INTRODUCTION:

Actinomycetes have been broadly acknowledged as a pre-eminent source of biologically active secondary metabolites producing a diverse array of bioactive compounds notably antibacterial, antifungal, anticancer and immunosuppressive agents^1,2. The antibiotic resistance among bacterial pathogens has risen considerably in recent years and has become a major impediment in the course of treatment for such infections³. The extensive exploitation of terrestrial actinomycetes in search of advanced antimicrobial metabolites has motivated the researchers to explore the new groups of actinomycetes from pristine habitats anticipating that they may serve as a source of potent antimicrobials.

Over the last few years, cave ecosystems have been attracting considerable attention for their unique environmental conditions and thus offer a possibility of unexplored microbial diversity with distinctive secondary metabolites^4-6. Though the cave microbiota is being explored across the globe, yet is at its infancy. The studies accentuating diversity of actinomycetes from cave habitats in Indian Sub-continent are insignificant. Garhwal Himalayan region which abodes several pristine caves remains yet to be fully explored for their microbial diversity. The study has been designed to provide us an insight into the bioactive spectrum of indigenous actinomycetes from cave habitats of Garhwal Himalaya. To the best of our knowledge, till date, no such reports exist in the literature.

MATERIALS AND METHODS:

Sampling:

A number of samples viz., microbial mats, soil, speleothem, wall scrapings and water were collected for the isolation of actinomycetes from three different caves located in three different regions of Garhwal Himalayas (Figure 1). Samples were immediately transported to the laboratory in an insulated ice-box and processed immediately.

Figure 1: Geographical location of the sampling sites in Garhwal Himalaya

Recovery of the isolates:

All the samples except water were air dried (pre-treatment) for 10 to 12 days preceding use to enhance the isolation of slow growing actinomycetes. Samples were then serially diluted in normal saline to 10^-5 and 0.1 ml of diluted samples was spread on agar plates under aseptic conditions. Different selective media viz., actinomycete isolation agar, glycerol asparagine agar, starch casein agar^7,8 supplemented with a concentration of 25 µg/ml and 75 µg/ml of cycloheximide and nalidixic acid respectively were used. All the plates were incubated at 28 ± 2 °C for a period of 7 to 30 days. Plates were maintained in triplicates and were observed regularly for appearance of the colonies.

Cultural and morphological characterization of the recovered isolates:

The morphological characteristics of the colonies were studied according with the help of light microscopy by conventional gram staining⁹, slide culture technique¹⁰ and scanning electron microscopy¹¹.

Screening for antibacterial activity:

The primary screening of isolates for antibacterial activity against selected bacterial pathogens was carried out by right angle streak method¹². All the recovered actinomycetes were inoculated as a straight line on Muller Hinton agar plates and incubated for 6 to 8 days at 28 ± 2 °C to attain a copious growth. Afterwards, 18 h old culture of test organisms was streaked perpendicularly to the actinomycetes and incubated at 37 ± 2 °C for 24 h and the plates were examined for the inhibition near the line of streak. The test pathogens A. baumannii (MTCC 12889), ESBL E. coli (MTCC 443), MRSA (MTCC 7443), VRE faecalis (MTCC 439) were obtained from IMTECH (Chandigarh). E. coli (Clinical isolate) was isolated from a clinical urine sample.

Production of bioactive metabolites and secondary screening:

All the isolates giving positive results in primary screening were subjected to secondary screening by agar well diffusion method¹³. The active isolates were grown in modified Gause’s fermentation media in an orbital shaker at 28 ± 2 °C and 180 rpm for a period of 7 to 10 days. The fermentation medium was withdrawn aseptically at regular intervals to check its antibacterial activity. The medium was centrifuged at 10,000 rpm for 10 mins to separate the supernatant from the mycelium. Organic solvents (chloroform, ethyl acetate, methanol, and petroleum ether) with varying polarities were added in equal volumes (1:1 v/v) to extract the extracellular metabolites from the fermented medium¹⁴. The organic fractions were rota-evaporated and used to determine their antimicrobial potency.

Approximately 100 µL of crude extract dissolved in methanol was loaded into wells bored with a sterile cork borer of 6 mm diameter into pre-inoculated Muller Hinton agar plates (using 0.5 McFarland turbidity standard). The plates were kept at room temperature for 20 mins in order to allow the extract to diffuse properly and afterwards incubated at 37 ± 2 °C for 24 h. The plates were examined for the presence of a zone of inhibition surrounding the wells. The antimicrobial activity of the standard antibiotics for each test organism was also determined.

