The antimicrobial effect of chemical sanitizers on Pseudomonas spp. biofilm

1. Institute of Chemical Technology Prague Department of Food Preservation and Meat Technology The antimicrobial effect of chemical sanitizers on Pseudomonas spp. biofilm Petra Sedláčková Supervisor: Dr. Ing. Miroslav Čeřovský

2. Biofilm – biologically active matrix of cells and extracellular substances in association with a solid surface. • Food-borne pathogens and spoilage microorganisms accumulate as biofilm on stainless steel, aluminium, glass, Buna-N, teflon steel and nylon materials typically found in food-processing. • Biofilm causes serious problem – cross contamination and post-processing contamination.

3. Mechanism of biofilm formation • conditioning of a surface – the accumulation of macromolecules at the solid-liquid interface on food-contact surface • adhesion of cells – attachment of microorganisms to the conditioned surface • formation of microcolony – layer of cells covering the surface • biofilm formation – continuous attachment of the bacterial cells with associated EPS production

4. Bulk fluid (c) (a) (b) Solid surface Reversible adherence. Planktonic cells (a) in the bulk fluid come in contact with the solid surface (b). The cells then desorb from the surface and become planktonic cells again (c).

5. Bulk fluid (a) polysaccharide (b) Solid surface Irreversible adherence. Planktonic cells (a) in the bulk fluid come in contact with the solid surface. If the forces that hold the cell on the surface are strong enough, the cell is not easily removed and may produce exopolymers that help anchor the cell to the surface (b).

7. Quantification of attachment • Direct methods • removal of the cells and counting by traditional plating methods • direct counting of the cells using microscopic techniques • Indirect methods • measuring cellular components that have accumulated on the surface - protein, ATP or exopolysaccharides.

8. Materialsand methods • Bacteria and biofilm formation • culture of Gram-negative bacteria Pseudomonas spp. was kept on GSP agar (25°C, 72 hr) • suspension of bacteria was prepared in MPbroth (25°C, 24 hr) • the inocula used for biofilm formation contained about 107 -108 CFU*cm-3 • biofilms were grown on plastic materials (polystyrene, polyethylene) and stainless steelsurfaces for 5, 13 days in MP brothand liquid egg

9. Biofilm was treated with: • chlorine baseddisinfectant • active ingredients  sodium hypochlorite in-use concentration < 5% • factors affecting the elimination of microbes  chemical and mechanical effect, holding time and temperature • ultrasound (46 kHz)

10. Evaluation of the sanitizersefficacy • the bacteria were scraped from the test surfaces with the cotton-wool swab →transferred into thetest tube containing 10 ml physiological solution → was stirred for1 min to release the cells into the solution • cultivation: GSP agar for 72 hr, 25°C

11. Evaluation of bacterial quantity and viability in 5,13-day-old Pseudomonas spp. biofilm grown on polystyrene surface after treatment with a chlorine based disinfectant and ultrasound.

12. Evaluation of bacterial quantity and viability in 5-day-old Pseudomonas spp. biofilm grown on stainless steel surface after treatment with a chlorine based disinfectant.

13. Evaluation of bacterial quantity and viability in 13-day-old Pseudomonas spp. biofilm grown on stainless steel surface after treatment with a chlorine based disinfectant.

14. Conclusion • biofilmthe source of contamination in food processing mechanical treatment is needed  to remove the soil (EPS matrix) before an effective disinfectant treatment • 13-day-old biofilm  more resistant than 5-day-old biofilm • the ability to develop as biofilm on polyethylene surfaceis insufficient • ultrasound  the efficacy of chemical methods is more successful