Persistence of listeria monocytogenes in equipment and premises

Severe listeriosis (which can cause meningitis, septicemia, or still birth) is an infrequent foodborne illness. Yet, because of its high lethality (between 15 and 30 per cent) its causal agent, Listeria monocytogenes, is perceived as a major threat. Outbreaks of listeriosis were not overly common over the last 30 years, but they caused fear for the general population. In spite of strict regulations and the numerous precautions taken by the food business operators in Europe, and after a sharp decrease of incidence from 1987 to 2002, the number of cases per year again began to increase over the last five years. Therefore, two questions need to be asked: how can L. monocytogenes persist in food industry equipment and premises and cause the contamination of food, and what measures could be taken to combat its persistence?

L. monocytogenes is able to grow within a large temperature range (between slightly below 0°C and 45°C), over a large pH range (4.6 to 9.5) and at relatively low water activity (0.90). It can therefore grow in almost any food premises and equipment. The installation of L. monocytogenes is likely the easiest in refrigerated locations where the majority of other bacterial species cannot multiply. By using DNA fingerprinting methods, it has been extensively demonstrated that strains of L. monocytogenes may be repeatedly found for months or years in a same food processing plant. Places where L. monocytogenes are frequently found are floors, drains and more generally, locations where they find water and nutriments, even in minute amounts. What is worrying is that persistence is observed even where the cleaning and the disinfection are done right. Therefore, several researchers have hypo – thesised that persistent and sporadic strains possess different phenotypes.

Attachment

One of the first hypotheses that was tested by scientists was that persistent strains attach and grow in greater numbers on surfaces. Although strains of L. monocytogenes have different potential in this regard, most studies found no differences between sporadic and persistent strains. Furthermore, when considering strains without taking into account whether they are persistent or not, it is striking to note that L. monocytogenes is more frequently found in conditions that disadvantage its adhesion, which are those that favour its growth: presence of organic soil, neutral or alkaline pH, relative humidity >70 per cent, temperature <10°C. Low temperatures can be considered as favourable because L. monocytogenes which is a psychrotrophic poor competitor bacterium can supersede mesophilic bacteria only at low temperature.

Resistance and tolerance to disinfectants

Persistent and sporadic strains were also compared regarding their resistance to disinfectant. Before describing the comparisons done, a few explanations on the word ‘resistant’ are necessary. Many scientists use the word resistance to designate quite different situations. They measure the minimum inhibitory con – centration, or MIC, which is the concentration above which the cells are unable to multiply (usually within one day incubation at 37°C). When the minimal concentration to inhibit the growth of a strain is greater to what is expected, one can say that the strain is adapted to the disinfectant but the phenomenon is often interpreted as an increase of resistance. Disinfectants are not designed to inhibit growth but to kill microbial cells. We therefore recommend using the word tolerance to describe adaptation to a disinfectant and the word resistance when survival after application of bactericidal concentration during a short contact time (usually between five and 20 minutes) is greater than expected.

It should be noted that the word resistance has another important meaning. When high numbers of attached bacterial cells of the same strain are submitted to minimal disinfectant concentrations that would kill the expected fraction of planktonic cells (cells in suspension within a liquid), a subpopulation survives and it is thus usual to say that attached cells or biofilm cells are more resistant than their planktonic counterpart is. This is the reason why standard methods to test disinfectants efficacy on attached cells are used to choose the bactericidal concentrations to be applied in food factories.

The answer to the question ‘Are persistent strains more tolerant to disinfectants?’ is that differences were found in some studies but one research team observed that the difference disappeared after a two hour sublethal exposure to the disinfectants tested (including quaternary ammonium compounds) suggesting that all strains have the same potential for adaptation.

Regarding resistance to disinfectants (survival to bactericidal concentrations), none of the published studies found any significant differences whether cells were compared in suspension or attached.

Other stresses

L. monocytogenes is able to survive desiccation for months and likely more as survival curves end by an almost horizontal tail; but persistent strains were not shown to have greater resistance to desiccation. Similarly, resistance to acidic stress or heat did not show striking diff er ences between persistent and sporadic strains. Harbourage sites From the above we can conclude that it is unlikely that some strains of L. monocytogenes have unique properties leading to persistence. Before explaining our hypothesis it is worth remembering that whatever cleaning and disinfection process is applied, there are locations where it is inefficient because cleaning and disinfection solutions cannot penetrate well and no mechanical action is possible. These are retention zones, hard to clean places or harbourage sites where the cells can find a shelter, a refuge, a niche. Crevices, cracks, holes, etc. in new or worn materials, or interstices at the junction of equipment pieces, are such retention zones. The fact that there are holes in new gaskets, flooring materials (Figure 1 opposite), welds, etc. remains unknown to most people and is seldom accounted for.

