7 Advances in Environmental Biology, 7(1): 7-11, 2013 ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE

Corresponding Author

Ahmed, Risikat Nike, Department of Microbiology, University of Ilorin, Ilorin, Nigeria. E-mail : anrisikat @gmail.com, TEL: +2348063109301.

Soil Screening For Antibiotic - Producing Microrgansms 1Ahmed, Risikat Nike, 2Sani, Al Hassan,3Ajijolakewu, 4Alamu, folake bosede

1,2,3,4Department of Microbiology, University of Ilorin, Ilorin, Nigeria.

Ahmed, Risikat Nike, Sani, Al Hassan, Ajijolakewu, Alamu, folake bosede; Soil Screening For Antibiotic - Producing Microrgansms.

ABSTRACT Naturally soil is rich in microorganisms capable of antibiotic synthesis but the frequency with which synthesis occurs at ecologically significant levels in has been much less clear. Over the past decade, however, genetic and molecular techniques, have been applied to demonstrate conclusively that microorganisms synthesize a variety of antibiotics, even under field conditions, in the rhizosphere. Soil sample from the Post Graduate Hostel of the Permanent Site campus,University of Ilorin, Nigeria was screened for antibiotic-producing microorganisms by agar sensitivity assay. Seven bacterial species and one fungus were isolated. The bacterial species were identified by their cellular characteristics, colonial morphology and biochemical tests. The bacterial isolates include; Staphylococcus aureus, Proteus vulgaris, Bacillus spp., Pseudomonas aeruginosa, Micrococcus luteus, Escherichia coli and Micrococcus varians. The fungus was identified to be Rhizopus stolonifer using lactophenol in- cotton blue staining technique and possessed inhibitory action against test isolates. Only Bacillus spp exhibited antibacterial activity of all the bacteria isolated. Key words: Soil screening, Antibiotic production, Sensitivity. Introduction Technically defined, antibiotics include a chemically heterogeneous group of small organic molecules of microbial origin that, at low concentrations, are deleterious to the growth or metabolic activities of other microorganisms. Over the past decade, however, genetic and molecular techniques, have been applied to demonstrate conclusively that microorganisms synthesize a variety of antibiotics, even under field conditions, in the rhizosphere i.e. that portion of the soil enriched in carbon and energy resources released by plant roots. That Soil is rich in microorganisms capable of antibiotic synthesis is well accepted, but the frequency with which synthesis occurs at ecologically significant levels in nature has been much less clear [1]. Actinomycetes are best known for their ability to produce antibiotics and are gram positive bacteria which comprise a group of branching unicellular microorganisms. Among actinomycetes, the Streptomycetes are the dominant. Important species of bacteria known to produce antibiotics is the Bacillus spp. Over 5000 antibiotics have been identified from the culture of gram positive, gram negative organisms and filamentous fungi, but only a few have been commercially used to treat human, animal and plant

diseases. The genus Streptomycete is responsible for the formation of more than 60% of known antibiotics [7]. The trend of search for antibiotics in the past and in recent times as a result of drug resistance by microbial species has required combing the earth for various sources of antibiotics including the soil. Materials and Methods Sample Collection: The debris from the soil surface of the sampling sites was first removed before commencement of sampling. A hand trowel sterilized with 70% ethanol was used for the collection of soil samples from the front of Block C at the Post Graduate Hostel, University of Ilorin (Main Campus). The sample was collected in a sterile polythene bag and taken to the laboratory for analysis. Isolation of Microorganisms from Soil Sample: One gram of the soil sample was weighed and diluted serially up to 10-5 with distilled water. Dilutions of 10-2 and 10-4 were taken and introduced into differenly labelled sterile Petri-dishes. Unto the petri dishes, molten agar was added and swirled evenly to ensure homogeneity of the mixture and to discrete colonies. The plates were left to set for some

8 Adv. Environ. Biol., 7(1): 7-11, 2013

minutes and incubated at 370C for 24 hours for bacteria while in the case of fungi incubation was done at room for about 48-72 hours.Distinct colonies were selected from the cultured plates after incubation onto sterile culture media to obtain a subculture of each colony, using a sterile inoculating loop and inoculating needle for the bacterial species and fungal species respectively. Slants were prepared as stock cultures and kept in the refrigerator for further use. Characterization and Identification Of Bacterial/ Fungal Isolates: The characterization and subsequent identification of isolates were carried out based on colonial and microscopic observation according to Fawole and Oso [4]. Screening Of Soil Isolates For Antibiotic Activity: A sensitivity assay was carried out to determine the antibiotic producing ability of some of the isolates (Bacteria and fungi) against the other test organisms isolated. This technique was aimed at demonstrating the Alexander Flemings observation of the discovery of penicillin. For the bacterial sensitivity test, the isolate being screened for antibiotic production was streaked horizontally at a particular portion on sterile nutrient agar while the other organisms on which the activity was tested against were streaked vertically very close to the screened one. Plates were incubated for 24 hours at 370C ,during which any substance produced by the screened isolate was expected to lyse the tested organisms growing perpendicularly and produce zones of inhibition. For the fungal isolate, the Sabouraud dextrose agar plates were seeded with the test organisms to be inhibited on one end of the plate and the isolate to be screened was smeared nearby the other. The plates were sincubated at room temperature for 42-72 hours.

