Exploring the Potential of Myxobacteria from Himachal Region for Antimicrobial Activity

 

Pooja Thakur1,#, Chirag Chopra1,#, Daljeet Singh Dhanjal1,#, Reena Singh Chopra1,*

1School of Bioengineering and Biosciences, Lovely Professional University, Phagwara -144411, India.

*Corresponding Author E-mail: reena.19408@lpu.co.in

#Equal First Author (Authors have contributed equally to the work)

 

ABSTRACT:

Myxobacteria being a gram -ve, rod-shaped, slow growing bacteria along with omnipresent producer of bioactive compound with anti-bacterial, anti-fungal, anti-viral as well as anti-tumor activity, has attained the attention of many researchers. Diverse areas and habitats are being explored for isolating this bacterium but, till date there is no reported documentation of this bacteria from the Himachal region. In this study, the focus was given on isolating the Myxobacteria from Himachal region and to evaluate its anti-microbial activity of isolated strains. The pure strains were isolated from the soil sample by visualizing the unusual properties of Myxobacteria, following the purification method, recognition and characterization on the basis of colony formation, observing the fruiting bodies under the stereomicroscope. After that cell extract as well as supernatant of the isolated strain was assessed for the presence of anti-microbial agent. From the obtained result, it was found that isolated strains were able to inhibit the growth of test organism such as E. coli, B. subtilis, Clostridium acetobutylicum, Stenotrophomonas maltophilia, Pseudomonas sp., and Trichoderma reesei.

 

KEYWORDS: Myxobacteria, Antimicrobial Activity, Himachal Region, Dung-baiting, E. coli baiting.

 

 

 


INTRODUCTION:

Investigation of the novel antimicrobial agents and metabolites has rendered the researcher to examine those areas which were not yet explored and identify those bacteria that are still under intervention, such as gram -ve, rod-shaped, slow growing bacteria Myxobacteria1. Myxobacteria are profoundly found in the soil of dumping area, animal feces, dead plant debris and bark2,3. Furthermore, the growing behavior of this bacterium is as unique as its isolation procedure, as it shows gliding movement onto the solid surface forming a swarming pattern4.

 

These are distinctive feature that separate Myxobacteria from the other bacteria like growing in swarm pattern, the formation of myxospores under unfavorable conditions, cellulase degradation ability of some strains, antibacterial and antifungal, anti-viral and anti- cancer activity against other bacteria and fungi by synthesizing extracellular enzyme and antibiotics1,5-6. Due to the ability to produce a vast variety of bioactive compounds of the remedial value which comprises anti-bacterial, anti-fungal, anti-viral and anti-tumor compounds6-8. Myxococcus xanthus is reported to have anti-bacterial activity because of the compound TA and comprises a cluster of 18 genes that encode for the synthesis of secondary metabolites5. The antibacterial agent, Myxovirescin made up of 28-macrolactam  lactone and found to be active against gram +ve, gram -ve bacteria and facilitating in preventing biofilm infection such periodontal disease, is also synthesized by the Myxococcus xanthus strain9-10. Another strain, Sorangium cellulosum produces antibiotic thuggacin, which causes restrain in the respiratory tract of the bacteria and another compound, Ratjadone A that has been reported to exhibit anti-viral activity against the HIV-AIDS11-12. The strain Stigmatella WXNXJ-B has been reported to synthesize anti-tumor compound13. Certain strain of Myxobacteria synthesizes diverse compounds such as Epothiolone which helps in stabilizing the microtubilin during mitotic division, inducing apoptosis in a cell that was unable to assemble functional mitotic spindles and there is another compound named Cruentaren, which has the anti-fungal activity14.

 

The current study focusses on the isolation and identification of the Myxobacteria species from the Himachal region on the basis of its growth pattern and 16s rDNA sequencing. Evaluation of the antimicrobial and antifungal activity of isolated strain against the certain selected microbes.

 

MATERIAL AND METHODS:

Collection of samples:

The soil sample was collected with scrapper from tree bark area of Jathia Devi, Shimla, Himachal Pradesh (31 6' 11" N, 77 10' 19”) from the depth of 20 cm in January 2016. The sample was collected in zip-lock bags and was stored in 4ºC for further use.

 

Isolation of Myxobacteria:

Two methods were employed for isolation of Myxobacteria:

1.       Dung Baiting:

Casitone media consisting of Tryptone (0.5g/100ml), Peptone (0.1g/100ml) and Agar (2%) was poured in the Petri plates. After solidification of media, moisted soil was added to the media in the center of the plate. Dried and autoclaved rabbit dung pellets were embedded onto the soil and the plates were incubated at 30ºC for 1-2 weeks looking for white color patches on the rabbit dung pellet1.

