Potentiality Test in Antimicrobial Activity and Antibiotic Sensitivity of Subterranean Streptomyces Strains Isolated from Kotumsar Cave of India
The almost high and stable environmental factors always represent a subterranean cave as one of the most vulnerable environments on Earth. In such conditions, the microbial communities that survive definitely reveal strong antimicrobial and other relevant biological activities. In the present study, the antimicrobial activity and the antibiotic sensitivity of seven Streptomyces strains isolated from various depth dependent microhabitats of a subterranean cave has been tested. Antimicrobial activity was found maximum against E. coli than Staphylococcus aureus and Pseudomonas aeruginosa. Further, the strains isolated from the deeper habitats of the cave have revealed much antagonistic activities as compared to the strains of anterior habitats. Some interesting results have also been revealed from antibiotic sensitivity tests which altogether indicate the possibilities for occurrence of high potential Streptomyces strains from this particular cave, useful for biotechnological tools.
to cite this article:
Yogita Rajput, Jayant Biswas and Vibhuti Rai, 2012. Potentiality Test in Antimicrobial Activity and Antibiotic Sensitivity of Subterranean Streptomyces Strains Isolated from Kotumsar Cave of India. International Journal of Biological Chemistry, 6: 53-60.
Received: February 22, 2012;
Accepted: March 12, 2012;
Published: May 29, 2012
The subterranean caves represent an extreme as well as fragile environment
due to its several particularities existing among biotic and abiotic factors.
The high constancy in several geophysical factors makes it one of the most vulnerable
environments on earth. Nevertheless, the energy-starved conditions possibly
encourage the competition among its microbial community that definitely promote
the production of substances such as antibiotics and hydrolytic enzymes that
inhibit the growth of their compatient (Rajput and Biswas,
2012). Subterranean caves are characterized by almost stable temperature
with high humidity and these factors were already pleaded to favor the growth
of heterotrophic bacteria, from which actinomycetes predominate (Groth
and Saiz-Jimenez, 1999). In some cases, Streptomyces species are
particularly found to be abundant. The Streptomyces are well-known producers
of antibiotics arising from their unlimited capacity to produce secondary metabolites
with diverse chemical structures and biological activities. Unfortunately, till
date no serious attempt has been taken to study the potentiality of cave microbes
from Indian subcontinent. Thus, the study of subterranean cave microbes is always
interesting for discovering potential microorganisms, important for several
Today, the life in highly contaminated/polluted environment is giving birth
to several kinds of pathogens at one end, whereas on other end the same is also
increasing the resistance capacity of the existing pathogens against available
antibiotics. Thus, now the situation has developed for the urgent need of new
antimicrobial agents, to check the resistance to the bacterial pathogens and
change in the spectrum of pathogens, together with the emergence of new diseases
(Davis and Webb, 1998; Zahner and Fielder,
1995). Following the same issue, in the present study, we tested the drug
sensitivity and resistant capacity of some subterranean microhabitat (depth
dependent) Streptomyces strains against ten well-known antibiotics; chloramphenicol,
erythromycin, gentamicin, kanamycin, neomycin, novobiocin, penicillin-G, polymyxin-B,
streptomycin and vancomycin. Further, potentiality for their antimicrobial activities
were also tested against three major human pathogenic bacteria; E. coli,
Staphylococcus aureus and Pseudomonas aeruginosa.
MATERIALS AND METHODS
Soil-sediment collection, strain isolation, preservation and re-culturing of isolated Streptomyces strains: Various strains of Streptomyces studied in this piece of study were isolated from various subterranean depth dependant habitats of Kotumsar cave India. The cave is lying in the Kanger Valley National Park (18°5209 N; 81°5605 E) at an altitude of 560 m. The main tunnel of the cave is nearly 500 m long and has several lateral and downward passages leading to several irregular chambers. The ambient external surface of this cave is surrounded by deciduous to mixed forest vegetation. The cave is subjected to frequent flooding during the monsoon season which generally begins in the middle of June and continues till the mid of October. The chambers of the cave are always wet, floored with either rocks or pebbles of various dimensions or by surface-derived soil/clay sediments.
