Microbiological Indicator Testing: Overview, Considerations, and FAQ

Introduction

Eurofins’ laboratories are frequently asked questions about microbial indicator testing. This whitepaper will attempt to answer the most common questions and give an overview of the uses and benefits of testing for microbial indicators. But first, we must review some basic concepts and background on pathogens and indicator microorganisms:

What are pathogens?

Pathogens are agents that cause disease. We identify two major categories of pathogen: cellular pathogens, which include bacteria, yeasts, and molds; and non-cellular pathogens, predominantly viruses. Cellular pathogens can grow and multiply independent of a host and most of them are amenable to being cultured in the laboratory. Culture methods offer the “gold standard” for detection, though many may be very slow and expensive. Viruses need to infect a host and take over its cellular machinery to multiply so most are impossible to detect by culture.

What is an indicator organism?

Microbial indicator organisms are generally harmless microorganisms that are associated with potential for presence of a pathogen or other hazard.

Why do we use indicator microorganisms instead of testing directly for pathogens?

Traditionally we test for indicators when it is more effective than testing for the primary pathogen(s) of concern. It may also be safer, quicker, easier and/or cheaper.

Where did the concept of microbiological indicator testing originate?

The concept of indicator testing arose in the late 1800s. The germ theory of disease causation was broadly accepted, though in many cases the specific pathogen (germ) was not yet identified. The pathogens causing cholera and typhoid had been identified (8) and shown to spread through water contaminated with feces, and progress was being made on treating public water supplies to ensure their safety. Testing those water supplies was the next logical step but it was difficult to isolate Salmonella Typhi and Vibrio cholerae, both of which could present a significant public health risk even when present in very low concentrations. A sensitive indicator for fecal contamination would allow potentially unsafe water to be identified.

According to Jay et al (6), in 1892, Shardinger suggested Escherichia coli as an indicator of fecal pollution of water, based on the work of Theodor Escherich, and in 1895, T. Smith proposed a practical method of testing drinking water for E. coli. This armed public health authorities with a way to test water supplies for the presence of an organism whose detection indicated that the water had been contaminated with feces and was likely, therefore, to contain bacteria responsible for causing serious disease.

The concept was later extended to assessing the sanitary quality of milk and dairy products, with the passage of the Pasteurized Milk Ordinance in 1924 (14). Later, the indicator concept was extended to the safety of foods more generally. In those applications, E. coli might not be the most appropriate indicator. Therefore, it is important to be clear about our goals when implementing an indicator testing program. Determining the most suitable indicator organism or organisms requires at a minimum some understanding of the normal microbiological condition of the matrix and preferably some knowledge of the process or environment from which it came.

Over the years, other terms have been used such as index organisms, surrogates, sentinels, and marker organisms (14) but there is no authoritative source that defines all these terms in a way that allows consistent use. Indeed, Kornacki asked “Is there any wonder that confusion and disagreement among food safety professionals abounds when the term “indicator” is used? (7)”

What are indicator microorganisms?

For purposes of this article, we will use the functional definition “microbial indicators are generally harmless microorganisms that are associated with potential for some microbiological risk”.

Such risks could include:

• Inadequate sanitation
• A process failure
• Contamination from an external source

Any of these risks might lead to the presence of a pathogen or the (excessive) presence of a spoilage organism or other quality defect, as illustrated in the example above. E. coli was a useful indicator for fecal contamination in water and the risk that disease causing organisms such as Vibrio cholerae or Salmonella Typhii might be present, both of which would be harder to detect. We recognize the attempts of other authors to offer a more precise definition of indicator vs. index vs. surrogate (2) but see no need for such precision here.

Many different microorganisms may be useful indicators, depending on our application, and we will discuss them in more detail shortly. However, note that we cannot use the non-detection of our chosen indicator as an assurance of safety, or to overturn other evidence of a risk. For example, if we have records showing that a pasteurization process has failed to reach its minimum temperature, a passing indicator test result does not give us leave to ignore that process failure. Furthermore, no indicator test is useful if the hazard is better able to survive in the matrix or environment than the indicator. For example, coliform bacteria do not survive for as long as certain zoonotic parasites in water, so a passing coliform test is no defense if we have other evidence that fecal contamination of that water has occurred.

Selection of indicator organisms

What are the characteristics or criteria used to select indicator organisms?

