ANTIBIOTIC PRODUCTION BY MICROBES ISOLATED FROM SOIL

ANTIBIOTIC PRODUCTION BY MICROBES ISOLATED FROM SOIL

Sonia Sethi*, Ravi Kumar and Saksham Gupta

Dr. B. Lal Institute of Biotechnology, Malviya Nagar, Jaipur-302017, Rajasthan, India

ABSTRACT: Antibiotic is one of the most important commercially exploited secondary metabolites produced by bacteria, fungi and Streptomyces and employed in a wide range. Most of the antibiotics used today are from the microbes. Bacteria are easy to isolate, culture, maintain and to improve their strain. Bacillus species being the predominant soil bacteria because of their resistant endospore formation and production of vital antibiotic like polymyxin, bacitracin etc. are always found inhibiting the growth of the other organisms. In the present research study, screening of bacteria, fungi and Streptomyces with potential antibiotic activity was carried out. Among the microbes isolated and identified, Bacillus subtilis, Penicillium chrysogenum and Streptomyces spwere selected on the basis of their anti-bacterial activity. The inhibitory activities of the isolated microorganisms were checked against Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Klebsiella pneumonia (ATCC 4352). It was observed that Penicillium chrysogenum metabolites showed maximum antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia with a zone of inhibition of 17mm, 11mm, 19.8mm and 8.2mm respectively. Bacillus subtilis metabolites showed activity against Staphylococcus aureus and Pseudomonas aeruginosa measured as zone of inhibition of 13.4mm and 13.8mm respectively whereas Streptomyces sp showed minimum activity against all the four tested organisms.

Antibiotic, Bacitracin, Bacillus, Penicillium, Streptomyces, Antimicrobial activity

INTRODUCTION:Antibiotics, in one form or another, have been in use for centuries. The vast majority of novel antibiotics have been detected by screening of “wild isolates” obtained from soil and other natural habitats. Although a wide taxonomic range of microbes have the ability to produce antibiotics. Thus, over 55% of the antibiotics detected between 1945 and 1978 originated from the genus Streptomyces, representing a total of more than 5,000 compounds ¹.

With advances in organic chemistry many antibiotics are now also obtained by chemical synthesis, such as the sulfa drugs. Drugs used in the chemotherapy of infectious diseases are classified into two groups. Drugs that have been synthesized by chemical procedures in the laboratory are called synthetic drugs while those produced by bacteria and fungi are called antibiotics ².

The antibiotics are widely distributed in the nature, where they play an important role in regulating the microbial population of soil, water, sewage, and compost.

Of the several hundred naturally produced antibiotics that have been purified, only a few have been sufficiently non-toxic to be of use in medical practice.

Those that are currently of greatest use have derived from a relatively small group of microorganisms belonging to the genera Penicillium, Streptomyces, Cephalosporium, Micomonospora and Bacillus ³.

Antibiotics are low molecular-weight (non-protein) molecules produced as secondary metabolites, mainly by microorganisms that live in the soil. While many  antibiotics  are  known  to  exist,  efforts  to discover  new  antibiotics  still  continue.  Therefore, many species such as Streptomyces, Bacillus and Penicillium have been studied continuously for their ability to produce antibiotics ⁴.

In addition, due to the fact that Bacillus species have produced antibiotics in the  soluble  protein  structure  and  that  these  antibiotics have  been  found  to  be  cheaper  and  more  effective  in studies  conducted  to  date,  these  microorganisms  are preferable for commercial production.

Currently, the target is to produce antibiotics such as polymyxin and bacitracin from Bacillus ^{5, 6}.  It  was reported  that members  of  the  species  Bacillus  generally produced  polypeptide type  bacteriocines,  and  that  these antibiotics generally affect Gram positive bacteria ^{7, 8}. The apparent increase of the occurrence of antibiotic resistance among bacteria during the past years and its possible implication in public health has led to an intensified surveillance of bacterial resistance in many countries.

Treatment of infectious diseases caused by pathogenic bacterial and fungal strains was one of the most traditional problems in the clinical field ^{9, 10}. This necessity encouraged the investigators to synthesize novel and more potent inhibitory compounds (like azoles and quinolones derivatives) ^{11, 12} to fight them. However, the adverse effects and also appearance of bacterial or fungal resistances persuaded the investigators to study on natural products from microorganisms or herbal extracts to discover novel and safe lead compounds ^{9, 10}.

It was not until 1940 with the discovery of penicillin, the first, best-known and most widely used antibiotic ^{13, 14} in 1928 by an English Bacteriologist, late Sir Alexander Fleming that the first clinical trials of penicillin were tried on humans. This antibiotic was obtained from a blue green mould of the soil called Penicillium notatum.

