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

Summary

This protocol describes an approach combining static and dynamic methods to evaluate the efficacy of organic peroxyacids for eradicating biofilms in the dairy industry. This approach may also be used to test the effectiveness of new biological or chemical formulations for controlling biofilms.

Abstract

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their high resistance to most clean-in-place (CIP) procedures frequently used in processing plants. Therefore, it is imperative to develop new biofilm control strategies for the dairy industry. This protocol is aimed at evaluating the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a commercial peracetic acid-based disinfectant) for eradicating dairy biofilms using a combination of static and dynamic methods. All the disinfectants were tested on the strongest biofilm-producing bacteria in either a single or a mixed biofilm using the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method. A contact time of 5 min with the disinfectants at the recommended concentrations successfully eradicated both the single and mixed biofilms. Studies are currently ongoing to confirm these observations using the Center for Disease Control (CDC) biofilm reactor, a dynamic method to mimic in situ conditions. This type of bioreactor enables the use of a stainless-steel surface, which constitutes most industrial equipment and surfaces. The preliminary results from the reactor appear to confirm the efficacy of organic peroxyacids against biofilms. The combined approach described in this study may be used to develop and test new biological or chemical formulations for controlling biofilms and eradicating microorganisms.

Introduction

The dairy industry is a major industrial sector worldwide, including in Canada, where there are more than 10,500 dairy farms producing nearly 90 million hL of milk each year¹. Despite the strict hygiene requirements imposed in the dairy industry, including in processing plants, milk constitutes a great culture medium for microorganisms, and thus, dairy products are likely to contain microorganisms, including spoilage or pathogenic microorganisms. These pathogens can cause various diseases; for example, Salmonella sp. and Listeria monocytogenes can cause gastroenteritis and meningitis, respectively². Spo....

Protocol

The work contained in this article requires a biosafety level 2 laboratory and was previously approved (Project number 119689) by the Université Laval institutional biosafety committee.

NOTE: The flowchart in Figure 1 represents a summary of the methodology combining static and dynamic approaches that was used to evaluate the efficacy of organic peroxyacids for eradicating biofilms.

1. Preparation of materials

1. Microbial isolates
   1. Turn on the biological safety cabinet (BSC) 15 min before use, and clean it with a 70% (v/v) alcohol solution.
   ....

Results

The SEM analysis shows the presence of biofilms produced by P. azotoformans PFl1A on the biofilm microplate pegs (Figure 2A). A three-dimensional biofilm structure can be observed. The P. azotoformans PFl1A was previously identified as a strong biofilm producer (A₅₇₀ > 1.5) using 96-well microtiter plates¹².

In addition, the P. azotoformans PFl1A biofilm that formed on a stainless-steel slide using t.......

