Antibiofilm and antifouling activities of extracellular polymeric substances isolated from the bacteria associated with marine gastropod Turbo sp

The gastropod (Turbo sp.) was collected from the Kanyakumari coast (West Coast of India), kept in a sterile container covered with an icebox and brought to the laboratory. The sample was gently washed with sterile seawater (Millipore filtered and autoclaved) in order to remove loosely attached organisms and the remaining bacteria attached to the shell were scraped off and mixed in 1 ml of filter-sterilized seawater. Subsequently, the suspension was serially diluted and plated onto Zobell marine agar (HIMEDIA, India) and incubated at room temperature (24 h) for the development of colonies. The developed colonies were isolated and purified by repeated streaking on Zobell marine agar plates. The isolated colonies were maintained on Zobell marine agar slants at 4°C for further analysis.

A loop full of bacterial culture was inoculated into the flask containing 100 ml Zobell marine broth and incubated for 72 h in a shaker. Following the incubation, the culture broth was centrifuged at 10000 × g for 15 minutes at 4°C and the collected supernatant was added with an equal amount of cold absolute ethanol. After one day of incubation at room temperature, the precipitated extracellular polymeric substances (EPS) were dissolved in double distilled water and mixed with 95% ethanol (1:4 ratio, aqueous solution: ethanol). The resulting precipitate was filtered through a membrane filter and the clear final solution was dialyzed using 8 kDa membrane for 3 days. The resulting EPS fraction was used for antibacterial activity tests. The agar disc diffusion method was used to assess the antimicrobial activity: 50 μl of EPS was loaded on a sterile filter paper disc (6 mm, Himedia, India) and placed onto Mueller-Hinton agar (Himedia) plates, swabbed with biofilm-forming bacteria (Alteromonas sp. and Pseudomonas sp.) isolated from acrylic panels submerged in coastal waters (Satheesh et al. 2012). At the same time, the control disc was loaded with distilled water and maintained for cross reference. All plates were incubated at 37°C for 48 h and the zone of inhibition around the discs were measured after 24 h of incubation.

The overnight grown culture broth of biofilm-forming bacteria (Alteromonas sp. and Pseudomonas sp.) was centrifuged at 10000 × g (15 min) and the obtained cell pellets were re-suspended in PBS buffer to obtain OD₅₄₀ = 0.2 and kept at 4°C for further studies.

The bacterial EPS (50 μl) was collected in a test tube containing 5 ml of Zobell marine broth (ZMA) along with 500 μl of biofilm-forming bacterial (Alteromonas sp. and Pseudomonas sp.) cell suspension; control tubes were also prepared without the addition of EPS to compare the inhibitory effect. The tubes were incubated for 24 h at 37°C after taking the initial OD value of the culture medium in a spectrophotometer (UV-Vis.119, SYSTRONICS) at 600 nm. Following the incubation, the final OD values of the culture medium were measured at 600 nm and the growth inhibitory effect of EPS was calculated using the formula given below:

The bacterial EPS (20 μl) was transferred into a well of a polystyrene microtiter plate (Cat No. 911296) containing 200 μl of the bacterial cell suspension and the reference was prepared by adding 20 μl of saline in place of bacterial EPS. The plate was incubated at 37°C for 24 h. Following the incubation, the content of each well was gently removed by tapping the plates and washed with 0.2 ml of phosphate buffer saline (PBS pH 7.4) to remove bacteria. The biofilm formed by adherent bacteria was fixed with sodium acetate (2%) and stained with crystal violet (0.1% wv⁻¹). The excess stain was rinsed off with deionized water and the plates were kept for drying. Finally, the optical density (OD) of stained bacterial cells attached to the plates was determined with a micro ELISA auto reader at a wavelength of 570 nm.

To prepare antifouling coating, the epoxy primer (3%) was added with epoxy resin (1%), hardener (1%) and bacterial EPS (1%). The control coat was prepared by adding saline (1%) in place of EPS. After proper mixing, the antifouling and reference coatings were coated on the fiber plates (18 × 12 cm) and kept in a sterile chamber to dry for a week. The dried plates were firmly fitted on a frame (iron) and submerged into the sea (Colachel coast, the west coast of India) about 2 meters from the mean sea level for a period of 50 days (3/8/2014 to 21/9/2014). After submersion, the plates were retrieved at 10 day intervals and brought to the laboratory. The fouling community recruited on the plates was scraped off and dried overnight in an oven at 60°C. The biomass of fouling on the reference and plates coated with EPS was calculated by using the following formula:

where, IWFC–the initial weight of the fouling community; FWFC–the final weight of the fouling community; N–the number of measurements taken.

