Biofilm Formation and Disinfection on The Surface of Fermented Fish (Pla-Ra) Containers

Research Article

Austin J Microbiol. 2024; 9(1): 1048.

Biofilm Formation and Disinfection on The Surface of Fermented Fish (Pla-Ra) Containers

Anggita Ratri Pusporini¹; Patimakorn Klaiprasitti¹*; Wannee Samappito²; Chanika Tianwitawat¹*

1Department of Food Technology, Faculty of Technology, Khon Kaen University, Thailand

2Department of Food Technology and Nutrition, Faculty of Technology, Mahasarakham University, Thailand

*Corresponding author: Patimakorn Klaiprasitti & Chanika Tianwitawat Department of Food Technology, Faculty of Technology, Khon Kaen University, Thailand. Email: chanika.tian@gmail.com

Received: January 17, 2024 Accepted: February 27, 2024 Published: March 05, 2024

Abstract

Pla-ra is one of Thailand’s most common lactic-fermented fish products. Fermented food was considered safe globally, but outbreaks of foodborne diseases have emerged. Biofilms produced on food processing equipment and other food-contact surfaces serve as a persistent source of contamination. Clostridium perfringens, Escherichia coli, Listeria innocua, and Staphylococcus aureus were isolated from the two types of containers most commonly used for fermenting pla-ra, made of clay and PE, from three representative pla-ra manufacturers in Khon Kaen province. The purpose of this study was to determine the ability of each of the pathogenic bacteria to form biofilms using a microplate assay and to find out how effective the disinfectants (chlorine and PAA) were at reducing the level of bacterial contamination through the artificial contamination method.Keywords: biofilm; formation; disinfection; fermented fish (pla-ra).

Introduction

In terms of fermented foods, fish is one of the most common products used as a fermented product. Southeast and East Asian countries are the leaders in this production [14]. In Thailand, a fermented fish product called pla-ra is the most popular lactic fermented fish product consumed as a condiment while eating papaya salad [26]. Even though known as a fermented product that is globally safe, Rattanasuk et al. (2015) revealed that from representative 20 samples of pla-ra in Roi Et province, Escherichia coli, Staphylococcus aureus, and Vibrio cholerae were found in the product by the rate 15 (75%), 20 (100%), and 3 (15%) of total samples, respectively. Some pla-ra products have also contained Clostridium perfringens and Salmonella spp [24]. Fish itself, however, is very perishable. It provides microorganisms with strong nutrient abundance coupled with a high water activity (aw) and moderate pH [23]. Besides, pla-ra making is classified as spontaneous fermentation, in which microorganisms on the raw materials are utilized for the direct fermentation process. It is difficult to control the environmental parameters that can lead to poor quality products [14]. Diverse microorganisms not only present in the fish and other ingredients used for making pla-ra, but also may exist in the food contact surface used during fermentation [13,28]. Among all foods, fermented products require the most extended time contact of the food with the fermenting equipment's surface, which can increase the potential of cross-contamination [13,17]. Multispecies bacteria can grow on food matrixes and along with food industry infrastructures. This growth may give rise to biofilm [9]. The biofilm development process is initiated with single cells attaching to a surface or each other, then followed by the formation of clustered cells or micro-colonies [2]. Over time, the micro-colonies are surrounded by a protective layer of protein-rich substances referred to as Extracellular Polymeric Substances (EPS) [28]. Previous research has suggested that almost all bacteria can form a biofilm and that once the transition from planktonic cells to their biofilm state is initiated, this becomes the optimum form for the existence of the bacterial cell [10,13,20].

