Actinomycetes isolation, Study Guides, Projects, Research of Pharmaceutical Microbiology

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Name : Xolani Paulos

Surname : Mfundisi

Student number : 219087865

Course : Biotechnology

Course code : ABBPPA3A

Experiment :Project 1

Lab Instructor : Mr. N.Laloo

TITLE: Determination of antibiotic production from actinomycete

isolated from soil and plant material.

INTRODUCTION

Secondary metabolites are produced by some organisms such as bacteria, fungi, plants, actinomycetes and so forth. Among the various groups of organisms that have the capacity to produce such metabolites, the actinomycetes occupy a prominent place. Actinomycetes are prokaryotes of Gram- positive bacteria but are distinguished from other bacteria by their morphology, DNA rich in guanine plus cytosine (G+C) and nucleic acid sequencing and pairing studies. They are characterized by having a high G+C content (>55%) in their DNA. Actinomycetes are of universal occurrence in nature and are widely distributed in natural and man- made environments. They are found in large numbers in soils, fresh waters, lake, river bottoms, manures, composts and dust as well as on plant residues and food products. However, the diversity and distribution of actinomycetes that produce secondary metabolites can be determined by different physical, chemical and geographical factors. Actinomycetes provide many important bioactive substances that have high commercial value. Their ability to produce a variety of bioactive substances has been utilized in a comprehensive series of researches in numerous institutional and industrial laboratories. This has resulted in the isolation of certain agents, which have found application in combating a variety of human infections. That is why more than 70% of naturally occurring antibiotics have been isolated from different genus of actinomycetes. Out of these different genus, Streptomyces is the largest genus known for the production of many secondary metabolites, which have different biological activities, such as antibacterial, antifungal, antiparasitic, antitumor, anticancer and immunosuppressive actions. Some antibiotics like penicillin, erythromycin, and methicillin which used to be one-time effective treatment against infectious diseases, are now less effective because bacteria have become more resistant to such antibiotics. Antibiotic resistant pathogens such as methicillin and vancomycin resistant strains of Staphylococcus aureus ( S. aureus ) and others cause an enormous threat to the treatment of serious infections. To avoid this happening, immediate replacement of the existing antibiotic is necessary, and the development of novel drugs against drug resistant pathogens is significant for today. Thus, finding and producing new antibiotics as well as using combined antibiotic therapy have been shown to delay the emergency of microbial resistance and can also produce desirable synergistic effects in the treatment of microbial infection. Antibiotic synergisms between known antibiotics and bioactive extracts are a novel concept and have an important activity against pathogens and host cells. Research in finding newer antibiotics and increasing productivity of such agents has been a very important activity. This is because some important drugs are expensive and/or have side effect to the

Aim:

To determine some antibiotic producing actinomycete strains from soil and plant material isolates.

Objective:

1. To isolate antibiotic producing microorganisms from soil and plant rhizosphere that may be antibiotic producers
2. To determine spectrum for antimicrobial activity of the isolated antibiotic-producers against clinically important bacteria
3. To execute biological essay for screening of antimicrobial activity
4. To report screening

Materials and Method:

Part 1: Isolation of actinomycete from soil and plant material

Requirements:

o Dilution bottle containing 9ml of sterile saline o Soil o Leaves o Bacteriological agar or agar-agar o Petri dishes o Calcium carbonate, potassium dihydrogen phosphate, zinc sulphate and sodium chloride. o Gram stain reagents, slides and a microscope o Inoculation equipment and Bunsen burner

Procedure:

To prepare soil isolation agar, a soil supernatant was prepared first by boiling 100g of soil for 5 minutes in 600ml of distilled water and allowed to cool and settle.To 500ml of soil supernatant agar containing the following was added: o Calcium carbonate - 0,001g o Potassium dihydrogen phosphate - 0,25g o Manganese sulphate - 0,001g o Zinc sulphate - 0,001g

o Iron sulphate - 0,001g o Sodium chloride - 0,002g o Agar - 1,5g The mixture was boiled, poured into bottles and autoclaved, the poured the mixture into plates and stored in the refrigerator as required, To prepare the plant isolation agars, a 1L bottle was filled with plant leaves and boiled in 600ml of distilled water for 5 minutes and allowed to cool and settle then used the supernatant.

Isolation of soil organisms

1g of soil was diluted to 10^-7 in sterile saline and spread plated samples from 10^-3 to 10^-7 dilutions. The plates were incubated at 25˚C until growth is observed (after 3 days). Colonies were picked from surface of the plate, gram stained and identified morphologically under the microscope. Once it was determined that the organism in the colony is a gram positive (+ve) it was streaked out on a soil isolation agar plates and once a pure culture was obtained it was transferred aseptically to a soil isolation slant, incubated at 25˚C and maintained viable.

Isolation of plant organisms

Two plant isolation agar plates were prepared, the plant isolation agar plates were prepared by pressing a semi-dried leaf (37˚C for 1 day) into surface of the agar and plates were incubated at 25˚C for 2 days. Once growth was observed colonies were “picked” and gram stained to determine morphology. Once it was determined that the colony picked is a gram +ve, it was streaked out on other plant isolation agar to obtain a pure culture. Once a pure culture was obtained a colony of pure culture was transferred to a plant isolation slant, incubated at 25˚C and maintained.