The sediment samples from various habitats belong to four different zones of
the cave i.e., the entrance zone, twilight zone, transient zone and the deep
zone (Biswas, 2010) were collected during the month
of May when the environmental conditions of the cave remain maximum stable.
Isolation of Streptomyces strains was done by dilution plate (Waksman
and Fred, 1922) and direct plate (Warcup, 1950)
techniques, using starch casein agar medium (soluble starch, 10.0 g; casein
hydrolysate, 0.3 g; K2HPO4, 2.0 g; KNO3, 2.0
g; NaCl, 2.0 g; MgSO4.5H2O, 0.05 g; CaCO3,
0.02 g; FeSO4.7H2O, 0.01 g; agar, 20 g; distilled water,
1000 mL; pH 7.2) (Williams and Cross, 1971). The sediment
samples were diluted by adding approximately 1 g of sediment to 9 mL of quarter
strength Ringers solution (NaCl, 8.5 g; KCl, 0.2 g; CaCl2,
0.2 g; NaHCO3, 0.01 g; distilled water, 1000 mL, pH 7.0) which was
further stirred for 10 min on a reciprocal shaker. In the next step to separate
spores from vegetative cells, 4 mL of the resultant 10-1 dilution
was taken in a test tube and placed in a water bath (Sonar, India) for 16 h
maintained at 45°C. The heat-pretreated was diluted to obtain 10-2,
10-3, 10-4, 10-5 and 10-6 dilutions
in a similar manner. Following the method forwarded by Porter
et al. (1960) and Williams and Davies (1965)
0.1 mL aliquots of 10-3, 10-4 and 10-6 dilutions
were plated in triplicates onto starch casein agar plates supplemented with
cycloheximide (25 μg mL-1; Sigma chemicals, USA) and nystatin
(50 μg mL-1), then incubated at 28°C for 7 to 15 days. The
selected colonies of Streptomyces from mother culture plates were transferred
onto respective agar plates and incubated at 28°C for 7-15 days. Plates
containing pure cultures were stored at 4°C until further examination. Isolated
strains once characterized and taxonomically identified by the using of Probabilistic
Identification of Bacteria (PIB) Win software (Bryant, 2003;
Langham et al., 1989) were re-grown on starch
casein agar medium at 28±1°C and maintained by subculturing every
fortnightly at 4°C.
Antimicrobial activity of Streptomyces strains by Cup plate diffusion
method: Sample preparation: Different Streptomyces sp. cultures
i.e., S. prasinosporus KCA 3, 8 and 22, S. aurantiacus KCA6,
S. roseus KCA13, S. longisporoflavus KCA18 and S. luridus
KCA23 were inoculated in starch casein broth medium of pH 7, incubated at 28°C
for 8 days. Cell free culture filtrates were obtained by filtering through Whatman
filter No. 1 and the same were transferred through syringe filter (Axiva, dia
2.5 cm, rating, 0.2 μm) for removal of bacteria. Thus, the obtained final
filtrates were used for antimicrobial activity.
Test organism: Test organisms used for screening of antimicrobial activity of isolates, were obtained from Microbial type culture collection and gene bank (MTCC), IMTECH, Chandigarh, i.e., Staphylococcus aureus (MTCC 96), E. coli (MTCC 1667) and one from J.N. Medical College, Raipur, Pseudomonas aeruginosa (JNMC). Bacterial cultures were inoculated in 5 mL of sterilized nutrient broth for preparation of fresh inoculums. Further, the same were incubated at 37°C for 24 h.
Cup plate diffusion method: (Harris and Ruger, 1953):
For each case, 0.1 mL of test organism was transferred on nutrient agar medium
plate (peptone, 5 g; beef extract, 3 g; NaCl, 5 g; agar 20 g; distilled water
1000 mL; pH 7.2) and with the help of sterilized cotton swab the same was uniformly
disseminated. Slug was removed by means of a sterile cork borer and than 100
μL of culture filtrate was transferred on seeded plate. Diameter of inhibition
zone was measured after 24 h of incubation at 37°C.
