Antibiotic potentials and isolation of metabolomes from microorganisms of mesophilic soil of Rajasthan , India

The present of research work was focused on mesophilic soil samples from four different sites of Jaipur (The Pink City) in Rajasthan. Rajasthan is a semi arid region and famous for its sandy soil, heat and temperature which reach up to 50°C in summers. Twenty nine colonies were isolated from the soil samples on nutrient agar media and three distinct isolates were selected and characterized after analyzing coloration of colony formation and zone of inhibition around and appearance of the colony. On the basis of biochemical tests, the isolates are suspected to belong to the genera, Bacillus, Actinomycetes and Staphylococcus. Antibiotic activity of the isolates was studied against some common nosocomial infection causing pathogens and promising results were observed. Out of 29 colonies, only 3 colonies viz. S-IA, S-IIA and S-IIIC showed appreciable biological activity against the above mentioned 2 test pathogens E. coli and Pseudomonas aeruginosa. Further, inhibition zones were calculated in S-IA (IZ = 12 mm; AI = 1.5); S-II A (IZ = 42 mm; AI = 4.2) and S-III C (IZ = 12 mm; AI = 1.1).


INTRODUCTION
In nature, microorganisms are the most widely distributed group of organisms, and abundantly present in soil.A number of continuous methods are in process for search of new bioactive compounds with high commercial value.Soil microbes provide near about 2/3 rd of naturally occurring antibiotics, and many of them have great medical significance.A study shows that approximately 82% of the worlds' antibiotics are known to come from soil origin (Bobbarala, 2012).Antibiotic resistance in pathogens is the major threat for the medical world these days.These antibiotic resistant microorganisms are known as multidrug resistant strains, that is, multidrugresistant strains (MDRS).Comparative studies show that emergence of MDRs is faster than the search of new drug and antibiotics.Continuous, improper and over usage of antibiotics/drugs is the main reason behind the *Corresponding author.E-mail: ekta.menghani@jecrcu.edu.in.Tel: +91 9829275441.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License evolution of MDRs.The pathogens get adapted, change their metabolism, alter the morphology accordingly, and prepare themselves against antibiotic mode of action.Due to this reason, new and untouched habitats attract scientists as well as pharmaceutical industries for the microbial strains isolation (Raghunath, 2008;Maragakis et al., 2008;Loomba et al., 2010) It is also noted in the past years that gram negative antibiotic resistant pathogens are spreading in community.These pathogens are threatening patients in hospitals and communities with multi-drug resistance, also incorporate resistance to first, second and third generations of penicillin's and cephalosporin's (Urban et al., 2003) Antibiotic resistance in pathogens is the major threat for the medical world these days.These antibiotic resistant microorganisms are known as multidrug resistant, that is, MDRs.Comparative studies show that emergence of MDR strains is faster than the search of new drug and antibiotics (Memish et al., 2012).Continuous, improper and over usage of antibiotics/drugs is the main reason behind the evolution of MDRs.The pathogens get adapted, change their metabolism, alter the morphology accordingly, and prepare themselves against antibiotic mode of action.Due to this reason, new and untouched habitats attract scientists as well as research and development departments of pharmaceutical industries for the isolation and screening of microorganisms for novel antibiotic production (Zoran et al., 2010).
Nosocomial infections (NI) are one of the most critical issues faced by clinicians and practitioners in daily health practices, while dealing with the severely ill patients.In India NIs are becoming a burden for the patient's health as well as for the country's economy, along with prolonged hospitalization time, increased morbidity and mortality.There has been prolonged research on infections associated with clinics and hospitals in India and several concerning facts have been revealed (Emori and Gaynes, 1993).
Exploitation of microorganisms for one beneficial product to another is increasing day by day, and there is a need of constant effort in the field of medical research for finding novel significant microbes.It is still necessary to find novel bioactive compounds of microorganism origin against MDR strains causing Nosocomial or any other sort of infection.Improved methods and strategies should be used to study the uncommon and less studied class of microorganisms for production of effective antibiotics, with reproducible strains known to produce bioactive metabolites.
Therefore, exploration of semi arid microflora for isolation of strains with potential antimicrobial metabolites.The aim of present investigations was isolation of strains from different soils samples of Jaipur District against MDR and Nosocomial infections of clinical prevalence.