To be useful, indicator organisms share certain characteristics (6, 14)

• They have some connection with the indicated hazard: ideally a unique connection.
• They should survive at least as well as the indicated hazard in the environment or matrix of interest.
• They should be readily detectable (or quantifiable).
• The method for detection (or quantification) should be reliable, faster, and no less safe than the method for the hazard itself.
• If the method is quantitative, results should be proportional to the level of the indicated hazard.
• Results can be used to initiate action to mitigate the indicated hazard with no delay or need for additional testing.

Common indicator organisms

Several different microorganisms have been used as indicators for testing water, foods, and processing environments (9). Let us briefly review some options.

Enterobacteriaceae

Several classic indicators are closely related, Gram negative, bile tolerant bacteria within the family Enterobacteriaceae (Figure 1). The Enterobacteriaceae would be considered “mostly harmless” [with apologies to Douglas Adams (1)] but does include Salmonella and Shigella species, and Shiga-toxigenic strains of E. coli, which are all individually pathogenic. Many members of the Enterobacteriaceae are not normally associated with feces but as common environmental organisms they can be good indicators of poor sanitation, and contamination events in processed foods. Hence, an Enterobacteriaceae test could be of value when one is not concerned solely with fecal contamination, but rather broader risks of sanitation.

Coliforms

Coliforms, (sometimes represented as “total coliforms”) are a functional group within the Enterobacteriaceae defined by their ability to rapidly ferment lactose producing acid and gas. They are typically cultured at 35 °C in the U.S. and at 37 °C in many other parts of the world. Many species within this broader group are associated with natural environments and not good indicators of fecal contamination but they can be excellent indicators of external contamination in treated water supplies, pasteurized milk, or other heat-processed foods.

Fecal coliforms

Fecal coliforms, also known as thermophilic coliforms, are a subset of coliforms able to grow at temperatures of 44.5–45.5 °C. This group includes E. coli and most, but not all, are associated with the intestines of mammals and birds and are therefore more indicative of potential fecal contamination than total coliforms.

E. coli

E. coli is the original safety indicator for water supplies and even today, detection of E. coli in a sample from a U.S. public water system is considered a maximum contaminant level violation (20), though total coliforms are considered the primary indication of contamination by an external source (not necessarily fecal in origin).

“Currently, [coliforms, fecal coliforms and E. coli are] all… used as indicators but in different applications. Detection of coliforms is used as an indicator of sanitary quality of water or as a general indicator of sanitary condition in the food-processing environment. Fecal coliforms remain the standard indicator of choice for shellfish and shellfish harvest waters; and E. coli is used to indicate recent fecal contamination or unsanitary processing.” (21).

Enterococci

Enterococci are Gram positive bacteria and generally more durable and heat-resistant than members of the Enterobacteriaceae. This durability can make them useful indicators of process failure and they are often used in place of Salmonella when validating thermal processes. The US Environmental Protection Agency has guidelines for maximum concentrations of enterococci in recreational waters (19) but currently neither the FDA BAM nor the USDA MLG has a method for enterococci in food.

Coliphage

Coliphage is a virus, known as a bacteriophage, that infects cells of E. coli. Following such infection, the cell makes multiple copies of the virus before dying and releasing the new virus particles into the environment to “hunt” for another E. coli to infect. This property means that coliphage is much more numerous than E. coli, hence it has been suggested as a more sensitive indicator of fecal contamination (10). It is also considered a better indicator of the persistence of enteric viruses in the environment than coliform bacteria or E. coli which is particularly valuable in recreational waters where most gastrointestinal illnesses contracted are viral in origin. However, methods for coliphage detection are not currently well developed.

Bacteroides

A genus of anaerobic bacteria, Bacteroides species are the numerically dominant microorganisms in feces and exclusively associated with the gastrointestinal tract of mammals and birds. That makes them good candidates for use as indicators of fecal contamination, but they can be challenging to culture because they must be kept away from oxygen. Molecular methods for detection of Bacteroides are available but not commonly in use for testing foods and food ingredients.