Determination of minimum inhibitory concentration:

The agar dilution method described by Wiegand et al. (2008)¹⁵ was employed to determine the antimicrobial susceptibility of test pathogens to the crude extracts of SCM1 and RCM1. A concentration ranging from 16 µg/mL to 1024 µg/mL of both the extracts was added to individual flasks containing approximately 25 mL of autoclaved MHA. Agar plates without any crude extract were kept as control. The test cultures were spot inoculated on the agar plates. The bacterial suspensions adjusted to 10⁸ CFU/mL were diluted to 1:10 to get a microbial density of 10⁴ CFU/spot. Plates were incubated at 37 ± 2 °C for 18 to 24 h. Agar plates with crude extract showing no visible growth of the test culture were read as MIC of the crude extract against the particular test culture. Plates were maintained in triplicates for the accuracy of the results.

RESULTS AND DISCUSSION:

Isolation and characterization of the recovered isolates:

In the present work, 30 samples were collected from all the three caves and a total of 103 actinomycetes were isolated with the help of selective isolation techniques. A preliminary identification to the genus level was based on the specific cultural and morphological characters and the colonies exhibiting growth pattern indicative of actinomycetes were chosen for further analysis. The colonies were aerobic, slow growing, chalky with substrate and aerial mycelia of varying colors producing an earthy odor. Streptomyces-like actinomycetes with white to grey aerial mycelium were found to be dominant in most of the samples.

Actinomycetes isolation agar yielded maximum number of actinomycetes as this medium besides other selective constituents contains glycerol which promotes efficient growth of actinomycetes. Several previous studies have also highlighted the significance of selective constituents of culture media used for the isolation of actinomycetes^16-17.

Screening for antibacterial activity:

In this study 16.5 % gave positive results in primary screening inhibiting either one or both the groups of test pathogens while only 23.5 % isolates showed broad-spectrum activity. Isolates RCM1, RCM14 and SCM1 showed significant antagonistic activity against gram-positive test pathogens in secondary screening. Benhadj et al.¹⁸ have also reported the increased susceptibility of gram-positive pathogens to organic extracts of rare actinomycetes which can be ascribed to the difference in their cell wall composition¹⁹.

The modified Gause’s medium was found to be most suitable for biomass and bioactive metabolite production. Singh et al.²⁰have also reported the influence of media components on the metabolite synthesis by actinomycetes. Ethyl acetate extracts of active isolates were found to have maximum antibacterial activity against the test pathogens followed by methanol extracts. This is in conformity with other studies that have reported the enhanced activity of ethyl acetate extracts of actinomycetes against broad range of pathogens^21-22.

Crude extracts (5 mg/mL) of SCM1, RCM1 and RCM14 significantly inhibited multi drug resistant strains (Figure 2). These isolates were found to exhibit better inhibitory activity as compared to the conventional antibiotics (Table 1). Crude extract of RCM14 was predominantly active against drug resistant gram-positive pathogens showing maximum antagonistic activity against drug resistant S. pyogens followed by MRSA, VRE and A. baumannii respectively, however it did not inhibit any of the E. coli strains.

Figure 2: Antibacterial activity of crude extracts of RCM1, RCM14 and SCM1 against drug resistant pathogens

Ethyl acetate extracts of RCM1 and SCM1 manifested impressive broad-spectrum activity. Crude extract of RCM1 exhibited maximum inhibitory activity against ESBL E. coli with a zone of inhibition of 31.8 ± 0.6 mm which is far better than the activity of standard antibiotics ceftazidime and cefotaxime (Table 1). The isolate gave a zone of inhibition of 22.0 ± 1.0 mm against a drug resistant strain of E. coli. Aliero et al.²³ have reported similar activity of ethanol extract of actinomycete BRWDSc (SP) isolated from dump soil of Western Uganda against multidrug resistant E. coli (clinical isolate)-22.3 mm. Furthermore, it was also noticeably active against MRSA, S. pyogens and VRE evincing better inhibition (Figure 2) than the antibiotics of choice except ampicilin which produced comparatively larger zones of inhibition against MRSA and VRE (Table 1).

SCM1 was found to be the most active isolate remarkably inhibiting every pathogen that was tested. Ethyl acetate extracts of SCM1 exerted significant activity against drug resistant pathogens including ESBL E. coli, carbapenem resistant A. baumannii, MRSA, S. pyogens and VRE. faecalis producing prominent zones of inhibition (Figure 2).

