Antifouling activity of bacterial extracts associated with soft coral and macroalgae from the Red Sea

The soft coral Sarcophyton trocheliophorum and the brown macroalga Dictyota dichotoma were collected from Obhur Creek (latitudes 21°42′11″ and 21°45′24″ and longitudes 39°05′12″ and 39°08′48″E) in Jeddah, Saudi Arabia. The samples were transferred to the laboratory in a sterile container with filtered (Millipore, 0.45 μm) and sterilised (autoclaved) seawater (FSW). In the laboratory, they were rinsed thoroughly with FSW to remove the loosely attached microbes and other associated epifauna. Cotton swab sticks were used to thoroughly swab the surface of the organisms and were then placed in test tubes containing 5 ml of FSW; this was followed by vigorous agitation of the test tubes with a vortex mixer. The swabs were removed from the tubes while the agitated seawater was serially diluted. Then, 0.1-ml aliquots of 10⁶ diluent were transferred to Zobell marine agar plates (ZMA; HiMedia, India) via the spread plate method, and incubated at 30°C for 24–48 h. Bacterial colonies observed on the agar plates were subcultured individually and purified using the streak plate method. The purified bacterial colonies were kept in agar slants at 4°C for further studies. A preliminary screening of the isolated bacterial strains’ antagonistic activity against biofilm-forming bacteria was performed using the agar overlay method (Hockett & Baltrus 2017). Two strains (MBR1 and MBR4) were selected for further analysis based on their preliminary activity against the biofilm bacteria.

Loopfuls from the agar slants were transferred to Zobell marine broth (ZMB; Himedia, India) in 10-ml tubes and incubated in a shaker incubator at 30°C overnight. Aliquots of the overnight cultures were transferred to conical flasks containing ZMB and were incubated at 30°C for 3 days in an incubator shaker. The cultures were centrifuged for 15 min at 5000 rpm. The supernatant was collected in a tube and filtered through a filter paper (Whatman no. 1) to remove the cells. An equal volume of ethyl acetate was added to the cell-free supernatant collected and agitated for 4 h at 28°C (Rajan & Kannabiran 2014). A separating funnel was used to separate the mixture and the organic phase was evaporated under reduced pressure using a vacuum evaporator to get the culture supernatant extract (CSE). The crude CSE was used for antibiofilm and antisettlement assays as well as GC-MS analysis.

A growth inhibition assay of CSE from the two bacterial isolates was performed against two biofilm-forming bacterial strains: Vibrio harveyi (NCBI: KY266820) and Planomicrobium sp. (NCBI: KY224086). The bacterial strains were isolated from the aquaculture cage nets submerged in Obhur Creek, as previously reported (Balqadi et al. 2018). The biofilm-forming ability of these bacterial strains and barnacle larval settlement-inducing activity had previously been tested under laboratory conditions (Siddik & Satheesh 2019). The bacteria were grown overnight in ZMB. The OD of the overnight culture was adjusted to a cell density of 0.4 at 600 nm and used for the experiments. The CSE was adjusted to three different concentrations (5, 10 and 20 μg ml⁻¹) and the method of Ba-akdah & Satheesh (2021) was followed for the bacterial growth inhibition assay using the spectrophotometric method at 600 nm. In brief, each target bacterium was grown overnight and adjusted to an OD of 0.4 with a spectrophotometer at 600 nm by diluting the culture in ZMB. Next, 3 ml of the adjusted cultures were transferred to new tubes followed by the addition of the extract. Three different concentrations (5, 10 and 20 μg ml⁻¹) were used for each target bacterium. Separate tubes without the bacterial extract were used as controls. The OD was measured at 600 nm at baseline and again after 5 h of incubation at 30°C. The bacterial growth inhibition was calculated as a percentage following Equation 1 below.

Final OD is the final optical density at 600 nm after 5 h of incubation, while initial OD is the initial optical density at 600 nm at baseline.

The biofilm inhibitory activity of the CSE was tested against the two target bacteria, V. harveyi and Planomicrobium sp., using the microtiter plate method described previously by O'Toole (2011) with some modifications. The experiment was conducted in 96-well microtitre plates. The overnight culture for each strain was adjusted to a cell density of 0.4 OD at 600 nm. To each well of the plate, 500 μl of overnight biofilm-forming bacterial culture and 500 μl of CSE was added at a concentration of 5 μg ml⁻¹ in triplicate (n=3). The control wells contained culture only without the addition of CSE (n=3). The microtiter plates were incubated statically at 37°C for 24 h in an incubator. After that, the contents of the wells were decanted gently by turning the plates downwards; they were then rinsed with phosphate-buffered saline (PBS) to remove the unattached cells and other materials from the culture broth. The biofilm-forming bacterial cells attached to the wells of the microtitre plate were stained with crystal violet solution (0.4%) for 5 min. After that, the wells were rinsed gently with PBS to remove the excess stain. Next, the wells were air-dried at room temperature and ethanol (96%) was added. They were incubated at room temperature while being shaken for 10 mins. The contents were transferred to new plates and the absorbance of the dye eluted from the wells after the addition of ethanol was measured at 630 nm in a microplate reader (Biotek). The percentage of biofilm inhibition was calculated using Equations 1 and 2 below: Eq. 1 PI=100−PF PI = 100 - PF Eq. 2 PF=BFC ×100C PF = {{BFC\, \times 100} \over C} where

