This research was supported by the Consortium de Recherche et Innovations en Bioproc&#233;d&#233;s Industriels au Qu&#233;bec (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.

<ol>
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The authors declare that they have no conflicts of interest.

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&#39;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 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.

<table><tbody><tr><td>0.2 &amp;micro;m filters&amp;nbsp;</td><td>Corning</td><td>09-754-28</td><td>diameter: 50 mm, PTFE- Membrane</td></tr><tr><td>316 stainless-steel disc coupon</td><td>Biosurface Technologies Corporation</td><td>RD128-316</td><td></td></tr><tr><td>316 stainless-steel slide coupon</td><td>Biosurface Technologies Corporation</td><td>CBR 2128-316</td><td></td></tr><tr><td>96-microtiter plate</td><td>Corning</td><td>07-200-89</td><td>cell Culture-Treated, flat-Bottom Microplate&amp;nbsp;</td></tr><tr><td>Acetic acid</td><td>Sigma Aldrich</td><td>27225</td><td>store at RT</td></tr><tr><td>Aluminium stubs</td><td>Electron Microscopy Science</td><td>75830-10</td><td>32x5mm</td></tr><tr><td>Aqueous glutaraldehyde EM Grade 25%</td><td>Electron Microscopy Sciences</td><td>16220</td><td>store at -20 &amp;deg;C</td></tr><tr><td>AB204-S/FACT Analytical balance</td><td>Mettler Toledo</td><td>AB204-S</td><td></td></tr><tr><td>Bacterial Vent Filter (0.45 &amp;micro;m)</td><td>Biosurface Technologies Corporation</td><td>BST 02915</td><td></td></tr><tr><td>BioDestroy</td><td>Groupe Sani Marc</td><td>09-10215</td><td>commercial peracetic acid-based disinfectant, store at RT</td></tr><tr><td>Carboy LDPE 20 L</td><td>Cole Parmer</td><td>06031-52</td><td></td></tr><tr><td>CDC biofilm reactor</td><td>Biosurface Technologies Corporation</td><td>CRB 90</td><td>bioreactor</td></tr><tr><td>Cerium (IV) sulphate</td><td>Thermo Scientific</td><td>35650-K2</td><td>store at RT</td></tr><tr><td>Confocal laser scanning microscope&amp;nbsp; LSM 700</td><td>Zeiss</td><td>LSM 700</td><td></td></tr><tr><td>Dey-Engley neutralizing broth</td><td>Millipore</td><td>D3435-500G</td><td>store at 4 &amp;deg;C</td></tr><tr><td>EMS950x + 350s gold sputter&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td></td><td></td></tr><tr><td>Epoxy resin</td><td>Electron Microscopy Sciences</td><td>14121</td><td>with BDMA</td></tr><tr><td>Ethyl alcohol 95%, USP</td><td>Greenfield global</td><td>P016EA95</td><td>store at RT</td></tr><tr><td>Ferroin indicator solution</td><td>Sigma Aldrich</td><td>318922-100ML</td><td>store at RT</td></tr><tr><td>Filling/venting cap</td><td>Cole Parmer</td><td>RK-06258-00</td><td></td></tr><tr><td>FilmTracer LIVE/DEAD Biofilm Viability Kit</td><td>Invitrogen</td><td>L10316</td><td>fluorescent cell viability kit (SYTO 9: green fluorescent stain, Propidium iodide: red fluorescent stain), store at - 20 &amp;deg;C</td></tr><tr><td>Glass flow break</td><td>Biosurface Technologies Corporation</td><td>FB 50</td><td></td></tr><tr><td>Gold with silver paint&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>12684-15</td><td></td></tr><tr><td>Heating plate set</td><td>Biosurface Technologies Corporation</td><td>110V Stir Plate</td><td></td></tr><tr><td>Hex screwdriver</td><td>Biosurface Technologies Corporation</td><td>CBR 5497</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma</td><td>216763</td><td>store at 4 &amp;deg;C</td></tr><tr><td>Inoculating loops</td><td>VWR</td><td>12000-812</td><td>sterile, 10 &amp;micro;l</td></tr><tr><td>Lactic acid</td><td>Laboratoire MAT</td><td>LU-0200</td><td>store at RT</td></tr><tr><td>MASTERFLEX L/S 7557-04 W/ 7557-02 with EASY-LOAD II peristaltic pump and 77200-50 Head</td><td>Cole Parmer</td><td>77200-60</td><td></td></tr><tr><td>MBEC (Minimum Biofilm Eradication Concentration) assay biofilm inoculator with a 96-well base</td><td>Innovotech</td><td>19111</td><td>Biofilm microtiter plate</td></tr><tr><td>Oxford agar base</td><td>Thermo Scientific</td><td>OXCM0856B</td><td>store at 4 &amp;deg;C</td></tr><tr><td>Plastic coupon holder</td><td>Biosurface Technologies Corporation</td><td>CBR 2203</td><td></td></tr><tr><td>Plastic slide holder rod</td><td>Biosurface Technologies Corporation</td><td>CBR 2203-GL</td><td></td></tr><tr><td>Potassium iodide</td><td>Fisher Chemical</td><td>P410-500</td><td>store at RT</td></tr><tr><td>Precision slotted screwdriver (1.5 mm x 40 mm)</td><td>Wiha</td><td>26015</td><td></td></tr><tr><td>Propionic acid</td><td>Laboratoire MAT</td><td>PF-0221</td><td>store at RT</td></tr><tr><td>Sartorius BCE822-1S Entris&amp;reg; II Basic Essential Toploading Balance</td><td>Cole Parmer</td><td>&amp;nbsp;UZ-11976-3</td><td></td></tr><tr><td>Scanning electron microscope JSM-6360LV model</td><td>JEOL</td><td>JSM-6360LV</td><td>SEM and user control interface</td></tr><tr><td>Screw cap tube, 15 mL</td><td>Sarstedt</td><td>62.554.205</td><td>(Lx&amp;Oslash;): 120 x 17 mm, material: PP, conical base, transparent, HD-PE</td></tr><tr><td>Screw cap tube, 50 mL</td><td>Sarstedt</td><td>62.547.205</td><td>(Lx&amp;Oslash;): 114 x 28 mm, material: PP, conical base, transparent, HD-PE</td></tr><tr><td>Sodium Cacodylate Trihydrate</td><td>Electron Microscopy Sciences</td><td>&amp;nbsp;12300</td><td>store at -20 &amp;deg;C</td></tr><tr><td>Sodium thiosulfate</td><td>Thermo Scientific</td><td>AC124270010</td><td>store at RT</td></tr><tr><td>Sonication bath</td><td>Fisher</td><td>15-336-122</td><td>5,7 L</td></tr><tr><td>Starch solution</td><td>Anachemia</td><td>AC8615</td><td>store at RT</td></tr><tr><td>Sulfuric acid</td><td>Sigma Aldrich</td><td>258105-500ML</td><td>store at RT</td></tr><tr><td>Tryptic soy agar</td><td>BD Bacto</td><td>DF0369-17-6</td><td>store at RT</td></tr><tr><td>Tryptic soy broth</td><td>BD Bacto</td><td>DF0370-17-3</td><td>store at RT</td></tr><tr><td>Tubing Masterflex L/S 16 25&#039;</td><td>Cole Parmer</td><td>MFX0642416</td><td></td></tr><tr><td>Tubing Masterflex L/S 18 25&#039;</td><td>Cole Parmer</td><td>MFX0642418</td><td></td></tr><tr><td>Tygon SPT-3350 silicon tubing&amp;nbsp;</td><td>Saint-Gobain</td><td>ABW18NSF</td><td>IDx OD: 1/4 in.x 7/16 in.</td></tr><tr><td>Vortex</td><td>Cole Parmer</td><td>UZ-04724-00</td><td></td></tr><tr><td>Water bath&amp;nbsp;</td><td>VWR</td><td>89202-970</td><td></td></tr><tr><td>Zen software</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