Listeria species

We have been asked “Is Listeria an indicator organism?” To answer this question, we need to differentiate between Listeria monocytogenes and Listeria species. U.S regulatory agencies have deemed Listeria monocytogenes a “zero tolerance” pathogen. Detection of L. monocytogenes in a food, or on a food-contact surface within a production area, leads to a product recall. At the same time, we now know of 27 species of Listeria other than L. monocytogenes of which only L. ivanovii has been observed as an opportunistic human pathogen (11). All Listeria species have similar requirements for growth. For these reasons, the food industry in the U.S. has adopted a practice of testing for Listeria species as an indicator of risk from Listeria monocytogenes without incurring the same regulatory risk to the business. Testing food processing environments for Listeria species increases the chances of finding environmental niches within the facility that can function as Listeria harborage sites. Eliminating those sites reduces the facility’s risk from L. monocytogenes. The United States Department of Agriculture Food Safety and Inspection Service considers Listeria species an indicator organism (16) for purposes of verifying compliance with the Listeria Rule (3).

Note that if L. monocytogenes is present in a sample, it will lead to a positive detection on a Listeria species test. Cultural confirmation of that detection may identify an isolate as L. monocytogenes.

Staphylococcus aureus

Staphylococcus aureus is associated with the nose, skin and infected wounds of humans and other mammals, with around 1/3 of humans carrying S. aureus in their nose (15). If permitted to grow to large numbers, S. aureus can produce a toxin that causes food poisoning. However, S. aureus is readily killed by heat, so its presence in heat-processed foods can indicate not only a direct risk from S. aureus toxin but also general failures in personnel compliance with good manufacturing practices.

Virulence genes

There are many virulence factors in Salmonella (5), enteropathogenic E. coli (12) and other enteric pathogens. Some of the genes for these virulence factors are found in non-pathogenic members of the Enterobacteriaceae which means that detection of a virulence factor gene indicates the potential for a risk but does not, by itself, show the presence of a pathogen.  EPRI™ is a Eurofins-proprietary, multiplexed PCR assay (4) that contains PCR primers and probes for virulence genes associated with enteric pathogens, but a positive EPRI result does not prove the presence of a pathogen. This allows action to be taken to mitigate food safety risks without the regulatory consequences of a Salmonella or STEC detection. The method workflow integrates seamlessly into several Eurofins pathogen assays if confirmation of pathogen presence is desired.

Aerobic plate count (APC)

APC is often thought of as a total bacterial count, but it is more accurately a measure of the number of bacteria in a sample able to grow in air at 35 °C (usually) on a general-purpose nutritive medium. It is useful as an indicator when we know the history of a product or environment and can identify deviations from the normal range or trends in results. We are sometimes asked “what happens if the APC is high?” The answer depends on the specific circumstances. At the simplest level, if the APC is higher than normal it indicates that something has changed. We probably want to investigate to understand why and determine what corrective actions, if any, are needed.

Mold and yeast count

Like the APC, the mold and yeast count could be considered a “general purpose” indicator if we know something of the history or expected condition of the product or environment. Incubated at 25 °C, this test takes longer than an APC but can be good for products or environments that are normally dry. Molds and yeasts will be the first microorganisms to begin to grow if these are allowed to pick up moisture.

What is a good indicator?

One of the most common questions that we are asked is “what is a good indicator organism?” As with anything in microbiology, the first answer is “it depends”. We need to consider the purpose for using the indicator and the nature of the matrix or environment to assess what would be most appropriate test (9). If we want primarily an indicator of microbiological safety for an early stage in a food processing operation, we may choose microorganisms that have a close association with specific pathogens, for example fecal coliforms or Listeria spp. If we are primarily concerned about the risk of premature product spoilage, then prior to any lethality process, elevated levels of relevant spoilage organisms such as lactic acid bacteria, yeasts (including osmophilic strains) or molds would indicate that a raw material is of poor quality. For both safety and quality at a later stage in the process, after any lethality step has occurred, we may prefer a general indicator of process failure, contamination, or other adverse event, such as an APC.

If we need to monitor the effectiveness of a plant sanitation program, indicators such as Listeria species, APC, coliforms, or Enterobacteriaceae can all be useful. Additionally, spoilage microorganisms relevant to the operation such as lactic acid bacteria, yeasts (including osmophilic strains) or molds can be important. The value of this testing is increased if the counts at each testing location are trended over time and action taken on indications of an upward trend, even before any limit that we established has been breached. It is still important to test directly for pathogens including Salmonella, and to follow up detections of Salmonella or Listeria species with actions to find their source.

