BROAD SPECTRUM ANTIBIOFILM AND ANTIQUORUM SENSING POTENTIAL OF CRUDE SECONDARY METABOLITE EXTRACTS OF MICROBIAL ISOLATES OBTAINED FROM POLLUTED WALDHUNI RIVER

BROAD SPECTRUM ANTIBIOFILM AND ANTIQUORUM SENSING POTENTIAL OF CRUDE SECONDARY METABOLITE EXTRACTS OF MICROBIAL ISOLATES OBTAINED FROM POLLUTED WALDHUNI RIVER

P. Lokegaonkar^* and B. M. Nabar

Department of Microbiology, Smt. Chandibai Himathmal Mansukhani College, Ulhasnagar - 421003 Maharashtra, India.

ABSTRACT: Bacterial biofilms due to antibacterial resistance are causing serious health issues with respect to infection control, its prognosis and treatment of disease. The current scenario demands need of new and alternative antibacterial compounds for treatment of such infections. The current study highlights the use of polluted river, Waldhuni, as a source of potent microorganisms with antibiofilm properties. 186 microorganisms were isolated from Waldhuni River, and tested for quorum sensing inhibitory activity (QSI) against C. violaceum MTCC 2656. Seven isolates exhibited QSI activity, out of which 1 isolate PB2 was selected for further study based on its high QSI potential. PB2 displayed broad spectrum antibiofilm activity against eight strains of biofilm forming pathogens. The effective minimum biofilm eradication concentration (MBEC) of crude extract of PB2 secondary metabolite (SME) was found to be 500ppm. The extract reduced exo-polysaccharide (EPS) by 94.16%, viability by 90% and biofilm density by 96% in 6h treatment. It also rendered to inhibit extracellular DNA of Streptococcus mutans MTCC497, Enterococcus faecalis MTCC 439 and Pseudomonas aeruginosa MTCC 2453. PB2 isolate was identified to be Pseudomonas aeruginosa. Thus, Pseudomonas aeruginosa PB2 with antiquorum sensing and broad spectrum biofilm inhibitory activity was isolated from highly polluted Waldhuni river.

Biofilm, Quorum sensing, Exopolysaccharide, eDNA, Secondary Metabolites

INTRODUCTION: Biofilm is a complex matrix made up of microbial consortium where cells strongly adhere to each other. It has been estimated that approximately 70% to 80% of microorganisms form a biofilm and almost 80% of the microbial infections that occur in a human body are biofilm derived ¹.

Biofilm mediated infections are responsible for a number of clinical challenges, which mainly include diseases involving unculturable pathogens, chronic inflammation, delayed wound healing, rapid antibiotic resistance, and systemic infections. Biofilm forming pathogenic bacteria exhibit an innate ability to resist disinfectants and host defenses. Due to ability to form a biofilm and persist, microorganisms are significantly less susceptible to antibiotics and host defenses than their planktonic forms ². Most common bacteria forming a biofilm are Staphylococcus aureus, Staphylococcus epidermidis, Vibrio cholerae, Enterococci spp., Pseudomonas aeruginosa and Klebsiella pneumoniae ^{3 - 7}.

Staphylococci, are a leading cause of nosocomial infections worldwide, is the etiologic agent of a wide range of diseases, ranging from relatively benign skin infections to potentially fatal systemic disorders ³. Pathogens such as Pseudomonas aeruginosa, Streptococcus mutans, Klebsiella pneumoniae have rendered the ability to form biofilm on indwelling medicinal devices with increasing rates of persistence ^{8 - 9}. Emerging cases of V. cholerae colonizing intestine to form a biofilm leading to a development of fatal disease have been reported recently ^{10 - 11}.

Conversely microorganisms are also known for production of various antimicrobial compounds. Actinomycetes, Bacillus species, Pseudomonas species are commonly known microorganisms which produce antimicrobial and antifungal compounds ¹². Soil and water are rich resources which offer abundant supply of such potentially novel microorganisms. In spite of decades of research, 99% of the diverse bacterial species from soil and water bodies are yet unexplored ¹³.

In the current study soil and water samples were collected from highly polluted Waldhuni river. Waldhuni river flows through four major cities of Thane District, Maharashtra state. It is polluted mainly due to industrial and domestic waste disposal. Such polluted environments tend to exhibit presence of highly stress tolerant microorganisms significant to human. These microorganisms can be tested for their biotechnological potential, such as bactericidal properties.

Present study intends to isolate the microorganisms from Waldhuni river and to study their antibiofilm potential against selected standard strains of pathogens.

