Objective: To study the genomic organization of vancomycin resistance in a local isolate of vancomycin resistant Staphylococcus aureus (VRSA).
Study design: Experimental study.
Place and duration of study: Department of Microbiology, University of Karachi, January 2008 through December 2010.
Methodology: A vancomycin-resistant Staphylococcus aureus (VRSA-CP2) isolate (MIC 16 μg/ml) was isolated from a local hospital of Karachi. Species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc gene. The vancomycin MIC was re-confirmed by E-test. For the genetic determination of vancomycin resistance, in-vitro amplification of vanA cassette was performed by using plasmid DNA of CP2, CP2's transformant as template on MWG Thermo-Cycler. Amplified products of vanR, vanS, vanH, vanA, vanY, orf2, orf1D, orf2E, orf-Rev and IS element genes were subjected to Sanger's electrophoresis based sequence determination using specific primers. The Basic Local Alignment Search Tool (BLAST) algorithm was used to identify sequences in GenBank with similarities to the vanA cassette genes.
Results: The vancomycin-resistant isolate CP2 was found to be resistant to oxacillin, chloramphenicol, erythromycin, rifampicin, gentamicin, tetracycline and ciprofloxacin, as well. The isolate CP2 revealed four bands: one of large molecular size ~56.4 kb and three of small size ~6.5 kb, ~6.1 kb and ~1.5 kb by agarose gel electrophoresis indicating the presence of 3 plasmids. The plasmid DNA of isolate CP2 was analyzed by PCR for the presence of the van cassettes with each of the vanA , vanB and vanC specific primers. It carried vanA cassette, which comprises of vanR, vanS, vanH, vanA, vanY, and orf2. The vanA cassette of isolate CP2 also carried an insertion element (IS). However, it did not show the PCR product for orf1. Vancomycin resistance was successfully transferred from the donor CP2 to a vancomycin-sensitive recipient S. aureus. The MIC of vancomycin for the transformant was 16 μg/ml, similar to the parent isolate CP2. Nucleotide sequencing of the PCR product showed similarity with van genes of enterococci and other VRSA reported from different parts of the world.
Conclusion: Sequence of vanA cassette of CP2 showed partial homology with vancomycin resistant enterococci, VRSA vanA cassette element recorded in gene bank NCBI.