Listeria Monocytogenes: Factory Cleaning, Thermal Inactivation and Acid Tolerance Response

Listeria Monocytogenes:  Factory Cleaning, Thermal Inactivation and Acid Tolerance Response
By Eben van Tonder
6 January 2018 (Update:  2/2018)

Summary

In light of the unprecedented listeria outbreak in South Africa, we examine processes and procedures at the cutting plant, factory cleaning, thermal inactivation and acid tolerance response of Listeria monocytogenes.  We question everything we do and examine the nature of the bacteria in order to ensure that we have the right cleaning program and processing hurdles in place.

Key conclusions of the study are the importance of sanitising stations in the cutting department or plant, deboning and slicing departments.  We also advance a suggestion for hand and boot washing stations before each department is entered.  We suggest an alkali detergent -> an acid-based sanitising sequence in cleaning and not acid first and then alkali.  Strict procedure must be enforced to prevent cross-contamination.

In order to achieve thermal inactivation in bacon production, at least three hours smoking/ thermal treatment is suggested at a core temperature of > 55 deg C.  A case will be made for maintaining our current NaCl levels (at least 4% NaCl to the meat) and processing it within 24 hours, while maintaining a meat temperature of < 7 deg C as an effective hurdle to prevent L. monocytogenes growth.

The approach is to set up multiple hurdles for eradicating and retarding the growth of bacteria, including L. monocytogenes.

Background

The current listeria outbreak in South Africa is the largest to date in recorded history with over 60 deaths as a result of listeriosis.  By 19 December there were 647 confirmed cases (550 on 5 December) i.e. almost 100 more in a question of 14 days.  It is reported that neonates are the most affected. The numbers indicate that it is one of the worst listeriosis cases in global history. “A large percentage (74%) of all the clinical isolates belong to the same sequence type i.e. ST6 – this means that these isolates originate from a single source, most likely a food product on the market.
By the end of December, statistics indicated that the outbreak has not yet peaked. (Anelich Consulting)

L. monocytogenes is a Gram-positive bacterium, in the division Firmicutes (Latin: firmus, strong, and cutis, skin, referring to the cell wall).  “This facultative intracellular bacterium can cause listeriosis, a severe invasive illness in humans, which may result in death. The risk of contracting listeriosis is high in immuno-compromised persons, the elderly, pregnant women and neonates. Thirteen serotypes of L. monocytogenes have been identified, but only three serotypes (1/2a, 1/2b and 4b) are associated with the majority of sporadic cases of listeriosis; serotype 4b is linked to almost all recent outbreaks.”  (Thévenot, et al., 2006)

The fact that it is Gram-positive, gives us a clue to its dangerous toxin formation.  The classification as Gram-positive has nothing to do with gram, as in a measure of weight as I first thought when I heard the word many years ago.  It refers to the Danish scientist Hans Christian Gram (1853 – 1938) who “devised a method to differentiate two types of bacteria based on the structural differences in their cell walls. In his test, bacteria that retain the crystal violet dye do so because of a thick layer of peptidoglycan (a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of most bacteria, forming the cell wall) and are called Gram-positive bacteria. In contrast, Gram-negative bacteria do not retain the violet dye and are coloured red or pink.”  (Diffen)

Generally, Gram-positive pathogens produce exocellular substances that typically account for most, if not all, of its ability to cause disease (i.e., virulence factors).  An exception to this is L. monocytogenes which is a Gram-positive intracellular pathogen.  Virulent strains, such as L. monocytogenes, are known to secrete a number of exotoxic factors.  Exotoxic factors are molecules produced by bacteria, viruses, fungi, and protozoa that add to their effectiveness and enable them to achieve a number of results such as obtaining nutrition from the host and colonization.  In the case of L. monoctytogenes, the exotoxin factor produced enables the organism to spread from cell to cell with the aid of listeriolysin O (LLO) which it secretes.  LOL is thiol-activated and pore-forming but is not the cause per se of foodborne gastroenteritis syndrome.  It must also be noted that in the case of L. monocytogenes, unlike other syndromes caused by Gram-positive bacteria (with the exception of Clostridium prefingens), “the ingestion of viable cells is necessary for listeric infection to occur” (Jay, et al.; 2005:  532, 533)

Distribution

Listeria is widely distributed in nature.  It is found in decaying vegetation, soil, animal faeces, sewage, silage (fodder), and in water.  Its association with dairy products is well known.  (Jay, et al.; 2005:  598)

Evaluating Control Measures

I examine factory cleaning protocols, thermal inactivation and acid tolerance response.   Nesbakken et al. (1996), as reported by Thévenot, et al. (2006), “found L. monocytogenes at every stage of the fresh pork meat industry, with increasing prevalence from the slaughterhouse to the cutting room.”  The widespread occurrence of the bacteria, however, makes it likely that contamination can occur at any stage in the processing process with an increased risk at every stage of processing.