Penicillin was discovered accidentally in 1928 by Fleming, who showed its efficacy in laboratory cultures against many disease producing bacteria. This discovery marked the beginning of the development of antibacterial compounds produced by living organisms.

Another antibiotic, streptomycin was isolated in 1944 by Waksman, a Microbiologist, from a species of soil bacteria, called Streptomyces griseus, particularly tubercle bacilli, and has proved to be very valuable against tuberculosis. A vigorous search for more antibiotics was on at this time and in 1947, another antibiotic, chloromycetin was discovered by Burkholder ^{14, 15}. It was isolated from S. venezuelae. It has a powerful action on a wide range of infectious bacteria both Gram positive and Gram negative.

Most of the peptide antibiotics produced by Bacillus are active against Gram positive bacteria ¹⁶. However, compounds such as polymyxin, colistin, and circulin exhibit activity almost exclusively upon Gram-negative forms, whereas bacillomycin, mycobacillin, and fungistatin are effective agents against molds and yeasts ¹⁷. As more antibiotics were discovered, designed and studied, scientists found that they had different properties. Some of these properties include their source, range of activity and their kinds. These were used to classify those ¹⁴.

The objective of the present study is production, extraction and assay of antimicrobial metabolites from bacterial, fungal and Streptomyces isolates using soil as source.

MATERIAL AND METHODS:

Isolation and screening of microbial isolates:In the present study, soil sprinkle technique was used to isolate antibiotic producing bacilli. For this purpose about 20-30 particles of soil were sprinkled on the surface of nutrient agar plates seeded with the test organism one Gram positive Staphylococcus aureus (ATCC 29213) and three Gram-negative Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumonia (ATCC 4352). The plates were incubated at 30°C for 24 hours. Antibiotic activity was checked by zone of inhibition, surrounding a colony.

Different colonies having zones of inhibition were picked and streaked on separate nutrient agar plates to get pure cultures. These isolates were used as the source of antibiotic producing microbes. All strains were stored at 4°C and subcultured periodically.

The soil fungi were isolated by both the Direct Soil Inoculation and the Soil Dilution Techniques using the pour plate method. The Media used for the isolation were Potato Dextrose Agar (PDA). Plates were incubated for 5 days at 28˚C.  Pure cultures of fungal isolates were identified using both macroscopic (cultural) and microscopic (morpho-logical) features with reference to Barnett and Hunter¹⁸.

Each fungal isolate was streaked on Nutrient Agar as a straight line and incubated at 30˚C. After two days of incubation, the test organisms, one gram positive Staphylococcus aureus (ATCC 29213) and three gram-negative Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumonia (ATCC 4352) were streaked perpendicular to the streaked line of the growing fungus.

This was then incubated at 37˚C for 24 hours, after which the zone of inhibition of each test organism from the streaked line of the growing fungus was measured.

For  Streptomyces,  1g  of  the  soil  were suspended in 100 ml of physiological water (NaCl 8.5  g/1)  then  incubated  in  an  orbital  shaker incubator at 28 ⁰ C with shaking at 200 rpm for 30 min. Mixtures were  allowed  to  settle,  and  serial dilutions  up  to  10^-5 were  prepared  using  sterile physiological water  and  agitated with  the  vortex at maximum speed. An aliquot of 0.1 ml of each dilution  from  10^-2 to 10^-5 was  taken  and  spread evenly  over  the  surface  of  starch  casein  agar  medium.

The  media  are  added  to  antibiotic to  inhibit  bacterial  and  fungal  contamination,  respectively. Plates  were  incubated  at  28^o  and  37^oC  and monitored  after  48,  72,  and  96  h.  Repeated streaking  on  starch  casein  agar  plates  led  to purify  bacterial  colonies  that  showed  an actinomycetes  like  appearance. The isolated strains are preserved at 4^oC during two months and maintained for longer period by serial subculture.

 Identification of antibiotic producing soil microbes:Isolated bacterial strain was identified morphologically (shape, gram staining, spore staining, spore shape, sporangium dilatation and motility) and biochemically (sugar utilization,  starch utilization, casein hydrolysis, indole production, citrate utilization, methyl red-voges proskauer (MR-VP), oxidase production, catalase production, nitrate reduction, gas production from glucose according to the Bergey’s Manual of Determinative Bacteriology ¹⁹.

Pure cultures of fungal isolates were identified using both macroscopic (cultural) and microscopic (morphological) features with reference to Barnett and Hunter¹⁸.

For  Streptomyces,  a medium containing  10 g of yeast extract per liter and 10 g of glucose per liter and pH 7.2 (yeast  extract-glucose medium) was used  for general cultivation  of  actinomycetes strains. The media used for morphological characterization of strain were those described by Shirling and Gottlieb²⁰. Cultural characteristics of cells in various media were recorded after incubation for 14 days at 30°C and morphological observations were made with a light microscope by using the method of Shirling and Gottlieb²⁰.