The analysis of functional groups present in the bioactive EPS was performed by FT-IR (2000, SHIMADZU) analysis. A small quantity of bioactive EPS was placed on the face of a highly polished K Br salt plate, and another KBr plate was positioned on the top to spread the compound in a thin layer.

The bacterial strain was cultured in marine broth at 37°C, and the total genomic DNA of the strain was extracted using the phenol chloroform method. The 16S rRNA was amplified by polymerase chain reaction (PCR) using primers 16S (5`-AGAGTRTGATCMTYGCTWAC-3`) and 16S (5`-CGYTAMCTTWTTACGRCT-3`). The PCR product was sequenced using the same PCR primers and other internal primers to confirm the sequence. The obtained 16S rRNA sequence of the bacterial strain was analyzed using the Basic Local Alignment Search Tool (BLAST) and the phylogenetic tree was constructed using the 16SrRNA sequence of others obtained from the NCBI Gen Bank.

Student’s T-test (P value < 0.05 was considered as significant) was applied to analyze the difference between the reference and the treatments in antibiofilm and antifouling assays.

The EPS isolated from the marine bacteria associated with Turbo sp. was screened against biofilm-forming bacteria Alteromonas sp. and Pseudomonas sp. Five out of 13 strains showed inhibitory activity and the results are presented in Table 1. Among them, the EPS of strain KT1 showed strong antibacterial activity against both Alteromonas sp. and Pseudomonas sp. and the zone of inhibition were 12 and 13 cm, respectively.

The growth rate of biofilm-forming bacteria on the culture medium treated with bioactive EPS was much smaller than the control medium. The growth rate of Alteromonas sp. and Pseudomonas sp. in the control medium was 36.4% and 40%, respectively. Whereas, the bioactive EPS-treated medium significantly reduced the growth of Alteromonas sp. to 29.2% (Student’s T-test, t stat=16.5, df=2, P<0.001) and Pseudomonas sp. to 30.6% (Student’s T-test, t stat=5.1, df=2, P<0.05) (Fig. 1).

Figure 1.

The biofilm formation of the bacterium Alteromonas sp. on the microtiter plate control well was 0.350±0.015, and it was significantly reduced, i.e. to 0.286±0.007 (Student’s T-test, t stat=6.41, df =4, P<0.001) in the well treated with bioactive EPS. Similarly, the biofilm formation of Pseudomonas sp. on the control well was 0.305±0.011, but it was significantly reduced to 0.269±0.017 (Student’s T-test, t stat = 2.9, df = 4, P<0.05) in the well treated with bioactive EPS (Fig. 2).

Figure 2.

The test panel coated with antifouling coating significantly (Student’s T-test, t stat=2.2, df =5, P<0.05) reduced the recruitment of the fouling community. The recruitment of the fouling community on the control panel was 40.4% and 24.51% on the panel coated with antifouling coating prepared by incorporating the bacterial EPS (Fig. 3).

Figure 3.

The FT-IR spectrum of EPS showed the presence of alcohol, alkanes, alkynes, amines, carboxylic acid and ethers. The broad O-H stretch between 3400-3300 and the C-O-H bending between 1550-1220 and the C-O stretch between 1260-1000 cm⁻¹ indicate the presence of alcohol. Similarly, the C-H stretch between 3000-2840, the CH2 bending mode near 1450 and the CH3 bending absorption near 1375 cm⁻¹ indicate the presence of alkanes. Likewise, the C-H stretching frequency near 3300 and the C-C stretch near 2150 cm⁻¹ indicate the presence of alkynes. Furthermore, the N-H stretch between 3500-3300, the N-H bending between 1640-1560 and the C-N stretch 1350-1000 cm⁻¹ indicate the presence of an amine. Moreover, the O-H broad peak between 3400-2400, the C=O stretch at 1730-1700 and the C-O stretch between 1320-1200 cm⁻¹ indicates the carboxylic acid group. The C=O stretch at 1300-1000 and the C-O-C asymmetric stretch near 1250 and the symmetric stretch near 1040 cm⁻¹ revealed the presence of ethers (Fig. 4).

Figure 4.

A total of 1,158 consensus nucleotides representing the 16S rRNA genes were obtained from the amplified DNA fragments of the good active strain KT1, and the phylogenetic tree constructed showed 99% similarity with Pseudomonas aeruginosa (available sequences in the NCBI). The nucleotide sequence data have been deposited at GenBank (Fig. 5).

Figure 5.