MATERIALS AND METHODS:

Test cultures and growth conditions: Eight standard strains of pathogens were selected for the study. The selected bacterial strains were S. mutans MTCC 497, S. aureus MTCC 3160, S. epidermidis MTCC 3165, E. faecalis MTCC 439, K. pneumoniae MTCC 3384, V. cholerae MTCC 3906, E.coli MTCC 687 and P. aeruginosa MTCC 2453.  For the quorum sensing inhibitory activity violacein producing C. violaceum MTCC 2656 was used. All strains were maintained in Brain heart infusion (BHI) broth medium at 37 °C, while C. violaceum MTCC 2656 was incubated at 30 °C. For all assays test culture suspension was prepared by using overnight growth with optical density adjusted to 0.1(OD_{610 nm}).

Collection of Soil and Water Samples: Soil and water samples were collected from 18 locations throughout the Waldhuni river. Based upon the earlier reports, locations with elevated levels of COD were selected in this study. Total 36 samples comprising 18 soil and 18 water samples, were collected from the river. The details of locations are listed in Table 1.

TABLE 1: DETAILS OF THE SAMPLING LOCATION

Isolation of Microorganisms: All samples were subjected to enrichment for isolation of Bacteria, Fungi and Actinomycetes. 1 gm of soil sample and 10ml of water sample was inoculated in Luria Bertani (LB) medium, Potato dextrose broth (PD) and Actinomyces broth (supplemented with 80µg/ml of cylcoheximide and 80µg/ml of nalidixic acid). After enrichment period, broth was inoculated on to LBA, PDA and Starch casein agar (SCA). Colonies thus obtained were preserved and were utilized to study their quorum sensing inhibitory activity.

Screening for Quorum Sensing Inhibitory (QSI) Activity: The QSI activity of isolates were determined by using method described by Packiavathy et al., with minor modifications ¹⁴. Isolates were inoculated into respective broth media, incubated for 2-3 weeks and centrifuged at 15000 rpm, for 20 min to obtain cell free supernatant (CFS). CFS was subjected to the qualitative analysis to find out their QSI potentials against C. violaceum MTCC 2656. The violet coloured pigment violacein is produced by C. violaceum MTCC 2656 as a response to its QS signal molecule N-hexanoyl-L-homoserine lactone (HHL). 10 μl of overnight culture suspension of C. violaceum MTCC 2656 (adjusted to 0.1 OD at 610nm) was added into wells of sterile microtiter plates (MTP) containing 180 μl of LB broth and incubated along with 10 μl of individual CFS. The plates were incubated overnight at 37°C and observed for the reduction in violacein pigment production. Only the CFS which exhibited significant QSI activity against C. violaceum MTCC 2656 alone was further considered for subsequent experiments in this study.

The percentage of violacein inhibition was calculated by following the formula:

Percentage of violacein inhibition = (Control OD_580nm − Test OD_580nm/Control OD_580nm) ×100

Preparation of Secondary Metabolite Extracts (SME): The secondary metabolite extracts (SME) were prepared from the isolates which QSI activity^15-16. Isolates were inoculated to the respective Broth media and then filtered, centrifuged at 15000 rpm, for 20 min to obtain cell free supernatant (CFS). Equal volume of ethyl acetate was added to CFS and kept under shaker conditions for 1 hour. In a separating funnel secondary metabolite was obtained as a layer between organic and aqueous layers. The layer of secondary metabolite was collected in a petridish and dried to evaporation. The dried secondary metabolite was dissolved in dimethyl sulfoxide (DMSO) and used to study the Antibiofilm properties.

Antibiofilm Properties:

Biofilm Control Assay: A microtiter plate based biofilm assay was carried out to identify the minimum biofilm eradication concentration (MBEC) of secondary metabolite extracts (SME) ¹⁷. Test cultures (20µl) were inoculated in 180µl BHI broth supplemented with 2% glucose. After 48h incubation at 37 °C, medium was removed aseptically. SME were added to well with final concentration 5000, 500, 50, 5 & 0.5 ppm, with 2% Glucose containing BHI medium, and incubated for 6h. Medium was then aseptically removed, and each well was washed with sterile phosphate buffered saline (PBS) (pH 7.0). After three washings, each well was stained with 200 µl of 0.2% crystal violet aqueous solution for 45 minutes. Wells were washed five times gently with sterile distilled water. 200 µl of 95% ethanol to was added to the wells. 100 µl from each well was transferred to a new microtiter plate OD was measured at 595 nm. The control for each test culture was also maintained (i.e. untreated 48h old biofilm). The percentage reduction in biofilm was calculated by using following formula:

Percent reduction in biofilm = (Control OD_{595 nm} –Test OD_{595 nm})/Control OD_{595 nm} x 100

Reduction in Exopolysaccharide Content (EPS): Exopolysaccharide content was estimated by total carbohydrate assay ¹⁸. Mature 48h old biofilm of each test culture was developed on 1 × 1 cm PVC slide. These biofilms were treated separately with MBEC of secondary metabolite for 3h.