In-vitro screening of Streptomyces strains for drug resistant
study: Following the Kirby-Bauer (Bauer et al.,
1966) the antibiotic sensitivity was tested against each Streptomyces
strain. Ten antibiotic discs viz., chloramphenicol (30 μg), erythromycin
(10 μg), gentamicin (10 μg), kanamycin (30 μg), neomycin (30
μg), novobiocin (30 μg), penicillin-G (10 units), polymyxin-B (100
units), streptomycin (10 μg), vancomycin (30 μg) used in this piece
of study were obtained from Hi-Media Pvt. Ltd., India. As per the specification,
the concentration of each antibiotic was maintained and each plate was incubated
at 28°C for 48 h during the study. After incubation, the occurrences and
sizes of inhibition zones around the discs of the different antibiotics were
tabulated. On the basis of forwarded specifications against each antibiotic
by the Hi-Media Pvt. Ltd., India, the isolates were either considered as sensitive
(S), intermediate (I) or resistant (R) to an antibiotic.
RESULTS AND DISCUSSION
Serious infections caused by bacteria are gradually becoming resistant to commonly
used antibiotics and which needs a major global attention in the present century.
However, instead to go for boundary-less expensive chemical resources, it is
imperative to search for new, efficacious and safe antibiotics from natural
resources to combat the menace of drug-resistant infections. To avoid the redundancy
for exploration of common compounds and to overcome the new drug resistance
in several microbial pathogens, new unexplored source of bioactive products
must be discovered (Ghadin et al., 2008). Streptomyces
shows almost reluctant activity against human pathogens, thus, the medical importance
of it could not be overruled. In search of bioactive antibiotics; Streptomyces
strains have been isolated from various types of soils starting from rice
paddy, lake-mud/water, deciduous forest, tropical forest, wasteland and cave
soils (Bhattacharya et al., 2007; Saadoun
and Gharaibeh, 2003). However, the caves are the most attractive place to
look for new actinomycete species (Streptomyces strains) that might be
a source of novel bioactive compounds. Actinobacterial growth is known to be
distributed all over the caves, starting from the dripstone formations (Laiz
et al., 2000; Yamac et al., 2011)
to the soil sediment contents (Groth et al., 1999;
Nakaew et al., 2009). The earthy smell of caves
is always due to the mixture of organic products being produced when Actinomycetes
decompose organic material (Jachymova et al., 2002;
Scholler et al., 2002) which is applicable for
elsewhere too (Raja and Prabakarana, 2011).
Antibacterial activities against all the three human pathogenic bacteria, used
in present study; E. coli, S. aureus and P. aeruginosa,
have already been well documented by various Streptomyces sp. which were
either isolated from Egyptian soil samples (Rizk et al.,
2007), termites gut contents (Khucharoenphaisan
et al., 2012), marine sediments (Kumar et
al., 2011) or the normal soil (Manjula et al.,
2009). Further, more or less the same types of reports supporting
the antimicrobial activities by Streptomyces sp. have also been revealed
by few other workers against E. coli and P. aeruginosa (Streptomyces
sp. isolated from sewage water; Rabeh and Fareed, 2008),
E. coli and S. aureus (Streptomyces sp. isolated from marine
actinomycete; Reddy et al., 2011), S. aureus
and P. aeruginosa (Streptomyces sp. isolated from marine actinomycete;
Devi et al., 2006), S. aureus and P.
aeruginosa (Streptomyces sp. isolated from Andaman seacoast; Peela
et al., 2005) etc. However, present study testified the antimicrobial
activities for the isolated Streptomyces strains against almost all the
three pathogens. Interestingly, the strains isolated from the inner zones of
the cave exhibited much sensitivity as compared to the strains isolated from
outer zones of the cave (Table 1, Fig. 1).
The strains of Streptomyces prasinosporus, isolated from most of the
microhabitats of the Kotumsar cave, are an unusual green-spored Streptomycetes,
abundantly found in the normal soil(s) of India (Tresner
et al., 1966). While testing the antimicrobial activity of this strain
against three major human pathogens; S. aureus, P. aeruginosa
and E. coli, maximum activity in the form of the clear inhibition zone
were exhibited against E. coli (16 mm = KCA3; 12 mm = KCA8; 13 mm =
|| Antimicrobial activity of Streptomyces isolates by
agar diffusion method
In the present study, the Streptomyces roseus (KCA13); the only strain isolated from the transient zone of the cave exhibited maximum diameter of inhibition against at least two treated pathogens (E. coli and P. aeruginosa). The environmental status of transient zone for any cave reflects an intermediate state between its ambient outer (variable) and inner (constant) conditions, where light remains completely absent but the atmospheric temperature, humidity and other geophysical factors vary up to some extent, depending on the influence of its outer world. However, in the present status we are not in a position to speculate any specific reason regarding the high potency of this specific strain.
