Article pp.116-119 du Vol.23 n°1 (2003)

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116-119 Sci. Aliments 23(1), 2003 P. Chavant et al.

FOCUS : JSMTV

Growth of Listeria monocytogenes in biofilm according to the surface and the temperature and effects of different treatments on its survival

P. Chavant¹, P. Folio and M. Hébraud^1,21

In the laboratories, the studies on bacteria are generally carry out from planktonic cells, growing in a liquid medium. However, in the natural environments, more than 90% of the micro-organisms have a sessile mode of growth, adhered to a biotic or an abiotic surface. They can then form biofilms which correspond to microbial aggregates surrounded by a matrix of exopolymers.

Under this mode of growth, it is known that the micro-organisms are more resistant to environmental variations and stresses, to cleaning disinfection treatments or to antibiotics.

Listeria monocytogenes is an ubiquitous Gram-positive rod that can be found in the ground, the hydraulic networks or on vegetables as well as in the food industry environments. This bacterium can also be present in the gastroin- testinal tract of animals and humans as asymptomatic carriage strain. This germ is the causative agent of the listeriosis, a foodborne disease which can be very serious for high-risk groups such as old persons, pregnant women, neonates, immunocompromised adults. Although the frequency of listeriosis outbreaks decreased thanks to the efforts of the food industry working people and to the implementation of efficient system of control, the sanitary risk remains important. So, during year 1999, two hundred seventy cases were registered in France (1) with a mortality rate of 23%. For the food industry, the economic consequences linked to a foodborne listeriosis outbreak are dramatic because, besides the direct loss due to the withdrawal and the destruction of the incrimi- nated product, the indirect losses due to the deterioration of the brand image of the product can be fatal to the company.

In pure culture, L. monocytogenes is able to form biofilms on numerous abiotic surfaces such as stainless steel, glass or polytetrafluoroethylene (PTFE or teflon) (2-6). In the plants of foodstuff production and transformation, L. monocytogenes can be isolated within pluri-microbial biofilms formed on the surfaces of equipments. So, it represents a potential source of contamination of food products.

1. Station de Recherches sur la Viande – Microbiologie¹, Plate forme protéomique², INRA de Theix, 63122 Saint-Genès Champanelle

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Growth of Listeria monocytogenes in biofilm according to the surface… 117

The aim of our work was on one hand to assess the ability to form biofilms according to the surface properties of the bacterium and the adhesion support and, on the other hand to compare the resistance of L. monocytoge- nes to treatments applied at different stages in planktonic or sessile mode of growth.

The surface physico-chemical properties play a role in the bacterial adhesion. So, these properties were characterized by two methods: the electropho- retic mobility and MATS (Microbial Adhesion To Solvents) measurements (7).

They showed that the strain L. monocytogenes LO28 possessed a global charge of their surface always negative with slight variations according to pH (pH 2-7), temperatures (37˚, 20˚ and 8˚C) or to tested physiological stages (exponential and beginning of stationary phase). Besides, the strain presented a hydrophilic tendency, due to a pronounced basic character, which increased with the temperature decreasing. The hydrophilic character of the strain allows to think that bacteria can adhere in a more important way to hydrophilic surfaces such as stainless steel or glass than to hydrophobic surfaces such as PTFE or PVC, and this, especially when the temperature is low.

In order to confirm this hypothesis, L. monocytogenes LO28 was grown at 37˚, 20˚ and 8˚C on two types of surfaces, stainless steel and PTFE. The kine- tic of growth in biofilms was followed by enumeration of sessile bacteria, by epifluorescence microscopy to assess the rate of contaminated surface and by scanning electron microscopy. The stainless steel or PTFE chips (3 by 1 cm) were submerged during 2 hours with a bacterial suspension, then they were rinsed to eliminate the non adherent bacteria, and recovered with sterile medium which was renewed daily. The enumerations and the microscopic observations revealed:

– the absence of matrix because the strain produced very few exopolymers,

– a biofilm with an important three-dimensional structure which develo- ped in some hours on both types of surface at 37˚C and phenomena of detachment of cellular aggregates which took place on PTFE after few days,

– a colonization slightly slower of the two surfaces at 20˚C with the formation of a three-dimensional structure at the end of 2 days on stainless steel and 3-5 days on PTFE but without detachment even after 7 days, – a very slow colonization of stainless steel at 8˚C by lengthened cells with

the formation of few aggregates after 7 days and the adhesion without colonization, of PTFE with even some cell detachment after 1-2 days.