Discussion

The MBEC assay (biofilm microplate assay) was the first method to be recognized as a standard biofilm eradication test by the ASTM¹⁷. Our study and others have shown that there are two critical steps when using this assay: the sonication step (time and power) and the disinfectant treatment time¹⁸. Stewart and Parker also suggested other parameters that could influence the outcome of the assay, such as the microbial species, biofilm age, surface area/volume ratio, etc.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 µm filters	Corning	09-754-28	diameter: 50 mm, PTFE- Membrane
316 stainless-steel disc coupon	Biosurface Technologies Corporation	RD128-316
316 stainless-steel slide coupon	Biosurface Technologies Corporation	CBR 2128-316
96-microtiter plate	Corning	07-200-89	cell Culture-Treated, flat-Bottom Microplate
Acetic acid	Sigma Aldrich	27225	store at RT
Aluminium stubs	Electron Microscopy Science	75830-10	32x5mm
Aqueous glutaraldehyde EM Grade 25%	Electron Microscopy Sciences	16220	store at -20 °C
AB204-S/FACT Analytical balance	Mettler Toledo	AB204-S
Bacterial Vent Filter (0.45 µm)	Biosurface Technologies Corporation	BST 02915
BioDestroy	Groupe Sani Marc	09-10215	commercial peracetic acid-based disinfectant, store at RT
Carboy LDPE 20 L	Cole Parmer	06031-52
CDC biofilm reactor	Biosurface Technologies Corporation	CRB 90	bioreactor
Cerium (IV) sulphate	Thermo Scientific	35650-K2	store at RT
Confocal laser scanning microscope  LSM 700	Zeiss	LSM 700
Dey-Engley neutralizing broth	Millipore	D3435-500G	store at 4 °C
EMS950x + 350s gold sputter	Electron Microscopy Sciences
Epoxy resin	Electron Microscopy Sciences	14121	with BDMA
Ethyl alcohol 95%, USP	Greenfield global	P016EA95	store at RT
Ferroin indicator solution	Sigma Aldrich	318922-100ML	store at RT
Filling/venting cap	Cole Parmer	RK-06258-00
FilmTracer LIVE/DEAD Biofilm Viability Kit	Invitrogen	L10316	fluorescent cell viability kit (SYTO 9: green fluorescent stain, Propidium iodide: red fluorescent stain), store at - 20 °C
Glass flow break	Biosurface Technologies Corporation	FB 50
Gold with silver paint	Electron Microscopy Sciences	12684-15
Heating plate set	Biosurface Technologies Corporation	110V Stir Plate
Hex screwdriver	Biosurface Technologies Corporation	CBR 5497
Hydrogen peroxide	Sigma	216763	store at 4 °C
Inoculating loops	VWR	12000-812	sterile, 10 µl
Lactic acid	Laboratoire MAT	LU-0200	store at RT
MASTERFLEX L/S 7557-04 W/ 7557-02 with EASY-LOAD II peristaltic pump and 77200-50 Head	Cole Parmer	77200-60
MBEC (Minimum Biofilm Eradication Concentration) assay biofilm inoculator with a 96-well base	Innovotech	19111	Biofilm microtiter plate
Oxford agar base	Thermo Scientific	OXCM0856B	store at 4 °C
Plastic coupon holder	Biosurface Technologies Corporation	CBR 2203
Plastic slide holder rod	Biosurface Technologies Corporation	CBR 2203-GL
Potassium iodide	Fisher Chemical	P410-500	store at RT
Precision slotted screwdriver (1.5 mm x 40 mm)	Wiha	26015
Propionic acid	Laboratoire MAT	PF-0221	store at RT
Sartorius BCE822-1S Entris® II Basic Essential Toploading Balance	Cole Parmer	UZ-11976-3
Scanning electron microscope JSM-6360LV model	JEOL	JSM-6360LV	SEM and user control interface
Screw cap tube, 15 mL	Sarstedt	62.554.205	(LxØ): 120 x 17 mm, material: PP, conical base, transparent, HD-PE
Screw cap tube, 50 mL	Sarstedt	62.547.205	(LxØ): 114 x 28 mm, material: PP, conical base, transparent, HD-PE
Sodium Cacodylate Trihydrate	Electron Microscopy Sciences	12300	store at -20 °C
Sodium thiosulfate	Thermo Scientific	AC124270010	store at RT
Sonication bath	Fisher	15-336-122	5,7 L
Starch solution	Anachemia	AC8615	store at RT
Sulfuric acid	Sigma Aldrich	258105-500ML	store at RT
Tryptic soy agar	BD Bacto	DF0369-17-6	store at RT
Tryptic soy broth	BD Bacto	DF0370-17-3	store at RT
Tubing Masterflex L/S 16 25'	Cole Parmer	MFX0642416
Tubing Masterflex L/S 18 25'	Cole Parmer	MFX0642418
Tygon SPT-3350 silicon tubing	Saint-Gobain	ABW18NSF	IDx OD: 1/4 in.x 7/16 in.
Vortex	Cole Parmer	UZ-04724-00
Water bath	VWR	89202-970
Zen software	Zeiss