The Ultimate Buddy System

Peanut butter and jelly. Salt and pepper. Grilled cheese and tomato soup. All great partners. All things that we incorporate simultaneously for maximum gratification. In a striking comparison of a gratuitous duet, food-borne/environmental pathogens and indicator organisms are providing the food and beverage industry a cost and time-effective validation of the safety of their products and environment.

While the idea of using indicators and surrogates seems a minimal task, understanding how to choose these indicators, how to assign a probable range for the growth of these organisms to signal “pathogen presence,” and how to correctly measure for these organisms can be quite complex.


What is indicator testing?
Indicator testing is the use of microorganisms for a variety of purposes in food systems, including evaluating quality or safety of food products and food processing or holding environments. The growth of these microorganisms signals, or indicates, that a related pathogen may also be growing or persisting in the same environment. Broadly, the growth of these organisms—or the compounds that are produced via their growth—are used to assess, validate, or verify effectiveness of microbial control measures. The indicator can be a specific microorganism, a metabolite produced by the organism, or even a fragment of DNA. In order to comply with these parameters, there are a few basic characteristics that indicator organisms must comply with in order to be effective in respect to pathogen indication.

They include:

1. The non-pathogenic organism must have a history of presence in foods at any time that a target pathogen or toxin may be present.
    
2. The concentrations initially, and after any growth opportunity (enrichment), are directly related to that of the target pathogen or toxin being evaluated.
    
3. The organism or compound should be absent from food when the pathogen target is not present. Also, these organisms or compounds should be absent after a process that would eliminate the target (i.e. pasteurization).
    
4. The organism should be easily and quantitatively detected, even at low concentrations among other microorganisms and food components.
    
5. The organism should be measureable in a short period of time, preferably less than the routine holding time of any product being processed.
    
6. The organism should be resistant to cellular injury or decrease in concentration from stress (handling, processing) unless the equivalent effect would occur with the target. In simpler terms, if you are validating your drying cycle with an indicator for Salmonella, using an organism that couldn’t withstand the drying process as Salmonella does would be an inefficient indicator.

Keeping these ideal characteristics in mind, the indicators are charged with providing insight to the potential growth of like pathogens. Ideally, the absence or a low concentration of a specific indicator means that food has not been exposed to conditions that would permit contamination by a specific target pathogen or present the opportunity for its growth.


How does surrogate testing differ from the use of indicators?
Surrogate organisms are used to indicate the efficacy of a process, as opposed to the possibility of growth of a like pathogen. Surrogate organisms are implanted within the process and evaluated to determine if the kill step, processing aide, or sanitizer is efficacious. These organisms are also non-pathogenic and are an invaluable resource in validation of decontamination processes, kill-step validations, and the like without the introduction of harmful organisms into the production facility.

Some popular examples of surrogates commonly used today are Clostridium sporogenes in the low-acid canning industry. This non-pathogenic spore-former is so similar in nature to its sister organism, Clostridium botulinum, that the organism proves to establish the destruction of these spores in the process. Similarly, the flat-sour thermophillic organism Bacillus stearothermophilus is also used in surrogacy for C. botulinum, generally for thermal process validation for sterility management.

As with indicators, surrogates have several criteria for desirability:

1. The organism must be non-pathogenic.
    
2. The inactivation characteristics and kinetics should be predictors of the pathogenic target organism.
    
3. It must display similar behavior to the pathogen in the testing environment. For example, similar responses to pH, temperature sensitivity, and oxygen tolerance should be standard between the surrogate and target pathogen.
    
4. The organism must have stable and consistent growth characteristics.
    
5. Similarly to indicator organisms, the organism must be easily enumerated and detected.

For most surrogacy tests, you are looking at setting up an experimental design that mimics your real-time process, while using the “worst case scenarios” of the process. For example, if looking at the efficacy of an oil roasting process on the reduction of Salmonella in peanuts, you would want to focus on the lowest possible temperature the oil could reach during roasting, the heaviest amount of peanuts that could be roasted in the process, and the shortest possible time that the process could conclude while producing a viable product. This failure mode-surrogacy testing is the best tool the processor would have on the realistic reduction under the worst possible processing conditions.

