Arthrobacter sp. CW01 produced two enzymes with antibiofilm activities, which were amylase and protease. However, the antibiofilm activity of Arthrobacter sp. CW01 was specific to the type of pathogenic bacteria that were inhibited, production media, inducers and concentration of the crude extracts used to treat the biofilm. It was also revealed that the utilization of different production media and inducers resulted in different metabolism which at the end affected the antibiofilm activity. The amylase and protease production were significantly induced by the addition of starch, casein and yeast extract. Nevertheless, the increase in amylase and protease enzyme activities were not always followed by the increase of biofilm destruction or inhibition activities, indicating that both of the enzymes had reached the maximum concentration to optimally destroy or disrupt the biofilm structure.
Methods
Antibiofilm producing bacterial strain
Arthrobacter sp. CW01 used in this research was obtained from Cunca Wulang River, West Flores, Indonesia. This bacterium was maintained on Glucose Yeast Malt Streptomyces (GYMS) agar and incubated at 28 °C for 7 days.
Biofilm-forming pathogenic bacteria
Two pathogenic bacteria used for antibiofilm assay, Pseudomonas aeruginosa (ATCC 27,853) and Staphylococcus aureus (ATCC 25,923) were procured from Microbiologics, Inc. Both of the bacteria were cultured on Brain Heart Infusion Agar (Oxoid) at 37 °C, overnight.
Screening for antibiofilm agent production media
Amylase as antibiofilm agent from Arthrobacter sp. CW01 was screened with five production media (Table 13) and then stained with iodine solution.
Arthrobacter sp. CW01 was spotted on all of those agar production media and incubated at 28 °C for 7 days. Each agar plate was stained with iodine solution for 5 min. Amylase activities on various media were compared by the size of the hydrolytic zone and calculated with this formula:
Hydrolytic zone = Diameter of the clearance zone – Diameter of the colony [21].
Production of antibiofilm agents
Arthrobacter sp. CW01 was grown on GYMS agar slant at 28 °C for 5 days. After five days, the inoculum in the agar slant was re-suspended with 5 mL of physiological saline. One mL of inoculum was transferred into 50 mL of GYMS broth in 250 mL Erlenmeyer flask and incubated at 28 °C with 120 rpm agitation for 5 days. This served as the seed culture for antibiofilm agent production. One mL of the seed culture was inoculated into 50 mL of each production broth medium in 250 mL Erlenmeyer flask. Each production medium was carried out in triplicate (with the total of 15 flasks). These flasks were incubated at 28 °C with 120 rpm agitation for one week.
After seven days, the cultures were centrifuged at 4 °C and 7800 x g for 10 min. Supernatants were transferred into new centrifuge tube and centrifuged again at 4 °C and 7800 x g for 10 min. Subsequently, the supernatants were filtered through 0.20 μm ultrafiltration membrane (Corning). The clear filtrates are referred to as crude extracts and can be stored at 4 °C for a week or -20 °C for a month.
Determination of amylase activity
Amylase activity was measured by the release of reducing sugar from soluble starch using 3,5-dinitrosalicylic acid (DNS). The reaction mixture contained 0.5 mL of crude extracts and 1 % soluble starch dissolved in 1 mL sodium phosphate buffer 0.1 M (pH 7.0). The mixture was incubated at 37 °C for 10 min. Subsequently, the reaction was stopped by the addition of 2 mL DNS and boiled for 10 min to develop colour. The absorbance of the mixture was measured at 540 nm. As control, 0.5 mL of crude extracts was first added with 2 mL DNS to inactivate the enzyme. After that, the mixture was added with 1 mL soluble starch solution and boiled for 10 min to develop colour.
The absorbance of the sample and control were calculated using standard curve equation to determine the glucose concentration. Standard curve was made using 0.5 mL D-glucose (150 µg/mL; 300 µg/mL; 450 µg/mL; 600 µg/mL; 750 µg/mL) mixed with 1 mL soluble starch solution. The glucose content of the sample was substracted by the glucose content of the control and used to determine the enzyme activity. One unit of enzyme activity is defined as the amount of amylase releasing reducing sugar equivalent to 1 µmol glucose per minute at 37 °C [23]. Amylase enzyme activity and specific activity were calculated using this formula:
$$\text{UA }\left(\text{I}\text{U/mL}\right)\text{=}\frac{\left[\text{Glucose sample}\right]\text{- }\left[\text{Glucose control}\right]}{\text{Glucose MW * Incubation time}}\text{* DF}$$
$$\text{SA (}\text{I}\text{U/mg)= }\frac{\text{UA (}\text{I}\text{U/mL}\text{)}}{\text{[Total protein] (mg/mL)}}$$
*UA represented Unit Activity of Enzyme, SA represented Specific Activity of Enzyme, DF represented dilution factor, MW represented molecular weight.
Determination of protease activity
Based on our preliminary study, there were other antibiofilm compounds from Arthrobacter sp. CW01 besides amylase which was identified as protease. In this study, protease activity measurement was done by measuring the release of tyrosine from casein hydrolysis using modified Sigma method. The reaction mixture for sample analysis contained 200 µL of crude extracts and 0.65 % (w/v) casein dissolved in 800 µL of sodium phosphate buffer 0.1 M (pH 7.0). The mixture was incubated at 37 °C for 10 min. Subsequently, the reaction was stopped by the addition of 500 µL of trichloroacetic acid 0.1 M. As control, 800 µL casein was incubated at 37 °C for 10 min and then was added first with 500 µL trichloroacetic acid 0.1 M. Subsequently, the control mixture was added with 200 µL of crude extracts.
