MLST Types of Vancomycin-Resistant Enterococcus faecium Strains Isolated from Blood Cultures

Abstract

Enterococci, particularly vancomycin-resistant enterococci (VRE), are important nosocomial pathogens with limited treatment options. Enterococci have low-level resistance to penicillins and aminoglycosides and are intrinsically resistant to cephalosporins. In addition, they can acquire high-level resistance to beta-lactam antibiotics, aminoglycosides and glycopeptides. The aim of this study was to determine glycopeptide resistance mechanisms and genetic relationships of vancomycin-resistant E.faecium strains isolated from blood cultures between 2003-2009 years by molecular epidemiologic methods. A total of 38 VRE strains isolated from blood cultures were included in this study. The isolates were identified by conventional methods and Phoenix 100 BD automated system (Becton Dickinson Diagnostic Systems, USA) and confirmed by sequence analysis of 16S rRNA amplicons. Antibiotic susceptibility tests were performed by the Kirby-Bauer disk diffusion method accor-ding to the CLSI standards. MIC values of vancomycin were determined in vancomycin resistant strains by E-test (AB Biodisk, Sweden) method. Vancomycin resistance genes included vanA, vanB, vanC, and vanD were investigated by polymerase chain reaction (PCR) method. Clonal relationship between strains was determined by pulsed-field gel electrophoresis (PFGE). Sequence analysis was performed for examples selected for multilocus sequence typing (MLST) of each pulsotype and subtype. Thirty eight strains of enterococci isolated from blood cultures were defined as E.faecium by phenotypic methods and confirmed by 16S rRNA sequence analysis. Vancomycin MIC values of strains were determined as > 256 µg/ml by E test. The vanA gene was detected in all isolates. Clonal relationship of 38 isolates E.faecium carrying the vanA gene was determined by PFGE and MLST methods. PFGE detected four pulsotypes (A-D) and one sporadic isolate. Twenty nine strains belonged to A pulsotype, three strains belonged to B pulsotype, two strains belonged to C pulsotype and three strains belonged to D pulsotype. Out of 29 isolates, eight strains were type A1, nine strains were type A2, six strains were type A3, two strains were type A4 and four strains were type A5. MLST identified four different sequence types (STs). Twenty nine A pulsotype and its subtypes belonged to ST117 (76.3%), three B pulsotype belonged to ST280 (7.9%), two C pulsotype belonged to ST18 (5.2%) and three D pulsotype belonged to ST17 (7.9%). In conclusion, bloodstream infections caused by VRE in our hospital arose from a dominant strain belonged to ST117. However, presence of different pulsotypes of this strain indicated that the strain had been present in the hospital for a long time and had accumulated genetic variations. In addition, infections caused by minor pulsotypes were also detected. Therefore for prevention and control of the spread of nosocomial infections caused by VRE, it is crucial to identify resistance patterns and clonal relationship of these organisms.