Anti-bacterial effect of marine sea grasses mediated endophytic actinomycetes against K. pneumoniae

2.1 Collection of samples

All the chemicals and glass wares of this study was purchased from ponmani@Co, Tiruchirappalli, India. All the media, PBS, and other solutions of this study were ordered from Ponmani@Co, Tiruchirappalli, India. The sample of the Sea grasses were collected from Gulf of Mannar Region (Latitude 9°15′41.88″N, Longitude 79°04′05.81″E), Rameshwaram, Southeast Coast of India, Tamil nadu, India. The young Sea grasses were collected from 10 m inside from the Sea shores. After collect, the samples were cleaned using Sea water and followed by fresh water. Then, the samples were taken to laboratory and cut in small pieces (6 mm) and processed surface sterilization according to the protocol of Ramachandrana et al. (2019). All the pieces were sterilized in 70% alcohol for 5 min followed by 1.5 M sodium hypochlorite (NaClO) for 5 min. Finally, the samples were washed by double distilled water for discard the more surface sterilants and keep in air dry until the remove of the wet condition. Next, the samples were slightly punched on the actinomycete isolation agar (AIA) plates supplemented with penicillin, nystatin and cycloheximide for suppress the contaminants. Finally, plates were maintained at incubator at room temperature seven days. Finally, the actinomycetes were emerged from inside of the Sea grasses of the samples were identified in AIA plates and confirmed the actinomycetes using phonotypical methods.

2.2 Detection of pure surface sterilization evidence

In endophytic actinomycetes isolation, the surface sterilization method is unavoidable experiment to detect the clear endophytes (Usha Nandhini et al., 2018). Shortly, the last step surface sterilized double distilled water was spread on the starch casein agar plates (SCA) and incubated at 28 ℃ at 48 h. Supportive for surface sterilization, the unsterilized Sea grass samples were also pressed on the AIA plates directly and incubated at 28 ℃ at 48 h. After incubation, no any microbial growths of the last wash water containing AIA plates, and fungal contamination of the unsterilized plates of the reports were noted for differentiation of pure or non-pure endophytes validation.

2.3 Extraction of crude compound

For detection of anti-bacterial efficacy, the isolated actinomycete strains were inoculated in actinomycete isolation broth and maintained 17 days to produce potential growth. After 17 days, the fermented broth culture was taken and centrifuged at 10, 000 rpm for 15 min with 4℃. After, the pellets were removed and supernatants were collected and filtered using Whatman NO.1 filter paper and stored at 4℃. Then, the filtered supernatant was extracted using polar and non-polar solutions such as alcohol, dichloromethane, acetic acid, methanol, ethyl acetate and petroleum ether (Lee et al., 2017).

2.4 Inactivation of bacterial cells

Bacterial cells inactivated of extracted crude compound against K. pneumoniae using agar well diffusion method followed by Usha Nandhini et al. (2018). Simplified, first day bacterial inoculated K. pneumoniae culture was spread on the whole nutrient agar containing plate and followed by punch of wells into the agar. Next, different concentration of different solvent extracted actinomycetes crude compound was added into the wells and rotates the plates evenly. All the plates were maintained at room temperature one day. Next day, the plates were seen and calculated the inhibition zones to detect the effect of actinomycetes.

2.5 Minimum inhibition concentration

To identify the minimum inhibition concentration of extracted actinomycetes crude compound against K. pneumonia was performed in 96-well plate, the protocol was chosen from previously evident of Lee et al. (2017). Simplified, evenly add 100 µg/mL of the crude actinomycetes extract into the 96-wells of the plates. Before, 100 µg/mL of the sterile broth was filled and also 10 µg/mL of K. pneumoniae culture. Then, the plates were kept in inside of the incubator with room temperature setup 1 day. After 1 day, the turbidity of the 96-wells was monitored visibly and noted the concentration, followed by taken spectrophotometer result to detect inhibition percentages. To detect the inhibition percentage, the resulted values were compared with untreated pathogen plus sterile media containing well of control. Then, the results were interpreted and identify the minimum inhibition percentage using following formula,

MIC (%) = Control-Test/Control X 100.