In evaluating the real risk of listeriosis from meat products, Thévenot reports that “the incidence of L. monocytogenes in meat products is generally low, even if the pathogen is present at low or moderate levels (Encinas et al. 1999; AFSSA 2000; FICT 2002). Even if a single bacterial cell has the potential to cause disease, epidemiological data indicate that foods involved in listeriosis outbreaks are those in which the organism has multiplied and in general, have reached levels significantly >1000 CFU g−1 (Ross et al.2002; Risk Assessment Drafting Group 2004).”  (Thévenot, et al.; 2006)

The approach is to set up multiple hurdles for eradicating and retarding the growth of bacteria, including L. monocytogenes.  Bacteria are able to resist small changes in an environment, “but severe or multiple changes stimulate complex stress responses that are generally directed to survival instead of growth (Booth 1998)”.  (Thévenot, et al.; 2006)

Focus areas for control measures

Cutting Plant and Chiller Rooms

Thévenot, et al. (2006) reports that “extensive study of meat contamination levels in the meat processing industry indicated that chilling and cutting significantly increased the contamination of pork meat (Nesbakken et al. 1996) while Van der Elzen and Snijders (1993) found that the environmental prevalence of the pathogen in chilling–cutting areas to be as high as 71–100%. These findings strongly suggest that postslaughter processing is a significant cause of meat contamination and that contamination is amplified in the chilling and cutting room environment (Nesbakken et al. 1996).”  (Thévenot, et al.; 2006)  This means that the first major area of focus is the cutting areas and chilling rooms where several measures can be implemented depending on the plant design.  These include meat decontamination wash with acetic or lactic acid and redesigning the continues cleaning, end of shift and weekly deep clean cleaning regimes to deal with L. monocytogenes effectively. I also suggest the creation of a dedicated sanitising station in this department where all bins, crates and other equipment are sanitized before they either enter or leave the area.  Likewise, equipment such as knives should be sanitised whenever a new batch is started and completed at an appropriate time interval (ex. 20, 40 or 60-minute intervals) during processing of any particular batch.

Processing Plant Contamination

Contamination in the processing plant can occur at several stages:

  • Raw material brought into the plant can be contaminated and insufficient hurdles may exist to deal with the contamination and detection.
  • The contaminated raw material can contaminate surfaces and people who spread it.  These are well-recognised routes of contamination.  I propose that in the event if receiving of meat involves sorting of meat, that the receiving department be viewed as a quarantine area of sorts and staff involved in receiving go through proper handwashing procedure before they can re-enter the processing area to avoid possible contamination of work surfaces and equipment.
  • Similarly, where a meat sterilizing step is used before injection, for example, handling of unsterilized meat should be restricted to a specific area and workers leaving this area should perform proper handwashing before entering the rest of the processing plant.
  • The distinction and clear separation between workers from production and slicing/ packing should be maintained and workers from production should undergo handwashing procedure before entering the slicing area to avoid cross-contamination from the possible handling of contaminated meat or working with contaminated equipment such as knives.
  •  A particular focus should be on knives and similar equipment which can be shared between different departments within a production or between production and slicing/ packing – the standard best practice should be enforced that no such transfer of equipment can happen unless it has not been sanitised.
  • “Poor personal hygiene, including simple procedures such as hand washing, has been identified as a causative mode of transmission of the pathogens (AFSSA 2000).”  (Thévenot, et al.; 2006)

Factory Cleaning *

In general, the following applies to factory cleaning:

Step 1:  Use water to remove gross, loose material such as meat.  “The cleaning step is important because it removes organic matter from processing surfaces (Chasseignaux et al. 2001; Thevenot et al.2005b). Besides harbouring bacterium, organic matter can reduce the efficiency of disinfectants [such as quaternary ammonium compounds (QACs)] or prevent disinfectants from reaching bacterium at the recommended concentrations (Gibson et al. 1999). Furthermore, Taormina and Beuchat (2002) showed that L. monocytogenes strains that were exposed to cleaning solutions which did not affect cell viability were more sensitive to subsequent treatment with sanitizing chemicals.”  (Thévenot, et al.; 2006)

In measuring results from last year June in the poultry industry Warnick Biersteker from Lionels Veterinary Supplies have demonstrated the superiority of a process where this step is performed without water.