Biofilm cells produce proteinaceous substances that allow synergic growth and protection from possible harsh environments it may encounter [2,15,20,28]. By such complex regulation systems, biofilm confers many advantages to the microbial cells in a food industry environment, such as physical resistance against desiccation, mechanical resistance such as liquid streams in pipelines, and chemical protection against antimicrobials and disinfectants used in the industry [9,13]. The age of biofilm, stress responses, or dormant cells are some of the factors that have been related to the increased resistance [19,28]. Of particular importance to the food industry is that some biofilm-forming species in food factory environments are human pathogens. These pathogens can develop biofilm structures on different artificial substrates common in the food industry, such as stainless steel, polyethylene, polypropylene, clay, wood, glass, rubber, and so on [3,9,13]

High diversity of the affected environments and the variety of colonizing bacterial species complicates biofilm eradication in the food industry and increase the risk of food contamination [28]. Especially in terms of the fermentation process, longer contact time is required and usually resulted in the transfer of more bacteria from surface to food. Even in only 300 s, bacterial transmission from food contact surface has happened [17]. Food-borne diseases associated with bacterial biofilms on food matrixes or factory equipment may arise via intoxications or infections [9,19]. Toxins, for example, can be secreted by biofilm found within food processing plants. From there, they can contaminate a food matrix, causing an individual or multiple intoxications [9]. Biofilm was involved 65% of all microbial diseases, according to NIH and the Centers for Disease Control and Prevention (CDC). Besides, most biofilm formation studies have revealed that they were resistant to commonly used sanitizers and disinfectants [13].

Many researchers have investigated regarding the microbiota, lactic acid bacteria, chemical and sensory analysis in the pla-ra product [14,24-26], however, there is no further study analyzed the potential of cross-contamination comes from biofilm that exists in pla-ra making containers. Besides, the fact that biofilm is commonly resistant to disinfectants urge this study to be conducted. The objective of the current study was to assess the ability of four pathogenic bacterial species from pla-ra making containers to form biofilm on 96 wells-plate and on the clay and polyethylene coupons, together with the biocide tolerance of the developed biofilms against two common food industry chemical disinfectants. Pla-ra substrate was also used to support the sessile development during the condition of artificial contamination on the coupons.

Materials and Methods

Bacterial Strains and Preparation of the Inocula

Four isolates consist of Clostridium perfringens, Escherichia coli, Listeria innocua, and Staphylococcus aureus were used in this study. All bacteria were collected from pla-ra making containers made from clay and polyethylene material from 3 different pla-ra manufacturers in Khon Kaen province, Thailand. The species identity was confirmed by 16s rRNA gene sequencing analysis (Kimura, 1980; Wang et al., 2008; Razzaq, 2013). Isolates maintained in Tryptone Soy Broth (TSB; Himedia Laboratories, LLC, India) with 15% glycerol stock were revived for incubation at 37°C for 24 hours (precultures) [8,23]. These temperatures were chosen to be closely to the optimum for quickly and successfully resuscitate the bacteria [23]. Working cultures were prepared by adding a 10-μl aliquot of each preculture to 10 ml of TSB and incubating for another 24 h at the appropriate temperature mentioned above. Cells from final workingcultures in stationary phase were harvested and re-suspended in sterile TSB. The bacterial suspensions of the four isolates were alsocombined and further diluted to yield mixed cultureinocula of approximately 107–8 CFU/mL, to be used for the subsequent artificial contamination study [10,23].

Categorization of Isolates Based on Biofilm-Forming Capacity

The heterogeneity in the biomass of the samples requires definition of a cut off value that would divide the samples in non-adherent, weak, moderate, and strong adherent. For this reason, all samples were tested in triplicate and calculated the OD average using negative controls (medium without inoculum).

The cut off value was defined for each species. The following criteria were used for biofilm gradation in clinical isolates (Singh et al., 2017) [4].

ODcut = ODavg of negative control + 3 × standard deviation (SD) of ODs of negative control.

OD = ODcut = Non-Biofilm-Former (NBF)

ODcut < OD = 2 × ODcut = Weak Biofilm-Former (WBF)

2 × ODcut < OD = 4 × ODcut = Moderate Biofilm-Former (MBF)

OD >4 × ODcut = Strong biofilm-former.

