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.
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.
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. Spoilage microorganisms can affect the quality and organoleptic properties of dairy products by producing gases, extracellular enzymes, or acids3. The appearance and color of the milk may also be altered, for example by Pseudomonas spp.4.
Some of these microorganisms can form biofilms on different surfaces, including stainless steel. Such biofilms enable the persistence and multiplication of microorganisms on the surface of the equipment and, thus, the contamination of the dairy products5. Biofilms are also problematic because of their ability to impede heat transfer and accelerate the corrosion of the equipment, leading to premature replacement of the equipment and, thus, to economic losses6.
Clean-in-place (CIP) procedures allow the food industry to control the growth of microorganisms. These procedures involve the sequential use of sodium hydroxide, nitric acid, and, sometimes, sanitizers containing hypochlorous acid and peracetic acid7,8. Although hypochlorous acid is highly effective against microorganisms, it also reacts with natural organic matter, causing the formation of toxic by-products9. Peracetic acid does not generate harmful by-products10; however, its effectiveness against biofilms in the food industry is highly variable10,11. Recently, other peroxyacids, including perpropionic and perlactic acids, have been studied for their antimicrobial activity, and they appear to be a good alternative for the control of microbial growth in biofilms12,13.
Therefore, this study aimed to evaluate the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a peracetic acid-based disinfectant) for eradicating dairy biofilms using an approach combining the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method, and the Center for Disease Control (CDC) biofilm reactor, a dynamic method that mimics in situ conditions. The MBEC assay is hereafter referred to as “biofilm microtiter plates” in the protocol. The protocol presented here and the representative results demonstrate the efficacy of organic peroxyacids and their potential application for controlling microbial biofilms in the dairy industry.
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
2. Formation of single and mixed biofilms
3. Quantitative evaluation of the efficacy of organic peroxyacids for eradicating biofilms
4. Qualitative evaluation of the efficacy of organic peroxyacids for eradicating biofilms
NOTE: After being treated with the disinfectants (step 3.1.1 to step 3.1.5), the P. azotoformans biofilms that formed on the pegs of the biofilm microtiter plate in the static method were prepared and analyzed by observation on scanning electron and confocal microscopes.
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 the bioreactor appeared to be very dense and displayed the morphological characteristics of a mature biofilm with a three-dimensional structure (Figure 2B). Furthermore, 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/cm2 and 7.86 log CFU/cm2 in TSB culture medium and sterile skim milk, respectively (Figure 2C).
Moreover, the results shown in Figure 3 obtained with the B. vesicularis isolate showed that certain factors can have an important influence on biofilm formation in the bioreactor. By varying some of the parameters, such as the stirring speed within the bioreactor, the medium flow rate, the medium nutrient concentration, and the duration of the continuous mode step, cell attachment can be enhanced, and denser biofilms can be observed. For instance, increasing the nutrient concentration from 100 mg/L to 900 mg/L (condition A compared to condition F) resulted in an increase in the bacterial count of the biofilms from 6.11 log CFU/cm2 to 8.71 log CFU/cm2. Furthermore, significant growth of biofilms was observed when the flow rate was reduced to 6.0 mL/min (in condition C), resulting in a longer residence time consistent with the growth rate of this isolate.
The microscopic observations of the biofilms formed on the biofilm microtiter plates or the bioreactor pre- and post-disinfectant treatment were complementary to the viable cell counts. Figure 4 shows that none of the biofilms contained detectable viable cells at the contact time (5 min) and concentrations (500 ppm with organic peroxyacids and 100,000 ppm with hydrogen peroxide) of disinfectants usually applied in dairy plants. These results were confirmed by microscopy (Figure 5). Figure 5A shows the three-dimensional structure of an untreated MBEC biofilm (control), while treated MBEC biofilms (with hydrogen peroxide, peracetic acid, perlactic acid, perpropionic acid, and a commercial disinfectant preparation) lose their three-dimensional conformation, as determined by SEM. However, although the biofilms were treated with disinfectant, an apparent biofilm structure remained, particularly with hydrogen peroxide. Nevertheless, the use of a live/dead technique, in this case, confocal microscopy (CM) with fluorescent viability staining, confirmed that the remaining biofilm structure was mainly lifeless after a disinfectant treatment (Figure 5B).