Our hypothesis is that persistence occurs when, by accident, L. monocytogenes cells reach a harbourage site in sufficient number for some cells to survive after several cleaning and disinfections. As the part of the adhering population that is resistant to disinfectant increases when the attached population ages and as surviving bacteria have then enough time to adapt to conditions of the harbourage site including presence of low disinfectant concentration, the trapped bacteria resume growth. Then, as long as reduction due to cleaning and disinfection is lower than the increase due to growth, eradication of the bacteria is impossible. The ability to grow thus appears to be the key factor that promotes L. monocytogenes persistence as corroborated by experienced food operators.

Synthesis

From the above we infer that:

● Some cells exposed to bactericidal concentration of disinfectant in appropriate killing conditions may still survive (disinfection is not sterilisation)

● Surviving cells may adapt to disinfectant at concentration higher than the initial MIC

● Surviving cells can then multiply in favourable conditions of temperature, humidity, pH and nutriment supply

Where else than in the retention zones are the cells exposed only to inhibiting concentration of disinfectant, find nutriments that the cleaning was unable to dislodge, and grow undisturbed so as to contaminate their surrounding?

A few recommendations

1. Use hygienically designed and well maintained equipment and premises

2. Do cleaning and disinfections at a frequency high enough to avoid survival of L. monocytogenes, cells recently deposited on surfaces and really exposed to recommended in-use concentration are the easiest to kill

3. Apply periodically, i.e. once a week, a more stringent cleaning and disinfection procedure (higher product concentrations, stronger disinfectant, greater contact time and / or temperature) so that it is possible to stop a possible persistence process. This should be done specifically where retention zones are known to exist and the equipment cannot be dismantled

4. Create a cool and dry environment. Limit the use of water to the greatest extent possible during cleaning and disinfection and during operations. Aim at the dry factory

5. Submit the places the most likely to shelter bacterial cells in harborage sites (e.g. floors) to cleaning and disinfection before the equipment. Thus, cells aerosolised onto the equipment during cleaning and disinfection are eliminated when the equipment is cleaned

6. Account for the above in Guides to Hygienic Practice and the Application of HACCP Principles, and in the operator training packages

References

• Carpentier, B (2011) A suggested method for assessing the cleanability of flooring materials. EHEDG Yearbook 2011/2012 16-20

• Carpentier, B. and Cerf, O. (2011) Persistence of Listeria monocytogenes in food industry equipment and premises. International Journal of Food Microbiology 145, 1-8

• European Hygienic Engineering and Design Group: www.ehedg.org

Biography

Brigitte Carpentier, PhD, has been working for 20 years in the field of hygiene of food contact and non-food contact surfaces in a French public institution which has been recently renamed ANSES. Her research is a succession of field and laboratory studies. Field studies are conducted to know what is happening in the real life and laboratory studies to try to understand bacterial behaviour on surfaces. After conducting research on the hygienic quality of flooring materials, she has been inter – ested by bacterial interactions on surfaces, notably interaction between L. monocytogenes and other bacteria found on food processing surfaces. After studies on bacterial transfer from surfaces to food, she has focused on bacterial persistence of several bacterial species. Apart from her research activities, she was a member of the Microbiology expert comity of the French Food Safety Agency for six years; she is a member of the editorial boards of two scientific journals and member of EHEDG, the European hygienic engineering design group.

Olivier Cerf has an MSc in Agronomy and PhD in Dairy Science. For 30 years, he was a scientist at the National Institute for Agricultural Research (INRA) as Head of the Laboratory of Engineering of Food Process Hygiene, before becoming Professor of Food Hygiene at Alfort Veterinary School (ENVA). He is the Vice-Chair, Scientific Panel on Microbiology, French Agency for Food, Environmental and Occupational Health Safety (ANSES). Olivier participated in the work on the emerging metrics for the management of microbial risks of the Codex Committee on Food Hygiene.