Detection of Antibiotic-Producing Streptomyces Inhabiting Forest Soils

Abstract

A modified agar overlay technique was developed for detecting antibiotic production by Streptomyces isolates and compared with two routinely used overlay procedures and the shake flask-disk assay method. Two types of inhibitory patterns could be discerned in the modified overlay method, and one of these was always exhibited by Streptomyces isolates which produced extractable antimicrobial substances. In a preliminary screening of 600 isolates, higher numbers of antibiotic-producing streptomyces were detected with the modified overlay procedure than with the streak or pour overlay method and the shake flask assay.The modified overlay distinguished between antibiotic production and other antagonistic effects. The inhibitory agents extracted after 6 days of culture growth on the modified overlay agar were of a higher concentration and more stable over longer periods of time than those recovered from shake flasks. It was observed that acidoduric streptomyces produced a greater proportion of antibiotics against gram-negative bacteria than did neutrophilic cultures, and this effect was greatest with the isolates from the most acidic soils. The neutrophiles were more active against gram-positive bacteria and, upon antibiotic classification, it appeared that the neutrophilic and acidoduric cultures from various soils produced different antimicrobial compounds.There are two widely used methods for examining Streptomyces isolates for antibiotic production. The first (7, 14) involves placing the test organism and the potential antagonist on an agar surface a specified distance apart, whereas in the second (3, 13) the streptomyces is grown on a basal agar and then the test organism is overlaid on top of the antagonist. Problems with these methods include obtaining a medium suitable for the growth of both antagonist and test organism and using a medium which enhances antibiotic production while differentiating between antibiotic activity and other antagonistic effects. Hsu and Lockwood (6) demonstrated that clear zones of inhibition may result from nutrient deprivation as well as antibiotic production, and they indicated that the only certain method for differentiating between the two effects was by preparing an extract from the inhibitory zone in the agar and examining it for retention of antimicrobial activity. In addition, inhibition can be caused by an alteration in the pH of the overlay layer and has been shown to be dependent upon both the composition and quantity of the overlay agar (9).After preliminary screening has detected iso-

Impact of interspecific interactions on antimicrobial activity among soil bacteria

“…For detection of antimicrobial activity, an agar overlay assay was performed on the 4th day of incubation (Nkanga and Hagedorn, 1978 ). The two target organisms E. coli WA321 and S. aureus 533R4 were grown overnight in liquid LB broth at 37°C, 220 rpm.…”

Suppression of Sensitivity to Drugs and Antibiotics by High External Cation Concentrations in Fission Yeast

“…Under natural conditions, S . pombe must compete for resources with other micro-organisms including lactic acid bacteria (LAB), non-LAB bacteria, other fungi and yeasts [ 6 , 7 ]. LAB produce lactic acid as a byproduct of fermentation.…”

“…LAB produce lactic acid as a byproduct of fermentation. They also produce bacteriocins (peptide antimicrobials) and other bacteriostatic molecules [ 5 – 7 , 15 ]. These substances are believed to confer competitive advantages to LAB in their natural environment [ 6 ].…”

“…Additionally S . pombe must also be able to resist the potential effects of yeast killer toxins [ 16 , 17 ], acetic acid [ 7 ] and antibiotics produced by non-LAB strains (e.g. Streptomyces spp .)…”

Mining into interspecific bacterial interactions

“…For detection of antimicrobial activity, an agar overlay assay was performed on the 4th day of incubation (Nkanga and Hagedorn, 1978). The two target organisms Escherichia coli WA321 and Staphylococcus aureus 533R4 were grown overnight in liquid LB broth at 37 °C, 220 rpm.…”