evaluation of the efficacy of organic peroxyacids for eradicating dairy biofilms using an approach combining static and dynamic methods

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 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 &#62; 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64619/64619fig01.jpg" /> 
Figure 1: Flowchart representing a combined static and dynamic approach to evaluate the effectiveness of organic peroxyacids for eradicating biofilms. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64619/64619fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64619/64619fig02.jpg" /> 
Figure 2: Analysis of biofilm formation by Pseudomonas azotoformans PFl1A.&#160;(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 &#181;m (300x), 100 &#181;m (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 &#181;m. 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 &#177; SD (n = 3). ****Significant difference (p &#60; 0.05) is based on Student&#39;s t-test. This figure has been modified from Niboucha et al.16 with permission. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64619/64619fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64619/64619fig03.jpg" /> 
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;&#160;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 &#177; SD (n = 3). Significant differences (different letters, p &#60; 0.05) are based on one-way ANOVA and Tukey&#39;s multiple comparison test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64619/64619fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64619/64619fig04.jpg" /> 
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 &#177; SD (n = 3). This figure has been modified from Goetz et al.12 with permission (Copyright 2022 American Society for Microbiology. All Rights Reserved.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64619/64619fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64619/64619fig05.jpg" /> 
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 &#181;m. 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 &#181;m. This figure has been modified Goetz et al.12 with permission (Copyright 2022 American Society for Microbiology. All Rights Reserved.). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64619/64619fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods at JoVE.com

