Molecular epidemiology of clinical Enterococcus faecium

Abstract: Enterococci are today the third most commonly isolated species from bloodstream infections, and problems with ampicillin and vancomycin resistance are increasing. Some putative virulence factors such as the enterococcal surface protein (Esp) and hyaluronidase (Hyl) have been described in Enterococcus faecium. Multilocus sequence typing (MLST) has revealed the existence of a genetic lineage, denoted clonal complex 17 (CC17), associated with hospital outbreaks and nosocomial infections worldwide. The general aim of the present study was to investigate if blood-stream infections caused by E. faecium were of endogenous origin or the result of nosocomial transmission, and also to determine the conjugation ability, presence of virulence determinants, antibiotic resistance, and to clarify if the globally spread CC17 occurred in Sweden. A total of 596 clinical E. faecium isolates consecutively collected during year 2000 to 2007 were included. All isolates originated from a blood-culture collection at the Karolinska University Hospital Huddinge, Sweden, deriving from hospitalized patients with blood-stream infections in the southern part of Stockholm. The conjugation ability was tested using in vitro filter mating. The genetic relatedness between isolates was analyzed using pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number tandem repeat analysis (MLVA) and MLST. Presence of virulence genes was determined with polymerase chain reaction (PCR). Antibiotic susceptibility to seven different antibiotics was also investigated. The vanA gene cluster was frequently transferred to E. faecium isolates in vitro. The conjugation frequencies among isolates harbouring esp were significantly higher compared to esp-negative isolates. Nearly half (43%) of the patients included were involved in cross-transmission events and the infecting strains disseminated both within and between all hospital categories. Clinical ampicillin susceptible isolates and normal microflora isolates displayed a similar and higher level of genetic diversity compared to esp-positive isolates. The esp gene was found in 56% of the isolates, hylfm in 4%, whilst the other virulence genes were only sporadically detected. The presence and spread of CC17 were found, which has not previously been described in Sweden. The most common MLVA type was MT-1 which was partly replaced in 2007 with MT-159 harbouring more antibiotic resistance and virulence determinants. The overall resistance to ampicillin increased from 68% to 86% whilst the levels of ciprofloxacin and imipenem resistance remained stable throughout the entire study period. In 2007, the resistance to gentamicin was significantly increased. The presence of esp significantly increased during the investigation period and 72% of the isolates harboured esp in 2007. The number of isolates harbouring hyl significantly increased during the study period, reaching a level of 35% in 2007. In conclusion, the E. faecium subpopulation belonging to the CC17 cluster, exhibiting enhanced properties for causing infections, are commonly occurring in the hospital environment in the Stockholm area, indicating a non endogenous origin for infection. The resistance to ampicillin and presence of the virulence genes esp and hyl increased markedly during the study period.