Step 2:  Apply alkaline detergent foam and leave on for 10 to 15 minutes.  Mechanically remove stubborn dirt and residual substances and rinse the detergent off.

Step 3:  Ensure that residue water has been removed and the area is as dry as possible.  Now foam with an antimicrobial chemical in order to sanitize surfaces.

Thévenot, et al.’s 2006 article refers to work by Holah et al. (2002) who “observed an increasing tendency for disinfectants to be left on surfaces and not rinsed off prior to recommencing production.”  They comment on this that despite the claims of manufacturers that residual disinfectants will prevent subsequent surface microbial development, although low, sublethal concentrations may, in fact, enhance resistant.”

After discussing this particular point at length, it was decided not to rinse it off due to poor water quality results reported over the previous few years.  In an environment where water quality is high, Holah’s observations will be valid, but in South African context, the possibility for re-contamination through water outways the arguments.

I have found the creation of a sanitising station in the deboning, production and slicing departments useful where dedicated staff is responsible for sanitising crates, bins, knives and any other equipment before they either enter or leave the processing or slicing departments.  In larger processing plants, these steps are hard-wired into the plant design.

Thévenot, et al., (2006) reports that an “acid treatment (pH = 5·4) followed by an alkaline treatment (pH = 10·5) was not very effective against L. monocytogenes, whereas the opposite combination led to a three-log reduction of the bacterial population.”  This makes me question the wisdom of our current approach and more work on this will follow.  Generally, they report that “the most efficient treatments were combinations of alkaline, osmotic and biocide shocks.”

Related to the formation of biofilm, Thévenot, et al. (2006) refers to work of  Norwood and Gilmour (2000) who concludes that “in most food-processing environments and more precisely in pork meat industries, the daily use of sanitizers at correct concentrations preceded by detergent-aided cleaning, will remove all adhering organisms or biofilms.

Control Points in Processing Steps

The processing steps must be evaluated and where possible adjusted to accommodate the reversal of any possible contamination that occurred.

-> Thermal Inactivation

Temperature is the most important hurdle against L. monocytogenes.  “It grows in a temperature range of +0·4 to 45°C, while the optimum growth temperature lies between 30 and 37°C (AFSSA 2000). Listeria monocytogenes grows in refrigerated foods (Augustin 1999), and this is of major importance for risk assessment of foodstuffs: even when initial contamination is low, the organism can multiply during refrigeration and reach levels up to 100 CFU g−1 (AFSSA 2000). Besides, the temperature home refrigerators are often closer to 9°C than 4°C (Sergelidis et al. 1997), which also favours L. monocytogenes growth.”  (Thévenot, et al.; 2006)

Several factors necessitate a careful evaluation of the heating step in processing due to the “organism’s high level of heat tolerance relative to those of other non-spore-forming foodborne pathogens.”  Not only is its heat tolerance high, but several factors which it is likely to encounter in the processing environment increases its heat tolerance.  For example, “previous studies involving L. monocytogenes have demonstrated that the composition of the heating medium affected the thermal inactivation of this organism. The heat resistance of L. monocytogenes was stronger in ground pork than in pork slurry, and its heat resistance was stronger in meat slurry than in phosphate buffer.”

Adding curing agents also increase its hear tolerance.  “In comminuted meats, the addition of phosphates, salt, or curing-salt mixes has been found to increase the thermotolerance of L. monocytogenes. The thermotolerance of L. monocytogenes is further increased when this organism has endured an environmental stress such as sublethal heat shock, osmotic stress, starvation, exposure to acid or alkali, exposure to ethanol, or exposure to hydrogen peroxide.  Therefore, heat-processing procedures for foods should be designed to destroy L. monocytogenes in its most heat resistant state and thus to provide an adequate margin of safety against this pathogen.” (Lihono, et al, 2003).