These results allowed to demonstrate that L. monocytogenes LO28 was able to develop and to formbiofilms as well on a hydrophilic surface than on a hydrophobic surface. However, there was a faster colonization and a the better stability of biofilms on stainless steel, which is in agreement with the hydrophilic properties of the strain. The detachments observed at 37˚C on PTFE could be due to the weak interactions with the support of a small num- ber of adherent bacteria at the basis of the voluminous aggregates. At 20˚C, the slower colonization and the presence of flagella allow to suppose a better adhesion and stability of the three-dimensional structure of the biofilms. It was

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118 Sci. Aliments 23(1), 2003 P. Chavant et al.

particularly interesting to note that at 8˚C, the colonization of the hydrophilic surface was dramatically reduced and moreover that it was impossible on the hydrophobic surface, probably due to the increase of the hydrophilic character of the strain at low temperature.

The physiological and molecular studies of L. monocytogenes cells growing in biofilms need great quantity of biomass. The conditions used on stainless steel at 20˚C give us this possibility with a biomass of 2 ✕ 10⁸ cfu/cm². From a practical point of view, the use of hydrophobic surfaces, such as PTFE, in conditions of low temperatures allows to limit the development of this bacterium in the workshops of foodstuffs production and transformation.

The second part of this study consisted to evaluate the resistance to different treatments of L. monocytogenes cells in a planktonic or sessile mode of growth. These treatments were applied to bacteria in exponential growth (6 hours) or in the beginning (1 day) or late (7 days) stationary phase, either in suspension in a liquid medium or adhered on stainless steel. The treatments consisted in 30 min challenges with solutions of acetic acid (pH 5.0, 1,8.10^–4 mol/l), or NaOH (pH 12.0, 0,009 mol/l), or Na₂SO₄ (10%), or quaternary ammonium (20 ppm) or monolaurine (75 ppm). The survival population was enume- rated by spreading cells onto TSA (Tryptone Salt Agar) plates and after a 2- days incubation period at 37˚C.

L. monocytogenes seemed to be very resistant to acetic acid, to salt as well as to monolaurine challenges whatever the growth conditions studied. On the contrary, the alkaline, the quaternary ammonium and the alkaline/salt combination treatments affected the viability of cells. For these three treatments, planktonic cells in exponential phase of growth were always the most sensitive (∆log from 4 to 8). The resistance of cells in biofilms during the alkaline and alkaline/salt challenges slightly decreased little with ageing (∆log of 0.7 and 0.68 at 6 h and of 2.6 and 1.6 at 7 d, respectively). During these 2 treatments, planktonic cells in stationary phase showed a similar resistance to aged biofilms one. Nevertheless, the quaternary ammonium treatment had a more important effect on planktonic cells in stationary phase (∆log of 4.8) than on sessile cells, these last ones being more resistant with ageing (∆log of 2.3, 1.9 and 0.3 at 6 h, 1 d and 7 d, respectively). The effect of the different treatments on 7 d biofilms with or without daily renewing of the culture medium did not show significant differences on the mortality rates of cells.

However, the adherent population treated was 2 logs lower after 7 d growth without medium renewing.

These results confirm that the mode and the phase of growth are important parameters to take into account for the application of cleaning disinfection treatments and that some of these treatments have low efficiency on L. monocytogenes lethality.

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Growth of Listeria monocytogenes in biofilm according to the surface… 119

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