• Subject objective: Each student should be able to

• To isolate antibiotic-producing microorganisms (Bacillus, Penicillium and Actinomycetes colonies) from soil that may be antibiotic producers.

• To determine the spectrum of the antimicrobial activity of the isolated antibiotic against some clinical important bacteria (Staphylococcus epidermidis as a Gram positive and Escherichia coli as a Gram-negative bacteria).

• Biological assay for screening of antimicrobial activity.• Reporter screening

Principle:• The antibacterial effect of penicillin was discovered by Alexander Fleming in

1929. He noted that a fungal colony had grown as a contaminant on an agar plate streaked with the bacterium Staphylococcus aureus, and that the bacterial colonies around the fungus were transparent, because their cells were lysing. Fleming had devoted much of his career to finding methods for treating wound infections, and immediately recognised the importance of a fungal metabolite that might be used to control bacteria. The substance was named penicillin, because the fungal contaminant was identified as Penicillium notatum. (See Figure A) Fleming found that it was effective against many Gram positive bacteria in laboratory conditions, and he even used locally applied, crude preparations of this substance, from culture filtrates, to control eye infections.

• The phenomenal success of penicillin led to the search for other antibiotic-producing microorganisms, especially from soil environments. One of the early successes (1943) was the discovery of streptomycin from a soil actinomycete, Streptomyces griseus. actinomycetes are bacteria that produce branching filaments rather like fungal hyphae, but only about 1 micrometer diameter. They also produce large numbers of dry, powdery spores from their aerial hyphae. Actinomycetes, especially Streptomyces species, have yielded most of the antibiotics used in clinical medicine today. Some examples are shown in the table (1).

• Other bacteria, including Bacillus species (see Figure E: Agar plate showing inhibition of fungal growth by a contaminating colony of Bacillus species.), have yielded few useful antibiotics. Fungi also have yielded few useful antibiotics. Apart from penicillin, the most important antibiotics from fungi are the cephalosporins (beta-lactams with similar mode of action to penicillin, but with less allergenicity).

Figure E. The constant search of soils throughout the world has yielded an abundance of antibiotics of great value for the treatment of many infectious diseases. Pharmaceutical companies are in constant search for new strains of bacteria, molds, and Actinomyces that can be used for antibiotic production. Although many organisms in soil produce antibiotics, only a small portion of new antibiotics are suitable for medical use. In this experiment an attempt will be made to isolate an antibiotic-producing Bacillus, Actinomyces and Penicillium from soil. Students will work in group. Figure 1 illustrates the procedure.

• Antimicrobial agents: are substances that are naturally produced by a variety of microorganisms (primarily Actinomycetes, fungi and bacteria), or have been synthesized in the laboratory, or a combination of both.

• Antibiotic: refers only to those antimicrobial substances produced by microorganisms, but the term is often used interchangeably with antimicrobial agent. Antimicrobial agents have inhibitory or lethal effects on many pathogenic organisms (especially bacteria) that cause infectious diseases.

• Antibiotic producer such as:Antibiotics are the best known products of actinomycete. Over 5000 antibiotics have

been identified from the culture of gram positive, gram negative organisms and filamentous fungi, but only100 antibiotics have been commercially used to treat human, animal and plant disease. The genus Streptomycete is responsible for the formation of more than 60% of known antibiotics. While further 15% are made by number of related Actinomycete, Micromonospora, Actinomadura, Streptoverticillium and Thermoactinomycetes

1. Streptomyces spp.: produce (Chloramphenicol, Erythromycin, Kanamycin, Neomycin, Nystatin, Rifampin, Streptomycin, Tetracyclines, Vancomycin)

2. Micromonospora: produce (Gentamicin)3. Bacillus:Produce (Bacitracin, polymxins)4. Fungi:• Penicillium griseofulvum: produce (Griseofulvin)• Cephalosporium: produce Cephalosporins

Some clinically important antibiotics

AntibioticProducer organismActivitySite or mode of

action

PenicillinPenicillium chrysogenumGram-positive bacteriaWall synthesis

CephalosporinCephalosporium acremoniumBroad spectrumWall synthesis

GriseofulvinPenicillium griseofulvumDermatophytic fungiMicrotubules

BacitracinBacillus subtilisGram-positive bacteriaWall synthesis

Polymyxin BBacillus polymyxaGram-negative bacteriaCell membrane

Amphotericin BStreptomyces nodosusFungiCell membrane

ErythromycinStreptomyces erythreusGram-positive bacteriaProtein synthesis

NeomycinStreptomyces fradiaeBroad spectrumProtein synthesis

StreptomycinStreptomyces griseusGram-negative bacteriaProtein synthesis

TetracyclineStreptomyces rimosusBroad spectrumProtein synthesis

VancomycinStreptomyces orientalisGram-positive bacteriaProtein synthesis

GentamicinMicromonospora purpureaBroad spectrumProtein synthesis

RifamycinStreptomyces mediterraneiTuberculosisProtein synthesis

• Why the few antibiotics are clinically useful?

