Isolation and identification of Escherichia coli (E. coli)

Introduction

E. coli overview

Escherichia coli, also referred to as E. coli, is a facultative anaerobic, Gram-negative member of the Enterobacteriaceae family of bacteria. E. Coli is a common environmental microbe that is also present in warm-blooded animals’ intestines. It is a key indicator organism in microbiology. Due to its dual position as a commensal bacterium in the human gastrointestinal tract and a potential pathogen, this bacterium has attracted a lot of research.

Also Read: E. coli – A Comprehensive and Easy to understand notes

The ability to ferment lactose and its rod-shaped morphology are two characteristics of Escherichia coli. While most E. Coli strains in the human digestive system are harmless and even beneficial, some pathogenic strains can result in a variety of disorders, such as urinary tract infections, gastrointestinal infections, and more serious disorders.

Isolation Techniques

Sample Collection

Types of Samples

• Water: Water sources often contain E. Coli, which is a sign of fecal contamination. Lakes, rivers, and drinking water sources are examples of potential sampling locations.
• Food: Food samples are essential for identifying possible sources of foodborne E. coli because the bacteria is frequently found in raw or undercooked meat, unpasteurized dairy products, and contaminated vegetables.
• Clinical Specimens: Stool or urine samples from patients with gastrointestinal or urinary tract infections, respectively, that show signs of E. Coli-related illnesses.

Enrichment Medias

Enrichment media to support the growth of E. coli are:

1. Luria-Bertani Broth (LB Broth
2. EC Broth (E. coli Broth
3. Lauryl Tryptose Broth (LST
4. Brilliant Green Bile Broth (BGBB
5. mTEC (Modified Tryptone Bile X-Glucuronide Agar)

Escherichia coli in all above broth medium typically exhibits robust growth and reaches a dense, cloudy, and turbid culture due to its efficient utilization of nutrients in the medium.

Isolation on Solid Media

1.       Nutrient Agar

On nutrient agar, Escherichia coli (E. coli) displays general colony morphology and growth patterns typical of a well-nourished bacterial culture:

Colony Morphology:

• Color: Creamy or off-white colonies.
• Appearance: Smooth and convex colonies.
• Size: Medium-sized colonies.
• Texture: Moist or mucoid appearance.

Growth Patterns:

• Growth Rate: Rapid growth. E. coli is known for its fast-doubling time, and nutrient agar provides the necessary nutrients for robust growth.
• Colonial Characteristics: Individual colonies may coalesce to form a continuous lawn of growth, especially in confluent cultures.

2. MacConkey Agar

On MacConkey agar, Escherichia coli (E. coli) exhibits distinctive colony morphology and growth patterns:

Colony Morphology:

• Color: Pink to red colonies.
• Appearance: Smooth and slightly mucoid.
• Size: Small to medium-sized colonies.
• Texture: Glistening appearance.

Growth Patterns:

• Lactose Fermentation: Positive. E. coli is a lactose-fermenting bacterium, and its ability to utilize lactose results in the production of acid.
• Color Change: The colonies of lactose-fermenting bacteria turn pink to red due to the acid production, which causes the pH indicator (neutral red) in the agar to change color.

3.       Eosin Methylene Blue (EMB) Agar

On Eosin Methylene Blue (EMB) agar, Escherichia coli (E. coli) exhibits specific colony morphology and growth patterns:

Colony Morphology:

• Color: Dark purple colonies with a metallic green sheen.
• Appearance: Smooth and moist colonies.
• Size: Medium-sized colonies.
• Texture: Glistening or mucoid appearance.

Growth Patterns:

• Lactose Fermentation: Positive. E. coli is a lactose-fermenting bacterium, and its ability to utilize lactose results in the production of acid.
• Color Change: The colonies of lactose-fermenting bacteria turn dark purple with a distinctive metallic green sheen. This color change is due to the interaction of acid production with the dyes in the medium.

