Antibiotics play a large role in the medical field; without them, humans are unable to treat many microbial diseases that afflict us and animals.  A large percentage of these antibiotics are naturally produced by bacteria, such as Streptomyces, and fungi. There is the potential for these microbes to become resistant to these antibiotics, leading to more virulent strains. One way to combat this scenario maybe to find an antibiotic that can kill off these resistant microbes before they can evolve.

       Streptomyces is known to produce antibiotics and is commonly found in soil.  It does not require unusual nutrients or growth conditions to grow and sporulate.  Therefore, we hypothesized that by meeting these basic growth requirements, we would be able to isolate and enrich for pure  Streptomyces colonies, several of which would produce antibiotics. Streptomyces, a large contributor to the discovery of antibiotics, may give rise to a powerful strain that could be isolated and used to kill the most resistant microbes.

       We were successful in isolating many species of the genus Streptomyces from the soil. All samples of soil were collected from various parts of the University of Maryland’s College Park campus. For example, sand like soil obtained from near the stream and loose dirt from McKeldin Mall were isolated and subsequently tested. However, the soil yielding a Streptomyces species  was found around the construction site surrounding the Denton Community. The soil surrounding this site was to be used for planting purposes, either around shrubs or trees. Therefore, the soil was probably some type of purchased potting soil, or manufactured compost. Characteristics of the soil samples included a dark brown-black phenotypic color, which was accompanied by a strong manure-like odor.

       Minor adjustments were made after our first attempt at isolating our organism. References encouraged incubations at room temperature which is typically 25° Celsius. After incubating the samples at both temperatures our results confirmed that room temperature was lower than what was required for optimal growth. We observed optimal growth at 29° Celsius. Preliminary soil samples were air dried for at least eight hours. Plates were initially either sprinkled with the sample or spread using a serial dilution. The results from these plates favored the serial dilution method although large amounts of mold were found on both plates. To suppress mold growth soil samples were dried at 106° Celsius for at least 1.5 hours. Finally, cyclohexamine was added to starch-casein plates as an anti-fungal agent.

    After the various soil cultures were diluted and spread on starch-casein plates. The plates were incubated in a dark environment at 29° Celsius. Single colonies that exhibited similar characteristics to that which were identified in the book were selected and streaked for pure colonies. These colonies exhibited an opaque, rough, and non-spreading morphology. Colonies were extremely difficult to remove and produced a musty soil odor (Brock). Gram staining results of these individual colonies proved to be inconsistent after two separate trials. However, it did reveal the organism had filamentous branching of aerial mycelium, which is a very distinct characteristic structure of Streptomyces. MacConkey’s Agar was then used to confirm the Gram stain results. MacConkey’s Agar selects against gram-positive organisms.

    Motility stabs were conducted to test for non-motile spores that are characteristic of Streptomyces. The majority of the motility stabs provided clear results. One questionable tube revealed a distinct stab line interrupted at various spots with bursts of circular growth resembling a pom-pom. This growth could be the result of a mixed culture, which is strongly disfavored because the growth would be more confluent throughout the tube instead of looking “arranged”. Another explanation could be attributed to the distinct characteristics of Streptomyces. In liquid media, Streptomyces form mycelium that after prolonged growth, forms clumps that seem to branch out from the cells. (See figure 1). Therefore, the growth in the motility stab was considered to be consistent with previous results.

    The candidates were also tested for the presence of the enzyme catalase, which allows the organism to break down toxic products of oxygen. The positive results were coherent with the fact that the catalase enzyme is present in species of Streptomyces. Also, the candidates were tested for growth under anaerobic conditions. The majority of the candidates could not grow without oxygen. This result was consistent with the description of Streptomyces as aerobic organisms, however one candidate that exhibited antibiotic characteristics in a later test was shown to be anaerobic. The result of candidate A led us to consider two scenarios: Either candidate A is a facultative anaerobe or it is simply another filamentous organism that produces an antibiotic. The inability to conduct further tests limits our ability to commit to either conclusion.

         Sugar fermentation tubes were used in the absence of oxidation/fermentation tubes to test for an oxidative type of metabolism. The results showed that the candidates were all able to utilize glucose both in the presence and absence of oxygen. These results indicate that the candidates are facultative anaerobes. References state that Streptomyces are aerobic, however the absence of the word “strict” allows for the possibility that some species may be facultative anaerobes. Until further tests can be conducted such as thioglycolate tubes, oxidative/fermentation tubes, and agar deeps, it is unreasonable to use this data to exclude a candidate from the possibility of being labeled a Streptomycete.

     Streptomyces species produce antibiotics as a means of inhibiting the growth of other organisms that may compete with it for limiting resources, thereby increasing its rates of survival. “Close to 50% of all Streptomyces isolated have proved to be antibiotic producers” (Brock). The test conducted to test for antibiotic production revealed that two distinct species of Streptomyces had been isolated. Broad-spectrum antibiotics are capable of reacting on both gram positive and negative bacteria.  Examples of such antibiotics would include tetracycline, which is produced by Streptomyces aureofaciens. Macrolide antibiotics such as erythromycin are commonly used in place of penicillin, which is primarily active against gram-positive bacteria. Erythromycin is commonly produced by Streptomyces erythreus.

    Candidates A, B, G, H, and I were observed for antibiotic production in the presence of four different microorganisms. Three of the microorganisms were gram negative while one, Serratia marcenes, was gram positive. Candidates H and I showed antibiotic inhibitory effects on the growth of the gram-negative microorganisms. However, it was noted that candidate H had a stronger effect on the growth of these gram-negative microorganisms. These candidates along with A, B, and G all showed antibiotic inhibition on the growth of gram-positive microorganisms. The ability of candidates H and I to inhibit the growth of both gram negative and positive microorganisms led to the conclusion that these candidates were species of Streptomyces that produced broad-spectrum antibiotics. The most popular broad-spectrum antibiotic, tetracycline is produced by Streptomyces aureofaciens. Therefore, it is possible that H or I might be members of this species. Candidates A, B, and G could be members of Streptomyces erythreus because of their ability to inhibit gram-positive but not gram-negative microorganisms. More comprehensive tests would be needed in order to make a formal conclusion.

    We concluded that an antibiotic-producing species of Streptomyces was successfully isolated and enriched for from the environment. The exact species of Streptomyces that was isolated is unknown. The specific species could be determined with continued testing with the possibility of isolating an omnipotent antibiotic-producing strain of Streptomyces.