How should an indicator or surrogate be chosen?
The first thing to think about when choosing an indicator or surrogate, above its inherent “best characteristics” as previously described, is the overall suggestive power that these microorganisms have on truly indicating for your target pathogen. Fecal coliforms have long been proposed and used as indicators of fecal contamination, as well as conditioning of manure or biosolids in agricultural practices. A low level of fecal coliforms would then suggest that the pathogenic microorganism (in this case, E. coli O157:H7) has diminished.

The use of coliforms or “generic” E. coli as indicators of possible enteric contamination in food products is also prevalent. Other examples of common indicator tests include Enterobacteriaceae and total plate counts. While one is focusing on the true indication power of these tests, they should be aware of the limitations as well. Focusing just on the genera of E. coli would limit the ability to indicate for Salmonella and other gram-negative pathogens. Focusing on total plate count may flabbergast the analyst, as this test is presumably tracking every aerobic organism that could possibly grow at a potential area or on a certain food/beverage testing sample when grown on appropriate agar at appropriate temperatures.

Conversely, if the processor is trying to elucidate the possibility of growth of Listeria monocytogenes, none of these above tests would indicate for this organism. The most common organism used for indication, as well as surrogacy, for L. monocytogenes is Listeria innocua. Alternative indicators for this gram-positive pathogen have not been adopted.

Using a risk-based approach of which pathogens may be contributing to the microbial breakdown in your facilities will be a starting point to choosing your indicator.

Factors to consider when deciding on the use of an organism for surrogacy testing are the number of cells to use as inoculums. Generally, historical data on the relative abundance of the target organism at the same step is used as a determinant. Determinants may also be the type of product itself or the point at which it has been previously processed or fabricated that may cause a reduction, or increase, in the relative abundance of probable pathogens. For example, when using surrogacy testing to identify a process decontaminant on fresh cantaloupe vs. cut and sliced cantaloupe, the relative abundance of pathogen may differ from the freshly picked fruit vs. the cut, handled, and perhaps previously treated cut and sliced fruit.

How can we be assured we are testing correctly?
As we stated earlier, the ideal indicator should be present and detectable at any time that the target pathogen may be present. Any change in the pathogen population, where it is spatial, temporal, or seasonal, must be considered and also applicable to the indicator. One issue that compromises the validity of these indicator organisms to truly indicate toward a pathogen would be ensuring recovery testing for injured cells.

In most food manufacturing processes, there are treatments and times during the production that the pathogen could be stressed or injured. Unfortunately, the root cause of most food-borne outbreaks is due to the persistence of these stressed and injured cells to remain in the products and remain viable to produce foodborne illness. The indicator, then, should be able to withstand the same treatment and likewise persist and remain in the products in a similar fashion. For instance, if you are evaluating a high-acid product for the presence of acid-adapted E. coli, and the indicator being used is not likewise acid-adaptable; the lack of recovery of the indicator will not be a realistic interpretation of the likelihood of E. coli being present. When choosing an indicator, focus on the process, as well as the products that you are identifying with, and incorporate those into your indicator or surrogate strains.

Therefore, it is extremely important to not only focus on things such as the time at which the indicator sample is taken, where in the process it’s taken, or the ambient temperature, but also on the up and down-stream treatments and interactions the product has at the point of sampling. Focus on things like pH, change in consistency, tenacity, viscosity, ingredient, or sanitizer addition, and cooling rate and time.

In summary, both indicators and surrogates can be novel ways to identify the validity of processes and food safety in the processing environment. Correct sampling design, stringency, and critical evaluation are critical to developing the use of these in your current programs. Challenges should be clearly identified for the selection of an indicator or surrogate for the specific situation and conditions of the food or beverage items. Clearly, a risk-based approach will help this buddy system prove its dual vivaciousness in every food safety program.



The author is the Director of Microbiology at AIB.