2.6 Size and shape damages

Size and shape damages of the K. pneumoniae were detected after treatment with crude actinomycete compound using chemical fixation method. The fixed cells of damaged or undamaged K. pneumoniae cells were confirmed by SEM micrograph. Initially, first day culture was taken and centrifuged at 2,000 rpm for 5 min. Pellet was collected and mixed in 2 mL of PBS and further centrifuged at same procedure. The unwanted cells were removed form PBS and attached cells were again mixed with 2 mL new PBS and fixed on glass slide. Then, the slide was fixed using glutaraldehyde with desired concentration. The fixation was kept at room temperature for 4 h and then washed with PBS initially followed by double distilled water. Then, ethanol serious of 30–90% was used step by step using 10 min time interval. Next, the slide culture was washed by t-butanol and treated by new t-butanol and kept at room temperature 6 h. Finally, the slide was maintained in 4 ℃ in refrigerator. In end, the slide was monitored in 40x SEM instrument, and the treated and untreated cells were differentiated and noted.

3.1 Isolation and identification of actinomycetes

Clear white, pale yellow, slightly yellow mixed powdered colonies, and spore producing ability of 10 different types of actinomycetes strains were identified around the Sea grasses punched places in 7th day. Some strains shown with sparse spore formation producing ability, and also little, half and entire colonies were shown with pigment formation. The areal mass and reverse sides of the colonies looking with clear pigment (Fig. 1a). All the phenotypic evidences are clearly stated that the isolated colonies were actinomycetes. Next, all the clear and differentiated means pigmented colonies were screened on respective AIA plates and allowed to grown till 7 days. After, all the strains were stored separately streaked slant format and stored in 4 ℃.

Close

Fig 1

Isolation and identification of endophytic actinomycetes from marine Sea grasses (a) and pure culture of actinomycete strains in actinomycete isolation agar (b).

Export to PPT

Further, all the screened strains were endophytic or not was confirmed the surface water validation process was also indicated that the strains were emerged from internal tissue of the Sea grasses. When could seen the last surface sterilized water spread plates, the plates did not contain any growth. Also, there was no any contamination was also observed. This information was clearly stated that the sterilization of Sea grasses samples were clear and uncontaminated samples (Fig. 1b). Finally, all the interpretation of the result was proved that the isolated strains were actinomycetes and also it arisen from inside of the Sea grasses samples. This confirmed evidence was confirmed by surface sterilization validation process. Finally, all the isolated strains were named as endophytic actinomycetes starins 1–10. The result was cross checked with previous result of Javee et al. (2020), and the isolated colonies were actinomycetes due to the phenotypical evidences of color, mass, pigment production (Fig. 2). The followed procedure of Bunbamrung et al. (2020) was supported to current study for confirm the surface sterilization. The result was approved by previous reports of Ramachandrana et al. (2019), the endophytic actinomycetes and surface sterilization was original and also pure.

Close

Fig 2

Validation of endophytic actinomycete by spread plate method using chemical surface sterilization method.

Export to PPT

3.2 Extraction of anti-bacterial compounds

After 15 days fermentation, all the endophytic actinomycete strains were extracted using liquid–liquid extraction experiment using various solvent (Fig. 3). After careful consideration, the solvent phase of the RMN 10 strain of ethanol, ethyl acetate, methanol, dichloromethane phases were exhibited some zones against K. pneumoniae containing plate. Surprisingly, ethyl acetate extract of the RMN 10 strain solvent evaporated phase was exhibited increased anti-bacterial activity compare with other strains. In addition, the zones were very high in increasing concentration compared with some lowest concentration. In 250 µg/mL, the RMN 10 extract of actinomycete solvent phase was exhibited with 16 mm zone of inhibition, 10 µg/mL was exhibited 4 mm zone of inhibition against K. pneumoniae (Fig. 4a, b). This anti-bacterial activity result was suggested that the RMN 4 strain of endophytic actinomycetes has anti-bacterial activity against various bacteria. The similar result was reported by Jeeva et al. (2020); marine algae mediated endophytic actinomycete has excellent anti-bacterial activity against various bacteria. This result was more correlated with our previous results (Parama Cita et al., 2017; Khan et al., 2019), and marine algae mediated endophytic actinomycetes has excellent anti-bacterial and anti-cancer activities against multi drug resistant bacteria and cancer cells. Recently, Bunbamrung et al. (2020) reported that the marine sources of endophytic actinomycetes acquired the nutrient sources from algae, plant and respective sources. Sometimes, the marine environmental sources pH, temperature, carbon, nitrogen, minarals and other sources are also influenced the activities of the actinomycetes (de Felício et al., 2015).