Lihono et al from Iowa State University used a quadratic linear response model in 2003 to “describe the combined effects and interactions of different parameters on the thermal inactivation of starved L.monocytogenes” which involved D-values (times [reported in minutes] required for a 90% reduction in viable cells), “modelled as a function of heating temperature, SPP concentration, and NaCl concentration”.  The model is based on starved L.monocytogenes since “water used for cleaning and rinsing food contact surfaces generally provides a low-nutrient environment for microorganisms.  Certainly, the stress from prolonged deprivation of nutrients can induce increased microbial resistance to subsequent chemical and physical challenges.”  (Lihono, et al, 2003)

In their study, they reported that “the addition of 3 and 6% NaCl to pork slurry significantly (P, 0.05) increased the D-values for the organism.”  Let’s look at their findings more carefully.  (Lihono, et al, 2003)

At a NaCl and SPP T concentration of 0, and temperature of 57.5 deg C, the observed D-value was 2.79 minutes (2.93, calculated).  When the salt % was increased to 3%, the D-value increased by 177% to 7.75 (observed) and 6.4 (predicted).  At 6% NaCl, it rose by another 88% to an astounding 14.59 observed minutes and 13.95 predicted.   Adding the phosphates (SPPT), at a 6% salt level and a phosphate concentration of 0.5%, reduce the D-value to 8.2 observed and 8.91 predicted.  At a salt concentration of 3%, and SPPT at 0.5%, the D-value is reduced to 4.03 observed and 4.16 predicted.

In their report, they site Juneja and Eblen who “demonstrated that the addition of NaCl (at 1.5 to 6%) to beef gravy protected L. monocytogenes against thermal inactivation at all temperatures (55 to 65 deg C) tested in their study.  Similar findings were reported by Yen et al., who demonstrated that the heat resistance of L.monocytogenes increased when the organism was heated at 60 deg C in ground pork with added NaCl.”  They commented on their own work and said that generally, their results on “the thermal inactivation of starved L. monocytogenes in pork slurry with added NaCl are consistent with those of other studies on the thermal inactivation of this pathogen in broth, pork, and beef. The investigators who conducted those studies reported an increase in the heat resistance of L. monocytogenes in various meat blends containing 3 to 4% NaCl. Therefore, the results of the present study can be used to predict the thermal inactivation of L. monocytogenes as affected by added NaCl in meat products.”  (Lihono, et al, 2003)

Faber, as reported by Jay, et al., found the same results in sausage-type meat.  The D-value ar 62 deg C was 61 seconds, “but when cure ingredients were added, the D value increased to 7.1 minutes, indicating heat-protective effects of the cured components, which consisted of nitrite, dextrose, lactose, corn syrup and 3% (w/v) NaCl.  (Jay, et al.; 2005:  602)

Thévenot, et al., (2006) reports that “products which are cooked at fairly low temperatures (50–60°C) may not be L. monocytogenes free even after if cooking times are long (FICT 2002).”  (Thévenot, et al.; 2006)  This leaves us with the question of an optimum cooking temperature for bacon.  It is my experience that bacon starts changing to a pale cooked colour as opposed to the pinkish-reddish cooked cured colour at a temperature of 48 deg C.  Faber’s 62 deg C is therefore out of the question.  Jay, et al states that cooking meat to an internal temperature of 71 deg C for 2 minutes will destroy L. monocytogenes, (Jay, et al.; 2005:  602) which in the case of bacon production is even more unworkable.  I am pitching the core temperature for bacon at 55 deg C for at least three hours (our standard smoking/ thermal processing time) based on the work of Lihono, et al. (2003) and will continue to search the literature for more precise guidance and submit this suggestion to academics for comment.