Figure 3 E. coli Colony Morphology on EMB Agar – Source: CliniSciences

4.       CLED Agar

On Cysteine Lactose Electrolyte Deficient (CLED) agar, Escherichia coli (E. coli) exhibits specific colony morphology and growth patterns:

Colony Morphology:

• Color: yellow to orange colonies.
• Appearance: Smooth and convex colonies.
• Size: Medium-sized colonies.
• Texture: Moist or mucoid appearance.

Growth Patterns:

• Lactose Fermentation: Positive. E. coli is a lactose-fermenting bacterium, and its ability to utilize lactose results in the production of acid.
• Color Change: The colonies of lactose-fermenting bacteria turn yellow to orange due to the acid production.

5.       Chrom Agar

Chromogenic agar, such as Chrom agar, is a type of selective and differential medium that contains chromogenic substrates to aid in the identification of specific bacterial species based on the color of their colonies:

Colony Morphology:

• Color: Typically, E. coli colonies on Chrom agar may appear as pink to mauve or reddish colonies.
• Appearance: Smooth and convex colonies.
• Size: Small to medium-sized colonies.
• Texture: light moist or dry appearance.

Growth Patterns:

• Chromogenic Substrate Utilization: Chrom agar contains chromogenic substrates that are specific for certain enzymatic activities of bacteria. In the case of E. coli, the color change is often due to the utilization of specific substrates, resulting in the production of colored compounds.

Microscopic Examination

1.       Gram Staining

• E. coli, being a Gram-negative bacterium, appears pink/red under the microscope after Gram staining.

2.       Morphological Characteristics Under the Microscope

• E. coli is characteristically small rod-shaped or bacillus in morphology. Cells may occur singly or in pairs.
• E. coli is often motile due to the presence of flagella, which can be observed using techniques like the hanging drop method or by employing motility media (SIM medium)
• Microscopic examination allows for the estimation of cell size, typically 2 micrometers in width and 0.5 micrometers in diameter.

Biochemical Tests

1.       SIM Test

The SIM (Sulfide, Indole, Motility) test is often used to assess three key characteristics of Escherichia coli:

• Sulfide Production: Negative (no blackening of the medium)
• Indole Production: Positive (indole production observed)
• Motility: Positive (diffuse growth away from the stab line)

2.       Methyl Red (MR) Test

• Escherichia coli, known for its ability to produce significant amounts of acidic end-products, typically exhibits a positive MR test (stable red color formation).

3.       Voges-Proskauer (VP) Test

• Escherichia coli typically yields a negative VP test (absence of red color), as it preferentially follows the mixed acid fermentation pathway.

4.       Citrate Utilization Test

• Escherichia coli is generally citrate-negative (no color change of media).

5.       Catalase Test

• Escherichia coli is known to be catalase-positive, contributing to its identification (rapid bubbles formation).

6.       Oxidase Test

• Escherichia coli is oxidase-negative (no color change is observed).

7.       Urea Hydrolysis Test

• E. coli hydrolyses urea and thus gives urea hydrolysis test positive (media turn pink).

8.       Triple Sugar Iron (TSI) Agar Test

• E. coli ferments the sugar in the TSI agar and produces acid in the slant thus producing a yellow slant and the butt of the TSI agar slant with the gas formation indicated by cracks in the media or the media as a whole raised from the bottom of the test tube.

Biochemical profile of E. coli

Basic Characteristics	Properties (E. coli)
Capsule	Capsulated
Gram Staining	Negative (-ve)
Oxidase	Negative (-ve)
Catalase	Positive (+ve)
Coagulase	Negative (-ve)
Citrate	Negative (-ve)
Gas	Positive (+ve)
Flagella	Flagellated
Motility	Motile
Indole	Positive (+ve)
H2S	Negative (-ve)
MR (Methyl Red)	Positive (+ve)
VP (Voges Proskauer)	Negative (-ve)
Nitrate Reduction	Positive (+ve)
TSIA (Triple Sugar Iron Agar)	Acid/Acid, Gas +ve
Urease	Negative (-ve)
Hemolysis	Negative (-ve)
Growth in KCN	Negative (-ve)
Gelatin Hydrolysis	Negative (-ve)
OF (Oxidative-Fermentative)	Fermentative
Pigment	Negative (-ve)
PYR	Negative (-ve)
Shape	Rod
Spore	Non-Sporing

