B01006: Why do bugs stick to what they stick to

Study Duration: April 1998 to March 2001

Contractor: Institute of Food Research

The prevention of contamination caused by pathogenic microorganisms during manufacture, processing and packaging of food is of considerable importance to public health, and consequently a major issue for industry. In particular, there is increasing concern associated with the contamination arising from bacterial biofilms which develop on materials used during food manufacture. These communities of bacteria, often embedded in a matrix of organic polymers exuded by the cells, can be extremely difficult to remove. Complete eradication of the pathogens is difficult, time consuming and expensive.

In general, the formation of bacterial biofilms is believed to take place over at least three stages: a reversible adsorption step, primary adhesion of microorganisms to a surface, and colonisation. The rates of these processes vary widely depending on the environmental conditions and the type of microorganism but the adhesion and colonisation stages are considered to be relatively slow compared to the first step of cell adsorption. In principle, it should be possible to retard, if not prevent, the formation of biofilms on substrates by using materials to which bacteria cannot initially attach. Such a material or surface coating would be of considerable commercial interest. However, in practice, synthetic materials that are capable of preventing bacterial adsorption have proved rather elusive. Properties of the substrate such as hydrophobicity (repelling water), hydrophilicity (attracting water), steric hindrance (molecules which prevent bacteria attaching), roughness and the existence of a �conditioning layer� at the surface are all thought to be important in the initial cell attachment process.

The objective of this project is to carry out a comparative study of the effect of polymer phase transitions on bacterial adhesion. This will be achieved using a range of surface-coated or surface-grafted polymers with pre-determined phase transition characteristics. In this way the physical state of surfaces (otherwise chemically identical) could be assessed for their effect on the adsorption of the representative food pathogens Listeria monocytogenes, Bacillus cereus and Salmonella typhimurium.

This research project aims to carry out a comparative study of the effect of polymer phase transitions on the adhesion of bacteria to surfaces modified or coated with the polymer. This will be achieved by the synthesis of a range of polymers with pre-determined phase transition characteristics, which are subsequently either covalently linked to, or spin-coated onto, glass substrates before incubation with cultures of Gram positive or Gram negative bacteria. Assessment of the number of cells still present following incubation and rinsing will show how readily the synthetic surfaces were colonised by the bacteria. In this way the importance of the physical state of surfaces (otherwise chemically identical) will be assessed for their effect on the adsorption of the representative food pathogens Listeria monocytogenes, Bacillus cereus and Salmonella typhimurium.

The study will be concerned with two distinctly different phase transitions of polymers: (1) the glass transition (Tg), which is property of the bulk polymer and describes the transition from a brittle, glassy material to a fluid-like or rubbery state and (2) the transition occurring at the cloud point or lower critical solution temperature (LCST). The latter phenomenon is a property exhibited by certain water-soluble polymers, which form stable solutions at temperatures below the LCST, but precipitate rapidly above the LCST i.e. they show inverse solubility behaviour.

Polymers of varying glass transition (Tg) were prepared by co-polymerisation of n-butyl acrylate and t-butyl acrylate, with the t-butyl content varying between 54 and 93%. The polymers were spin-coated from solution onto cleaned glass slides and advancing water contact angle measurements confirmed that all polymer surfaces were hydrophobic to a similar degree (θA = 95
2
). Incubation for 24 hours with culture of ethidium bromide stained Salmonella typhimurium and Bacillus cereus following by cell counting showed no significant difference between the numbers of cells adhering to the surface above or below the Tg.

Polymers for the LCST study were prepared by the polymerisation of N-isopropylacrylamide (NIPam) or by the co-polymerisation of NIPam with N-t-butylacrylamide or with acrylamide. This gave polymers with TLCST of 32
C, 21.3
C and 42.3
C respectively. The addition of 3-mercaptopropanoic acid as a chain transfer agent during the polymerisation and the use of 4,4�-azobis (4-cyanovaleric acid) as initiator, ensured that a large proportion of the polymer chains were terminated with a carboxylic acid at one end of the polymer backbone. This allowed the polymers to be covalently grafted to an amino-derivatized glass surface using a carbodimide reagent. Titration confirmed that 98% of the amine groups were derivatized with polymer. Measurement of advancing water contact angle at 10
C and 50
C showed the change from a hydrophilic surface at low temperature (θA = 34 � 48 o) to a hydrophobic surface at high temperatures (θA = 52 � 61o) with the largest change occurring for the polymer with the lowest TLCST.

Measurements of bacterial adhesion were made at two temperatures, 20
C and 37
C. Under this regime the polymer surfaces were either TLCST at both temperatures, above TLCST at both temperatures or, in the case of poly(NIPam), below TLCST at 20
C and above TLCST at 37
C. Cell numbers were estimated by scintillation counting of radiolabelled L. monocytogenes after incubation with the immobilised polymer films and washing. Overall cells adhered to the polymer films in greater numbers as the TLCST of the polymers increased, however the most striking difference was seen in the comparison of cell numbers on the same polymer at each of the two temperatures of incubation. In extreme cases of low TLCST and high TLCST polymers, cell numbers were very similar at both temperatures, with a slightly higher count at the higher temperatures of incubation; but in the case of poly(NIPam), whose phase transition sits in-between the two temperatures of incubation, there were significantly fewer cells (approximately 35% less) adhering to the film at the higher temperature of incubation. This suggests that, at least in the case of L. monocytogenes, the cells can �see� the difference in the surface characteristics of the polymer film and find it more difficult to attach themselves to the polymer in its collapsed, hydrophobic state than in the solvated, hydrophilic state.

This effect, while not enormous, clearly shows that bacteria are sensitive to the physical state of the underlying polymer surface and are less able to colonise the polymers at temperatures above the critical solution temperatures. While this phenomenon would not be sufficient on its own to prevent biofilm formation, it may suggest a new approach to the engineering of non-adherent surfaces. Coatings that undergo phase transition with small increments in temperature could provide a �moving target� for bacteria to get a foothold, and temperature cycling of such a coating could assist in the more thorough cleaning of surfaces prone to contamination by pathogens.

Final report is available from the Agency's Information centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: infocentre@foodstandards.gsi.gov.uk).

Contact: For any enquiries concerning this research project, please contact the relevant Programme contact or email science@foodstandards.gsi.gov.uk