Coliform Bacteria- Characteristics, Examples, Identification

Coliform bacteria are a group of Gram-negative, aerobic, and facultative anaerobic rod-shaped bacteria containing the enzyme – galactosidase that is capable of fermenting lactose-producing acid and gas. They are also called Coli-aerogenes bacteria.

Coliform Bacteria
Coliform Bacteria

Coliform Definition

WHO defines coliform as “Gram-negative, rod-shaped bacteria, capable of growth in the presence of bile salts or other surface-active agents with similar growth-inhibiting properties, and able to ferment lactose at 35–37°C with the production of acid, gas, and aldehyde within 24–48 hours. They are also oxidase-negative, non-spore-forming and display β-galactosidase activity.”

The group originally contained only 4 genera of the Enterobacteriaceae family: only the strains from genera Escherichia, Enterobacter, Klebsiella, and Citrobacter.  Initially, the classification was based on the morphological features and the ability to ferment lactose, so only these 4 genera were considered coliforms. 

Recently the module for classification changes, and the coliform group is now defined with the ability to produce enzyme – galactosidase based on the ONPG (o-Nitrophenyl-β-galactopyranoside) test and other molecular features and biochemical tests like IMViC. Now there are 19 genera in the coliform group, and all of them are members of Enterobacteriaceae.  Aeromonas, which belong to the family Aeromonadaceae, is also referred to as a member of the coliform group but is not accepted widely. Aeromonas also have the capacity to produce – galactosidase, and many species ferment lactose at 37°C but are oxidase-positive; however, all other coliforms are oxidase negative.  

The following table contains all the coliform bacteria;

Arsenophonus spp.
Budvicia spp.
Buttiauxella spp.
Cedecea spp.
Ewingella spp.
Kluyvera spp.
Leclercia spp.
Moellerella spp.
Pantoea spp.
Rahnella spp.
Trabulsiella spp.
Yokenella spp.
Citrobacter spp.
Enterobacter spp.
Escherichia spp.
Hafnia spp.
Klebsiella spp.
Serratia spp.
Yersinia spp.
Source:  LECLERC ET AL. Annual Review of Microbiology, 55(1), 201-234.
**Aeromonas spp. are also included in some literature.

Among the above-listed genera, Escherichia, Klebsiella, Citrobacter, and Enterobacter cover over 90% of commonly isolated genera.  

Characteristics of Coliform Bacteria

  1. Gram negative non-sporing bacilli
  2. Aerobic or facultative anaerobic
  3. Lactose fermentation, along with the production of gas and acid at 37°
  4. Possesses – galactosidase enzyme
  5. All are oxidase negative (excluding Aeromonas from the group).

Habitat/Ecology of Coliform Bacteria

Coliforms are ubiquitous in nature and are cosmopolitan in habitat. They are commonly found in soil, water, decaying vegetation, and in/on the body of living organisms (insects, mollusks, vertebrates, plants) as commensals or pathogens. They are major genera in the gastrointestinal tract of warm-blooded animals; hence they are mostly isolated from fecal matter. In humans, they are mainly found in the GI tract. E. coli, Klebsiella, Citrobacter, Enterobacter, Hafnia, Serratia, and Yersinia species are found abundantly in the GI tract of vertebrates.   

Classification of Coliform Bacteria

The coliform group is sub-divided into 3 groups, namely, (i) Total Coliform, (ii) Fecal Coliform, and (iii) E. coli 

1. Total Coliform (TC)

TC includes all the 19 genera; the genera that are found in the free environment like in soil, water, decaying vegetation, plant surfaces, etc., and the genera that are found in the GI tract of warm-blooded animals. This group mainly contains species that are non-pathogenic (some are opportunistic) to humans. They usually contain strains that are mesophilic in nature and hardly tolerate bile salts and temperatures of 44°C.

 Their presence in water or any other edibles indicates contamination from any environmental sources or fecal matter, but not necessarily the fecal matter. However, their presence in any edibles indicates the risk of contamination from probable pathogens. 

2. Fecal Coliform (FC) / Thermotolerant Coliform

Fecal coliform is another sub-group of coliform or TC, which includes coliform genera that are found specifically in the GI tract and fecal matters of warm-blooded animals, including humans. They are now called “thermotolerant coliforms” because they can thrive well and ferment lactose at around 440C.

Their specific character is the ability to tolerate bile salts and other similar surface agents and ferment lactose, releasing acid and gas at about 44°C.

Their presence in water or other edibles indicates contamination with the fecal matter of humans or other warm-blooded animals. Their presence does not guarantee the presence of pathogens in the edibles but indicates a higher potential for the presence of pathogens. 

FC contains genera Escherichia which originates from feces; however, there are other genera like Klebsiella, Enterobacter, and Citrobacter, which not necessarily are found in feces. Hence, it is now called thermotolerant coliform.

3. E. coli

E. coli is a sub-group of thermotolerant coliform, including strains of E. coli bacteria only. It is the major coliform species that are widely isolated from water and other edibles. E. coli is generally found in the GI tract and fecal matters of warm-blooded animals; hence their presence strongly indicates fecal contamination. Several strains of E. coli are pathogenic, and their presence strongly suggests a higher probability of the presence of the pathogens.

