. Therefore, this procedure is suitable when the research aims at a mere presence of a single

The weight of the sample and the volume of diluent used in making the sample suspension may affect the probability of bacteria recovery. Large sample weight normally increases the sensitivity of the isolation procedure. For example, in *E*. *coli* studies to isolate nonsorbitol-fermenting Shiga toxin-producing *E*. *coli* (NSF STEC) whereby *E*. *coli* broth was used to enrich fecal samples, different prevalence measure was obtained. When 10 g of sample was suspended in 90 ml of *E*. *coli* broth, the prevalence of Shiga toxin-producing *E*. *coli* (STEC) obtained was 1.3% [9], while the suspension of 20 g in 180 ml of same diluent resulted into a prevalence 11.1% NSF STEC [10]. Purification of *E*. *coli* colonies can be done in nondifferential media such as blood or nutrient agars. Depending on the degree of colony density, a series of inoculations can be desired until

These are procedures undertaken to validate the accuracy of the bacteria isolates. Among the measures of quality control in isolation of *E*. *coli* include incubation of uninoculated media

. This is because the bacteria growth rate depends on initial cell density in the

, will give lower number of bacteria than low dilutions, for

**3.1. Isolation of** *E. coli*

whereby higher dilution, like 10<sup>5</sup>

specific strain and not its variants.

pure, single, or solitary colonies are obtained.

**3.2. Quality control**

example, 101

sample [8].

*E*. *coli* isolates can be confirmed biochemically by the use of a traditional method called IMViC tests. This is a set of four tests that are used to differentiate members of the family Enterobacteriaceae. IMViC is an abbreviation that stands for the Indole, Methyl red, Voges-Proskauer, and Citrate utilization tests. In Indole test, the bacteria are tested for their ability to produce indole from tryptophan (amino acid) using the enzyme tryptophanase.

The indole reacts with the aldehyde in the Kovac's reagent to give a red or a pink ring at the top of the tube. Peptone water in a tube, which contains tryptophan, is inoculated with bacteria isolate to be tested. The mixture is incubated overnight at 37°C. Then, a few drops of Kovac's reagent are added to the mixture and formation of a red or a pink colored ring at the top is a positive reaction. *E*. *coli* are indole-positive bacteria.

Methyl red test detects the ability of a bacterium to produce acid from glucose fermentation. Methyl red, a pH indicator, remains red in color at a pH less or equal to 4.4. The bacterium to be tested is inoculated into glucose phosphate (MRVP) broth, which contains glucose and a phosphate buffer and incubated at 37°C for 48 h. Three to five drops of MR reagent are added to the tube. Red color development is a positive reaction that occurs when the bacteria have produced enough acid to neutralize the phosphate buffer. Yellow discoloration occurs to MR-negative bacteria. *E*. *coli* are MR-positive bacteria.

Voges-Proskauer test is used to detect the presence of acetoin in the bacteria-containing media. Acetoin is oxidized to diacetyl in the presence of air and sodium hydroxide. Diacetyl, in the presence of alpha-naphthol, reacts with guanidine to produce red color. In order to perform VP test, the test bacterium is inoculated into glucose phosphate (MRVP) broth in a tube and incubated for 72 h.

Addition of 15 drops of alpha-naphthol to the test broth is followed by shaking. Then add five drops of 40% potassium hydroxide (KOH) to the broth and shake well. Allow the tube to stand for 15 min to see a positive red discoloration, after 1 h of no color change the isolate is categorized as VP negative. *E*. *coli* is VP negative.

incubation, but some may require additional reagents. Mark each test as positive or negative on the lid of the tray and score them. Add up the scores, the maximum score being seven, to

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Strict selective media that check for specific enzymatic activities in *E*. *coli* can be used to confirm *E*. *coli* isolates. For instance, brilliance *E*. *coli* agar or Petrifilm Select *E*. *coli* count plate can be used to check for presence and activity of β-glucuronidase enzyme. Beta-glucuronidase enzyme, which is specific to *E*. *coli*, cleaves glucuronide substrate resulting in purple and bluegreen colonies in Brilliance *E*. *coli* agar and Petrifilm *E*. *coli* Select count plates, respectively. Non-*E*. *coli* coliforms have ß-galactosidase only, which enable them to break down lactose, whereas most of *E*. *coli* have both β-galactosidase and β-glucuronidase. However, *E*. *coli* O157 are glucuronidase negative; therefore, these media are not appropriate for initial screening of *E*. *coli* population but can be used to differentiate *E*. *coli* O157 from confirmed *E*. *coli* population.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALD-TOF) mass spectrometry is a rapid and accurate method for microorganism identification. Principally, the biomolecules are allowed to gain or lose electrons (ionization) and then sorted based on their mass to charge ratio, when subjected to electric or magnetic field. The spectrum generated is analyzed and compared to stored profiles using software. These spectra, which are species-specific, can be used to confirm microorganism, for example, *E*. *coli* or discriminating closely related spe-

*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

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

study objectives, bacteria characteristics, skill, and resource availability.

get a 7-digit code that is used to identify the bacteria by using the online database.

**4.3. Enzymatic activities**

**4.4. MALD-TOF mass spectrometry**

cies such as *E*. *coli* and Shigella.

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

Citrate utilization test detects the ability of the bacteria to use citrate as its sole source of carbon and energy. Citrate agar media contains a pH indicator called bromthymol blue. The agar media changes from green to blue at an alkaline pH. Streak a loopful of bacteria onto a citrate agar slant without stabbing the butt and incubate at 37°C for 24 h with a loose cap. Citrate in the media breaks down to oxaloacetate and acetate due to action of an enzyme citritase. Oxaloacetate is further broken down to pyruvate and CO<sup>2</sup> . Production of Na<sup>2</sup> CO<sup>3</sup> from sodium citrate changes the media into alkaline pH, and hence color change from green to blue. Blue color formation is a positive reaction, whereas the slant remaining green colored is a feature for negative test. *E*. *coli* is citrate negative.

