France
June 6, 2012
Bacteria adhering to the surface of equipment are a major source of food contamination in industry. Inhibiting their adhesion during the production phase, and encouraging their detachment during cleaning, constitute two major public health challenges. By means of the Interspore project, INRA researchers in Lille and their partners have acquired crucial data on the mechanisms that govern bacterial adhesion by accurately estimating the interaction forces in play between bacteria and materials. These results clearly demonstrate the inefficacy of mechanical cleaning on the detachment of Bacillus spores or Listeria cells. On the other hand, their findings evidence the ease of transfer of adherent bacteria to foods.
The research paths currently being explored to inhibit the adhesion of bacteria to inert surfaces, or facilitate their detachment, are numerous and varied: the definition of anti-adhesive or antimicrobial materials, the design of more hygienic equipment, new cleaning processes (enzymes, pulsed flow systems, etc.). In order to understand all aspects of this issue, it is necessary to obtain in-depth knowledge of the interactions that occur between bacteria and materials. However, the models used until now have failed to take account of either the heterogeneity of bacterial populations or the complexity of the surfaces (presence or not of an appendage) or the mechanical properties (deformability, fragility) of bacteria.
Interspore, a project to model phenomena at the bacteria/material interface
Interspore is a project that is being funded by the French National Research Agency (ANR) for 3 years; its aim is to characterise the interaction forces between bacteria and receptor surfaces, to clarify the role played by these interaction forces in bacterial resistance to detachment and to identify the bacterial properties affecting these phenomena.
Hydrophobicity, a key property in adhesion
The surface properties of a panel of Bacillus spores with or without an exosporium (outer envelope) were characterised and their effect on adhesion assessed. Contrary to what is generally accepted, the adhesion capacity of spores appeared to be little affected by the presence of an exosporium, but could be directly explained by their hydrophobic nature and the presence of appendages. Another surprise was that this hydrophobicity varied both between Bacillus species and between strains belonging to the same group. Thus the hydrophobicity of Bacillus spores seemed to be linked to the presence (or not) of a hyperglycosylated glycoprotein (BclA) on the exosporium surface, and also the composition of this glycoprotein.
Particularly strong interaction forces between bacteria and materials and their effects on the mechanisms underlying the detachment of adherent spores
The interaction forces between bacteria and materials (mainly stainless steel) were estimated from the forces enabling the detachment of four strains of Bacillus selected for their contrasting properties. Using an atomic force microscope (AFM), the maximum force required to detach a spore ranged from 100 to 900 picoNewtons, depending on the strain, which suggested that extremely strong interaction forces were in play.
These results were confirmed by detachment tests performed under conditions similar to those encountered in the agri-food industry (turbulent flow). Indeed, thanks to the construction of a test loop in which wall shear stress values reached 500 Pa, the scientists showed that adherent spores were extremely resistant to mechanical detachment alone; between 10% and 50% of spores were not detached after 10 minutes at 500 Pa. However, the use of 0.5% soda at 60°C was able to detach more than 90% of B. cereus 98/4 spores after 30 minutes with a mean wall shear stress of 8 Pa, thus demonstrating the crucial role of chemical action in cleaning.
Easy transfer to foods
Adherent Bacillus spores were detached by successive contacts with agar-coated plates. More than 40% of them were detached by the first contact, which suggests an ease of transfer to foods. Despite the very strong interaction forces present, the mechanisms in play during this transfer still need to be identified.
Results confirmed with Listeria monocytogenes
The properties of Bacillus spores that were identified as affecting the interaction forces between bacteria and a substrate were then studied in a non-sporulating bacterial model, Listeria monocytogenes. Once again, hydrophobicity predominated among the factors explaining adhesion. Bacteria-material interaction forces, although usually slightly weaker than with Bacillus, nevertheless remained very strong. In terms of their transfer to foods, 20% to 50% of strains were detached by a single contact with an agar-coated plate, suggesting once again an important risk of cross-contamination with foods.
Pursuit of this work may soon result in the optimisation of cleaning procedures for agri-food production systems, with the additional benefit of probable reductions in water use.
Scientific leader:
Christine Faille
INRA
UR638 PIHM - Processus aux Interfaces et Hygiène des Matériaux
369 rue Jules Guesde
BP 39
59651 VILLENEUVE-D'ASCQ CEDEX
For further information:
E. Galopin et al (2010) Selective adhesion of Bacillus cereus spores on heterogeneously wetted silicon nanowires. Langmuir 26: 3479‐3484.
C. Faille et al (2010) Viability and surface properties of spores subjected to a cleaning‐in‐place procedure. Consequences in their ability to contaminate surfaces of equipment. Food Microbiol. 27:769‐776.
C. Faille, et al (2010) Morphology and physico‐chemical properties of Bacillus spores surrounded or not with an exosporium. Consequences on their ability to adhere to stainless steel. Int. J. Food Microbiol. 143:125‐135.
Y. Lequette, E. Garénaux, G. Tauveron, S. Dumez, S. Perchat, C. Slomianny, D. Lereclus, Y. Guerardel, C. Faille (2011) Role played by exosporium glycoproteins in the surface properties of Bacillus cereus spores and in their adhesion to stainless steel. Appl. Environm. Microbiol. 77: 4905-4911.
Y. Lequette, E. Garénaux, T. Combrouse, T. Del Lima Dias, A. Ronse, C. Slomianny, X. Trivelli, Y. Guerardel, C. Faille (2011) Domains of BclA, the major surface glycoprotein of the Bacillus cereus exosporium. Glycosylation patterns and role in spore surface properties. Biofouling 27 :751-761.
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