Isolation and identification of beta-lactam producing microorganisms using PCR based methodologies

Krallis, Myrsini (1997) Isolation and identification of beta-lactam producing microorganisms using PCR based methodologies. Masters thesis, Rhodes University.

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The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for β-lactam producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (IPNS) gene in S. clavuligerus, S. tanashiensis, S. grise us, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the IPNS probe revealed the presence of lPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known β-lactam producing organisms; one unidentified soil isolate; and two non-j3-lactam producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The IPNS gene was also detected in a β-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profIles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of β-lactam producers and in the elimination of duplicate strains.

Item Type:Thesis (Masters)
Uncontrolled Keywords:Beta lactum antibiotics, Bacterial genetics, Fungi, Polymerase chain reaction, Microbial enzymes
Subjects:Q Science > QD Chemistry
Divisions:Faculty > Faculty of Science > Biochemistry, Microbiology & Biotechnology
Supervisors:Kirby, Ralph
ID Code:3317
Deposited By: Mrs Carol Perold
Deposited On:06 Sep 2012 10:08
Last Modified:06 Sep 2012 10:08
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