Uninoculated minced beef was placed in vacutainers and allowed to equilibrate in water-baths pre-adjusted to 50, 55 or 60 deg C (McMahon 1997), reported by Bolton, et al (2000).  They reported the D-values for Listeria monocytogenes in minced beef using a laboratory water-bath.  At 50 deg C, the D-value was 32.7, 32.7 and 36.1 minutes respectively and at 55 deg C, 3.4 and 3.2 minutes.  It is interesting that they report that at 48 deg C, the solid beef sample showed a D-value of 88.6 minutes and 92.6 minutes respectively, heated in a commercial retort.  This, I take to predict the ineffectiveness of a smoker setting of 48 deg C for core temperature requirement as far as inactivating L. monocitogenes.  It must also be noted that due to the variable factors enhancing the heat resistance of this bacteria in a factory setting, one must opt for an as high temperature as can be tolerated. (Bolton, et al.; 2000)

Frankfurters that was processed to an internal temperature of 71.1 deg C has been shown to effect at least a 3-log cycle reduction of strain Scott A..  (Jay, et al.; 2005:  602)

Why does the presence of salt increase the D-value?

Lihono, et al. says “the influence of salts on the thermal inactivation of microorganisms is largely due to reduced water activity and increased osmotic pressure of the heating medium.”  (Lihono, et al, 2003)

->  Acid Tolerance Response

It is reported that the pH minimum for the growth of two strains was 3.5 and 4.0 in a chemically defined medium using HCl.  On beef treated with 2% lactic and acetic acid, acid adaption did not protect L. monocytogenes strain Scott A.   The importance for us is that we have implemented a carcass wash procedure with lactic acid sprayed with very low pressure as one of many control measures.  Another consequence of its “acid adaption is that this characteristic provides protection not only the ability to develop acid resistance but also resistance against HHP” as well as freezing.  (Jay, et al., 2003:  530) This is of concern since HPP is currently being considered for several new product formulations.  This warrants further investigation.

“In general, the minimum growth pH of any bacterium is a function of temperature of incubation, general nutrient composition of growth substance, water activity, and the presence and quality of NaCl and other salt inhibitors.”  (Jay, et al.; 2005:  595)

->  Combined effect of pH and NaCl

The interaction between pH, NaCl and incubation periods have been studied extensively.  Time to reach visible growth (expressed in days) is, in one study, representing the number of days required to achieve a 100-fold increase in the number of L. monocytogenes. The findings from this study are instructive.  “At pH 4.66, time to visible growth was 5 days at 30 deg C with no NaCl added. This changes to 8 days with 4.0% NaCl at 30 deg C and 13 days with 6% NaCl.  At a temperature of 5 deg C, and a pH of 7.0, growth occurs only after 9 days with no NaCl added, 15 days at 4% NaCl and 28 days for 6.0% NaCl.  (Jay, et al.; 2005:  597)

This would indicate that adding 4% NaCl to the meat and processing it within 24 hours, maintaining a meat temperature of < 7 deg C is an effective hurdle to prevent L. monocytogenes growth.

Conclusion

This article is a “work in progress” and a discussion document.  Work on it will continue and updates will be made available after every update for comment.

*  I rely on Warnich Biersteker from Lionels Veterinary Supplies in Cape Town for the evaluation of this section.  Thank you for your tireless input and your forensic approach.  Warnick can be contacted at warnich7@gmail.com

References

Bolton, D. J., McMahon, C.M., Doherty, A.M.,  Sheridan, J.J., McDowell, D.A.,  Blair, I.S. and Harrington, D.. 2000. Thermal inactivation of Listeria monocytogenes and Yersinia enterocolitica in minced beef under laboratory conditions and in sous-vide prepared minced and solid beef cooked in a commercial retort.  Journal of Applied Microbiology 2000, 88, 626-632

Jay, J. M., Loessner, M. J., Golden, D. A..  2005.  Modern Microbiology, Seventh Edition.  Springer.

Lihono, M. A., Mendonca, A.F., Dickson, J.S., and Dixon, P. M..  2003.  Predictive Model To Determine the Effects of Temperature, Sodium Pyrophosphate, and Sodium Chloride on Thermal Inactivation of Starved Listeria monocytogenes in Pork Slurry.  Journal of Food Protection, Vol.66, No.7, 2003, Pages 1216–1221 Copyright (c) International Association for Food Protection.

Thévenot, D., Dernburg, A., Vernozy‐Rozand, C. 2006.  An updated review of Listeria monocytogenes in the pork meat industry and its products.  Journal of Applied Microbiology, Volume 101, Issue 1 July 2006, Pages 7–17.

http://anelichconsulting.co.za/index.php/industry-news

https://www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria

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