Study location and experiment design
This prospective study was undertaken in Department of Microbiology, JECRC University, Jaipur, India some experiments were also performed at S.P. Institute of Biotechnology and Department of Microbiology, SMS Medical College, Jaipur, India during a period of 8 months (August 2013 to April 2014).GC-MS analysis was performed at Advanced Instrumentation Research Facility (AIRF), School of Physical Sciences (SPS), Jawaharlal Nehru University, New Delhi, India.
A total of four samples of Mesophilic strains were collected from soil of Jaipur region and five Nosocomial infection causing pathogens (NICP/ test pathogens) were acquired from SMS Medical College and Hospital, Jaipur, India.Isolates were obtained mesophilic soil and biological activity was tested against NICP.Compounds responsible for antibiotic activity were identified at AIRF and similarity was match with drug library.

Sample collection
Soil samples were collected in sterile air tight plastic bags by sterile showel from four locations of Jaipur, Rajasthan, that is, slum area of jawahar circle, SMS hospital, Durgapura and Mansarovar.The samples collected were named as S-I, S-II, S-III and S-IV, respectively.Selection of site was also based on various factors, temperature of the particular area, population load around the area, presence of any organic or inorganic additive/contaminant to the soil of the particular region, ease of repetition of sample collection, ease of accessibility of the area.Soil was collected from 4 cm deep each of these locations.It was then brought to the laboratories and stored at 4°C till further processing.

Isolation of microorganisms
Suspension of all the four soil samples were prepared and followed by serial dilution method.Spread plate technique on nutrient agar plates was used for the isolation of microorganisms.Serial tenfold dilution of mesophilic soil samples were spread on sterile nutrient agar and incubated at 36°C for 24 h.Total 29 isolates were obtained out of which only six were morphologically dominant which were selected for further study.

Identification
Selected six isolated of microbial strains were performed for gram staining.Out of the six isolates, four isolates were gram positive, confirmed by gram staining.All the isolates were assigned a specific number according to sampling location, that is, S-I (Jawahar Circle) S-II (SMS) S-III (Durgapura) S-IV (Mansarovar) and according the isolated strain A, B, C,D.All the isolates were identified by some biochemical tests and microscopic morphological studies and assessment by Bergy's manual.Isolates were checked for the presence of enzyme like catalase, urease, oxidase and hydrolase by qualitatively standard methods.

Fermentation
The colonies screened from the dilutions of soil samples were inoculated in Luria broth media.Shaker treatment for 2 h daily at 700 rpm was provided for 30 days at different temperature ranges.Regular testing of metabolites (primary/secondary) was done after

Isolation of mixture containing bioactive compounds
After 27 days of incubation, each culture was centrifuged at 8000 rpm for 10 min and supernatant was collected separately.Metabolites were extracted using three solvents, that is, benzene, ethyl acetate and chloroform.Supernatant was mixed in 2:1 ratio with each of the three solvents, shaken and allowed to mix properly.
The mixture was left undisturbed to allow the separation of the solvent having the dissolved metabolites from the culture.The solvent was then decanted from the culture and allowed to vaporize at 40 to 50°C in the oven.The method used for separating bioactive compounds from extracellular secondary metabolites was liquidliquid extraction.Further, these extractives were named as LLE-I, LLE-II and LLE-III for benzene, ethyl acetate and chloroform respectively.

Antimicrobial analysis
The dried form of compounds collected was again mixed in 2 to 3 ml of respective solvents.Sterilized circular discs were cut and soaked in solvents containing bioactive compounds.Lawn of five common nosocomial infection causing pathogens, that is, Staphylococcus aureus, Klebsiella pneumonia, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli was prepared on nutrient agar.The antimicrobial activity was then tested against these pathogens using disc diffusion method (Saadoun et al., 2008).Plates were incubated at 38°C for 48 h.

Centrifugation
Isolates kept for fermentation were centrifuged at 8000 rpm for 10 min.Supernatants of S-IA, S-IIA and S-III C were performed for liquid extraction with benzene, ethyl acetate and chloroform as LLE-I, LLE-II and LLE-III respectively and processed for antimicrobial screening and three active rich fractions were collected and stored at 4°C for further studies.