Table 1: Zone of inhibition of standard antibiotics against drug resistant pathogens

Test pathogens	Standard antibiotics
Test pathogens	Amp	Caz	Ctx	Ery	Imp	Lvx	Mem	Met	Tmp	Van
A. baumannii	NA	NA	NA	NA	7.7±1.7	NA	5 ±0.5	NA	NA	NA
E. coli (c.iso.)	-	-	3±0	-	NA	-	NA	NA	-	NA
ESBL E. coli	14.8±1.0	21.7±1.8	27.0±0.5	-	NA	23.3±0.6	NA	NA	19.8±0.8	NA
MRSA	27.6±0.4	NA	NA	22.6±0.6	NA	NA	NA	-	NA	10.2±1.0
S. pyogens	5.4±0.8	NA	11.4±0.5	10.3±1.2	NA	NA	NA	NA	4.4±1.2	NA
VR E. faecalis	23.4±1.3	NA	NA	18.8±0.3	NA	NA	NA	-	14.5±0.7	13.8±0.7

All values are expressed (in mm) are mean of the triplicates with standard deviation

Amp: Ampicilin; Caz: Ceftazidime; Ctx: Cefotaxime; Ery: Erythromycin; Imp: Imipenem; Lvx: Levofloxacin; Mem: Meropenem; Met: Methicillin; Tmp: Trimethoprim; Van: Vancomycin; NA: Not used-: No zone

It also significantly inhibited a multi drug resistant strain of E. coli isolated from a clinical sample which was very surprising considering none of the antibiotics used could inhibit this isolate (Table 1). Likewise, Nakaew et al.⁵ have also reported the inhibitory effect of ethyl acetate extract of Spirillospora sp. isolated from Thai cave soils against MRSA with a zone of inhibition of 32.0 mm. A similar study by Yucel and Yamac¹ on the Streptomyces isolates from Karstic caves in Turkey has also highlighted the activity of a heat stable active component from one of their isolates against multidrug resistant pathogens including MRSA, VRE and A. baumannii. Cheeptham et al.⁴have also reported the antimicrobial potential of actinomycetes isolated from caves of Helmcken Falls, British Columbia against MDR pathogens.

The MIC values of both the crude extracts against selective pathogens ranged between 128 µg/ml to 512 µg/ml (Table 2). Crude extract of SCM1 was highly efficient against most of the drug resistant pathogens suppressing their growth at a concentration of 128 µg/ml as compared to RCM1. However, an MIC of 256 µg/ml was observed against A. baumannii and E. coli (c. iso.). Similar results have been put forth in various other studies^24,25.

Table 2: Zone of inhibition and minimum inhibitory concentration of crude extracts of RCM1 and SCM1 against test cultures

Test cultures	Zone of Inhibition (mm)		MIC (µg/ml)
Test cultures	RCM1	SCM1	RCM1	SCM1
A. baumannii	17.0	21.0	512	256
E. coli (⁸c. iso.)	22.0	23.3	256	256
ESBL E. coli	31.7	34.7	128	128
MRSA	23.3	34.3	512	128
S. pyogens	20.3	30.7	256	128
VR E. faecalis	21.3	31.3	256	128

^*Clinical isolate

The single factor one-way ANOVA used to analyze and compare the inhibitory zones of different extracts of the isolates in the present study revealed that the results were statistically significantly different (P< 0.05).

Preliminary identification of isolates:

The isolates RCM1 and SCM1 were found to have moderate growth comparatively with white and grey aerial mycelium, respectively and the individual colonies were rough, powdery and elevated in appearance. The cultures produced sporulation on AIA within 2-3 days of incubation with no diffusible pigmentation on the medium. Both the isolates produced abundant sporulation on diverse solid media used with rapid growth in broth culture (half strength). Microscopic studies showed the presence of branched network of mycelia. Scanning electron microscopy revealed the arrangement of spores in chains (Figure 3). These findings strongly suggest that both these isolates belong to genus Streptomyces.

Figure 3: Morphological characteristics of isolates RCM1 (A) and SCM1 (B): (i) Slide-culture, (ii) Gram staining, (iii) Scanning electron micrograph

CONCLUSION:

This study has given us a preliminary idea on the antibacterial activity of indigenous cave actinomycetes of Garhwal Himalayan region against multi drug resistant pathogens, simultaneously prompting us to expand our understanding on the metabolic potential of cave microflora. Since this is our first attempt to study the bioactive spectrum of cave actinomycetes from this region, the novelty of antibacterial compounds from these isolates needs to be studied. Further studies on the purification and characterization of these metabolites are under progress. Considering the noteworthy outcome of the study, there is a high probability that the cave actinomycetes of this region could be a source of new biologically active and effective compounds.

ACKNOWLEDGEMENTS:

Authors acknowledge the financial assistance and facilities provided by University Grants Commission, India and Dept. of Botany and Microbiology, H. N. B. Garhwal University Srinagar, Uttarakhand respectively to carry out the study. Authors also acknowledge the contribution of Dr. Shairy Chaudhary, Dept. of Geography, H. N. B. Garhwal University in designing the geographical location of study sites.

CONFLICT OF INTEREST:

Authors declare that there is no conflict of interest regarding this study

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Received on 01.07.2021 Modified on 18.10.2021

Research J. Pharm. and Tech 2022; 15(9):3893-3897.

DOI: 10.52711/0974-360X.2022.00652