After incubation slides were transferred to the glass tubes. 1ml of 5% phenol and 5ml of H₂SO₄ containing 1% hydrazine sulphate was added to each of the tube and incubated for 1 h in dark. Absorbance was measured at 490 nm. The control of biofilm for each test culture was maintained separately.

The percentage reduction in EPS content of biofilm was calculated by using following formula:

Percent reduction in EPS = (Control OD_{490 nm} –Test OD_{490 nm})/Control OD_{490 nm} x 100

Quantify Biofilm Density Reduction: To quantify the effect of SME on biofilm density, method described by Lokegaonkar and Nabar was used ¹⁷. The biofilm (48h) of test cultures were grown on 1x1 cm PVC slides. These slides were exposed to MBEC of secondary metabolite extracts and incubated for 3h. After incubation the biofilm was scraped off from the slide and suspended in the 5 ml of phosphate buffer (pH 7.6). The 0.1ml of fluoresce in diacetate (FDA) in acetone, was added to 3 ml of the suspension. The mixture was incubated for 1 h at 37ºC.

Reaction was stopped using 3ml Acetone and absorbance was read at 490nm. 0.3ml of the scraped suspension was used to determine the bacterial titer by spread plate technique on LB agar.

The results were compared with the untreated biofilm of each test culture respectively. The biofilm density was calculated by following formula:

Surface density of biofilm =

(Bacterial concentration x Buffer Volume)/ Area of slide

Effect on Viability: The biofilm was developed in a microtiter plate by inoculating 20µl of test culture in 180µl of 2% Glucose containing BHI medium. Plates were incubated for 48h, and then medium was decanted. The biofilm was washed with sterile PBS (pH 7.2) to remove debris. Biofilm is exposed to MBEC of extract for 3h. After the treatment biofilm washed to remove residual SME. The biofilm was then disrupted and suspended in a sterile saline. The load of viable cells was determined by serial dilution technique using spread plate method. The positive control of untreated biofilm of test pathogens were also maintained. Reduction in viability was evaluated by comparing the load of bacterial viable cells of SME treated biofilm with control biofilm.

Reduction in Viability = (Control CFU/ml – Treated CFU/ml)/Control CFU/ml x 100

Effect on Extracellular DNA: DNAse activity of crude SME was estimated by using sub MBEC levels SME. In a micro centrifuge tube 200µl of test pathogens was inoculated in 1800µl 2% glucose containing BHI medium along with sub MBEC concentration. Tubes were incubated for 24h, at 37 °C. After incubation tubes were centrifuged at 5000 rpm for 10 minutes. Supernatants were subjected to agarose gel electrophoresis to check the presence of DNA. Electrophoresis was carried out with 1% Agarose gel in Tris Borate buffer with ethidium bromide as a visualizing agent. Agarose gel is observed under UV transilluminator to check the presence of DNA band. Absence of DNA in supernatant indicated the DNAse activity of crude extracts. The test pathogens without SME was kept as a control for each test culture.

Identification of Potential Microorganisms: Isolate with significant QSI activity, and antibiofilm properties was identified on the basis of colony characters, Gram’s staining and biochemical analysis.

Statistics: Each assay was performed in triplicate and the values were expressed as the means ± SD. One-way analysis of variance (ANOVA) was performed using SPSS version 17.

RESULTS AND DISCUSSION:

Isolation of Microorganisms: As per the recent study done by Nabar et al., Waldhuni river is highly polluted with respect to its physical, chemical as well as biological aspects ¹⁹. Though COD value was reported 28931mg/L, high MPN levels indicate persistence of microorganism in such stressed locations ¹⁹. Thus the locations were selected on the basis of COD and MPN values.  From 36 collected samples total 186 isolates were obtained. Among the isolated microbes bacterial population was found to be highest, i.e. 127 (124 mesophiles, 3 thermophiles) while fungi as well as actinomycetes were relatively moderate i.e. 22 (mesophilic) and 37 (18 mesophilic, 19 thermophilic) respectively.

Presence of “Psychrophiles” was not evident among all sampling sites. The thermophilic microbes were obtained from the above selected polluted sites. All the isolates were screened for their ability to reduce violacein production by Chromobacterium violaceum MTCC 2656.