 

2.       E. coli Baiting:

E. coli culture was grown in Luria broth and after 24 hr at 37ºC, the culture was spread on the surface of casitone agar media. On to the centre, the soil was streaked in a cross pattern and incubated the plates at 30 ºC for 1-3 week. Later the plates were observed for swarm growth as well as the fruiting bodies1. After culturing of Myxobacteria by both the methods the bacteria was inoculated in to separate test tube of casitone broth and incubated at 30 ºC in shaking incubator at 120 rpm for 1-2 weeks while looking for yellow clump formation, a characteristic feature of Myxobacteria in broth.

 

Subculturing of Myxobacteria:

The pure colonies were isolated by using the culture from casitone broth that contained Myxobacteria isolated by both Dung as well as E. coli Baiting method. The inoculum was taken and a straight line was streaked onto casitone agar media and incubated the plates at 30ºC for 1 week. After that plates were observed for swarm growth.

 

Screening of Myxobacteria:

For confirming the isolate to be Myxobacteria, the standard procedure of gram staining was performed. To further examine the morphology(swarm pattern) and fruiting bodies, stereomicroscope studies were carried out as suggested previously 1-5

 

Genomic DNA Isolation and Amplification:

For the isolation of genomic DNA, the bacterial cells were grown in casitone broth for 36-48 hrs further followed the procedure as described previously by 15. After isolation of genomic DNA, gel electrophoresis was performed to check the quality of DNA. After confirmation, PCR amplification of Myxobacterial 16S rRNA genes was performed. Myxo-specific forward primer Myxo2F (5-AMG ACG SGT AGC TGG TCT- 3) and reverse primer 1492R (5- TAC CTT GTT ACG ACT T-3) were used for the same. The amplification protocol was as follows: Pre-denaturation was done at 94ºC for 3 min, followed by 30 cycles of denaturation at 94ºC for 30 secs, annealing of primers at 43ºC for 1 min and extension was performed at 72ºC for 1 min. After the completion of 30 cycles, the final extension was given at 72ºC for 5 min. Then the reaction mixture was held indefinitely at 4 ºC.

 

Antimicrobial Assay:

For the assessment of antimicrobial activity, the pure culture was inoculated into 50 ml casitone broth and incubated at 30ºC for 4-5 days. After that, the suspension culture was centrifuged at 6000g for 10 mins. For lysis of cell, 1ml of lysis buffer (50mM Tris-HCl: pH 7.5, 150mM NaCl, 5mM EDTA) was added and 1/3rd volume of glass beads were added. After that solution was homogenized for 1 hr with intermittent cooling. After vortexing the solution was centrifuged at 12000g for 10 min and cell extract was collected into a new tube 9-16.

 

The test strains of bacteria (E. coli, B. Subtilis, Pseudomonas sp., Clostridium acetobutylicum, and Strenoterophomonas maltophilus) were cultured in Luria broth. The test strain Trichoderma resei (fungus) was cultured in nutrient broth. All the bacterial tubes were kept in shaking incubator at 37ºC and Fungal tubes were kept at 27 ºC for 24 hours.

 

The assay plates were prepared according to test strains and 100 µl of culture was spread per plate with a sterile spreader. Agar diffusion method was employed for assessment of antimicrobial activity for which three well were made on each plate with puncturing syringe. 50µl of supernatant and 50µl of cell extract was added to two different wells. The one well was kept left undisturbed to serve as a negative control. The plates were kept at 30ºC for 24 hours. After that plates were observed for the zone of inhibition around the well 17.

 

RESULTS:

Isolation and Screening of Myxobacteria:

Applying the above-mentioned baiting methods to the soil sample collected from tree bark and organic-rich area, provided us two Myxobacterial strains. The dung-baiting method using sterile rabbit dung pellets was found to be more effective and quicker method, in comparison to E. coli Baiting method which provided us with a few colonies (mostly false positive). White patches formed on the dung pellets were collected (Fig. 1A) and inoculated into the casitone broth which resulted in the formation of a yellow ring and provided us with the two Myxobacterial strains named myxo010 and myxo001. Typical identification of Myxobacteria can be made by plating them on Water Agar plates containing 25ug/mL cycloheximide. Myxobacteria Myxo001 and Myxo010 showed the typical swarming growth pattern, almost conclusive that the strains belonged to the myxobacteria clan (Fig. 1B, 1C). Further investigation was carried out on these strains for confirming Myxobacteria. Gram staining of these strains was done which confirm these strains to be gram -ve as it retained the counter stain (safranin). Myxobacterial origin was further confirmed as they showed formation of fruiting bodies under the stress-conditions (nutrient deficiency of water agar). This was seen under a stereomicroscope at 4X magnification as suggested by Reichenbach and Dworkin (1992) and explained in the Bergey’s Manual of Systematic Bacteriology (Fig. 1D). The growth pattern and morphological parameters were quite similar to the previous reports 18-20.

 

Extraction of Genomic DNA and 16s rRNA Gene Amplification:

The genomic DNA of both the strains was isolated according to standardized procedure and run on the 0.8% agarose gel and the DNA isolated was observed to be of high molecular weight (Fig. 2). Thus, genomic DNA from both Myxo010 and Myxo001 were used as template for amplification of DNA. On amplification with forward primer Myxo2F and reverse primer 1492R, the amplicon was run on the 1.2% agarose gel. Forward primer Myxo2F and Reverse primer 1492R used for the amplification were then employed for the sequencing of the 16S-rDNA region. These are myxo-specific primers and mere successful amplification meant that the cultures belonged to Myxobacteria family.