Part 2: Giant colony technique

Requirements:

o Pure Actinomycete cultures isolated from soil and plant material o Nutrient agar plates o Test tubes containing nutrient broth for growing the test organism. o Test organism for evaluating antibiotic production: Klebsiella, E.coli, Staphylococcus, Proteus. o Dessicator o Chloroform

Procedure:

Two nutrient agar plates were inoculated with a single line of purified actinomycete culture down the center of the plate (Use loopful of organism). One plate for the soil isolate and the other for the plant

Figure 2: Plant isolation agar plate with colonies. Figure 3: purified soil sample isolated colonies. Figure 4: pure culture from plant isolation agar

Figure 5: Gram stain results from soil isolation at 1000X oil immersion objective Figure 6: Gram stain results from plant isolation agar at 1000x oil immersion under microscope

Part 2: Giant colony technique

All the isolates were found to be positive in gram staining and had different morphological structures. The biochemical properties of actinomycetes isolates were recorded. The isolates were screened for their inhibitory activity against the human pathogenic bacteria. Both the primary and secondary screening methods were used to screen the actinomycetes for antibacterial activity. The first screening was used to select the antibacterial isolates and determine the range of microorganisms that were sensitive to the antibiotics. The secondary screening method was crucial to select the isolates for further studies. The screening may be qualitative or quantitative in its approach. The qualitative approach is used to determine the range of the microorganisms that are sensitive to a potential antibiotic. The antibacterial activity of secondary metabolite extract of some isolates confirmed through secondary screening that were able to inhibit the extracellular growth of filaments in the test organism. Single colonies of the grown actinomycetes cultures were examined with the microscope, to detect diversity in appearance. Four indicator microorganisms ( Escherichia coli, Proteus, klebsilla, and Staphylococcus ), were spread plated as broth cultures on separate plates. actinomycetes were then deposited for each indicator organism, as described in the lab manual. All processes were performed aseptically, to avoid contamination. Some actinomycetes isolated showed antibiotic producing activity in primary screening. These were subjected to secondary screening and were analysed by cup plate method. From the results of primary and secondary screening we can say that more numbers of actinomycetes were active against gram positive bacteria ( Staphylococcus) than those against gram negative bacteria ( Escherichia coli). The reason behind this may be due to the presence of outer polysaccharide membrane carrying the structural lipopolysaccharide components in gram negative bacteria. This makes the cell wall impermeable to lipophilic solutes. The gram positive bacteria are more susceptible because it carries only outer peptidoglycan layer which is not an effective permeability barrier. Out of the isolates studied for secondary screening 75% isolates were active against Staphylococcus , 55% against E. coli.

Conclusion:

Antibiotics are the most important bioactive compounds for the treatment of infectious diseases. But now, because of the emergencies of multi-drug resistant pathogens, there are basic challenges for effective treatment of infectious diseases. Thus, due to the burden for high frequency of multidrug resistant pathogens in the world, there has been increasing interest for searching effective antibiotics from soil actinomycetes in diversified ecological niches.In the present study, the randomly selected soil and plant sample was teken from the University’s agricultural area for isolation of actinomycetes. The successful isolation of actinomycetes from environmental samples requires an understanding of the potential soil sample areas and environmental factors affecting their growth. Previous studies showed that selection of different potential areas such as rhizosphere soil samples were an important activity for isolation of different types of potent antibiotic producing soil actinomycetes. The finding of this study showed that the antimicrobial compound obtained from soil isolation agar plates has an antibacterial activity and also an antifungal activity. The data, in general, showed that the antimicrobial compounds obtained from soil samples demonstrate broad spectrum and a remarkable antimicrobial activity

against bacterial and Staphyloccocus. Actinomycetes isolates recovered from rhizosphere samples showed the potential to produce antimicrobial bioactive compounds. It is also suggested that the other isolates should be further processed to fully realize their antibiotic property on different test microorganisms. There is need for further studies to optimize the production conditions of the bioactive compounds from the potent actinomycetes isolates. (Assefa.F.2018)

References:

Bizuye.A., Moges.F,.Andualem.B. 2018. APJTD. Isolation and screening of antibiotic producing actinomycetes from soils. Ncbi.nlm.nih.gov/pml/articles/PMC70275/. 23 Apr 2020 WAssefa,F., Maleta, A. 2018. African journal of biotechnology. Isolation and screening of antibiotic producing actinomycete from rhizosphere and agricultural soils. Academicjournals.org/journal/AJB/article-full-text-pdf/7A5853857299. 23 Apr 2020 Sundaramoorthi C, Vengadesh PK, Gupta S, Karthick K, Tamilselvi N. Production and characterization of antibiotics from soil-isolated actinomycetes. Int Res J Pharm. 2011;2(4):114–118. 23 Apr 2020 Wadetwar RN and Patil AT: Isolation and characterization of Bioactive Actinomycetes from soil in and around Nagpur. Int J Pharm Sci Res 2013; 4(4); 1428-1433. 23 Apr 2020 Jeffrey, L.S. (2008) Isolation, characterization and identification of actinomycetes from agriculture soils at Semongok, Sarawak. Afr. J. Biotechnol., 7: 3697-3702. 23 Apr 2020 Wang Y, Zhang ZS, Ruar TS, Wang YM, Ali SM. Investigation of Actinomycetes diversity in the tropical rainforests of Singapore. J Ind Microbiol Biotechnol. 1999;23:178–87. 23 Apr 2020