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. Adherent bacteria are known to be highly resistant to cleaning and disinfection.  Food borne microorganisms display a broad spectrum of resistance. Impermeable outer membrane Impermeable outer membrane Mechanism for antibiotic-inactivation Mechanism for antibiotic-inactivation Biofilm formation on surfaces – protect colonies from cleaning and sanitation Biofilm formation on surfaces – protect colonies from cleaning and sanitation

3.  Biofilm formation was studied in stationary systems.  The adhesion ability of Pseudomonas spp. to polystyrene and polyethylene surface and ability to develop as biofilm was compared.  Effect of biofilm age on chlorination resistance was studied on Pseudomonas spp.

4. Materials and 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 10 7 - 10 8 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 disinfectant and ultrasound.

10. Results  Surface-attached cells are more 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.