Thin layer chromatography (TLC)
Glass plates of 18 cm ×18 cm were used to perform TLC, so that approximately 4 samples could be run together.Slurry was made with silica gel and water.Mixing and shaking of silica gel in water should be proper for homogenous and adhesive mixture.Thin layer on glass plates were formed and kept for 3 to 4 h on the plain surface for drying and later in the oven at 70°C.The temperature of the oven was raised to 110°C for 1 h for activation of the plates.Taken the plates out and allowed to cool.Mobile phase (solvent) prepared and poured in TLC glass chamber in which plates spotted with extract were placed.The plates were placed in the chamber till it run or develop upto ¾ of the TLC plate and then again kept in oven for drying.Plates were sprayed with specific reagents and spots were observed.The developed plate was baked at 110°C for 30 min and observed under UV light chamber and the displacement of development (mobile phase) and extract were measured and recorded (Sahin and Ugur, 2003).

GC-MS analysis
Shimadzu model QP-2010 plus, column-Rtx -Ms, 30 m × 0.25 mm i.d.× 0.25 µm film thickness was used for detection.Samples were prepared accordingly for analysis.Extracts were collected in 100 ml beaker and mixed with methanol.Mixture was filtered properly to remove any crystal particle.Homogenous solution was collected in ependrof after testing it with microinjection.Samples were loaded in injector and processed.Chromatograms with compounds detected in solvent were recorded and compared with compound library (Ceylan et al., 2008).

RESULTS
Isolation, selection and biochemical testingwere performed for primary screening of microorganisms.Main objective is to find a compound which can be useful in manufacturing drug against pathogens which causing nosocomial infections.After plating methods, microbes were selected on the basis of their growth pattern and morphological features.29 colonies were targeted as different among all the colonies.These 29 colonies were further sub-cultured to isolate as pure and for further   5.All the selected strains were performed for biochemical test and microscopic morphological studies (Table 6) as per Bergey's manual.Isolates were checked for the presence of enzyme like catalase, urease, oxidase and hydrolase by qualitatively standard methods (Table 2).For secondary screening, the above mentioned 3 isolates were processed through fermentation process with regular shaker treatment for a specific duration and different incubation period and temperature.
Centrifugation followed by extraction with solvents benzene, ethyl acetate and chloroform as LLE-I, LLE-II and LLE-III, respectively (Table 3).Above strategies and tests confirmed that the isolates S-IA, S-IIA and S-IIIC are having the compounds which need to be targeted.For detailed information the compounds were processed through thin layer chromatography (TLC) (Table 4), contact microbial autobiography and GC-MS (Figures 2 to 4).
GC -MS study elaborated every bit of information about the compounds extracted from isolates, like molecular weight, RT time, boiling point, structural formula, linear formula, IUPAC name.The results are given in Tables 7, 8 and 9 for S-I A, S-II A and S-III C, respectively.Applications and medical significance of every compound was analyzed with chem library (Ceylan et al., 2008).
GC -MS analysis of S-IA, S-II A and S-III C revealed that these samples possessed seven, seven and two antibacterial compounds, respectively (Table 5).Chromatograms showed the number of detected peaks in each solvent.The RT of the Peaks reveals the presence of different compounds.The area covered by the peaks is

Inhibits phagocytosis Antibacterial
directly proportional to the amount of compound present in the solvent.The peaks were selected and compared with standard which automatically generated the list of compounds.From the consortium of compounds detected and we focused on the ones which could be responsible for antibacterial, antimicrobial, and antifungal activities.S-IA showed seven prominent compounds with potentials antibacterial activity (Table 7).1H-pyrrole show anti-inflammatory and antitumor activity, and inhibits retroviral reverse transcriptase activity.Similarly, cyclobutene-3,4-dione, 2-piperidinone, perhydroquinolin-4-yl ester, silane, pentacosane, palmitic acid compounds show antifungal activities.Heneicosane.heptadecane, nonacosane, pentadecane, pentacosane, tricisane and  8).There were two common attributes: all the compounds are aliphatic hydrocarbons, and all possess antibacterial activities.The later makes them responsible for acting against nosocomial infection causing pathogens.The samples contained variable amounts of extracts.Heneicosane, tricosane and heptadecane are the major constituents among all biological active compounds found in the extracts.Three of the compounds that showed antimicrobial and anti-bacterial activities, which were found in S -III C sample (Table 9), also have larvicidal, asnitiseptic and chemo-therapeutic activities.All these facts make the isolate to fight Nosocomial infection causing pathogens.Dibutyl phthalate is colorless to faint yellow oily liquid, Heneicosane is aromatic compound and 1-Heptacosanol is of fatty acid nature.From the experimental results we can concluded that the detected compounds in active rich fractions of S-IA, S-II and S-III C were responsible for bioactivity against infection caused by nosocomial pathogens.

