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Molecular Screening Methods for the Determination of Vancomycin Resistance in Selective Competitive Exclusion Product CF3 (Preempt) Bacteria


1) To isolate, identify, and biochemically characterize vancomycin-resistant bacteria present in a commerially available competitive exclusion product CF3. 2) To develop a rapid PCR method of the detection of vancomycin-resistance determinant genes, namely, the VanAO, VanB, VanC and D-ala-D-lac ligase gene Ddl. 3) To characterize plasmid DNA Profile and plasmid-mediated drug resistance transfer. 4) To conduct genetic fingerprinting of the vancomycin-resistant microorganisms present in an Preempt culture. 5) To condut nucleotide sequence analysis of the PCR products of the vancomycin-resistant determinant genes showing interesting restriction profiles.

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FY 2000 Accomplishments: 1) Ten vancomycin-resistant bacteria belonging to genus lactobacillus were isolated from the competitive exclusion (CE) product Preempt. 2) Developed individual PCR assays for the detection of VanA, VanB, VanC1, VanC2 and VanC3 genes in ATCC control strains. 3) Developed multiplex-PCR assay to simultaneously detect VanA, VanB, VanC1, VanC2 and VanC3 genes in a single-reaction tube. This provides a fast detection procedure and also cuts down the cost of reagents used. 4) Developed PCR assay for the detection of ddlA/B genes in lactobacilli.

FY2001 Plans: 1) PCR products of vancomycin-resistance determinant genes will be digested with restriction enzymes and if different restriction profiles are obtained for the same gene, the nucleotide sequence analysis of the variants will be carried out. 2) Study the plasmid profile of the vancomycin-resistant bacteria isolated from the Competitive Exclusion Product. If any plasmids are detected, their ability to transfer the resistance markers to enterococcal, lactobacillus, and staphylococcal strains of the poultry and human origin will be tested. 3) Genetic fingerprinting and strain typing of vancomycin-resistant organisms isolated from the CE product will be carried out by pulse-field gel electrophoresis and molecular probing. 4) A collaborative research protocol with the FDA CVM on "The fate and degradation of antimicrobials, oxytetracycline (OTC), sulfadimethoxine-ormetoprim (Romet-30) from aquaculture environmental samples" will be intitiated.

Khan, Saeed
DHHS/FDA - National Center for Toxicological Research
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