Quorum Sensing Inhibitory Activity Assay: The mechanism of QS regulation is established target for developing alternative biocontrol strategies ²⁰. O’ Loughlin et al., reported to show that the anti-QS approach is a promising bio control method against P. aeruginosa infections ²¹. Recent studies indicate that the soil and plant bacterial isolates produce degradation enzymes rendering efficient QSI activity ^{19, 22 - 24}. In current study all isolates were screened for violacein inhibition. As the experiment was conducted in dose independent manner, relative potential of each isolate to inhibit violacein was tested. 7 isolates were found able to reduce violacein pigmentation significantly, which include one fungal isolate, 2 Bacterial isolate and 4 Actinomycetes.

Maximum reduction in violacein, with highest significance was showed by bacterial isolate PB2. Hence this isolate was selected for further studies. Fig. 1 shows the percentage reduction in violacein synthesis in presence of CFS. Table 2 represents statistical analysis of mean percent reduction in violacein pigmentation.

In the present study QSI (i.e. violacein inhibition) activity is considered as indicator of quality antibiofilm properties. Hence further study emphasizes on biofilm eradication, EPS reduction and other biofilm parameters. The secondary metabolites were extracted using ethyl acetate precipitation method and studied for its antibiofilm potential.

Detection of MBEC of PB2 Crude Secondary Metabolite Extract: The percentage reduction in biofilm formation was checked by performing microtiter plate based quantitative assay. MBEC of PB 2 bacterial isolate was found to be 500ppm for selected Gram negative standard strains of pathogens. The effective MBEC for Staphylococci was found to be 500ppm, while for S. mutans and E. faecalis MBEC of crude SME was more than 500ppm.  Table 3 represent the statistical analysis of MBEC determination assay of PB2.

FIG. 1: PERCENTAGE REDUCTION IN VIOLACEIN

TABLE 2: DESCRIPTIVE ANALYSIS OF REDUCTION IN VIOLACEIN

Results are represented as the percentage reduction in Violacein pigmentation. Mean values of triplicate individual experiments and Standard Deviations are shown.  **Indicates statistical significance (p<0.01), * indicates statistical significance (p<0.05)

TABLE 3: DETERMINATION OF MINIMUM BIOFILM ERADICATION CONCENTRATION (MBEC) OF PB2 AGAINST STANDARD PATHOGENS

One way ANOVA was performed using SPSS 17., * indicates significance value less than 0.05, Confidence interval : 95%

Reduction in Exopolysaccharide (EPS): The EPS degradation ability of crude SME was studied by exposing a mature biofilm of test pathogens to PB2 extract. Total carbohydrate content of treated and untreated biofilm was compared to evaluate the reduction EPS. Hydrogen peroxide (H₂O₂) is also used as standard antibiofilm agent, to compare the test result. SME reduced EPS content of test pathogens upto 94.16%. While in case of S. mutans MTCC 497 76% of EPS of biofilm was degraded compared to control biofilm. In case of Gram negative standard strains of pathogens more than 81% EPS reduction is observed. Crude SME degraded EPS of Gram positive pathogen biofilms significantly. Hydrogen peroxide (H₂O₂) (100ppm) was more efficient against Gram positive pathogens with respect to EPS reduction of a biofilm. Results showed SME have wide range of EPS reduction, degrading EPS of Gram positive as well as Gram negative pathogen biofilms Table 4.  From Fig. 2 it is clear that broad spectrum effect on biofilm EPS was exhibited by crude extract of secondary metabolites.

FIG. 2: EFFECT ON EPS CONTENT OF BIOFILM AFTER 6 h TREATMENT WITH PB2 SME AND 100ppm H₂O₂

FIG. 3: EFFECT ON VIABLE LOAD OF BIOFILM AFTER            

FIG. 4: EFFECT ON BIOFILM DENSITY AFTER 6 h TREATMENT WITH PB2 SME AND 100ppm H₂O₂ 6 h TREATMENT WITH PB2 SME AND 100ppm H₂O₂

FIG. 5: EFFECT ON eDNA RELEASE WITH SUBMBEC CONCENTRATIONS OF PB2 SME

1.=S. mutans MTCC 497, 2.=S. aureus MTCC 3160, 3.=S. epidermidis MTCC 3165, 4.=E. faecalis MTCC 439, 5= K. pneumoniae MTCC 3384, 6=V. cholarae MTCC 3906, 7=E.coli, 8=P. aeruginosa MTCC 2453, 9= Control. Image was captured using digital camera, and processed with Microsoft paint to add number to each well

TABLE 4: EFFECT OF SECONDARY METABOLITES ON THE VIABLE LOAD, EPS CONTENT AND BIOFILM DENSITY OF STANDARD STRAINS OF PATHOGENS

Results are presented in Mean values with the standard deviation obtained with the help of SPSS 17. All experiments are performed in triplicates.