DISCUSSION
Total 29 microbial strains were isolated from different locations of Rajasthan for screening of antimicrobial efficacy against Nosocomial infections causing pathogens.These isolates were performed for antimicrobial screening.Out of which, three were selected for present studies possessing antimicrobial potentials.Further, these selected strains were examined for morphological and biochemical test using Bergeys manual which indicates that these are gram positive bacteria and belong to genus Bacillus (Shirling and Gottlieb,966).All the three isolates showed appreciable efficacy.S-IA, S-IIA and S-IIIC showed biological activity against selected test pathogens E. coli and P. aeruginosa.Further, inhibition zones were calculated in S-IA (IZ = 11 mm; AI = 1.5);S-II A (IZ = 42 mm; AI = 4.2) and S-III C (IZ = 12 mm; AI = 1.2) (Figure 1).
Further, studies reveals that active rich fractions showed antimicrobial efficacy against gram negative bacteria and it is noteworthy to mention that the activity index is more than one.The inhibition zone of one of them is higher than 40 mm against P. aeruginosa.Pseudomonadaceae has received huge attention in last year's due to its role as pathogenic microorganisms in an increasing number of clinical syndromes (Robin and Janda, 1996).
This pathogen cause various infections and diseases in  clinical areas such as bacteremia, infections of the respiratory and urinary tracts, skin and soft tissue infections, biliary tract infections, conjectivitis, endocarditis, serious wound infections meningitis (Fisher et al., 1981;Denton and Kerr, 1998) cystic fibrosis and central nervous system infections.Pseudomonsa sp. has also been noted as an important nosocomial pathogen (Denton and Kerr, 1998).The treatment of infections caused by this microorganism is difficult because P. aeruginosa is frequently resistant to almost all the widely used antibiotics (Vartivarian et al., 1994;Liu et al., 1995;Skaehill, 2000;Krueger et al., 2001).Therefore, production of antibiotics from isolated strains can be further used for large scale production of antimicrobial compounds especially against Nosocomial infections causing pathogens.
The compounds are Dibutyl phthalate (6.95%), Heneicosane and 1-Heptacosanol (19.12%).Thus, high burden of multidrug resistant pathogens in the current world there is increasing interest for searching of effective antibiotics from soil microflora from diversified ecological niches (Rajasekar et al., 2012).All the seventeen compounds possess antibacterial, antifungal and antioxidant activity.Antibiotics are important bioactive compounds for the cure of infectious disease.Due to emerging of multiple drug resistant, it is difficult to cure

Figure 1 .
Figure 1.Clear zones showing antimicrobial activity against lawn of test pathogens.

Figure 2 .
Figure 2. Chromatogram showing peaks of extract S -I A detected in GC-MS analysis.

Figure 3 .
Figure 3. Chromatogram showing peaks of extract S -II A detected in GC-MS analysis.

Figure 4 .
Figure 4. Chromatogram showing peaks of extract S -III C detected in GC-MS analysis.

Table 4 .
TLC Rf value and color of spot.

Table 6 .
Morphological features and Gram staining.

Table 7 .
Name , retention time (RT), molecular formula, molecular weight, peak area %, structure, nature and biological activity of the compounds present in sample S -I A.RT Name

Table 8 .
Name, retention time (RT), molecular formula, molecular weight, peak area %, structure, nature and biological activity of the compounds present in sample S -II A.

Table 9 .
Name, retention time (RT), molecular formula, molecular weight, peak area %, structure, nature and biological activity of the compounds present in sample S -III C.
hexadecane were detected in sample S-IIA (Table