Sugar Fermentation Profile of E. coli

Lactose	Positive (+ve)
Glucose	Positive (+ve)
Maltose	Positive (+ve)
Sucrose	Variable
Glycerol	Variable
Mannose	Positive (+ve)
Mannitol	Positive (+ve)
Sorbitol	Positive (+ve)
Malonate	Negative (-ve)
Tartrate	Positive (+ve)
Trehalose	Positive (+ve)
Xylose	Positive (+ve)
Arabinose	Positive (+ve)
DNase	Negative (-ve)
Arabitol	Negative (-ve)
Cellobiose	Negative (-ve)
Melibiose	Positive (+ve)
Raffinose	Variable
Rhamnose	Positive (+ve)
Mucate	Positive (+ve)
Myo-Inositol	Negative (-ve)
Salicin	Variable
Dulcitol	Variable
Erythritol	Negative (-ve)
Adonitol	Negative (-ve)

Enzymatic reactions profile of E. coli

Beta Lactamase	Positive (+ve)
Lipase	Negative (-ve)
Arginine Di hydrolase	Negative (-ve)
Ornithine Decarboxylase	Variable
Lysine Decarboxylase	Positive (+ve)
ONPG (β-galactosidase)	Positive (+ve)
Acetate Utilization	Positive (+ve)
Esculin Hydrolysis	Variable
Aesculin Hydrolysis	Variable
Phenylalanine Deaminase	Negative (-ve)

Molecular Techniques for E. coli Identification

Polymerase Chain Reaction (PCR)

Target Genes for E. coli Identification

Polymerase Chain Reaction (PCR) is a highly efficient method for amplifying specific DNA sequences, and several target genes are employed for the identification of Escherichia coli:

• 16S rRNA Gene: The 16S ribosomal RNA gene is a widely used target for E. coli identification. This gene is part of the bacterial small ribosomal subunit and possesses conserved regions for general bacterial identification as well as variable regions for species-specific identification. The primers designed for the amplification of the 16S rRNA gene can specifically target E. coli DNA.
• uidA Gene (Beta-Glucuronidase): The uidA gene encodes for beta-glucuronidase, an enzyme characteristic of E. coli. Detection of this gene is indicative of the presence of E. coli.
• malB Gene (Maltose Transport System): The malB gene, associated with the maltose transport system, is another target for E. coli identification.
• fliC Gene (Flagellin): The fliC gene encodes for flagellin, a protein involved in flagella formation. This gene is specific to certain pathogenic strains of E. coli.

References

• El Ayis, A. A., Elgaddal, A. A., & Almofti, Y. A. (2015). Isolation, identification and enterotoxin detection of Escherichia coli isolated from calf diarrhea and their virulence characteristics. J Appli and Indust Sci, 3(4), 141-149.
• Beneduce, L., Spano, G., & Massa, S. (2003). Escherichia coli 0157: H7 general characteristics, isolation and identification techniques. Annals of microbiology, 53(4), 511-528.
• De Boer, E., & Heuvelink, A. E. (2000). Methods for the detection and isolation of Shiga toxin‐producing Escherichia coli. Journal of applied microbiology, 88(S1), 133S-143S.
• Zinnah, M. A., Bari, M. R., Islam, M. T., Hossain, M. T., Rahman, M. T., Haque, M. H., … & Islam, M. A. (2007). Characterization of Escherichia coli isolated from samples of different biological and environmental sources. Bangladesh Journal of Veterinary Medicine, 25-32.
• Caballero, M., Rivera, I., Jara, L. M., Ulloa-Stanojlovic, F. M., & Shiva, C. (2015). ISOLATION AND MOLECULAR IDENTIFICATION OF POTENTIALLY PATHOGENIC Escherichia Coli AND Campylobacter Jejuni IN FERAL PIGEONS FROM AN URBAN AREA IN THE CITY OF LIMA, PERU. Revista Do Instituto De Medicina Tropical De Sao Paulo, 57(5), 393–396. Https://Doi.Org/10.1590/S0036-46652015000500004