Effect on Viability: Reduction in viable load of bacteria in biofilm was studied by treating the 48h old biofilm for 6h with SME. The control biofilm of each test pathogen was considered as 100% viable mass in a biofilm. After 6h treatment significant reduction in the viability of bacterial population of biofilm was observed. 90% reduction was achieved after only 6h of exposure to the extract.

H₂O₂ was found to be more bactericidal towards Gram negative biofilm than that of Gram positive pathogens (Fig. 3). PB2 proved to more consistent with respect its bactericidal properties.

Effect on Biofilm Density: The biofilm density is a measure of bacterial population per biofilm area. The efficient antibiofilm agent not only affects virulence properties, but also impacts on physiology and structure of biofilm, i.e. biofilm density. Result data is shown in Table 4. Highest reduction (i.e. 96%) in biofilm density was observed in case K. pneumoniae MTCC 3384 by PB2 SME in 6h treatment. Though EPS content and viability of V. cholare MTCC 3906 was reduced efficiently, PB2 reduced its biofilm density only by 66% Fig. 4. H₂O₂ reduced the biofilm density upto 66% in case Gram negative pathogens, and upto 60% in case of Gram positive pathogens.

Extracellular DNA: Bacteria in a biofilm stage release DNA, which enhances adhesion of cells to biotic and abiotic surfaces. This eDNA thus considered to be a potent virulence factor in transformation of planktonic stage to compact biofilm. Compounds having DNAse activity are proven to control the biofilm development. The effect of SME on eDNA release by pathogens have been analysed during the biofilm formation. Hence pathogens were incubated along with subMBEC levels of PB2 SME. After centrifugation, supernatants were electrophoresed on agarose gel and checked for presence of DNA Fig. 5. Well no. 9 shows the presence of control DNA. Well no 1, 4 and 8 does not show presence of DNA band.

Extracellular DNA was not obtained from S. mutans MTCC497, E. faecalis MTCC439, and P. aeruginosa MTCC 2453 in presence of SME of PB2.  Recently many studies have reported the significance of eDNAin Streptococcus mutans, S.aureus, P. aeruginosa, V. cholerae ^{25 - 27}. Chiang et al elaborated eDNA mediated aminoglycoside resistance in a biofilm forming Pseudomonas aeruginosa ²⁸.

Identification of Isolate PB2: Isolate PB2 showed significant quorum sensing inhibitory activity and broad spectrum antibiofilm properties. It was identified on the basis of Bergey’s manual of determinative bacteriology, colony characters, Grams characters and biochemical tests. Table 5 represents the details of identification. Bacterial isolate PB2 was identified as Pseudomonas aeruginosa. It will be further subjected to molecular level identification tests such as 16S rRNA.

TABLE 5: IDENTIFICATION OF PB2 BASED ON BIOCHEMICAL TEST ANALYSIS 

Key: + = Positive test, - = Negative test, v= variable

Results of Standard Pseudomonas aeruginosa are derived from Bergey’s manual determinative bacteriology 9^thEdⁿ.

CONCLUSION: Polluted conditions not only affect the water environment of a water body but also the soil ecosystem nearby it. Also pollutants are gradually absorbed in soil compared to water, making conditions less vulnerable for soil microbial flora and fauna to survive. Microorganisms adapt diverse metabolical mechanism while growing in such stressed environment.

Waldhuni river, heavily polluted with chemicals and a biological waste, lead microorganisms to grow in stressed environment. The adapted microbial flora was isolated and tested for Quorum sensing inhibitory activity. Pseudomonas aeruginosa PB2, isolated from Waldhuni river exhibits significant QSI activity. Crude SME P. aeruginosa PB2, rendered broad spectrum antibiofilm properties against the selected standard strains of pathogens. Its high efficiency to degrade EPS, reduce biofilm density, bactericidal properties and denaturing eDNA proves it to be antibiofilm agent. Though purification of active component from SME is needed P. aeruginosa PB2 isolates highlights consideration of polluted environments as a potent source of microorganisms.

ACKNOWLEDGEMENT: Nil

CONFLICT OF INTEREST: The authors declare no conflict of interest.

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    How to cite this article:

    Lokegaonkar SP and Nabar BM: Broad spectrum antibiofilm and antiquorum sensing potential of crude secondary metabolite extracts of microbial isolates obtained from polluted Waldhuni River. Int J Pharm Sci Res 2018; 9(6): 2378-86. doi: 10.13040/IJPSR.0975-8232.9(6).2378-86.

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