The methods of inactivation and removal of bacterial biofilm

1. The methods of inactivation and removal of bacterial biofilm Petra Sedláčková

2. Biofilm – source of product contamination • Biofilm - interest in the context of food hygiene. Adherent bacteria are known to be highly resistant to cleaning and disinfection. • Food borne microorganisms display a broad spectrum of resistance. • Impermeable outer membrane • Mechanism for antibiotic-inactivation • Biofilm formation on surfaces – protect colonies from cleaning and sanitation

3. Biofilm formationwas studied in stationary systems. • Theadhesionability of Pseudomonas spp. to polystyrene and polyethylene surface and ability todevelop as biofilmwas compared. • Effect of biofilm age on chlorination resistance was studied on Pseudomonas spp.

4. 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 MPA broth (25°C, 24 hr) • the inocula used for biofilm formation contained about 107 -108 CFU*cm-3 • biofilm was grown on polyethylene and polystyrene surface for 4, 7 and 13 days in MPA broth

5. Biofilm was treated with: • commercial disinfectant - chlorine based product used in the food processing • active ingredients in the disinfectant is sodium hypochlorite in-use concentration < 5% • factors affecting the elimination of microbes – chemical and mechanical effect, holding time and temperature • disinfectants must be effective, safe and rinsable, easy to use and they should not affect the sensory qualities of the product • ultrasound (46 kHz)

6. Conventional cultivation • the bacteria were scraped from the test surfaces with a cotton-wool swab→transferred into test tube containing 10 ml physiological solution→was stirred for 1 min to release the cells into the solution • the samples were cultivated at 25°C on GSP agar for 72 hr

7. Evaluation of bacterial quantity and viability in 7-day-old Pseudomonas spp. biofilm grown on polyethylene surface after treatment with a chlorine based disinfectant.

8. Evaluation of bacterial quantity and viability in 7-day-old Pseudomonas spp. biofilm grown on polystyrene surface after treatment with a chlorine based disinfectant.

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

10. Results • Surface-attached cells aremore resistant to disinfectant treatment than cells of the same microorganism grown in suspension. • No growth was detected after 5 min treatment (0,02% sodium hypochlorite) at the polyethylene surface→ the ability to develop as biofilm of Pseudomonas spp. to polyethylene surface is insufficient. • The cultivation measurement showed that the CFU were not reduced efficiently by a 10 min treatment using the chlorine-based disinfectant (0,02% and 0,5%) at polystyrene surface.

11. For biofilm removal of polystyrene surface mechanical treatment is needed → to remove the soil (EPS matrix) before an effective disinfectant treatment. • The efficacy of chemical methods of inactivation and removal of biofilm is more successful with use of ultrasound.