Inoculum preparation:The inoculum of Bacillus species, identified as Bacillus subtilis, selected on the basis of maximum activity against the test organisms, was prepared in Tryptic Soy broth (pH7.3) at concentration of 10% (v/v) by incubating at 30°C for 72 hours.

The fungus was sub-cultured on PDA plates and incubated at 30 ⁰C for 3-5 days to obtain the spores used for antibiotics production. Spores were washed into a sterile beaker using 0.1% Tween 80 in 0.1M potassium phosphate buffer at pH 7.0. The spore suspension was standardized such that 1 in 10 dilutions has an absorbance of 0.48 at 530nm.

The inoculum of Streptomyces specie, selected on the basis of maximum activity against the test organisms, was prepared in Streptomyces growth medium was used at concentration of 10% (v/v) by incubating at 28°C for 4 days.

 Production medium for antimicrobial compounds:

1. For Bacteria: About 50 ml of the Synthetic medium (g/L); L-glutamic acid 5.0; KH₂PO₄ 0.5; K₂HPO₄ 0.5; MgSO₄.7H₂O 0.2; MnSO₄.H₂O 0.01; NaCl 0.01; FeSO₄.7H₂O 0.01; CuSO₄.7H₂O 0.01; CaCl₂.2H2O 0.015; Glucose 10; and pH 7), was taken in 250 ml flask as production medium. After sterilization of synthetic media concentrated glucose solution previously sterilized by 0.2 μm pore size filter paper, was added to give a final concentration of 1% in the medium.
2. For Penicillium: About 50 ml of the Synthetic medium (g/L); 6.0g Ammonium acetate, 0.5g NaSO₃, 0.02g ZnSO₄.7H₂O, 0.25g MgSO₄.7H₂O, 6.0g KH₂PO₄, 0.02g FeSO₄.7H₂O, 0.5g Phenylalanine and 10.0g carbon source (lactose) (pH-6.0) was taken in 250 ml flask used as production medium. The flasks were incubated with intermittent shaking for 21 days after which the contents were sieved through cotton wool and filter to remove cells.
3. For Streptomyces: About 50 ml of the Synthetic medium (g/L); 40g Sucrose, 12.8g H₃C₆H₅O₇.H₂O, 6.0g (NH₄)₂SO₄, 0.25g MgSO₄.7H₂O, 0.15g K₂HPO₄, 11g CaCO₃,0.25g MnSO₄.4H₂O, 0.04g ZnSO₄.7H₂O, 0.016 g K₂Cr₂O₇was taken in 250 ml flask used as production medium. After sterilization of synthetic media then add 5% mycelium inoculums. And incubate at 28^oC with intermittent shaking (200 rpm) for 6-7 days.

Antibiotic production condition and Purification:Shake flask fermentation method was used for bacterial antibiotic production. Seventy two hours old inoculum was prepared in Tryptic Soy Broth (pH 7.3) at concentration of 10% (v/v). Inoculum was added to the production medium and incubated for 24 hours in orbital shaker at 120 rpm. After incubation, culture was centrifuged to get cell free supernatant and was further used for antimicrobial activity.

The purification of penicillin from the production media began with filtration of the broth. In the first stage, large solids and microbial cells were separated by filtration, as filtration is the most versatile method for removing the insoluble from the broth.

Penicillin rich aqueous broth was treated with activated charcoal to remove pigments and impurities. After filtration and carbon treatment, penicillin recovery was done by liquid-liquid extraction (solvent extraction). Penicillin was extracted from an aqueous phase into the solvent butyl acetate. Potassium ions were added to precipitate the salt of penicillin and this was further used for antimicrobial activity.

The purification of antibiotic from the production media of Streptomyces began with filtration of the broth. In the first stage, large solids and microbial cells were separated by filtration, as filtration is the most versatile method for removing the in soluble from the broth and cell free filtrate was further used for antimicrobial activity.

Antimicrobial activity by Agar diffusion assay:Agar well diffusion method was used to check the cultures for the production of antimicrobial metabolites. Twenty-four hours fresh cultures of one Gram positive Staphylococcus aureus (ATCC 29213) and three Gram-negative Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumonia (ATCC 4352) were diluted with pre-sterilized normal saline. A sterilized cotton swab was dipped in the diluted cultures and lawns were prepared over the agar surface.

Wells were made in the inoculated plates using sterile corn borer. About 80 μl cell free supernatants were added in the wells and the plates were incubated at 37ºC for 24 hours. After 24 hours, the zones of inhibition were observed. The diameter of the zone of inhibition was measured in mm with well size of 6mm.