A common approach to selecting indicator organisms is to review what we believe to be true about the product, process, environment, etc. We then make an educated guess about which indicators to choose. However, since the cost of genetic sequencing has dropped rapidly and dramatically, it is now economically viable to investigate the entire microbiome of the product, process or environment and use the knowledge gained to make an informed selection of appropriate indicators (9).

Some more questions from our files

This whitepaper has attempted to address our clients’ most frequently asked questions but there were a few more in our files that have not yet been specifically addressed. If your question has not been answered yet, perhaps it’s below.  

What are the benefits of using a food safety indicator?

Pathogens can be present sporadically and at low concentrations that are hard to detect. An appropriate indicator is easier to detect and shows that pathogens are likely to be present, thus avoiding a false sense of security. The ability to quantify some indicators provides a further benefit if data are plotted over time to identify and react to any negative trends before a safety incident arises or to improve quality and safety over time by identifying positive trends and capturing any positive changes in the process that caused them.

Why should I test for indicator organisms in an environmental monitoring program?

In an environmental monitoring program, indicators can show that cleaning and sanitation has not been effective, even if no pathogens are detectable.

What are indicator organisms in milk?

The Grade “A” Pasteurized Milk Ordinance (17) sets limits for APC in raw milk and for APC and coliforms in pasteurized milk and milk products.

Is E. coli an indicator organism?

E. coli is the original indicator organism for assessing the safety of water supplies and is still used for both drinking water and recreational waters. However, a few Shiga-toxigenic (STEC) strains including E. coli O157:H7 are pathogenic, and these would not be considered indicators.

What is the indicator organism for Salmonella?

There is no direct indicator organism for Salmonella, rather we look for organisms that indicate conditions that might increase the likelihood that Salmonella is present. Closely related organisms including E. coli, coliforms or Enterobacteriaceae are potential candidates. However, even unrelated organisms including enterococci, or yeasts and molds may be useful depending on the matrix and storage conditions, for example when examining dry products or environments high mold or yeast counts suggest past exposure to water and potential for Salmonella to grow or persist (9).

I’m testing my plant environment for APC and Enterobacteriaceae as indicators, do I still need to test for pathogens?

Trending the data for quantitative indicators such as APC and Enterobacteriaceae is a good way to see if something is changing in the plant environment and to take actions leading to continuous improvement in the results. However, pathogens may be present at concentrations too low to noticeably affect the quantitative test results. Testing directly for pathogens using sensitive, qualitative tests is the only way to directly detect presence of pathogens and then to find and eliminate their source by testing adjacent locations and applying directed sanitation to pathogen positive locations.

What if my water test is positive for coliform?

This is usually a concern only with private wells. In the first instance, stop using the water or use it only after boiling for 1 minute. Then get professional assistance to disinfect the well or add a water treatment unit to the supply line. If the water comes from a municipal supply, immediately notify the water supplier.

Can you have coliforms in water but no E. coli?

E. coli is probably the best-known member of the coliform group, but there are many other bacterial species also considered coliforms. As mentioned above, many coliforms are associated with natural environments, and it is possible to find coliforms in natural waters without the presence of E. coli. Accordingly, the US Environmental Protection Agency (EPA) standards for recreational waters focus on enterococci and E. coli, not coliforms (19).

What is the normal coliform count in drinking water?

The US EPA National Primary Drinking Water Regulations require that for a public water supply to be in compliance, not more than 5 % of water samples tested in a month (or one sample, if only 40 or fewer tests are done during the month) may contain coliforms (total coliforms) and none may contain fecal coliforms or E. coli (18).

How do you test for indicator organisms?

We have many different highly regarded microbiological methods available from AOAC, FDA, USDA-FSIS, ISO and other reputable sources and accredited within our laboratories. We also have a proprietary multiplex PCR test known as EPRI™ that detects virulence genes from enteric pathogens without identifying pathogens directly. We offer the most appropriate method for the chosen indicator.

Where to get help

Your local Eurofins Microbiology laboratory will be able to help you using their own expertise or tapping deeper into the Eurofins network

Connect with an expert.

Acknowledgements

Christopher Crowe, Ph.D., Daniel DeMarco, Ph.D., Andrzej Benkowski, and Scott Moosekian all provided helpful comments on the manuscript.

References

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