Single Cell Level Survey on Heterogenic Glycopeptide and β-Lactams Resistance

Abstract

Antibiotic Resistant Bacteria-A Continuous Challenge in the New Millennium been found in E. faecalis. Vancomycin resistance is usually determined by one of the two related gene clusters, vanA and vanB, that encode a dehydrogenase (VanH or VanHB) and ligase (VanA or VanB). Expression of VanA-and VanB-type resistance is regulated by the VanRS and VanRBSB two-component regulatory systems, respectively (Arthur et al., 1992; Evers and Courvalin, 1996). www.intechopen.com Single Cell Level Survey on Heterogenic Glicopeptide and -Lactams Resistance 2. Material and methods The study was performed on reference and clinical strains listed in Tab 1. Fourteen MRSA strains were isolated from patients of various wards of the University Clinical Centre in Gdańsk and 12 Enterococcus faecalis from patients of Kościerzyna Medical Centre. The staphylococcal strains were isolated in the hospital laboratory during the period March 2008 to March 2009 and were sent to the Microbiology Department of the Medical University of Gdańsk for epidemiological typing. One isolate per patient was included in the study. Bacteria were cultured from swabs: wound (4 isolates), nose (2 isolates) and fluids: tracheostomy tube fluid (3 isolates), blood (1 isolate), abscess fluid (1 isolate), pus (1 isolate), bronchial fluid (1 isolate), urine (1 isolate). The data about clinical recognitions were not available. The isolates were cultured on sheep blood agar and were identified as S.aureus by colony morphology, a positive plasma coagulase reaction and by biochemical tests (API, bioMerieux, France). Resistance to methicillin was primary tested using disc diffusion method with cefoxitin disc (Clinical and Laboratory Standards Institute,2006) and then was confirmed by detection of mec A gene by the Polymerase Chain Reaction (PCR) as described previously (Barski et al., 1996). DNA of bacterial isolates was extracted according to the previous report (Barski et al., 1996). For further analyses, the isolates were subcultured on nutrient broth and stored with glycerol at-70°C. Spa typing and BURP analyses. The polymorphic X region of the protein A gene (spa) was amplified from the isolates by PCR with primers and according to procedure described by Kobayashi (1999). Spa types were determined by using Ridom Staph Type software, according to Harmsen et al (2003). The spa types were clustered into spa-CCs (clonal complexes) using the algorithm BURP-based upon repeat pattern (Rupptisch et al., 2006). Phage typing was performed according to Blair and Williams (1961). Two sets of phages were used as follows: a basic set of 23 phages with additional phages 88,89,187 and an additional set of phages MR8, MR12, MR25, 30, 33, 38, M3, M5, 622, 56B supplied by the Central Public Health Laboratory in London for use on MRSA strains (Richardson et al., 1999). The phages were used in concentrations at routine test dilution (RTD) and 100xRTD. Reactions for phages were noted as described by Blair and Williams (1961). The phage type was defined by all the phages with strong reacti...

Changes of PBP5 Gene Expression in Enterococcal Isolates from Renal Transplantation Recipients

“…Because of analyzing many more cells than by conventional methods, rare cell types are more likely to be detected and the results are accessible to statistical analysis. By combining flow cytometry and FISH technique the Flow-FISH one may obtain excellent method for detection of gene expression [5]. …”