RESULTS AND DISCUSSION:

Isolation, Screening and Identification of microbial isolates:By using soil sprinkle technique, five different bacterial colonies having zones of inhibition were picked and streaked on separate nutrient agar plates to get pure cultures. Out of 5 bacterial isolates only one bacterial isolate showing maximum inhibition zone was selected and identified as Bacillus subtilis (Table 1).

TABLE 1: MORPHOLOGICAL AND BIOCHEMICAL TESTS FOR THE IDENTIFICATION OF BACILLUS SUBTILIS

By using soil dilution technique three different fungal cultures having zones of inhibition were picked and streaked on separate potato dextrose agar plates to get pure cultures. Out of three fungal strains, one isolate showing maximum inhibition zone was selected and characterized as Penicillium chrysogenum by its shiny blue green mold surrounded by white mycelium on potato dextrose agar. Such colonies were visualized under light microscope using phenol blue to confirm the mycelium and conidia of the fungi.

By using soil sprinkle technique seventeen different Streptomyces colonies were obtained. But five species having zones of inhibition were picked and streaked on separate starch casein agar plates to get pure cultures. Out of five species only one Streptomyces species (Isolate 3) showing maximum inhibition zone was selected (Table 2).

TABLE 2: MORPHOLOGICAL IDENTIFICATION OF STREPTOMYCES BASED ON PIGMENTATION CHARACTERISTICS

 Antimicrobial activity by Agar diffusion assay: Samples drawn during batch fermentations were subjected to agar well diffusion assay, using Staphylococcus aureus as test organisms. Antimicrobial activity was measured in terms of zone of inhibition. The incubated samples were evaluated and optimum antimicrobial activity of inoculum of Bacilius species was ensured at 48hours.

It was observed that Penicillium chrysogenum metabolites showed maximum antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia with a zone of inhibition of 17mm, 11mm, 19.8mm and 8.2mm while Bacillus metabolites showed activity against Staphylococcus aureus and Pseudomonas florescens measured as zone of inhibition of 13.4mm and 13.8mm and Streptomyces sp showed minimum activity against all the four tested organisms with inhibition zone of 7.2mm, 7.5mm, 10.6mm and no zone was observed in  case of Klebsiella pneumonia (Table 3).

Schallmey ²¹, isolated soil bacteria which shows antibiotic activity under normal growth condition and was found inhibiting some gram positive and some gram negative organism both  Bacillus lentus and Bacillus alvei also shows antibacterial activity against Staphylococcus aureus. Bacillus pumillus only show slight zone of inhibition on Proteus spp while it is inactive against others.

The culture extracts of P. chrysogenum exhibited significant antibacterial effects against isolates. The results show that the organism produces antibiotics that are effective against the bacterial isolates.   This   is   consistent with   the   fact   that most   strains   of Penicillium   chrysogenum produce   β-lactam antibiotics, mainly penicillins²².

Out  of  22,500  microbial  bioactive  metabolites,  10,100  metabolites  are  produced  by Actinomycetes ²³. Streptomyces are especially prolific, producing around 80% of total antibiotic products²⁴.

The present study was focused on antimicrobial metabolites production from soil microbial isolates.  The strains showed good inhibition against all bacteria in cross streak method were selected.

 TABLE 3: ANTIMICROBIAL ACTIVITY OF MICROBIAL METABOLITES AGAINST TEST ORGANISMS

 CONCLUSION: In search of new antibiotics, relatively simple and rapid methods have been developed for screening microorganisms for antibiotic producing ability. Soil samples are commonly employed in the isolation of antibiotic producing organism.

The  detection  of  these  antagonistic  substances  revealed interesting properties that justify its importance and its study on the potential  application  in  biological  control  of  pathogenic microorganisms  and  spoilage  food.  For  these  reasons,  the biochemical  nature  and  the  best  conditions  to  production  of  the substances  studied  in  this  work  are  being  investigated  to  further purification experiments. Many  microorganisms  have  been  evaluated  for  the  production  of  antimicrobial  substance. However the high cost and low yields have been the main problem for its industrial production²⁵.

In the present study, the microbial isolates with antimicrobial activity from soil were isolated. Among all screened isolates, Penicillium chrysogenum metabolite showed maximum inhibition against both gram positive as well as gram negative bacteria. Production of antibiotic by microorganisms from soil is affected by many factors including nitrogen and carbon source. Therefore there is a great need to optimize with different substrates that provides maximum production of antimicrobial substance.

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    How to cite this article:

    Sethi S, Kumar R and Gupta S: Antibiotic production by Microbes isolated from Soil. Int J Pharm Sci Res 2013: 4(8); 2967-2973. doi: 10.13040/IJPSR. 0975-8232.4(8).2967-73

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