PI is the percentage of biofilm inhibition,

The adult barnacles (Amphibalanus Amphitrite = Balanus amphitrite), along with the attached substratum, were collected from Obhur Creek and brought to the laboratory. The method described by Salama et al. (2018) was used to keep the adults and rear barnacle larvae. In the laboratory, the barnacles were kept in a tank containing FSW. The tank was supplied with moderate aeration, and the salinity and temperature of the tank water were maintained during the experiment at 39 PSU and 27°C, respectively. The adult barnacles were fed a diet of Artemia nauplii and microalga (Chaetoceros calcitrans). The nauplii released by the adults were collected using a hand net and transferred to small glass tanks. In the nauplii stage, the larvae were fed a microalgal diet (Chaetoceros calcitrans) and were reared in the laboratory until stage III for the toxicity assay. The nauplii were also reared to the cypris stage (settling stage) for the antisettlement assay.

A toxicity assay was conducted in order to study the effects that the secondary metabolites present in the CSE had on the survival of the barnacle larvae. Six-well plates were used for the toxicity assay. Each well was filled with 5 ml of FSW and 25 nauplii (stage III) were added to the wells using a Pasteur pipette. Three different concentrations (5, 10 and 20 μg ml⁻¹) of CSE were added to the treatment wells. The control wells were maintained without the addition of CSE. The 6-well plates were kept at 28°C in an environmentally controlled chamber (walk-in type). The number of dead/alive nauplii in the wells was observed periodically for 96 hours. The toxicity assay was repeated in triplicate (n=3 for each concentration) and the mean ± SD values were recorded.

An antisettlement assay was conducted to understand the settlement-inhibiting activity of the CSE against the settlement of barnacle larvae. The nauplii larvae were reared to the cypris stage in the laboratory. This experiment was also conducted in 6-well plates, with 25 cypris larvae in each well. The CSE (5 μg ml⁻¹) was added to the treatment wells. The control wells were maintained without the addition of CSE. The 6-well plates were kept at 28°C in an environmentally controlled chamber (walk-in type) under dark conditions. The plate was observed periodically under a stereomicroscope for the settlement of larvae. The experiment was conducted in triplicate using different batches of barnacle larvae for a period of 48 h.

The bacterium isolated from the soft coral was labelled as MBR1, while the one from macroalga was labelled as MBR4. They were identified based on 16S rRNA gene sequencing. Previously outlined methods (Satheesh et al. 2012; Balqadi et al. 2018) were followed for the isolation of DNA and sequencing of the 16S rRNA. In brief, a loopful of bacterial culture was inoculated in marine broth in a conical flask and kept in an incubator at 30°C. The overnight bacterial culture was used for DNA extraction using a genomic DNA isolation kit (InstaGene^TM). The isolated DNA from the bacterial strains was confirmed using agarose gel electrophoresis. Universal 16S rRNA gene primers (27F:5′ - AGAGTTTGATCMTGGCTCAG - 3′ and 1100R: 5′ - GGGTTGCGCTCGTTG - 3′) were used for the amplification of the isolated DNA in a multigene thermal cycler (Labnet Inc). The PCR conditions were as follows: initial cycle at 95°C for 5 min, followed by 28 cycles at 95°C for 45 sec, annealing at 58°C for 45 sec, with extension at 72°C for 1 min 45 sec and a final extension step at 72°C for 10 min. The amplified PCR products were purified using a GeneJET PCR Purification Kit according to the manufacturer's instructions and then confirmed by agarose gel electrophoresis. The amplified PCR products were sent to Macrogen Inc. in South Korea for sequencing of the 16S rRNA gene. The resulting sequences were analysed and identified using the RDP classifier (http://rdp.cme.msu.edu/) and the Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi/), with the search option limited to type material in order to select the nearest match and using 99% as the threshold for identification at the species level, as reported previously (Abdulrahman et al. 2022a). The 16S rRNA gene sequences of all the strains were submitted to NCBI Genbank for the assignment of accession numbers. A phylogenetic tree was constructed with related strains from Genbank using MEGAX (Tamura et al. 2021) and annotated using Itol software, web version (Letunic & Bork 2021).

