Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

Biofilm forming microorganism are a major issue in the food industry, including the dairy industry. Because of their negative impact on the quality of the products, then it is important to develop biofilm control strategies. This approach combines a static high-throughput screening method and a dynamic method that mimics in situ conditions.

These two methods are complementary for studying the efficacy of disinfectants against biofilms. This approach can be used to test new biological or chemical formulations to control biofilms in the food, medical, or environmental sectors. Begin by vortexing the Pseudomonas azotoformans bacterial culture tubes.

Aseptically transfer 100 microliters of the bacterial culture into 10 milliliters of sterile tryptic soy broth or TSB medium to achieve the final concentration of approximately two times 10 to the seventh CFU per milliliter. Vortex the culture tube. Then using a multichannel pipette, transfer 150 microliters of the diluted bacterial culture in the biofilm microtiter plate wells in triplicate.

Load 150 microliters of TSB medium into three new wells as control. Next, incubate the biofilm microtiter plate at 30 degrees Celsius for 24 hours without agitation. Clean and air dry the parts of the bioreactor before placing the flat blade inside the one liter glass beaker attached to the holder by a magnetic bar.

Maintain the setup upright using the plastic bar attached to the inner side of the bioreactor lid. Use the screwdriver to place the stainless steel coupons or slides on their polypropylene rods. Insert coupons or slides into the holes in the lid without placing their alignment pins in the notches allowing steam to escape during sterilization.

Cover all the bioreactor vents with aluminum foil and wrap the rest of the equipment, namely the size 18 tubing and the size 16 tubing, the glass flow brake, the container caps, the screwdrivers, the forceps, and 0.2 micrometer filters with aluminum foil. Autoclave the bioreactor set up in a dry cycle at 121 degrees Celsius for 20 minutes. To perform the first step of the biofilm formation in the bioreactor in batch mode, inside a biological safety cabinet, connect one end of the size 18 tubing to the outlet spout of the bioreactor and keep the other end wrapped in aluminum foil.

Remove one coupon or slide holder from the bioreactor lid and place it in a sterile 50 milliliter tube. Then using a 50 milliliter serological pipette, fill the bioreactor beaker with 340 milliliters of 300 milligrams per milliliter TSB medium through the hole occupied by the rod. To inoculate the culture medium in the bioreactor, add one milliliter of 10 to the eighth CFU per milliliter Pseudomonas azotoformans bacterial solution through the orifice using a five milliliter pipette and place the rod back into its original position.

Position the rods already placed in the lid holes for the pins to fit into the respective notches. Then place a 0.2 micrometer bacterial air purge filter at the end of the smallest diameter tube located on the lid of the bioreactor. The other tube of the same diameter remains permanently plugged with a metal screw cap or a silicone plug that closes tightly.

Place the bioreactor for 24 hours over the heating plate set at 30 degrees Celsius and stir at 130 RPM. After 24 hours, perform the second step of the biofilm formation in the bioreactor in continuous flow mode. Place a carboy containing 18 liters of sterile distilled water in the biosafety cabinet.

Then add two liters of 1000 milligrams per liter TSB culture medium to obtain a final concentration of 100 milligrams per liter. Cover the container with its sterile cap to which two tubes are connected. The first one is a silicone tube fixed on the cap's interface to pump the medium.

The second one the size 16 tubing is connected to the external port allowing the liquid to flow toward the bioreactor. After connecting the size 16 tubing to the extremity of the glass flow brake, insert the latter into the largest tube on the lid of the bioreactor by its other end and then install the size 16 tubing in the peristaltic pump. Use another 20 liter carboy to collect the effluent from the bioreactor.

Attach the end of the tube connected to the bioreactor outlet spout to the cap of the waste container. Insert a 0.2 micrometer filter into the tube present on the lid of this container. Once done, start the peristaltic pump at a flow rate of 11.3 milliliters per minute and leave the system running for 24 hours.

After 24 hours, turn off the peristaltic pump and stop stirring and heating the bioreactor. To recover the bacterial biofilm, rinse the coupons or slides in 40 milliliters of PBS to eliminate planktonic bacteria. Then release them into a sterile 50 milliliter conical tube containing 40 milliliters of PBS.