While assessing the isolated Streptomyces strains for antibiotic sensitivity by subjecting them to different antibiotics, we found 85.71% isolates were resistant against polymyxin B, 57.14% against gentamicin, 42.85% against streptomycin, tetracycline, kanamycin and 28.57% against erythromycin, chloramphenicol and neomycin. Interestingly, in this study except, the strain S. prasinosporus (KCA3); isolated from the entrance zone of the Kotumsar cave we found that rest all the remaining strains which were isolated from the inner zones of the cave were resistant to Penicillin G. Nevertheless, except the strain S. prasinosporus (KCA3), isolated from the entrance zone of the Kotumsar cave, remaining all the other strains, isolated from various other deeper zones of the cave were found to be highly sensitive for the streptomycin and vancomycin. Further, the remaining tests handed us a mixed type of result (Table 2, Fig. 2).
|| Antibiotic sensitivity profile of Streptomyces sp.
|S: Sensitive, R: Resistant, I: Intermediate, Zone size diameters
(mm) are in superscript
||Exemplary image representing antibiotic sensitivity of test
by disc diffusion method in Streptomyces luridus KCA23, isolated
from sediments of innermost zone of Kotumsar cave
During isolation, we found the species; S. prasinosporus from three different microhabitats viz. (i) S. prasinosporus KCA3; from entrance zone or outside the cave, (ii) S. prasinosporus KCA8; from twilight zone which remain partially under the influence of external environmental conditions and (iii) S. prasinosporus KCA22; from deeper zone, the environmental conditions of which remain altogether independent from its ambient external environmental conditions. Interestingly, when we treated these strains separately with tetracycline and chloramphenicol, we found, the strain KCA3 that was isolated from the entrance zone was resistant to both of these antibiotic whereas the strains KCA8 and KCA22, isolated from the inner zones of the caves were sensitive to the same antibiotics.
Natural resistance towards the antibiotics is the most uncommon and unstable
characteristic found in most of the Streptomyces, which is possibly due
to its respective genetic instability (Crameri et al.,
1982). For any isolated strain of Streptomyces, the appearance of
natural resistance against streptomycin and vacomycin group (antibiotic) is
very hard, however the mutational techniques leading to its specific sensitivity
needs a high degree of biotechnological consummation. The steady spread of resistance
to the penicillin for various microbes is well-established fact now (Frere,
1995). The antibiotic property for any strain towards vancomycin is usually
antagonistic to the penicillin (Kuzin et al., 2000)
and the same is somehow strengthening our findings too. Resistant to tetracycline
and chloramphenicol is widespread in microbes, however the occurrences of antibiotic
sensitivity towards the same from those strains of S. prasinosporus,
which were isolated from the inner microhabitats of the cave is of great interest.
Absence of several pollutants and other relevant factors embody the cave ecosystem
an almost sterile atmosphere (Rajput et al., 2012)
due to which several physiological and biochemical alterations are just obvious
in its resident microbes that perhaps altogether absent in its external counterparts.
From the above study, it could be concluded that all the identified isolates belonging to Streptomyces group have antimicrobial and significant other biological activities too. The screening of any available collection of microorganisms may yield new information about the nature of the available gene pool. The isolation of the above Streptomyces group from a complete unusual habitat; cave also somehow strengthen the chances of the same. Conclusively, this piece of work certainly opens a new gate of invention for Streptomyces sp. from soil-sediment samples of different habitats of Indian caves.
We are highly grateful to the director of Kanger Valley National Park of Chhattisgarh, India for allowing us to conduct this study. Thanks are due to Dr. Anil Kumar Ramteke and Master Siddharth Biswas of National Cave Research and Protection Organization, India for collecting the sediment samples from caves for this study.
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