This conventional IMViC test method gives results (**Table 1**) that are similar to an agar plate IMViC method [11]. *E*. *coli* and *Proteus vulgaris* show the same IMViC pattern, but *Proteus* spp. are lactose-negative, motile, and show swarming behavior.

### **4.2. The API 20E system**

Analytical Profile Index 20E is a set of biochemical tests specific for differentiating between members of the Gram-negative bacterial family Enterobacteriaceae. It is used for rapid identification of already known bacteria. API 20E system is made up of 20 small reaction tubes that contain dehydrated substrates for detection of the enzymatic fermentation of sugars by the test isolates. This fermentation occurs during incubation, and the resulting pH change is detected by an indicator. It is important to confirm that the test culture is of an Enterobacteriaceae first, by doing a quick oxidase test. Enterobacteriaceae are oxidase negative.

Inoculate the suspension of a pure culture into each of the 20 reaction tubes and Incubate the tray at 37°C for 18–24 h. You can read the color change in some compartments right after


**Table 1.** MViC test results of some members of family Enterobacteriaceae (Adapted from Powers and Latt [11]).

incubation, but some may require additional reagents. Mark each test as positive or negative on the lid of the tray and score them. Add up the scores, the maximum score being seven, to get a 7-digit code that is used to identify the bacteria by using the online database.

## **4.3. Enzymatic activities**

Addition of 15 drops of alpha-naphthol to the test broth is followed by shaking. Then add five drops of 40% potassium hydroxide (KOH) to the broth and shake well. Allow the tube to stand for 15 min to see a positive red discoloration, after 1 h of no color change the isolate is

Citrate utilization test detects the ability of the bacteria to use citrate as its sole source of carbon and energy. Citrate agar media contains a pH indicator called bromthymol blue. The agar media changes from green to blue at an alkaline pH. Streak a loopful of bacteria onto a citrate agar slant without stabbing the butt and incubate at 37°C for 24 h with a loose cap. Citrate in the media breaks down to oxaloacetate and acetate due to action of an enzyme

from sodium citrate changes the media into alkaline pH, and hence color change from green to blue. Blue color formation is a positive reaction, whereas the slant remaining green colored

This conventional IMViC test method gives results (**Table 1**) that are similar to an agar plate IMViC method [11]. *E*. *coli* and *Proteus vulgaris* show the same IMViC pattern, but *Proteus* spp.

Analytical Profile Index 20E is a set of biochemical tests specific for differentiating between members of the Gram-negative bacterial family Enterobacteriaceae. It is used for rapid identification of already known bacteria. API 20E system is made up of 20 small reaction tubes that contain dehydrated substrates for detection of the enzymatic fermentation of sugars by the test isolates. This fermentation occurs during incubation, and the resulting pH change is detected by an indicator. It is important to confirm that the test culture is of an Enterobacteriaceae first,

Inoculate the suspension of a pure culture into each of the 20 reaction tubes and Incubate the tray at 37°C for 18–24 h. You can read the color change in some compartments right after

**Table 1.** MViC test results of some members of family Enterobacteriaceae (Adapted from Powers and Latt [11]).

. Production of Na<sup>2</sup>

CO<sup>3</sup>

categorized as VP negative. *E*. *coli* is VP negative.

is a feature for negative test. *E*. *coli* is citrate negative.

**4.2. The API 20E system**

are lactose-negative, motile, and show swarming behavior.

citritase. Oxaloacetate is further broken down to pyruvate and CO<sup>2</sup>

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

by doing a quick oxidase test. Enterobacteriaceae are oxidase negative.

**Bacterium Indole MR VP Citrate** *Escherichia coli* + + − − *Klebsiella pneumoniae* − − + + *Enterobacter aerogenes* − − + + Salmonella species − + − + Shigella species − + − − *Proteus vulgaris* + + − − *Proteus mirabilis* − + − + *Citrobacter freundii* − + − +

Strict selective media that check for specific enzymatic activities in *E*. *coli* can be used to confirm *E*. *coli* isolates. For instance, brilliance *E*. *coli* agar or Petrifilm Select *E*. *coli* count plate can be used to check for presence and activity of β-glucuronidase enzyme. Beta-glucuronidase enzyme, which is specific to *E*. *coli*, cleaves glucuronide substrate resulting in purple and bluegreen colonies in Brilliance *E*. *coli* agar and Petrifilm *E*. *coli* Select count plates, respectively. Non-*E*. *coli* coliforms have ß-galactosidase only, which enable them to break down lactose, whereas most of *E*. *coli* have both β-galactosidase and β-glucuronidase. However, *E*. *coli* O157 are glucuronidase negative; therefore, these media are not appropriate for initial screening of *E*. *coli* population but can be used to differentiate *E*. *coli* O157 from confirmed *E*. *coli* population.

### **4.4. MALD-TOF mass spectrometry**

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALD-TOF) mass spectrometry is a rapid and accurate method for microorganism identification. Principally, the biomolecules are allowed to gain or lose electrons (ionization) and then sorted based on their mass to charge ratio, when subjected to electric or magnetic field. The spectrum generated is analyzed and compared to stored profiles using software. These spectra, which are species-specific, can be used to confirm microorganism, for example, *E*. *coli* or discriminating closely related species such as *E*. *coli* and Shigella.
