Setting and study design
Chang Gung Memorial Hospital at Keelung in Taiwan is a 1088-bed, tertiary-care, teaching hospital. The prevalence of vancomycin resistance among enterococci spp. in this hospital rose from 10% in 2003 to 30% in 2009, and it was most pronounced in the nephrology ward. A VRE outbreak was suspected in the nephrology ward because the prevalence rate, 30%, was higher than the average rate,16%, in Taiwan . This research plan was approved by the Human Trial and Ethics Committee of Chang Gung Memorial Hospital on December 24, 2008 (reference number 97-2117B). Between February 2010 and February 2011, a VRE surveillance study was conducted on both hemodialysis and non-hemodialysis inpatients in the nephrology ward of this hospital. Rectal swab cultures for VRE were collected from all nephrology inpatients during admission after patient consent was obtained. Colonization was defined as VRE isolation from rectal swabs in the absence of infection symptoms or signs. Infection was defined as VRE isolation from a sterile or non-sterile site along with the presentation of fever, leukocytosis, and other signs caused by the VRE.
All VRE-infected patients were confined to a single room or a double room with two beds for 2 VRE-infected patients of the same sex. All health-care workers (HCW) who administered care on VRE-infected patients were asked to follow infection control policies during patient care, including hand washing and glove and gown wearing when necessary . During this study period, active surveillance was performed on inpatients of the nephrology ward after patient consent was obtained. Further, within this study period, potential VRE specimens were collected both from HCWs after they provided consent and from the nephrology ward environment, including bedrails, pillows, tables, door handles, blood-pressure cuffs, ventilator monitor surfaces, and the surfaces of medical devices such as EKG monitors. All VRE strains isolated from inpatients, HCWs, and the environment were stored until needed for epidemiological and antibiotic susceptibility studies.
Identification of VRE
All rectal swabs were cultured on blood agar plates, which were examined after 48 h of incubation at 37°C. The colonies were identified as those of Enterococcus spp. based on known enterococcus characteristics, including the presence of gram-positive cocci, optochin resistance, bile-esculin color change to black, and growth in 6.5% sodium chloride (NaCl) [1, 2]. Specific enterococcus spp. were identified by differential utilization of arginine, sorbitol, arabinose, and raffinose and by the rapid 32 Strep kit test (bioMerieux Vitek Inc., Hazelwood, Missouri, USA) [1, 2]. VRE presence was confirmed by growth in brain heart infusion agar that contained 6 μg/mL vancomycin [13, 14].
Pulsed-field gel electrophoresis
For PFGE analysis, the isolates were inoculated into 5-mL nutrient broth and incubated for 3 h at 37°C with shaking to achieve exponential growth. Agarose plugs were prepared from the cultures, and within the plugs, the bacterial cells were lysed by proteinase K. Extracted genomic DNA was digested with the restriction endonuclease SmaI [15, 16]. The resulting restriction fragments were separated by PFGE using the MAPPER system (Bio-Rad Laboratory, Hercules, CA, USA). Band patterns were analyzed to determine clonal identity. Previously described criteria were used for the analysis of genomic DNA [15, 16].
Multilocus sequence typing
VRE isolates were typed by MLST. With the use of the Ibis T5000™ Biosensor System (Abbott, USA), we amplified 7 selected gene fragments that encode 7 housekeeping proteins by broad-range polymerase chain reaction (PCR). The base compositions of the amplicons were determined by electrospray ionization mass spectrometry. The base compositions of different target regions are shown by mass spectrometry and were used to create a signature that distinguished strains from one another .
Genotypic analysis of resistance pattern of VRE
To identify possible additional epidemiological markers, we investigated the presence of vanA, vanB, vanC1, and vanC2 genes by PCR. The PCR primer sequences were based on the published genes for Enterococcus faecalis, Enterococcus faecium, and Enterococcus gallinarum[18, 19].
The VRE isolate MICs for 8 antimicrobial agents, including daptomycin (Cubist Pharmaceuticals), fusidic acid (Leo), linezolid (Pfizer), mupirocin (GlaxoSmithKline), teicoplanin (Sanofi-Aventis), tigecycline (Pfizer), trimethoprim/sulfamethoxazole (Sandoz), and vancomycin (Eli Lilly), were determined by Etest (AB Biodisk, Solna, Sweden) according to the published guidelines [13, 14]. The MIC ranges for these antibiotics were as follows: daptomycin, 0.002–32 μg/mL; fusidic acid, 0.016–256 μg/mL; linezolid, 0.016–256 μg/mL; mupirocin, 0.064–1024 μg/mL; teicoplanin, 0.016–256 μg/mL; tigecycline, 0.016–256 μg/mL; trimethoprim/sulfamethoxazole, 0.002–32 μg/mL; and vancomycin, 0.016–256 μg/mL. As a control strain, E. faecalis ATCC 29212 was included with acceptable MIC limits according to CLSI M100-S19 (January 2009): daptomycin, 1–4 μg/mL; linezolid, 1–4 μg/mL; teicoplanin, 0.125–0.5 μg/mL; tigecycline, 0.03–0.12 μg/mL; and vancomycin, 1–4 μg/mL.