Bacterial Cultivation
Fourty actinobacteria isolates used in this research were obtained from the previous study [4,5] that isolated from aquatic environments in Indonesia. Actinobacteria were cultured onto Yeast Malt Extract Agar (YMEA) incubated at 28°C for 7 days. Indicator strains of quorum sensing activity were C. violaceum wild type and C. violaceum CV026 cultivated onto Luria Agar (LA) and incubated at 28°C for 24 h. While six pathogenic bacteria which were B. cereus ATCC 14579, S. aureus ATCC 29213, and E. faecalis ATCC 33186, P. aeruginosa ATCC 27853, S. typhimurium, V. cholerae cultivated onto LA and incubated at 37°C for 24 h.
Primary Screening of Antiquorum Sensing Activity
The assay was conducted using the overlay agar method [6] with modification. Actinobacteria isolates were inoculated into YMEA then incubated at 28°C for 3 days. C. violaceum were grown separately in Luria Broth (LB) and incubated at 28°C for 24 h. The indicator strain diluted within semisolid agar (0.75% LA) until OD600=0.132 and poured onto YMEA plates. These plates were incubated at 28°C for 24 h. Positive result indicating antiquorum sensing can be seen through the absence of violacein pigment production around actinobacteria isolates.
Preparation of Crude Extract
Each isolate was inoculated into Tryptic Soy Broth (TSB) supplemented with glucose (1% w/v). The culture was incubated at 28°C for 7 days at 125 rpm then centrifuged at 7,800xg for 15 minutes. The supernatant was mixed with ethyl acetate in a 1:1 ratio and shaken at 150 rpm overnight. The solvent phase was evaporated using rotary evaporator then dried using vacuum oven and resuspended in Dimethyl Sulphoxide (DMSO). Crude extracts were stored at -20°C for a month [7,8,9]
Antimicrobial Assay
Assay is conducted using agar well diffusion method [10]. Tested bacteria (OD600=0.132) were streaked onto Brain Heart Infusion Agar (BHIA). The crude extract (50 µL) of actinobacteria with various concentration (10 and 20 mg/mL) was added. DMSO (1% v/v) was used as the negative control, while streptomycin (10 mg/mL) was used as the positive control. The plates were incubated at 37°C for 24 h.
Detection of Antiquorum Sensing Activity
This assay was conducted based on [11] with modification. C. violaceum (OD600=0.132) used as indicator strain streaked onto BHIA. The crude extract (50µL) of actinobacteria with various concentration (10 and 20 mg/mL) was added. DMSO (1% v/v) was used as a control. The plates were incubated at 28°C for 24 h. Antiquorum sensing activity was determined qualitatively through the inhibition of violacein production around the wells [12]
Quantification of Antibiofilm Activity
Quantification of antibiofilm activity was conducted based [8] with modification. The antibiofilm activity was divided into inhibition and destruction activity. For inhibition activity assay, each pathogen was inoculated into Brain Heart Infusion Broth (BHIB) supplemented with glucose (2% w/v) and incubated at 37°C, 150 rpm for 24 h. Then 100 µL of bacterial culture (OD600=0.132) and 100 µL of crude extract was transferred to the 96-wells microplate to quantify the biofilm inhibition activity. For destruction activity assay, another 96-wells plate was filled with diluted culture and re-incubated at 37°C for 24 h until mature biofilm attached to the well. Crude extract (100 µL) was added to each well to quantify the biofilm destruction activity. Overnight culture of pathogens without any treatment was used as a positive control, while sterile BHIB was used as the negative control.
After incubation, planktonic cells and media were discarded. Adherent cells were rinsed twice with sterile water and stained using crystal violet (0.4% w/v) for 30 minutes. 96-wells were rinsed twice and air-dried for 5 minutes. About 200 µL of ethanol was added and resuspended. Absorbance was determined at 595 nm with a microplate reader (TECAN M200 PRO). This assay was performed in triplicates. Percentage of inhibition or destruction activity was calculated by the equation below:
Validation of Quorum sensing inhibition
Validation assay was conducted based on [11]. The culture of C. violaceum CV026 (OD600=0.132) was mixed with 100 µL of the crude extract and 1 µmol/mL of Hexanoyl-L-Homoserine-Lactone (HHL). The culture was incubated at 28°C for 24 h. Positive control contained culture and 1 µmol/mL of HHL without crude extract. The tested tubes were centrifuged at 1000rpm for 15 minutes. About 1 mL of DMSO (1% v/v) mixed and centrifuged. The absorbance of the supernatant was measured at 540 nm. Furthermore, the determination of protein compounds in the crude extract was performed. Each crude extract was treated with proteinase-K (100 µg/mL) for 2 h incubation in 37°C and heat loss method with 95°C for 1 h. The treated crude extract was used to do the validation assay. Total violacein produced by C. violaceum CV026 was compared [13]
Examination of Biofilm formation by Scanning Electron Microscope (SEM)
The effect of crude extract was investigated using SEM at Dexa Laboratory of Biomolecular and Science (DLBS), Cikarang, Indonesia. B. cereus and S. typhimurium were grown in BHIB supplemented with glucose (2% w/v) and incubated at 37ºC for 24 h. Bacterial culture (100 µL) were spotted to sterile cover glass kept in sterile petri dish and incubated at 37ºC for 24 h to form a mature biofilm. After the incubation period, 100 µL of crude extract with the highest destruction activity was added into the formed biofilm and incubated at 37°C for 24 h. Mature biofilm without crude extract was used as control [14].
Statistical Analysis
Statistical analysis was conducted by using IBM SPSS Statistics 23. The results were analyzed using the analysis of variances (ANOVA) and the Tukey test for significant difference were determined at P <0.05 and standard error was determined using Microsoft Excel 2016.