Both sample and control had the same treatment from this step onwards. The sample and control were incubated at room temperature for 30 min. After that, each of them was centrifuged at 13,000 rpm for 10 min. 400 µL of the supernatant was transferred into new microtube. One mL of Na2CO3 0.4 M and 200 µL of Folin-Ciocalteu reagent were added into the mixture. Each microtube was incubated at 37 °C for 30 min. Thereafter, the microtubes were centrifuged at 13,000 rpm for 10 min. The absorbance of the supernatant was measured at 655 nm and calculated using tyrosine standard curve equation. Tyrosine concentration of the sample was reduced by the tyrosine concentration of the control and used to determine the enzyme activity. One unit of protease activity is defined as the amount of enzyme that is needed to hydrolyze casein to produce 1 µmol of tyrosine product per minute at 37 °C. Protease enzyme activity and specific activity were calculated using this formula:
$$\text{UA}\left(\text{I}\text{U/mL}\right)\text{=}\frac{\left[\text{Tyrosine sample}\right]\text{-}\left[\text{Tyrosine control}\right]}{\text{Tyrosine MW * Incubation time}}\text{* DF}$$
$$\text{SA (}\text{I}\text{U/mg)= }\frac{\text{UA (}\text{I}\text{U/mL}\text{)}}{\text{[Total protein] (mg/mL)}}$$
*UA represented Unit Activity of Enzyme, SA represented Specific Activity of Enzyme, DF represented dilution factor, MW represented molecular weight.
Determination of protein content
Protein content of the crude enzyme was measured with Bradford method. The reaction was carried out by adding 0.4 mL of crude extracts with 8 mL of Bradford reagent and mixed thoroughly. This mixture was incubated for 5 min in room temperature. Blank was prepared by mixing 0.4 mL of deionized water with 8 mL of Bradford reagent. The absorbance of the crude enzyme was determined by using spectrophotometer (λ = 595 nm). Total concentration of the protein was calculated using standard curve equation of bovine serum albumin.
Antibacterial activity test
Antibacterial activity test was carried out using agar diffusion test (Kirby-Bauer method). Two pathogenic bacteria, P. aeruginosa (ATCC 27,853) and S. aureus (ATCC 25,923) were grown on BHI (Oxoid) until a turbidity of 0.5 McFarland was reached. The cultures were uniformly spread over the surface of Mueller Hinton Agar (Oxoid) plate using sterile cotton swab. Excess moisture was allowed to dry for 10 min. The sterile blank antimicrobial susceptibility discs (Oxoid) were placed over the swabbed plates and 10 µL of the crude extracts were loaded on the discs. The plates were incubated at 37 °C for 24 h and the diameter of inhibition zone is measured [24].
Biofilm eradication activity assay
Biofilm eradication activity assay was performed with static biofilm. Bacterial pathogens were grown in BHI (Oxoid) supplemented with 1 % D-glucose and then incubated at 37 °C until the absorbance value reached 0.132 (λ = 600 nm). Each well in 96-well microplates (Iwaki) was filled with 200 µL of cultures and incubated at 37 °C, overnight. Mature biofilm were then mixed with 5 % (v/v), 10 % (v/v), 15 % (v/v), and 20 % (v/v) of crude extracts from every production treatment and incubated at 37 °C for 30 min. After incubation, planktonic cells and the media were discarded. Adherent cells were rinsed twice using distilled water and air dried. These adherent cells supposed to be the biofilm, were stained with 0.4 % crystal violet and incubated for 30 min. Dyes were discarded and washed with distilled water for 5 times and then air dried. The stained biofilm were solubilized with 200 µL of 96 % ethanol for 5 min and then transferred into new microplate. The optical density of the solution in each well will be determined at 595 nm using microplate reader [25].
Biofilm inhibition activity assay
Bacterial pathogens were grown in BHI (Oxoid) supplemented with 1 % D-glucose and incubated at 37 °C until the absorbance value reached 0.132 (λ = 600 nm). Each well in 96-well microplates (Iwaki) were filled with 200 µL of pathogenic bacteria cultures along with 5 % (v/v), 10 % (v/v), 15 % (v/v) and 20 % (v/v) crude extracts from every production treatment. Microplates were incubated at 37 °C, overnight and assayed on the next day [25].
Determination of antibiofilm compound activity
Percentages of antibiofilm eradication and inhibition activity were calculated using this formula [25]:
$$\text{\% }\text{Activit}\text{y= }\text{1-}\frac{\text{OD sample-OD negative control}}{\text{OD positive control-OD negative control}}\text{*}\text{100\%}$$
*Positive control contained pathogenic bacteria without crude extracts, negative control contained broth medium without crude extracts, blank contained broth with crude extracts.
Statistical analysis
Enzyme and antibiofilm activities were analyzed using one-way analysis of variance (ANOVA). Antibiofilm activities were tested further with Tukey’s Honest Significant Difference (HSD) Test to seek the significant differences between specific groups. Every treatment was repeated 9 times and analyses were performed with 95 % confidence interval using IBM SPSS Statistics 24.