Results: In the screening of the soil sample taken from Post Graduate Hostel, University of Ilorin, Permanent Site, a total of Seven Bacterial species and a fungus specie were isolated employing the soil dilution plate technique. The bacterial isolates include; Staphylococcus aureus, Proteus vulgaris, Bacillus spp., Pseudomonas aeruginosa Micrococcus luteus, Escherichia coli and Micrococcus varians.. The result of the bacterial identification is presented in Table 1. The fungus Rhizopus stolonifer was identified based on cellular and structural morphology as shown in the list below. Characterization and Identification of Rhizopus stolonifer: Surface colour White Undersurface colour Yellow Filament colour White Colour of colour Black Shape of spore Circular The colonies on SDA were fluffy to cotton white at first, becoming grayish brown, spreading widely by means of aseptate stolons (completely filling the Petri-dishes within 36-48 hours). The mycelia appeared multinucleated and non-septate, sporangiophore erect, sporangia globose, shiny white at first then later turning black as spores matured. The sensitivities of the tested organisms to the screened bacterial isolates are presented in Table 2. Of all the screened isolates Bacillus spp. Was the only bacterial isolate that demonstrated antibiotic producing ability against the tested organisms, showing zones of inhibition around the colonies of two other tested bacteria. Table 3 shows the sensitivity test of the fungi to the screened fungus isolate. The sensitivity assays for both bacteria and fungi are illustrated in plates 1 and 2.

Plate 1: Clear zone around the colony of E. coli due to sensitivity to the surrounding Bacillus species. KEY:

9 Adv. Environ. Biol., 7(1): 7-11, 2013

A = Bacillus species (Screened isolate) B = Zone of inhibition around E. coli C = Pseudomonas aeroginosa D = Escherichia coli Table 1: Identification of Bacterial Isolates.

SHAPE

COLONIAL MORPHOLOGY

CELLULAR CHARACTERISTICS

BIOCHEMICA L TESTS

S/N

SHA

PE

SUR

FAC

E TE

XTU

RE

EDG

E

OPT

ICA

L C

HA

RA

CTEI

STIC

S

PIG

MEN

TATI

ON

ELEV

ATI

ON

CO

NSI

STEN

CY

GRA

MS

REA

CTI

ON

CEL

LULA

R

AR

RA

NG

EMN

T

MO

TILI

TY

CA

TALA

SE

CO

AG

ULA

SE

IND

OLE

CIT

RA

TE

STA

RC

HH

YD

RO

LYSI

SSP

OR

E ST

AIN

NIN

G

OX

IDA

SE

SUGAR FERMENTATION

IDENTIFIED ORGANISM G

LUC

OSE

SUC

RO

SE

LACT

OSE

1 Circular

Smooth

Entire

Opaque

Golden Yellow

Flat Butyrous

+ Clusters

_ + + + + + + + A A A Staphylococcus aureus

2 Irregular

Smooth

Undulate

Transparent

Pale

Convex

Butyrous

_ Single rods

+ + _ + + + _ + AG

AG

- Proteus vulgaris

3 Circular

Rough

Entire

Transparent

Yellowish

Raised

Granular

+

Rods

+ + _ _ + + + + A A A Bacillus spp.

4 Round

Rough

Entire

Transparent

Bluish Green

Flat Butyrous

+ Rods

+ + _ _ + + + + A AG

- Pseudomonas aeruginosa

5 Circular

Smooth

Entire

Opaque

Yellowish

Convex

Viscid

_

Clusters

_ + _ _ + + + + AG

AG

A Micrococcus luteus

6 Round

Glossy

Entire

Transpa=rent

Whitish

Raised

Butyrous

_ Rods

+ + _ + _ +

_ + AG

AG

AG

Escherichia coli

7 Irregular

Rough

Lobate

Transparent

Yellowish

Raised

Butyrous

+

Cluster

_ + _ + + + _ + AG

AG

A Micrococcus varians

KEY: + =Indicate Positive A=Acid Production -=Indicate Negative AG=Acid and Gas Production Table 2: Antibiotic activity of bacterial isolates from soil.

Screened Isolates

Sampled organisms to be inhibited Micrococcus luteus

Bacillus spp.

Escherichia coli

Staphylococcus aureus

Pseudomonas aeruginosa

Proteus vulgaris

Micrococcus varians

Staphylococcus aureus

_ _ _ _ _ _ _

Proteus vulgaris _ _ _ _ _ _ _ Bacillus spp. _ _ + + _ _ _ Pseudomonas aeruginosa

_ _ _ _ _ _ _

Micrococcus luteus

_ _ _ _ _ _ _

Escherichia coli

_ _ _ _ _ _ _

Micrococcus varians

_ _ _ _ _ _ _

KEY: + = inhibition, - = No inhibition.