Figure: Scheme of Sub-grouping of Coliform Bacteria

Coliform as Indicator Organisms

Coliforms are referred to as “indicator organisms” and are routinely used to indicate the microbiological quality of water, and also in dairy products, and other beverages. Coliforms are easy to isolate, purify and identify, and their presence is often associated with other potential pathogens; hence they are used as an indicator organism in water. Fecal coliforms, together with other fecal bacteria, are used as the major indicator organisms. Their presence indicates fecal contamination. E. coli is the most important coliform as an indicator organism. The presence of E. coli is very serious than the presence of other fecal coliforms. Most strains of E. coli are non-pathogenic, but there are pathogenic strains like E. coli O157:H7.

According to the guidelines of WHO, there must be any detectable E. coli or other thermotolerant coliforms in water intended for drinking or distribution in the community water supply system.

Pathogenicity of Coliform Bacteria

Most of the species in the coliform group are non-pathogenic to humans. Some species of genera like Escherichia, Klebsiella, Citrobacter, Enterobacter, Hafnia, Serratia, and Yersinia are pathogenic to humans causing several gastrointestinal infections, UTIs, septicemia, bacteremia, or other systemic infections. The most common infection is a gastrointestinal disorder like diarrhea. E. coli, and Klebsiella are the most common pathogens in this group.

Roles of Coliform on Spoilage of Foods and Drinks

Several species of coliforms are found responsible for the spoilage of perishable foods with high moisture content like milk and dairy products, fruit juices, vegetables, and fruits. They are also responsible for the spoilage of meat and unprocessed meat products. They usually contaminate the food from water and food handlers or are also already present in the source animals. They start producing acid and gas, giving off an odor, sliminess, and change in texture. E. coli, Yersinia spp., Klebsiella spp., Serratia spp., Kluyvera spp., Pantoea spp. are commonly associated with spoilage.

Reservoir and Source of Coliform Bacteria

Contaminated water is the main source of coliform bacteria. From the contaminated water, they are disseminated to foodstuffs and infect the human hosts. 

Mainly warm-blooded animals are the reservoir of fecal coliforms. Feces of human and other vertebrates are major reservoir that usually contaminates water bodies. Besides, soil, vegetation, and decaying matters are the reservoir of total coliforms.  

Detection of Coliform Bacteria

It is mandatory to detect coliform bacteria, especially thermotolerant coliforms, in drinking water; both processed and packed, as well as drinking water for distribution in the community via pipeline. Coliforms can be detected by the following methods:

1. Membrane Filtration Method

It is the most common and simple method used to isolate the coliforms from the water sample. It can detect the presence of total coliforms, but it confirms the presence of E. coli only directly. For confirming other coliform species, it must be followed by a series of biochemical tests.  

In this method, water is filtered through a membrane filter, usually a nitrocellulose filter paper with 0.45μm pore sizes, and the filter paper is placed over a solidified culture media and incubated at 370C for up to 24-48 hours and observed for the growth of microorganisms. The most common media used in this process are M-endo Agar, EMB (Eosin Methylene Blue) Agar, and VRBA (Violet Red Bile Agar) Medium. These mediums are preferred because they are selective for Gram-negative and are indicator mediums for E. coli and lactose fermenters.    

2. Most Probable Number (MPN) / Multiple Tube Fermentation Method

It is another commonly used technique for the detection of total coliform contamination of water. It is a more complex and laborious process of coliform detection than membrane filtration. However, it gives an approximate count of viable bacteria and can detect any type of coliform genera and other non-coliform bacteria too. 

In this method, sample water is analyzed in three different steps; (i) presumptive test, (ii) confirmatory test, and (iii) completed the test. The presumptive test is the primary screening step where only the presence or absence of acid and gas-producing lactose fermenter bacteria is studied. The approximate load of bacteria is also counted in this step. In the confirmatory test, it is confirmed that the lactose fermenter identified in the presumptive test is coliform bacteria because other non-coliform bacteria can also ferment the lactose-producing acid and gas. Finally, in the completed test, the result of the confirmatory test is once again verified and the presence or absence of E. coli is confirmed. If the coliform is other than E. coli, it needs further biochemical tests for identification. 

In this method, lactose broth (single and double strength), brilliant green lactose broth (BGLB) medium, indole medium (tryptone water), EMB agar or M-endo agar, and Nutrient Agar (NA) are mostly used. Its main drawback is the requirement of a longer time period, different media and instruments, process complexity, and the need for further tests for the detection of genera.    

3. Culture on Selective Media

A sample is inoculated in selective and indicator media like VRBA, EMB, MacConkey agar, Lactose broth, M-endo agar, Coliform agar, chromogenic coliform agar, etc. are used for detection of coliform, especially E. coli

4. Polymerase Chain Reaction (PCR)

It is one of the most advanced and accurate methods for detection and confirmation of coliform up to the level of subspecies, serovars, and sub-strains. The DNA or RNA of isolated bacteria is analyzed to confirm their identification. The – galactosidase coding gene is the key used to identify coliform by PCR method. 

