The debate: Coliforms, Fecal coliforms, and Enterobacteriaceae as Indicator Organisms

Which one Should you Use?

What are Indicator Organisms and why use them?

Indicator organisms are organisms used as a sign of quality or hygienic status in food, Dietary supplements, water, or the environment. The initial goal in finding a safety indicator was to find a group of bacteria that could indicate the presence of fecal material and serve as a surrogate for Salmonella, but was easier and simpler to detect. Such a group may signify the potential presence of pathogens, a lapse in sanitation as required in good manufacturing practices (GMPs), or a process failure.

The longest used indicator organism was the coliform group that was recommended for use in the early 1900s for water testing. Fecal coliforms and E. coli followed as more specific indicators of potential presence of pathogens. The Pasteurized Milk Ordinance includes a requirement of coliform testing of pasteurized for milk and milk products.

Many different types of safety indicators have been proposed for use in particular applications. A thorough review of the indicator organisms is given in Tortorello (2003).

Definitions

Coliforms

Coliforms are gram negative, oxidase negative, non spore-forming, aerobic or facultative anaerobic rod shaped bacteria. The coliform group is not a distinct valid taxonomic group, but is defined functionally as organisms that ferment lactose with both gas and acid production at 35°C. The coliform members include Citrobacter, Enterobacter, Escherichia, and Klebsiella. Some also add to the group Serratia and Hafnia to the coliform group. Many of these bacteria are found naturally in the intestines of humans and animals, and some are even found naturally in soil and water. However, of the 1% of coliforms found naturally in the human gut, E. coli represents the majority and is found exclusively in the intestines of humans and animals. It is important to note that many of the coliforms can be found also in plants and the environment, thus, a positive coliform test does not necessarily indicate fecal contamination.

Enterobacteriaceae

The family Enterobacteriaceae encompasses approximately 20 genera, including E. coli and all members of the coliform group; in addition it includes foodborne pathogens Salmonella, Shigella, and Yersinia. The family was originally proposed as an indicator alternative to the coliform group because testing for the entire family would be more inclusive for the pathogenic bacteria. The Enterobacteriaceae may be superior to coliforms as indicators of sanitation GMPs because they have collectively greater resistance to the environment than the coliforms. This group is more widely used as indicators in Europe than in the United States. The determining factor separating coliforms from Enterobacteriaceae is the ability of coliform to ferment lactose, while the Enterobacteriaceae family ferments glucose.

Fecal Coliform

These organisms are a subset of the total coliform group. The fecal coliforms have the same properties as the coliform group, except that the fermentation is able to proceed at 44.5°–45.5°C. They are considered a better indicator of fecal contamination than the coliform group.

E. coli

E. coli is present in all mammalian feces at high concentrations; it does not multiply appreciably, but can survive in water for weeks, and so it is useful as an indicator of fecal pollution of drinking water systems. E. coli meets all the criteria used for the definition of both total coliforms and fecal coliforms. In addition, the organism can be distinguished from other fecal coliforms by the lack of urease and the presence of B-glucuronidase enzymes.

When to test and how to test

There are some regulations in various industries that require testing of one or several of these organisms. In some industries, product manufacturers use these indicators to assure that there is no lapse in sanitation or process failure.

Coliform

This test is required in the dairy industry, bottled water and drinking water. Many producers in the food industry also utilize the coliform test, especially in the USA. The BioLumix Coliform vial (CC-C) is useful for water testing, for food, and in some cases also in dietary supplements. The coliform vial contains lactose as the sole carbon source and selective ingredient such as bile salts. The assay is simple to perform – just add the appropriate amount of the liquefied sample to the ready-to-use vial and run it in the instrument at 35°C for 16-18 hours.

Enterobacteriaceae

USP(ENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS) recommends the testing of Enterobacterial Count (Bile-Tolerant Gram-Negative Bacteria) in dietary supplements. Most European producers prefer to use this test instead of the coliform test for foods. The BioLumix Enterobacterial vial (ENT-C) is very similar to the Coliform vial, except the medium contains glucose in addition to lactose. It is mainly used for dietary supplement industry.

Fecal coliforms

The fecal coliform test is used instead of the coliform test in industries where it is considered to be more directly associated with fecal contamination from warm-blooded vertebrates than are other members of the coliforms, such as in seafood, nuts, etc. The BioLumix coliform vial is used for this application however is incubated at an elevated temperature of 44.0°C.

E. coli

E. coli testing is required in drinking water systems. Also, USPsuggests the testing of Dietary supplement for the absence of E. coli in 10 grams of product. In the meat industry there are regulations relating to the presence of generic E. coli on carcasses.

The BioLumix E. coli vial (EC) contains a highly selective medium and includes MUG that is the most commonly used fluorogenic substrate for the detection of E. coli. It detects the activity of glucuronidase by E. coli. For tests with a specified level of 10 cfu/g of product, the liquefied sample can be added directly into the EC vial and the vial is run in the instrument at 35°C for 18-20 hours. For tests with a specified level of absent in 10 grams, the samples are diluted 1:10 in TSB for 18-24 hours followed by the addition of 0.1 mL of sample into the EC vial.

BioLumix has them all!

The BioLumix system is ideal for testing of any of the indicator organisms. With the BioLumix automated microbiological system, users have control over their testing, leading to accurate results and accelerated product release while reducing costs. The BioLumix Advantage:

  • All assays can be performed on one Automated Microbial testing system
  • Fast, automated results of all assays
  • Can be operated by non-Microbiologist
  • Complies with FDA cGMP Regulations
  • System is validated during system installation
  • Designed to accelerate product release
  • Fully automated data archiving
References:
  • Ockerman, C. “Rapid Microbiological Testing of E. coli with the BioLumix Vial” (2012). BioLumix Blog.
  • Tortorello, M. “Indicator Orgnanisms for Safety and Quality – Uses and Methods for Detection: Minireview.” Journal of AOAC International 86 (2003): 1208-1217.
  • United State PharmacopeiaENUMERATION TESTS—NUTRITIONAL AND DIETARY SUPPLEMENTS (2005). The National Formulatory . Rockville MD.
  • United State PharmacopeiaMICROBIOLOGICAL ATTRIBUTES OF NONSTERILE NUTRITIONAL AND DIETARY SUPPLEMENTS. (2005). The National Formulatory . Rockville MD.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>