10 Adv. Environ. Biol., 7(1): 7-11, 2013

Table 3: Antibiotics activity of fungal isolate from the soil by seeding technique. Screened Isolate

Sampled organisms to be inhibited Trichophyton rubrum Aspergillus niger Epidermophyton

floccosum Trichophyton mentagrophytes

Rhizopus stolonifer _ _ + _ KEYS: + = indicates Inhibition _ = indicates No inhibition

Plate 2: Inhibition of the growth of E. floccosum by R. stolonifer KEY A = Epidermophyton floccosum B = Zone of inhibition C = Rhizopus stolonifer Discussion: The antibiotic - producing microorganism isolated from the soil sample taken from the Post Graduate Hostel of the University of Ilorin, Permanent Site was Bacillus sp. of all the bacterial species originally isolated. The results conform to many reviews of literature that Bacillus spp. are known to produce bioactive secondary metabolites (antibiotic). According to Prescott et al. [6] spore forming bacteria and other members of the Bacillus genus possess genes for the catabolism of diverse carbon source and antibiotic synthesis. Muaz and Shahida, [5] in a research discovered the production of bacitracin and subtilin by Bacillus sp. Prescott et al., [6] reported that Bacitracin produced by Bacillus sp. inhibits Escherichia coli and Staphylococcus aureus which corroborated the result in the present study. The other Bacterial isolates including Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris, Micrococcus luteus, and Micrococcus varians were found to be incapable of exhibiting antibiotic activity against the various test organisms employed for antibiotic sensitivity testing. This may be due to the fact that these strains of bacterial species do not naturally have the tendency to produce antibiotic substances. No actinomycetes antibiotic- producing organism was isolated. This may have been due to reasons such as the textures of soil and other prevailing environmental activities at the location

where the sample was collect. Also, Actinomycetes will take a longer time to grow than the other bacteria as it has been reported by some literatures. This could possibly be the reason why Actinomycetes did not grow on the media used. The results of antibiotic sensitivity testing of the fungal isolate by seeding method (from table 2) revealed that Rhizopus stolonifer did not inhibit majority of the test organisms which include; Trichophyton rubrum, Aspergillus niger, and Trichophyton mentagrophytes which may be because these organisms are not susceptible to the antimicrobial substance produced by Rhizopus stolonifer . However, it exhibited inhibitory effect against Epidermophyton floccosum. This is in agreement with some previous literatures where Rhizopus spp were reported to exhibit amylo-lytotic and antibiotic activity including Rhizopus stolonifer. Prescott et al., [6] reported that Rhizopus secretes a toxin. It was discovered that the toxin was produced by Burkholderia which was found growing within the fungus although this association is yet to be clarified. Hence its inhibition of E. floccosum may be as result of its production of antibiotics or toxin to which E. floccosum is susceptible. In conclusion, the extracellular substance produced by Bacillus spp. or Rhizopus stolonifer may equally become potent and exhibit marked effect as antibiotics if harnessed and purified as those of the commercially available antibiotics

11 Adv. Environ. Biol., 7(1): 7-11, 2013

The discovery of antibiotics is one of the most useful discoveries in the field of science and particularly in the field of microbiology. This intense search for antibiotics has paid off over the past few decades because more of them have been discovered,though surprisingly only a relatively small number have been applicable to chemotherapy , because finding a new antimicrobial substance is only a first step in drug development. References 1. Brun, Y.V. and L.J. Skimkets, 2000.

Prokaryotic development. ASM Press, pp: 11-31.

2. Catherine Boisvert-Bertrand, 2005. Journal of Young Investigators: Searching for narrow spectrum antibiotics from microbes in soil from Presque Isle, Pennsylvania.

3. Egorov, N.S., 1985. Antibiotic: A Scientific Approach. Translated from the Russian by Alexander Rosinkin.MIR publisher, Moscow, 9(12): 30-158.

4. Fawole, M.O., B.A. Oso, 2004. Laboratory Manual of Microbiology. Spectrum Books LTD Publisher. pp: 16-17; 89.

5. Muaz Mutaz Al-Ajlani and Shahida Hasnain 2010. Bacteria Exhibiting Antimicrobial Activities; Screening for Antibiotics and the Associated Genetic Studies. The Open Conference Proceedings Journal, 1: 230-238.

6. Prescott, M.L., P.J. Harley and Klein, A.D. 2008. Microbiology 7th edition. Publishing Group; 42-51, 232-233, 762-764.

7. Williams, S.T., R. Locci, A. Beswick, D.I. Kurtboke, V.D Kuznetsov, F.J Lemonnior, P.F. Long, 1993. Detection and identification of novel actinomycetes. Research in Microbiology; 144(8): 653-656.