Figure 1: Flowchart representing a combined static and dynamic approach to evaluate the effectiveness of organic peroxyacids for eradicating biofilms. Please click here to view a larger version of this figure.
Figure 2: Analysis of biofilm formation by Pseudomonas azotoformans PFl1A. (A) Scanning electron micrographs (300x and 2,000x) of the P. azotoformans PFl1A biofilm formed on pegs of the biofilm microtiter plate. This figure has been modified from Goetz et al.12 with permission (Copyright 2022 American Society for Microbiology. All Rights Reserved.). Scale bars = 50 µm (300x), 100 µm (2,000x). (B) Scanning electron micrographs (2,000x) of (1) a stainless-steel slide containing a P. azotoformans PFl1A biofilm grown in the bioreactor and (2) a biofilm-free slide (negative control). Scale bar = 10 µm. This figure has been modified from Niboucha et al.16 with permission. (C)Bacterial density of P. azotoformans PFl1A biofilms formed in the bioreactor in TSB culture medium and sterile skim milk and determined after their removal by ultrasonication from the stainless-steel slides. Data are presented as mean ± SD (n = 3). ****Significant difference (p < 0.05) is based on Student's t-test. This figure has been modified from Niboucha et al.16 with permission. Please click here to view a larger version of this figure.
Figure 3: Bacterial density of Brevundimonas vesicularis biofilms developed using the bioreactor under varying conditions of stirring, flow rates, time in continuous mode, and culture media (TSB). Condition A: 130 rpm stirring speed, 11.8 mL/min flow rate, 24 h continuous mode in 100 mg/L TSB medium (in accordance with the ASTM International protocol E2562-1714 for the formation of biofilms by Pseudomonas aeruginosa); Condition B: 60 rpm stirring speed, 11.8 mL/min flow rate, 24 h continuous mode in 100 mg/L TSB medium; Condition C: 60 rpm stirring speed, 6.0 mL/min flow rate, 24 h continuous mode in 100 mg/L TSB medium; Condition D: 60 rpm stirring speed, 6.0 mL/min flow rate, 48 h continuous mode in 100 mg/L TSB medium; Condition E: 60 rpm stirring speed, 6.0 mL/min flow rate, 48 h continuous mode in 300 mg/L TSB medium; Condition F: 60 rpm stirring speed, 6.0 mL/min flow rate, 48 h continuous mode in 900 mg/L TSB medium; Condition G:60 rpm stirring speed, 6.0 mL/min flow rate, 24 h batch mode in 2.7 g/L TSB and 48 h continuous mode in 900 mg/L TSB medium. Data are presented as mean ± SD (n = 3). Significant differences (different letters, p < 0.05) are based on one-way ANOVA and Tukey's multiple comparison test. Please click here to view a larger version of this figure.
Figure 4: Viable cell count in Pseudomonas azotoformans PFl1A biofilms before and after treatment with hydrogen peroxide, perlactic acid, perpropionic acid, peracetic acid, or commercial disinfectant. Each point represents the mean of triplicate counts obtained on three independent days for each isolate. Data are presented as mean ± SD (n = 3). This figure has been modified from Goetz et al.12 with permission (Copyright 2022 American Society for Microbiology. All Rights Reserved.). Please click here to view a larger version of this figure.
Figure 5: Microscopic observation of biofilm structure and viability. (A) Scanning electron micrographs (300x and 2,000x) of a Pseudomonas azotoformans PFl1A biofilm formed on the pegs of the biofilm microtiter plate before (control) and after treatment with hydrogen peroxide, peracetic acid, perlactic acid, perpropionic acid, and commercial disinfectant at their minimum biofilm eradication concentrations (MBEC). Scale bar = 10 µm. This figure has been modified from Goetz et al.12 with permission (Copyright 2022 American Society for Microbiology. All Rights Reserved.). (B) Viability of a biofilm formed by P. azotoformans PFl1A using fluorescent cell viability staining on the biofilm microplate peg before (left) and after (right) treatment with peracetic acid (500 ppm), visualized by confocal laser scanning microscopy (63x/1.40 oil differential interference contrast objective). The viable cells are stained green, and the dead cells are stained red. Scale bar = 20 µm. This figure has been modified Goetz et al.12 with permission (Copyright 2022 American Society for Microbiology. All Rights Reserved.). Please click here to view a larger version of this figure.
