Applying Surrogate Bacteria for Food Safety

Non-pathogenic surrogate microorganisms are typically used in food safety challenge studies to take the place of foodborne pathogens under conditions where the use of pathogens may be prohibited as in food processing facilities. They can also be used in biosafety level 2 research laboratories where there may be a likelihood of forming aerosols (aerosolization) and possibly the contamination of infection of support personnel as well as in biosafety level 1 laboratories, which cannot use pathogenic microorganisms. Some of the more common pathogens that surrogates have been used to supplant include Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Clostridium botulinum.

Surrogates may be specially selected laboratory-prepared strains added to a food prior to a processing treatment or storage study, or they can be naturally occurring strains that have been confirmed to exist at adequate levels in the food. Surrogate microorganisms must be carefully selected and validated in relationship to the targeted pathogen of interest before utilization. A surrogate that has been validated is one that reacts similar to the pathogen of interest with regard to growth parameters or inactivation kinetics along with accepted statistical analysis or mathematical modeling. Several criteria and approaches for selecting potential surrogate bacteria under given conditions should be considered. All of these concepts will be explored in this article, in addition to examining actual studies where these microorganisms have been used.

What does it mean to say Surrogate bacteria?

A surrogate bacterium models or replicates the behaviour of a given pathogen of interest; hence it has known characteristic and has been validated for a given food processing intervention or growth and survival storage study.

The FDA defines surrogate bacteria similarly; surrogate microorganisms as ‘a non-pathogenic species and strain responding to a particular treatment in a manner equivalent to a pathogenic species and strain;(FDA, 2001).

On this basis, surrogate bacteria are commonly used in place of foodborne pathogens in challenge studies conducted either in laboratories or food manufacturing plants to obtain pertinent inactivation/survival/growth date in those environments where pathogens cannot be introduced.

Surrogate bacteria: Basic Characteristics

The main features or basal desired characteristics of a surrogate bacteria are:-

• nonpathogenic (BSL-1 – biosafety level 1)
• behaviour, inactivation characteristics and kinetics that are used to predict those of the target pathogenic microorganism when exposed to similar environmental conditions
• susceptibility to injury similar to that of the target pathogen
• simple to prepare
• simple to detect
• genetically stable

In some cases, exceptions to the rule occur. Some BSL-2 pathogens can be used in the place of biothreat agents that require modelling such as Bacillus anthracis and Yersinia pestis where work with virulent BA or YP is not permitted. The surrogate examples that are BSL-2 are B. cereus and Yersinia pseudotuberculosis.

Some surrogates are not recommended in some situations. One example is Listeria innocua to replace Listeria monocytogenes in food processing environments which would show up as Listeria spp. or Listeria-like organisms in routine environmental testing. This surrogate would alter your testing regime results.

So, ideally a surrogate is an organism that would not test positive as a traditional indicator organism in routine tests. Some potential surrogates may be considered ‘out of range’ in that they are either significantly more resistant or less resistant than the target pathogen to the food processing intervention that they are being challenged by.

It is more straightforward to use validated surrogates reported in the literature, and these may be used in studies provided that:

1. the same target pathogen
2. the same food commodity
3. the same food processing treatment is used in the present study as was used in the validated study.

If a validated surrogate cannot be found for your three given conditions just stated, exploratory studies must be performed to validate a surrogate microorganism for the targeted conditions at hand.

The USDA did a study on E.coli O157:H7 and Salmonella by PEF treatment in orange juice (Gurtler et al., 2010). The objective was validating a surrogate for pulsed electric field treatment in orange juice with the targeted pathogens just described. The key finding was the level of inactivation (log CFU per ml units) of a series of microorganism which ranged from 0 to 5.5.  So 20 potential surrogate strains or bacteria and 3 pathogens are looked at. The pathogens of interest led to a 3.54 log reduction an 2.8 log reduction for 2 types of Salmonella. The most resistant pathogen was E.coli O157:H7 which had a 2.2 log reduction. The ideal suurogate is one which had a level of inactivation or less similar to these pathogens. There were three potential surrogates on the left of the E.coli pathogen. These were lactobacillus casei, Salmonella chi 3985 which is a vaccine strain,  and E.coli 35218 which is a non-pathogenic strain. The 35218 strain was chosen because it was equivalent in its inactivation to the targeted pathogen E.coli O157:H7. This surrogate E.coli is easy to use and work with in the lab as a generic E.coli.

Validated and Commonly-Used Surrogate Strains

One of the most commonly used surrogate strains is L. innocua which is used in stead of L. monocytogenes. Its widely discussed in research literature and is involved in assessing:-

• ozone
• liquid smoke
• vacuum packaging
• high hydrostatic pressure
• irradiation
• UV radiation
• thermal processing etc.