The secondary metabolites present in the CSE were analysed using gas chromatography-mass spectrophotometry (GCMS-QP2010 Plus, Shimadzu, Japan). The protocol outlined by Arulazhagan and Vasudevan (2009) was followed for the analysis. In brief, the capillary column (30 m × 0.25 mm; ID = 0.25 μm) was programmed as follows: the initial column temperature was set at 100°C for 1 min, then increased up to 160°C at the rate of 15°C min⁻¹. The column temperature was then set for 7 min at 300°C, the final temperature, at a rate of 5°C min⁻¹. Subsequently, with helium as the carrier gas at 1.0 ml min⁻¹, the GC injector was held isothermally at 280°C flow rate while the temperature at the GC/MS interface was maintained at 280°C for an uninterrupted period of 3 min. The mass spectrometry was set to operate in electron impact ionization mode with 70 eV electron energy. The possible metabolites were identified with the National Institute of Standards and Technology library using the similarity index (<92%) by comparing their retention index.

The inhibitory activity against bacterial growth of different concentrations of CSE was tested using the Kruskal–Wallis H test. The antibiofilm assay results and the anti-larval settlement data were tested using one-way analysis of variance (ANOVA) in order to determine the difference between the controls and the treatments. Furthermore, the barnacle larval toxicity of CSE was tested using two-way ANOVA. Concentration and bacterial strain were used as the factors for two-way ANOVA. A post hoc Tukey HSD test was carried out to determine the variation between the controls and the treatment groups in the antibiofilm and antisettlement assays. All the statistical tests were conducted in STATISTICA (ver. 13) and p<0.05 was set as the level of significance.

After the bacteria were screened using the agar overlay assay, one bacterium each was selected from soft coral and macroalgae. Based on its morphologic features and a comparison with the closest strain from the NCBI database, MBR1 had close similarity with more than 15 strains of Bacillus licheniformis and 99.72% with the type strain B. licheniformis ATCC 14580^T. MBR4 had similarity with Vibrio alginolyticus and a similarity of 99.91% and recorded the highest BLAST score with the type strain V. alginolyticus NBRC 15630^T. These strains MBR1 (from soft coral) and MBR4 (from macroalga) were identified as Bacillus licheniformis MBR1 (NCBI: OP379261) and Vibrio alginolyticus MBR4 (NCBI: OP379262), respectively. A phylogenetic tree showing the strains and other related organisms is presented in Figure 1.

Figure 1

The CSE from the two bacterial strains inhibited the planktonic growth of the biofilm-forming bacteria V. harveyi and Planomicrobium sp. The CSE extracted from MBR1 exhibited strong inhibitory activity against V. harveyi (Figure 2), whereas the CSE of MBR4 strongly inhibited the planktonic growth of both V. harveyi and Planomicrobium sp. (Figure 3). Among the concentrations of the CSE tested, no significant differences were observed in growth inhibiting activity.

Figure 2

Figure 3

The percentage of biofilm inhibition from the extract against the test strains ranged from 45% to 78%, as presented in Figure 4. Both the lowest and high activity was recorded by the extract from MBR1, and the two different extracts demonstrated higher inhibition activity towards V. harveyi than Planomicrobium sp. The extracts of both bacterial strains significantly (p<0.05) inhibited the attachment of biofilm-forming bacteria (V. harveyi: F=38.65; df=3,20; p<0.05; Planomicrobium sp: F=14.83; df=3,20; p<0.05). Post hoc analysis indicated significant variation in biofilm-inhibiting activity between the extract treatment and control (Table 1).

Figure 4

The CSE treatment did not show much effect on the survival of the nauplius larvae during the 96-h toxicity study (Figure 5). The two-way ANOVA results also confirmed the weak toxicity of the extracts against barnacle larvae, as evidenced by the insignificant variation (F=2.32; df=3,24; p=0.1) in mortality between the concentrations of the extracts.

Figure 5

The results indicated reduced settlement of cypris larvae on the 6-well plates due to CSE treatment. The CSE of strain MBR4 exhibited higher settlement inhibition than the extract of MBR1 (Figure 6). The one-way ANOVA results indicated a significant variation in cyprid settlement between the control and CSE treatment groups (F=25.86; df=3,8; p<0.05). Further, the post hoc Tukey test revealed that settlement of larvae in the wells with CSE was reduced significantly in comparison with the control (Table 1).

Figure 6

The GC-MS spectra of the CSE extracted from the two bacterial strains are presented in Figures 7 and 8. The secondary metabolites present in the CSE of MBR1 included n-tridecanol, 3-chloropropionic acid heptadecyl ester, hexadecanoic acid ester, hexacontane and tetrapentacontane (Table 2). The GC-MS analysis results of the CSE obtained from MBR4 showed the presence of compounds such as cyclopropane, octadecanoic acid ester, cis-anti-cistricyclo (2,6) dodecane −7,8-diol, 7-hexyltridecanol, tetrapentacontane, pentatriacontane, 2H-Pyran-2-one and 5-ethylidenetetrahydro-4-(2-hydroxyethyl) (Table 3).

Figure 7

Figure 8