Screening and Quantification of Antiquorum-Sensing Activity of Actinobacteria Isolates Against Gram-Positive and Gram-Negative Biofilm Associated Bacteria CURRENT STATUS: POSTED

Objectives: The purpose of the study is to screen antiquorum sensing activity compound from actinobacteria using Chromobacterium violaceum as indicator and quantify the antibiofilm activity against several biofilm-forming pathogenic bacteria, such as Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 14579, Enterococcus faecalis ATCC 33186, Vibrio cholerae , Salmonella typhimurium , Pseudomonas aeruginosa ATCC 27853. Results: Crude extracts from marine actinobacteria isolated from Indonesia were promising for treating biofilm-related diseases. Out of 40 isolates 10 were found to have great quorum quenching activity against C. violaceum. Moreover, these isolates were able to inhibit biofim formation by Gram positive and Gram negative pathogenic bacteria. Quorum quenching agents in extracts of 1AC, 14PM, 16PM, 18PM, and CW17 isolates were predicted as proteins or enzymes.


Introduction
Bacteria can communicate with each other using unique chemical compounds called autoinducer.
Communication activity of bacteria is known as quorum sensing. This mechanism is referring to the capacity of bacteria to monitor their cell density. Quorum sensing also affects the genes expression which influences microbial behaviors, such as regulating virulence factors and biofilm formation [ 1 ,2].
Biofilm formation is a process when flocked bacteria develop extracellular matrix that facilitate attachment. It gives rise to serious problems for public health because biofilm-associated organisms are more resistant to chemical agents and physical treatments. In this case, the probability of successful infections will increase [2]. Every activity to inhibit bacterial quorum sensing is known as quorum quenching. It has been proposed as novel therapeutics for treating biofilm-based diseases.
The biofilm-based diseases are usually treated by antibiotics. However, the use of antibiotics is not an effective solution and might lead to antibiotic resistance. Actinobacteria are fungi-like Gram-positive bacteria, well known as a supreme producer of secondary metabolite compounds [ 3 ], but research around antiquorum sensing and antibiofilm from these bacteria yet have not been much explored.
The objectives of this research are to screen antiquorum sensing activity compound from actinobacteria using C. violaceum as indicator and quantify the antibiofilm activity against several 3 biofilm-forming pathogenic bacteria.

Bacterial Cultivation
Fourty actinobacteria isolates used in this research were obtained from the previous study [4,5]

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 OD 600 =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 (OD 600 =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 4 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 (OD 600 =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 (OD 600 =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 5 Validation assay was conducted based on [11]. The culture of C. violaceum CV026 (OD 600 =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.

Bacterial Characteristic
Actinobacteria isolates have specific characteristics, such as calcareous-like, opaque big colonies, embedded or strongly attached to the agar medium, and produce earthy odor compounds called geosmin.

Screening for Antiquorum Sensing Activity
Inhibition of violacein pigment was determined as a positive result. According to this assay, The results showed that 10 out of 40 actinobacteria isolates had antiquorum sensing activity without inhibiting C.violaceum growth.

Antibacterial Activity Assay
There were found 3 out of the 10 actinobacteria crude extracts showed antibacterial activity against V. cholerae and non-antibacterial activity against other pathogens, while positive control inhibited the growth of tested pathogens.

Detection of Antiquorum Sensing Activity
All 10 isolates also performed great quorum quenching activity against C. violaceum shown with absence of violacein pigment observed around the wells. Presence of quorum quenching activity would inhibit violacein production.

Validation Test Antiquorum Sensing Activity and Protein Characterization
The results shown in Table 1 indicate that all 10 isolates showed quorum quenching activity compared with the control. Quorum quenching agents in extracts 1AC, 14PM, 16PM, 18PM, and CW17 showed a significant increase in absorbance value after Proteinase-K treatment, while extracts from 11AC, 15PM, 20PM, CW01, CW17, and TB12 showed lower absorbance value (Table 1).

Biofilm Inhibition and Destruction Assay
From this assay, all crude extracts were capable of inhibiting and destructing biofilm formation ( Table   2). Antibiofilm activity performed were broad spectrum. Antibiofilm against Gram positive bacteria was correlated with the concentration while results against Gram negative bacteria was varied. cholerae. Isolates showing quorum quenching activity are validated. All 10 isolates showed great activity in inhibiting C. violaceum quorum sensing activity as seen from lower pigment production.

Microscopic Observation using Scanning Electron Microscope
Quorum quenching agents in extracts of 1AC, 14PM, 16PM, 18PM, and CW17 isolates were predicted as proteins or enzymes because they showed a significant increase in absorbance value after proteinase-K treatment. The treatment might affect protein-based antiquorum sensing compounds produced by actinobacteria, such as AHL lactonase, acylase, decarboxylase, oxidoreductase, and deaminase [15]. Those enzymes were degraded by the treatment. In the absence of antiquorum sensing compounds, HHL (C6) signal will diffuse into bacterial cells and binds with LuxR receptor formed a protein-ligand complex. This complex will activate violacein production. The more violacein produced, the higher absorbance will be detected [16].
Crude extract from isolate 15PM, 20PM, CW01, and TB12 treated with proteinase-K showed better activity. It might indicate that proteinase-K degrades the competitive proteins which share similar receptor or degrades proteins that can inhibit the quorum quenching activity [17]. Other probabilities, the antiquorum sensing compounds were produced in inactive form hence reaction with Proteinase-K will activate the antiquorum sensing compound [18].
Antibiofilm activity usually correlates with concentration, but few isolates did not show any significant difference. It might be caused by several factors. For instance, lower concentration of crude extracts might degrade all the ECM. Crude extracts might also contain some inhibitors or analog components that match competitively to the receptor. According to [19] undesirable component might affect the effectiveness of the crude extracts. Some components in the crude extracts might work antagonistically or synergistically that can decrease or increase the activity [18,20].
Topographical observation using SEM showed the presence of biofilm. S. typhimurium biofilm before treated with crude extracts showed a rough surface, which means there is a presence of ECM. After 8 the treatment, Fig. 1d

Limitation
This research did not comprehend the specific compound in the crude extract that competent to perform antibiofilm and antiquorum sensing activity. species of actinobacteria have not identified, characterization of crude extract limited to protein compounds, and the specific mechanism of the quorum quenching activity of each isolate did not determine.