It was first studied 30 year ago (Fairchild & Foegeding, 1993). They used L. inoccua M1 which is available from the ATCC as strain 33091, as a thermal surrogate in a study for L. monocytogenes in milk. Its also been tried in thermal and UV studies in hamburgers patties, frank furters and chicken for example. It is a ubiquitous surrogate that deserves serious mention.

Two other commonly used strains are nonpathogenic and quality control strain E. coli ATCC 25922 and E.coli K12 in place of E.coli O157:H7 and other Shiga toxin-producing E. coli  (Amornkul & Henning, 2007). The latter, K12 is the most commonly used surrogate bacterium (Awuah et al., 2005; Duffy et al., 2000; Pata & Toklucu, 2011; Sirsat & Neal, 2013; Sommers & Rajkowski, 2008). These surrogate strains have been used with cheddar cheese manufacture with the lactoperoxidase system, radiofrequency heating of milk, UV treatment of apple cider and pomegranate juice, cold plasma treatment of packaged liquid food products, in soil-free versus in-soil grown lettuce, and in gamma or irradiation studies.

E.coli K12

Commonly used in food micro labs.  and food engineering labs for various validation intervention technologies where E.coli O157:H7 was to be inactivated. Its not always the best strain especially when it’s not validated. In the Gurtler study, where they looked art inactivation for various mos. The inactivation level of pathogenic E.coli O157 was 2.2. but for E.coli K12, the degree of inactivation was 5.06. If they hadn’t done the validation study, and only used K12, then the processing treatment which in their specific example was 20 kilovolts per cm, 55 deg. F for 70 microseconds was sufficient to kill E.coli O157 in orange juice. This was a misleading result underestimating the treatment needed. The treatment was not then seen as sufficient and could be a possible food safety issue further down the line.

Salmonella Typhimurium LT2

A non-pathogenic strain commonly used to replicate Salmonella in typical lab. studies and was validated for resistance to QACs (quaternary ammonia compounds), to UV light, ionizing radiation and in a storage study for the survival of Salmonella on cantaloupes.

The most commonly used strain is Enterococcus faecium NRRL B-2354 (a.k.a ATCC-8459)  in place of desiccation-resistant Salmonella Enteritidis PT 30 in thermal validation studies with almonds (Jeong et al., Yang et al., 2009, 2010). The E. faecium strain is especially useful with low water activity foods, dried and low moisture foods. Because of its high heat resistance, mathematical modelling is sometimes required to compare its death curve with Salmonella.

E. faecium (orginally known as Micrococcus freudenreichii, then Pediococcus B-2354) is also used as a thermal surrogate for survival in ground beef, dry pet food, model peanut butter, pecans, pistachios, corn meal, dried basil, peanuts, dried coconut, chia seeds, chicken patties, peppercorns etc. (Ma et al., 2007; Ceylan & Bautista, 2015; Enache et al., 2015). The safety of E. faecium NRRL-2354 has been questioned in the past because opf its genomic and functional characteristics (Kopit et al., 2014). However, safety was confirmed in terms of:

• lack of virulence factors
• biofilm formation
• high antibiotic susceptibility
• survival at low pH
• survival at high temperature
• survival in the presence of 8% ethanol

It is a very effective BSL-1 microorganism.

Clostridium sporogenes PA 3679 spores and are frequetly used in place of C. botulinum in the thermal processing of low-acid canned foods. PA 3679 has a reasonable margin of safety with average D-values of 1.28 minutes at 121.1 C in liquid media at neutral pH vs. 0.19 min for proteolytic C. botulinum (Diao et al., 2014). Its also been validated for C. botulinum in elevated temperature, high pressure processing of extra-lean ground beef (Zhu et al., 2008). The high D-value makes it an ideal surrogate.

There is a review available from Hu & Gurtler (2017) detailing surrgate microorganisms useful in the industry with tables etc. (Hu et al., 2017). These include S. enterica, shiga-toxin producing E. coli (STEC), B. anthracis, C. difficile, Y. pestis, B. cereus, L. monocytogenes and Mycobacterium avium subsp. paratuberculosis.

There are something like 22 different types of surrogates available to peruse:-

Hafnia alvei, Pediococcus spp, Lactobacillus spp., Lactococcus spp., Providencia spp. Enterobacter spp., L. innocua & ivanovii, Corynebacterium spp., Pantoea spp., B. amyloliquefaciens, B. atrophus, B. stearothermophilus, B. subtilis, B. thuringiensis, B. megaterium, B. mycoides, C. sporogenes, Thermoanaerobacterium spp. & Geobacillus spp. nonpathogenic E.coli and Salmonella. The review covers food products examined, types of treatment in each case, level of validation and any additional features.

Conclusion

Surrogate bacteria are highly useful tools in assess ing microbiological safety or quality of food products or process control methods; howver, attention must be given to validating utility of a given microorganism if conditions are not the same as in a validated system.