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.19. Lastly, it is important to note that the formation of biofilms around the pegs on the microplate lids might be heterogenous and will vary depending on the bacteria used.
The MBEC method can potentially be used with various types of disinfectants. The use of microscopy techniques, such as SEM and confocal microscopy, are complementary and extremely useful for studying the three-dimensional structures and viability status (fluorescent cell viability staining kits, see Table of Materials) of biofilms pre- and post-disinfectant treatment, respectively12. The ability of bacteria to form biofilms has been mainly studied with microtiter plates due to the ease and speed of execution. However, the use of this closed system with no shear forces, as observed in most situations, has limitations. Therefore, the use of a dynamic biofilm formation method, such as the CDC bioreactor, is highly recommended to confirm the observations made using the biofilm microplate assay.
According to the ASTM standardized method14,15 and published studies20,21, the bioreactor is extensively used to grow biofilms and to evaluate the efficacy of disinfectants. This device mimics certain environmental conditions of biofilm formation, such as in terms of the high shear force, specific and renewable nutrients, and surface materials. In this study, reproducible mature P. azotoformans PFlA1 strain biofilms were obtained on stainless-steel slides using this dynamic system. The protocol presented here was based on the ASTM standard protocol using Pseudomonas aeruginosa. However, this standardized biofilm method cannot be applied to all microorganisms, even if they have been shown to be strong biofilm producers using other techniques. This was the case for B. vesicularis, for which the protocol had to be optimized in order to reach a higher biofilm density.
Previous studies have demonstrated that some operational conditions (or parameters) are crucial in the process of biofilm development and must be adapted for each one of the bacteria tested22,23,24. For example, the nutrient requirements must be adjusted for each bacterium in terms of preference and concentration25. Moreover, it has been shown that the biofilms are thicker and have more significant biomass under continuous nutrient supply conditions in the bioreactor than when grown in the batch mode, which is considered a nutrient-limited environment26. Another important aspect is the residence time, which is controlled by the flow rate and should be adjusted in order for it to be shorter than the bacterial doubling time. In addition, the temperature and the shear mixing of the fluid have to be adjusted for each bacterium in order to provide optimum growth conditions27. The high shear stress created by the movement of the baffle promotes biofilm development and plays a role in controlling its structure and thickness28. However, if the stirring speed is increased too much, it can cause erosion or sloughing of the biofilm's surface, which leads to reduced bacterial density29,30.
The use of a bioreactor has some limitations, such as it being costly and time-consuming in addition to using a large volume of fluids as the nutrient medium. Nevertheless, it remains a highly repeatable and reproducible method for forming and studying biofilms. The use of a bioreactor also allows the simultaneous use of several coupons made of different materials. The bioreactor is designed to monitor the experimental conditions during biofilm growth, such as the temperature, the speed of stirring, and the flow rate, and it allows the recovery of homogeneous and representative biofilms for subsequent analysis. Overall, static and dynamic methods are complementary for studying in vitro the efficacy of disinfectants against biofilms isolated from the food and environmental sectors.
The authors have nothing to disclose.
This research was supported by the Consortium de Recherche et Innovations en Bioprocédés Industriels au Québec (CRIBIQ)(2016-049-C22), Agropur, Groupe Sani Marc, and the Natural Sciences and Engineering Research Council of Canada (NSERC) (RDCPJ516460-17). We thank Teresa Paniconi for the critical review of the manuscript.
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 Palmer | UZ-04724-00 | |
Water bath | VWR | 89202-970 | |
Zen software | Zeiss |