 

Fig.1 Showing different observations made for identifying myxobacterial strains. A: White Patches seen on Rabbit Dung pellets; B: Swarming pattern as seen in Myxo001; C: Swarming growth pattern as seen in Myxo010; D: Fruiting Bodies in the swarm visible under a stereomicroscope at 4X magnification.

 

Fig.2 Showing Agarose Gel Electrohoresis of Genomic DNA and PCR-amplified 16S rRNA gene. A (from left to right): 0.8% agarose gel showing two wells containing Myxo001 genomic DNA and next two wells containing Genomic DNA of Myxo010; B: 1%rose gel showing Myxobacterial 16S rRNA gene amplified from Genomic DNA of Myxo001 (Left) and Myxo010 (Right), using Myxo-specific Primers.

 

Antimicrobial activity:

After identifying the isolated strains were tested for their antimicrobial activity against 14 microbial test strains by using agar well-diffusion methods as discussed in the section of material and method. Test strains consist eight bacterial strains, five fungal strains, and one yeast strain. Zone of inhibition was observed against sensitive test strains. Sample (Myxo010) showed antimicrobial activity against bacterial strains, whereas sample (Myxo010) was active against bacteriaas well as fungus. The supernatant of Myxo010 was found to be more effective against the E. coli, B. subtilis, Clostridium acetobutylicum and Stenotrophomonas maltophilia whereas supernatant of Myxo001 was only mildly effective against E. coli, B. subtilis and Strenotrophomonas maltophila. Whereas the cell extract of Myxo010 was effective against bacterium Bacillus subtilis, the cell-free extract of Myxo001 did not kill any microbes. The results showed that antimicrobial activity was found mostly in the supernatant of Myxo010 and was only weakly seen in Myxo001 culture supernatant (Table 1 and Fig. 3).


 

Table 1: Assessment of antimicrobial activity of both the strains (myxo010 and myxo001) against various stress strains.


Test strains

Myxo010 Supernatant

Myxo010 Cell extract

Myxo001 Supernatant

Myxo001 Cell extract

Escherichia coli

+

-

+/-

-

Bacillus subtilis

+

+

+/-

-

Pseudomonas sp.

+

-

-

-

Clostridium acetobutylicum

+

-

-

-

Stenotrophomonas maltophilia

+

-

+/-

-

Trichoderma reesei

+

-

-

-

 

Fig. 3 showing Anti-microbial activity test of culture supernatant and cell-free extracts of Myxobacteria strain Myxo010. A. E. coli; B. Stenotrophomonas maltophilus; C: Clostridium acetobutylicum; D: Bacillus subtilis; E: Pseudomonas spp.; F: Trichoderma reesei.

 

 


DISCUSSION:

Myxobacteria being significant secondary metabolites producers, are considered as preferred source for conducting anti-microbial research which would help in synthesizing the numerous useful drugs which are effective in inhibiting various microbes 21-22. Due to this, Myxobacteria were isolated from the himachal soil, which showed similar morphological characteristics and growing pattern in agreement to the previous studies conducted on Myxobacteria isolated from organic-rich soil from different biogeographical regions 19,23-25. As no previous report was documented from this geographical region made it the targeted region for our study.

 

The method followed for isolating the Myxobacteria provide us with effective results, otherwise the Myxobacteria grow slowly and for that it has to compete with more rapidly growing bacteria and fungi 9,26. Thus, to prevent the chances of these contamination the media was provide with the antifungal and antibacterial agent (cycloheximide at a concentration of 25µg/mL) 27.

 

Further, isolated Myxobacterial strains were characterized on the basis of their morphological traits such as shape and size of vegetative cell, swarm growth pattern and formation of myxospores as well as fruiting bodies under unfavourable conditions 1,28.

 

The antibacterial activity of culture supernatant and cell-free extract of the two isolated strains, was assessed against different microbes. The antimicrobial activity shown by supernatant of Myxo010 was higher as compared to supernatant of Myxo001 as well as the cell extract of both the strains (Myxo001 and Myxo010). The Myxococcus xanthus strain produces antibacterial agent which has been reported to inhibit the growth of E. coli [3,9]. Whereas, different reports have documented Myxobacteria which have been tested against various eubacteria29. Thus, our study confirmed the presence of varied bioactive agents that has diverse role to play in the medicinal as well as biotechnology industry.

 

ACKNOWLEDGEMENTS:

The authors thank the senior administration of Lovely Professional University for providing the seed grant for initiating the project.

 

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Received on 17.05.2017             Modified on 08.06.2017

Accepted on 30.06.2017           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(8): 2628-2632.

DOI: 10.5958/0974-360X.2017.00466.8