5. Fluorescent in-situ Hybridization (FISH)

FISH is a new molecular technique that is being used to detect several species of coliforms, including E. coli, Klebsiella, Enterobacter, and Citrobacter. FISH uses specific oligonucleotide probes for the detection of complementary sequences in 16S rRNA of coliform. This method is very quick and more efficient than any other available method of coliform detection. However, it is expensive and requires fluorophore, fluorescent microscope, and other complex and expensive instruments, processes, and expert microbiologists. 

6. Commercial Coliform Detection Kits

Several commercial kits are available for the detection of coliforms in drinking water. These kits are based on the principle that coliforms will ferment lactose, releasing acid and gas. 

Roles of Coliform Bacteria  

  1. Positive Roles
  • Can be used as indicator organisms
  1. Negative Roles
  • Potential pathogens
  • The most common contaminants of water and foods
  • Can cause spoilage of milk and dairy, vegetables, and fruits

References

  1. Britannica, T. Editors of Encyclopaedia (2022, January 13). coliform bacteria. Encyclopedia Britannica. https://www.britannica.com/science/coliform-bacteria
  2. Coliform Bacteria – Examples, Characteristics, Fecal/Total Count Tests (microscopemaster.com)
  3. Coliform Bacteria (psu.edu)
  4. Coliform Bacteria – an overview | ScienceDirect Topics Tatsuya Tominaga, Masaharu Ishii, in Methods in Microbiology, 2020.
  5. Fecal Coliform – an overview | ScienceDirect Topics C.A. Batt, in Encyclopedia of Food Microbiology (Second Edition), 2014
  6. 5.11 Fecal Bacteria | Monitoring & Assessment | US EPA
  7. Coliform Bacteria: 5 Things You Should Know (culligannation.com)
  8. Coliform Bacteria in Drinking Water Supplies (ny.gov)
  9. Guidelines for drinking water quality, Fourth Edition. World Health Organization. Guidelines for Drinking-water Quality, Fourth Edition (who.int)
  10. Microorganisms in food Spoilage – Microbes in food spoilage (microbiologynote.com)
  11. Membrane Filtration Method, Types, Advantages, Disadvantages, Applications. (microbiologynote.com)
  12. Membrane Filtration – an overview | ScienceDirect Topics
  13. Membrane Filter Technique • Microbe Online
  14. Most Probable Number (MPN) Test: Principle, Procedure, Results • Microbe Online
  15. Most Probable Number Method – Definition, Principle & Method – Biology Reader
  16. Doyle, M. P., and M. C. Erickson. 2006. Closing the door on the fecal coliform assay. Microbe 1:162-163.
  17. Malcolm J. F. (1938). The classification of coliform bacteria. The Journal of hygiene, 38(4), 395–423. https://doi.org/10.1017/s0022172400011281
  18. Mishra, M., Arukha, A. P., Patel, A. K., Behera, N., Mohanta, T. K., & Yadav, D. (2018). Multi-Drug Resistant Coliform: Water Sanitary Standards and Health Hazards. Frontiers in pharmacology, 9, 311. https://doi.org/10.3389/fphar.2018.00311
  19. Korajkic, A., McMinn, B. R., & Harwood, V. J. (2018). Relationships between Microbial Indicators and Pathogens in Recreational Water Settings. International journal of environmental research and public health, 15(12), 2842. https://doi.org/10.3390/ijerph15122842
  20. Martin, N. H., Trmčić, A., Hsieh, T. H., Boor, K. J., & Wiedmann, M. (2016). The Evolving Role of Coliforms As Indicators of Unhygienic Processing Conditions in Dairy Foods. Frontiers in microbiology, 7, 1549. https://doi.org/10.3389/fmicb.2016.01549
  21. Leclerc, H., Mossel, D. A., Edberg, S. C., & Struijk, C. B. (2001). Advances in the bacteriology of the coliform group: their suitability as markers of microbial water safety. Annual review of microbiology, 55, 201–234. https://doi.org/10.1146/annurev.micro.55.1.201
  22. Eshamah, H. L., Naas, H. T., Garbaj, A. M., Azwai, S. M., Gammoudi, F. T., Barbieri, I., & Eldaghayes, I. M. (2020). Extent of pathogenic and spoilage microorganisms in whole muscle meat, meat products and seafood sold in Libyan market. Open veterinary journal, 10(3), 276–288. https://doi.org/10.4314/ovj.v10i3.6
  23. Kuo, Jong-Tar, Li-Li Chang, Chia-Yuan Yen, Teh-Hua Tsai, Yu-Chi Chang, Yu-Tang Huang, and Ying-Chien Chung. 2021. “Development of Fluorescence In Situ Hybridization as a Rapid, Accurate Method for Detecting Coliforms in Water Samples” Biosensors 11, no. 1: 8. https://doi.org/10.3390/bios11010008

Leave a Comment

This site uses Akismet to reduce spam. Learn how your comment data is processed.