Close

Fig 3

Extraction of anti-bacterial compounds from RMN 10 strain of actinomycetes using liquid–liquid extraction.

Export to PPT

Close

Fig 4

Anti-bacterial activity of actinomycete extract against K. pneumoniae using various solvent (a), Zones of inhibition against K. pneumoniae using ethyl acetate solvent by agar well diffusion method.

Export to PPT

3.3 Minimum inhibition concentration

After careful analysis, the more turbidity of the well was visibly detected at 250 µg/mL concentration and it was stated that the concentration was excellent anti-bacterial concentration. When seen in the all wells, the turbidity was shown with lowest to increase at concentration dependent mode. The result was suggested that the RMN 10 extract has anti-bacterial activity against K. pneumoniae at concentration dependent. The concentration dependent inhibition was validated by calculation of O.D values of control and treated results. In the O.D values, we have performed in microtitre plate reader at 640 nm. In microtitre plateanalysis, the inhibition concentration was initially exhibited at 25 µg/mL only. Before, it could not attain the inhibition level. When seen at 100 µg/mL, the inhibition range of 57% was observed. Finally, the complete death cell of 94% was exhibited at 250 µg/mL concentration (Fig. 5). At this concentration was initially shown with more turbidity, and same result was identified after calculate the O.D value. So, finally we have fixed 250 µg/mL concentration was MIC in this study. The similar result of actinomycete extract against bacteria at concentration dependent manner result reported by Ye et al. (2017). Our result was more accordance with previous evidences of Upasana et al. (2020), and marine actinomycete inhibited the bacteria increasing concentration with concentration dependent manner. This stamen also agreed by Soleck et al. (2012) and Rashad et al. (2015) result was also supported to current study. Previously, Law et al. (2019) and Tan et al. (2019) were reported that the marine mangrove environment of pH, temperature, salt stress, carbon, mineral and nitrogen sources have influenced the actinomycetes of bioactivities. Recently, Singh and Dubey (2018) was reported that the Sea grasses mediated endophytic actinobacteria compounds have more anti-microbial activity against various multi drug resistant bacteria. This result was supported by previous reports of Li et al. (2012) and Ravikumar et al. (2010) were reported that the endophytic bacteria were isolated from Sea grasses and they have shown excellent anti-bacterial activity against various pathogenic microorganisms.

Close

Fig 5

Minimum inhibition concentration of RMN 10 strain of actinomycetes extract against K. pneumoniae by microtitre plate method.

Export to PPT

3.4 Size and shape damages using SEM

The SEM pictogram of the actinomycete extract treated K. pneumoniae cells were shown with shape damages. The belbing formation was shown internal rod shape, and also rod shape was damaged. The shortened size and shortened shapes were indicated that the K. pneumoniae was affected by actinomycete extract. The original size and shapes were entirely modified and the modified growth was shown with non-rod shape (Fig. 6a, b). The distortion morphology of bacterial cells was shown after retaliated the endophytic actinomycete extract. The inhibited shapes of the rod were shown with endophytic actinomycete extract layers. The layer by layer attachment was shown on the surface of the bacterial shapes were observed. It was suggested that the endophytic actinomycete extract has excellent anti-bacterial activityagainst K. pneumoniae. The supportive evidences were reported by Zheng et al. (2000), the size and shapes were entirely changed due to the influence of endophytic actinomycete extract. Our previous report was closely related to current study, and the endophytic actinomycetes has excellent anti-bacterial activities due to the utilization of Also, the marine endophytic actinomycete compound has excellent anti-bacterial and anti-cancer properties. Recently, Ramachandran et al. (2018) was also reported that the endophytic actinomycete compound has anti-bacterial activity against multi drug resistant bacteria and it has better bioactivities compared with terrestrial due to the influence of marine environments including pH, temperature, salt stress, carbon, nitrogen and organic nutrients.

Close

Fig 6

Detection of size and shape of control (a) and damages of cells after treatment with RMN 10 strain of actinomycetes extract (b) against K. pneumoniae by scanning electron microscope analysis.

Export to PPT