Biofilm Formation Assay

Wells of 96-wells sterile polystyrene plates were each filled with 90 μL of another sterilized TSB and inoculated with 10 μL of the prior working cultures to develop biofilms on the surfaces of the microtiter plates [7,8]. Negative control wells containing only TSB were included in the assay. Each pathogenic bacterial strains was incubated at 37oC for 0, 6, 12, 24, 48 and 72 h. Removal of the culture medium from the microtiter plates were done three times by inverting the plates and shaking out the liquid and then gently submerged in a small tub of distilled water to wash off any remaining unbound cells or medium components. After air-drying in a laminar flow, wells were stained with 50 μL of 0.5% Crystal Violet (CV) for 5 min. Excess stain was removed by the same prior treatment for washing the plates five times with distilled water. Dye bound to adherent cells was de-stained by pipetting 50 μL of 95% ethanol. The concentration of crystal violet was determined by measuring the optical density at 595 nm (CV-OD595 value) using a spectrophotometer (SPECTROstar Nano, BMG Labtech, Germany) [7,21].

Artificial Contamination

The two mL of previous overnight working cultures(107–8 CFU/mL) of mono-species and mixed-species were transferred to each sterile clay and polyethylene coupons surfaces and evenly spread in perpendicular directions with a sterile cotton swab. To study the effect of pla-rasolution which accumulated during fermentation, the model substrate for the condition of with and without pla-ra solution were prepared. Pla-ra was bought from the same manufacturers took place for the isolation. The substrate was prepared as 50% pla-ra suspensions by mixing 50 mL of the pla-ra solution and 50 mL of sterile distilled water [23]. All the coupons were incubated under controlled temperature (37°C) in an incubator for 6, 12, 24, 48, or 72 hours based on the result of the highest biofilm production for each pathogenic bacteria [7,10,23].

Disinfection Treatment

Two common disinfectants were used in this study: (i) chlorine based disinfectant (Sodium Hypochlorite) (Vittayasom Sriracha Co., Ltd., Thailand), and (ii) Peracetic acid based disinfectant (Calgonit DS 658) (Calvatis Asia Pacific Co., Ltd, Thailand) Both disinfectants were used in concentrations advised by the manufacturer’s instructions: 200 mg/L (ppm) for chlorine suspension on food contact surface (22.72 g in 1000 mL sterile distilled water) and 400 mg/L (ppm) for the PAA suspension (500 mL of solution in 1250 mL of sterile distilled water). Artificial contamination was performed on both clay and polyethylene coupons surfaces with incubation under controlled temperature at 37 °C. All coupons were soaked into chlorine solution and PAA solution for 10 min based on the disinfection procedure. After the exposure time attained, pre-soaked cotton swab in TSB was used to detach the bacterial cells on the surface and was transferred to 10 mL of sterile TSB followed by homogenizing step for 10 sec on vortex mixer (Labnet International, Inc., USA).

Bacterial Quantification

Enumeration of the pathogenic bacteria in TSB after the artificial contamination and disinfection treatment was performed by growing the inoculates on each specific agar plates using sterile spreaders. The plates were incubated for 24-48 h at 37°C and the specific colonies were counted. The killing effect of each disinfectants was calculated by the reduction between the initial incubated coupons (artificial contamination treatment) and the coupons exposed to disinfectant streatment (Log CFU/cm2) [23].

Results and Discussion

The Estimation of Biofilm Formation of Pathogenic Bacteria by Microtiter Plate Biofilm Assay

All of the pathogenic bacteria (Clostridium perfringens, Escherichia coli, Listeria innocua, and Staphylococcus aureus) were tested for the biofilm formation, performed by 96-well microtiter plate assay. Two types of containers used for fermentation in most pla-ra manufacturers were clay as the common and traditional one, then Polyethylene (PE). The OD values of biofilm formation and each bacteria ability of each pathogenic bacterium to produce biofilms are shown in Table 1 and Table 2.