Isolation and identification of antibiotic producing microorganisms from natural habitats in the KwaZulu-Natal midlands.
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Date
2001
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Abstract
The search for new antibiotics continues in a rather overlooked hunting ground. In the course of
screening for new antibiotic-producing microorganisms, seventy-nine isolates showing
antimicrobial activity were isolated from soil samples from various habitats in the KwaZulu-Natal
midlands, South Africa. Existing methods of screening for antibiotic producers together
with some novel procedures were reviewed. Both modified agar-streak and agar-plug methods
were used in the primary screens. The use of selective isolation media, with or without antibiotic
incorporation and/or heat pretreatment, enhanced the development of certain actinomycete
colonies on the isolation plates. Winogradsky's nitrite medium (Winogradsky, 1949), M3 agar
(Rowbotham and Cross, 1977), and Kosmachev's medium (Kosmachev, 1960), were found to
be selective for actinomycetes. Statistical analysis showed highly significant interactions
between isolates, assay media and the test organisms. The diameters of inhibition zones were
found to be larger on Iso-sensitest agar (ISTA)[Oxoid, England] than in nutrient agar plates. Of
the 79 isolates that showed antimicrobial activity, 44 isolates were selected for confirmatory
screening. Of these, 13 were selected for secondary screening. Criteria for selection were based
on significant inhibition of at least two test organisms and/or the inhibition of the specifically
targeted organisms, Pseudomonas and Xanthomonas species. Following secondary screening
eight isolates were considered for further investigation. The isolates were tentatively identified .
on the basis of morphological features, using both light microscopy and scanning electron
microscopy(SEM); their ability to utilize various carbon sources; and selected physiological and
staining tests. Suspected actinomycetes were further characterized on the basis of selected
chemical properties using thin layer chromatography (TLC) and high pressure liquid
chromatography (HPLC) techniques. High pressure liquid chromatography analysis (Beckman
6300 analyzer) detected the presence of diaminopimelic acid (DAP) in whole-cell hydrolysates
of six of the isolates while TLC analysis confirmed the type ofDAP present. The isolates N2,
N12, N16, N19 and N35 were tentatively identified as Thermomonospora, Saccharopolyspora,
Nocardiodes, Corynebacterium and Promicromonospora, respectively. Isolate N30 was
identified as belonging to the coryneform group ofbacteria, possibly an Arthrobacter species.
Isolate, N8, tentatively identified as Actinosynnema, was unique among the isolates tested as it
showed good antimicrobial activity against all the Gram- positive and Gram-negative bacteria,
and yeasts used as test organisms in the present investigation.
Description
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.
Keywords
Anti-infective agents., Microbial metabolites., Antibiotics., Soil microbiology--Methodology., Theses--Microbiology.