After 30 seconds of vortexing, sonicate the tubes at 40 kilohertz for 30 seconds. Repeat this three times. Once done, collect the 40 milliliters of biofilm suspension in a sterile 50 milliliter conical tube and rinse the coupons or slides with two of sterile PBS solution.

Recover this rinsing liquid and add it to the already collected biofilm suspension. Take out the microtiter plate from 30 degrees Celsius. Add 200 microliters of PBS into three wells of a 96-well microplate.

To wash the biofilms and eliminate planktonic bacteria, transfer the lid of the biofilm microplate with biofilms formed on the pegs to the 96-well microplate containing the PBS for 10 seconds. Prepare the disinfectants at the required concentrations and add 200 microliters of each disinfectant concentration into the wells of a new 96-well microtiter plate in triplicate. Transfer the lid of the biofilm microtiter plate onto the 96 well-microtiter plate containing the disinfectants and incubate the plate at room temperature for the desired exposure time.

Next, add 200 microliters of Dey-Engley neutralizing broth to the wells of a new 96-well microtiter plate. Transfer the lid of the biofilm microtiter plate to the 96-well microtiter plate containing the neutralizing broth. Seal the microtiter plate with parafilm and place it in the bath sonicator at 40 kilohertz for 30 minutes.

After 30 minutes, from the first column of the sonicated plate, transfer 100 microliters of detached biofilms to the first row of a new 96-well microtiter plate. Then add 180 microliters of sterile PBS to a 96-well plate except for the first row. Next, transfer 20 microliters of the biofilm solution from the first row to the wells in the second row containing 180 microliters of PBS to achieve the dilution 10 to the minus one.

Then transfer 20 microliters from the second row to the wells in the next row to achieve the dilution of 10 to the minus two. Repeat this to obtain dilution between 10 to the minus five and 10 to the minus seven. Inoculate 100 microliters of the desired dilution on TSA and incubate the plates as per the growth parameters of the bacterium.

Form the Pseudomonas azotoformans PFLA 1 biofilms on the coupons in the bioreactor, remove the rod holding the coupons and rinse the rod inside a conical tube containing 30 milliliters of PBS to remove planktonic cells. Drop each coupon into a sterile 50 milliliter conical tube using a screwdriver. Then add four milliliters of the appropriate organic peroxyacid solution or PBS for the control.

After five minutes of incubation, add 36 milliliters of Dey-Engley neutralizing broth. After vortexing the conical tube for 30 seconds, sonicate them at 40 kilohertz for 30 seconds. Repeat the process three times to obtain the biofilm suspension.

Repeat the same procedure with the other rods, prepare serial delusions of the biofilm suspension, and plate the suspension on TSA medium as described earlier. The SCM analysis showed the presence of the biofilm by Pseudomonas azotoformans PFL1A on the biofilm microplate pegs. The Pseudomonas azotoformans PFL1A biofilm formed on a stainless steel slide using a bioreactor appeared very dense and displayed the morphological characteristics of a mature three-dimensional biofilm.

The results of the bacterial counts of the biofilms developed by this isolate in the dynamic system showed significant cell densities corresponding to 8.74 log CFU per centimeter square in TSB culture medium and 7.86 log CFU centimeter square in sterile skim milk. The results obtained with the B.vesicularis isolate showed that the increase in the nutrient concentration from 100 to 900 milligrams per liter resulted in an increase in the bacterial count of the biofilms from 6.11 to 8.71 log CFU centimeter square. Also, significant biofilm growth was observed when the flow rate was reduced to 6.0 milliliter per minute, indicating that certain factors could influence biofilm formation in the bioreactor.

None of the biofilms contained detectable viable cells at five minutes contact time in 500 parts per million with organic peroxyacids and 100, 000 parts per million with hydrogen peroxide concentrations of disinfectants usually applied in dairy plants. The SCM showed the three-dimensional structure of untreated minimum biofilm eradication concentration or MBEC biofilms, while the treated MBEC biofilms lost their three-dimensional conformation. It is essential to remain under the sterile condition while performing this protocol.

Also, user must remember that generated result are associated with a unique isolate and disinfectant. Combining disinfectant with mechanical vibration, ultrasound, or enzymatic treatments could be evaluated to improve the efficacy of biofilm eradication. This approach using static and dynamic methods to form biofilms paved the way for researchers to screen and study new anti-biofilm formulation to better control biofilms in various sectors.