• Several hundreds of compounds with antibiotic activity have been isolated from microorganisms over the years, but only a few of them are clinically useful. The reason for this is that only compounds with selective toxicity can be used clinically - they must be highly effective against a microorganism but have minimal toxicity to humans. In practice, this is expressed in terms of the therapeutic index - the ratio of the toxic dose to the therapeutic dose. The larger the index, the better is its therapeutic value. So the antibacterial product should be assessed by pharma and then decide to put in the market when it passes ADME/T test

• It will be seen from the table above, that most of the antibacterial agents act on bacterial wall synthesis or protein synthesis. Peptidoglycan is one of the major wall targets because it is found only in bacteria. Some of the other compounds target bacterial protein synthesis, because bacterial ribosomes (termed 70S ribosomes) are different from the ribosomes (80S) of humans and other eukaryotic organisms. Similarly, the one antifungal agent shown in the table (griseofulvin) binds specifically to the tubulin proteins that make up the microtubules of fungal cells; these tubulins are somewhat different from the tubulins of humans.

• Factors affecting antibiotic production:

1. Medium Composition: • Carbon source• Nitrogen source• Inorganic phosphates• Inorganic salts• Trace metals• Precursors• Inhibitors• Inducers

2. Fermentation Conditions:• pH• Temperature • Oxygen

• How can determine the target of inhibitor molecule which may inhibit one of the biological pathways?

•There are many different pathwaies can be applied such as reporter essay by using the reporter strains

Reporter screening

Reporter strain

This lab. Consist of three steps

Primary isolation Colony selection

&Inoculation

Evidence of antibiosis&

Confirmation

FIRST STEP:• (Primary Isolation)Unless the organisms in a soil sample are thinned out sufficiently, the isolation of potential

antibiotic producers is nearly impossible.

Materials per group of students:1. six large test tubes, one bottle of physiological saline solution2. Three Petri plates of glycerol yeast extract agar, Tryptic soy agar, Sabouraud

dextrose agar.3. L-shaped glass rod, beaker of alcohol4. six 1 ml pipettes, one 10 ml pipette

• Procedure: 1. Label six test tubes, and with a 10 ml pipette, dispense 9 ml of saline into each tube.2. Weigh out 1 g of soil and deposit it into tube 1.3. Vortex mix tube 1 until all soil is well dispersed throughout the tube.4. Make a tenfold dilution from tube 1 through tube 6 by transferring 1 ml from tube to

tube. Use a fresh pipette for each transfer and be sure to pipette-mix thoroughly before each transfer.

5. Label three Petri plates with your initials and the dilutions to be deposited into them.6. From each of the last three tubes transfer 1 ml to a plate of glycerol yeast extract agar.7. Spread the organisms over the agar surfaces on each plate with an L-shaped glass rod

that has been sterilized each time in alcohol and open flame. Be sure to cool rod before using.

8. Incubate the plates at 30° C for 7 days.

Figure 1

• SECOND STEP(Colony Selection and Inoculation)• The objective in this laboratory period will be to select Bacillus, Penicillium and Actinomyces-

like colonies that may be antibiotic producers. The organisms Penicillium sp and Actinomyces will be streaked on nutrient agar plates that have been seeded with Staphylococcus epidermidis, and Bacillus will be streaked on nutrient agar plates that have been streaked firstly by fungi, after incubation we will look for evidence of antibiosis. Students will continue to work in groups. Figure 2 illustrates the procedure.

• Materials per group of students:1. four trypticase soy agar pours (liquefied)2. four sterile Petri plates3. TSB culture of Staphylococcus epidermidis, Bacillus firmus, and Penicillium sp.4. 1 ml pipette5. three primary isolate plates from previous period water bath at student station (50° C)

Procedure: 1. Place four liquefied agar pours in water bath (50°C) to prevent solidification, and then inoculate

each one with 1 ml of S. epidermidis.2. Label the Petri plates with your initials and date.3. Pour the contents of each inoculated tube into Petri plates. Allow agar to cool and solidify.4. Examine the three primary isolation plates for the presence of Bacillus sp. Penicillium sp. and

Actinomyces-like colonies. Actinomyces have a dusty appearance due to the presence of spores. They may be white or colored. Your instructor will assist in the selection of colonies.

5. Using a sterile inoculating needle, scrape spores from Penicillium sp. and Actinomyces-like colonies on the primary isolation plates to inoculate the seeded TSA plates. Use inoculums from a different colony for each of the four plates.

6. Incubate the plates at 30° C until the next laboratory period.

• THIRD AND FOURTH STEPS(Evidence of Antibiosis and Confirmation)

• Examine the four plates you streaked during the last laboratory period. If you see evidence of antibiosis (inhibition of S. epidermidis growth and Fungal growth), proceed as follows to confirm results.

• Materials:1. 3 Petri plates of trypticase soy agar2. TSB culture of S. epidermidis, PDA culture of Penicillium and Aspergillus

sp.

• Procedure:1. If antibiosis is present for each of Actinomyces, Penicillium, Bacillus, use

three TSA plates and make two streaks on each of the TSA plates as shown in figure 2. Make a straight line streak from (antibiotic producer microorganisms)

2. cross-streak with organisms from a culture of S. epidermidis and Aspergillus sp.

3. Incubate at 30° C until the next period.

Figure 2