References

Amornkul, Y., & Henning, D. R. (2007). Utilization of microfiltration or lactoperoxidase system or both for manufacture of Cheddar cheese from raw milk. Journal of dairy science, 90(11), 4988-5000.

Awuah, G. B., Ramaswamy, H. S., Economides, A., & Mallikarjunan, K. (2005). Inactivation of Escherichia coli K-12 and Listeria innocua in milk using radio frequency (RF) heating. Innovative Food Science & Emerging Technologies, 6(4), 396-402

Ceylan, E., & Bautista, D. A. (2015). Evaluating Pediococcus acidilactici and Enterococcus faecium NRRL B-2354 as thermal surrogate microorganisms for Salmonella for in-plant validation studies of low-moisture pet food products. Journal of Food Protection, 78(5), 934-939

Diao, M. M., André, S., & Membré, J. M. (2014). Meta-analysis of D-values of proteolytic Clostridium botulinum and its surrogate strain Clostridium sporogenes PA 3679. International journal of food microbiology, 174, 23-30.

Duffy, S., Churey, J., Worobo, R. W., & Schaffner, D. W. (2000). Analysis and modeling of the variability associated with UV inactivation of Escherichia coli in apple cider. Journal of food protection, 63(11), 1587-1590

Enache, E., Kataoka, A. I., Black, D. G., Napier, C. D., Podolak, R., & Hayman, M. M. (2015). Development of a dry inoculation method for thermal challenge studies in low-moisture foods by using talc as a carrier for Salmonella and a surrogate (Enterococcus faecium). Journal of food Protection, 78(6), pp. 1106-1112.

Fairchild, T. M., & Foegeding, P. M. (1993). A proposed nonpathogenic biological indicator for thermal inactivation of Listeria monocytogenes. Applied and environmental microbiology, 59(4), pp. 1247-1250.

Gurtler, J. B., Rivera, R. B., Zhang, H. Q., & Geveke, D. J. (2010). Selection of surrogate bacteria in place of E. coli O157: H7 and Salmonella Typhimurium for pulsed electric field treatment of orange juice. International Journal of Food Microbiology, 139(1-2), pp. 1-8.

Gurtler, J. B., Bailey, R. B., Geveke, D. J., & Zhang, H. Q. (2011). Pulsed electric field inactivation of E. coli O157: H7 and non-pathogenic surrogate E. coli in strawberry juice as influenced by sodium benzoate, potassium sorbate, and citric acid. Food Control, 22(10), pp. 1689-1694

Hu, M., & Gurtler, J. B. (2017). Selection of surrogate bacteria for use in food safety challenge studies: a review. Journal of Food Protection, 80(9), pp. 1506-1536

Jeong, S., Marks, B. P., & Ryser, E. T. (2011). Quantifying the performance of Pediococcus sp.(NRRL B-2354: Enterococcus faecium) as a nonpathogenic surrogate for Salmonella Enteritidis PT30 during moist-air convection heating of almonds. Journal of food protection, 74(4), pp. 603-609

Kopit, L. M., Kim, E. B., Siezen, R. J., Harris, L. J., & Marco, M. L. (2014). Safety of the surrogate microorganism Enterococcus faecium NRRL B-2354 for use in thermal process validation. Applied and Environmental Microbiology, 80(6), 1899-1909.

Ma, L., Kornacki, J. L., Zhang, G., Lin, C. M., & Doyle, M. P. (2007). Development of thermal surrogate microorganisms in ground beef for in-plant critical control point validation studies. Journal of food Protection, 70(4), pp. 952-957.

Pala, Ç. U., & Toklucu, A. K. (2011). Effect of UV-C light on anthocyanin content and other quality parameters of pomegranate juice. Journal of Food Composition and Analysis, 24(6), 790-795.

Sirsat, S. A., & Neal, J. A. (2013). Microbial profile of soil-free versus in-soil grown lettuce and intervention methodologies to combat pathogen surrogates and spoilage microorganisms on lettuce. Foods, 2(4), 488-498.

Sommers, C. H., & Rajkowski, K. T. (2008). Inactivation of Escherichia coli JM109, DH5α, and O157: H7 suspended in Butterfield’s phosphate buffer by gamma irradiation. Journal of food science, 73(2), M87-M90.

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Yang, J., Bingol, G., Pan, Z., Brandl, M. T., McHugh, T. H., & Wang, H. (2010). Infrared heating for dry-roasting and pasteurization of almonds. Journal of Food Engineering, 101(3), 273-280.

Zhu, S., Naim, F., Marcotte, M., Ramaswamy, H., & Shao, Y. (2008). High-pressure destruction kinetics of Clostridium sporogenes spores in ground beef at elevated temperatures. International Journal of Food Microbiology, 126(1-2), pp. 86-92.