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

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 ...

evaluation-efficacy-organic-peroxyacids-for-eradicating-dairy

Nanyang Technological University

Research

JoVE Journal

Biology

1.6K Views.  Université Laval. Biofilmvormende micro-organismen zijn een groot probleem in de voedingsindustrie, inclusief de zuivelindustrie. Vanwege hun negatieve invloed op de kwaliteit van de producten, is het belangrijk om biofilmcontrolestrategieën te ontwikkelen. Deze aanpak combineert een statische high-throughput screeningsmethode en een dynamische methode die in situ omstandigheden nabootst.Deze twee methoden zijn complementair voor het bestuderen van de werkzaamheid van desinfectiemiddelen tegen biofilms...

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

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Immunology and Infection

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p &gt; 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

Environment

Rapid and Non-Destructive Characterization of Biofilm Elastic Properties

Quantifying Elastic Properties of Environmental Biofilms using Optical Coherence Elastography

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances (EPS). This paper presents a detailed account of the implementation of optical coherence elastography (OCE) measurements tailored for the elastic characterization of biofilms. OCE is a non-destructive optical technique that enables the local mapping of the microstructure, morphology, and viscoelastic properties of partially transparent soft materials with high spatial and temporal resolution. We provide a comprehensive guide detailing the essential procedures for the correct implementation of this technique, along with a methodology to estimate the bulk Young's modulus of granular biofilms from the collected measurements. These consist of the system setup, data acquisition, and postprocessing. In the discussion, we delve into the underlying physics of the sensors used in OCE and explore the fundamental limitations regarding the spatial and temporal scales of OCE measurements. We conclude with potential future directions for advancing the OCE technique to facilitate elastic measurements of environmental biofilms.

Author Spotlight: Characterizing Environmental Biofilm Mechanics Using Optical Coherence Elastography and its Applications in Wastewater Treatment

This paper highlights the optical coherence elastography (OCE) technique's efficacy in rapidly and non-destructively characterizing biofilm elastic properties. We elucidate critical OCE implementation procedures for accurate measurements and present Young's modulus values for two granular biofilms.

Biofilms are complex biomaterials comprising a well-organized network of microbial cells encased in self-produced extracellular polymeric substances ...

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

Systematic Approach to Identify Novel Antimicrobial and Antibiofilm Molecules from Plants' Extracts and Fractions to Prevent Dental Caries

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active compounds from plants are challenging because of the complex plant matrix and time-consuming isolation and identification procedures. Therefore, a stepwise approach for screening natural compounds from plants, including the isolation and identification of potentially active molecules, is presented. It includes the collection of the plant material; preparation and fractionation of crude extracts; chromatography and spectrometry (UHPLC-DAD-HRMS and NMR) approaches for analysis and compounds identification; bioassays (antimicrobial and antibiofilm activities; bacterial &#34;adhesion strength&#34; to the salivary pellicle and initial glucan matrix treated with selected treatments); and data analysis. The model is simple, reproducible, and allows high-throughput screening of multiple compounds, concentrations, and treatment steps can be consistently controlled. The data obtained provide the foundation for future studies, including formulations with the most active extracts and/or fractions, isolation of molecules, modeling molecules to specific targets in microbial cells and biofilms. For example, one target to control cariogenic biofilm is to inhibit the activity of Streptococcus mutans glucosyltransferases that synthesize the extracellular matrix&#8217; glucans. The inhibition of those enzymes prevents the biofilm build-up, decreasing its virulence.

Natural products represent promising starting points for the development of new drugs and therapeutic agents. However, due to the high chemical diversity, finding new therapeutic compounds from plants is a challenging and time-consuming task. We describe a simplified approach to identify antimicrobial and antibiofilm molecules from plant extracts and fractions.

Natural products provide structurally different substances, with a myriad of biological activities. However, the identification and isolation of active ...

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the ...

Detection of Biofilm Disassembly Through a Dispersion Assay

Source: Nickerson, K. P. et. al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57322">Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification.</a>&#160;J. Vis. Exp.&#160;(2018)
The video showcases a biofilm dispersion assay using a multi-well plate. Buffer and glucose treatment disassemble the biofilm, releasing bacteria, while bile salts induce stress, retaining bacteria in the protective extracellular polymeric substance. Higher dispersion with buffer and glucose confirms successful biofilm disassembly, estimated by colony forming units measurement.

1. Preparation of Reagents

	Bile salts medium: To prepare tryptic soy broth (TSB) containing 0.4% bile salts (weight/volume), resuspend 200 mg of bile ...

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

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