S230 17th ECCMID / 25th ICC, Posters Conclusions: 1) erm(B) was the most prevalent gene in our clinical isolates; 2) A bimodal distribution of ER MICs was noted in erm(A) isolales. 3) All clinical isolates resistant to TL harboured erm(B) gene. 4) Resistance to Q/D is common in our E. faecium. P887 Linezolid resistance in clinical isolates of Staphylococcus haemolyticus J. Calvo, M. Cano, M. Mar´ ın, E. Cercenado, L. Martinez-Martinez (Santander, Madrid, ES) Objectives: To characterise four isolates of linezolid-resistant S. haemolyticus obtained from different patients. Methods: Identification and preliminary susceptibility testing of organ- isms were performed with the WalkAway system. Linezolid MICs were determined by reference microdilution and Etest assays, according to CLSI guidelines and manufacturer’s instructions, respectively. Detection of the G2576T mutation was performed by PCR amplification and sequencing of the domain V of 23S rRNA gene (Tsiodras et al, Lancet 2001). Clonal relationship of the isolates was examined by PFGE of SmaI macrorestricted genomic fragments. Results: Four linezolid-resistant S. haemolyticus isolated in pure cultures from blood samples (n = 3) or bile (n = 1) were identified among 17 organisms of this species obtained in our laboratory in the period March-April 2006. All four isolates were from different patients admitted to ICUs of our hospital. Reference MICs of linezolid were 64 mg/L for 3 isolates and 128 for the other one. Etest yielded one twofold dilution lower in all isolates. The G2576T mutation was detected in all four isolates and all of them were homozygous. Similar resistance phenotype (WalkAway) was observed for all 4 isolates, being resistant to oxacillin, teicoplanin, ciprofloxacin, clindamycin, erythromycin, gen- tamicin, tobramycin, amikacin, rifampin, trimethoprim-sulfamethoxazol and susceptible to vancomycin, tetracycline and quinupristin-dalfopristin. Two closely related PFGE-patterns were observed for the 4 isolates, each one corresponding to two isolates. Conclusions: We have identified the emergence of multiresistant S. haemolyticus isolates presenting homozygous resistance to linezolid. P888 Macrolide efflux pumps mef(E) and mef(I) detected in clinical isolates of S. pyogenes isolated from PROTEKT 1999–2005 and PROTEKT US 1999–2004 J. Northwood, L. Cossins, M. Coley, A. Pantosti, M. Del Grosso, D. Farrell (London, UK; Rome, IT) Objectives: To determine the presence of the macrolide efflux mechanism of resistance, mef(A) subclass mef(E), in community- acquired lower respiratory tract infections of Streptococcus pyogenes, from a global collection of clinical isolates (n = 28,892) 1999–2005. Methods: Isolates of S. pyogenes with MIC values demonstrating the M phenotype of resistance to macrolides were screened for the mef(E) gene using a novel TaqMan TM assay designed for this project. Isolates which were positive for the presence of mef(E) using the TaqMan TM assay were confirmed by di-deoxy sequencing of a 300bp segment of the mef(E) gene. Results: Twenty-Two isolates (0.07%) were considered to be mef(E) although 3 isolates had 1 amino acid substitution from mef(E): M30I and 1 isolate had 3 amino acid substitutions from mef(E): C97V, M104I, and I105V (5 SNPs). These isolates may have additional substitutions outside of the 300 bp region examined, and further sequence analysis of the mef gene is required. Five isolates (0.02%) of S. pyogenes were consider to have the mef(A) subclass mef(I) gene present, all of these isolates had 1 amino acid substitution from mef(I): K39R. One isolate was considered to be a novel subclass of mef(A) due to the following variations: mef(A) 4 amino acid substitutions L52V, S89A, T92A and A125S; mef(E) 5 amino acid substitutions I59V, L60F, C97Y, I105V and A125S; mef(I) 4 amino acid substitutions L96F, V107I, V114I and A125S and mef(O) 3 amino acid substitutions L96F, V107I, A125S (14 SNPs). There was a global distribution of mef(E) in S. pyogenes whereas 4 out of the 5 isolates positive for mef(I) came from Belgium. Conclusions: This study definitively shows the presence of mef(E) in S. pyogenes and its global distribution, the previously un-described presence of mef(I) in S. pyogenes and the presence of a novel subclass of mef(A). P889 Emergence of linezolid-resistant Enterococcus faecalis in Spain and rapid characterisation by real-time PCR E. Cercenado, M. Marin, O. Cuevas, E. Bouza (Madrid, ES) Objectives: Linezolid was introduced in our institution in 2004. From May 2005 to September 2006 we determined the in vitro activity of linezolid against all staphylococci and enterococci isolated in our laboratory. We characterise the first linezolid-resistant (LNZ-R) clinical isolates of Enterococcus faecalis in Spain. Methods: MICs were determined by the broth microdilution method using commercialised MicroScan panels (Dade-Behring). CLSI guide- lines (2006) were followed for determination of breakpoints. LNZ-R isolates were confirmed by E-test. Detection of the G2576T mutation was performed by PCR and sequenciation of the 23S rRNA gene, as well as by real-time PCR (Woodford et al; JCM 2002; 40: 4298–4300) using a fluorescent dye-labeled detection probe. Results: Over the period of study, we tested 6,214 staphylococci (3,817 S. aureus; 2,407 coagulase-negative staphylococci), and 1,850 enterococci (1,439 E. faecalis; 340 E. faecium; 71 other species). All staphylococci were susceptible to linezolid (MIC  4 mg/L). Three E. faecalis isolates (0.16%) were LNZ-R (MICs 64, and 128 mg/L; 2 and 1 isolate, respectively. The isolates were recovered in 2006 and corresponded to 3 patients hospitalised in different wards that received linezolid for >15 days previous to the isolation of the LNZ-R isolates. PCR and sequenciation detected the G2576T mutation only in 2 isolates (MICs 64 mg/L). Real-time PCR was positive in all 3 isolates, being one strain (MIC = 128 mg/L) heterozygous, and the other 2 homozygous. Results were obtained in less than 2 h. The G2576T mutation in the heterozygous strain was not detected by PCR amplification and sequencing of the 23S rRNA gene. Conclusions: We characterise for the first time in Spain the emergence of LNZ-R E. faecalis isolates in patients previously treated with linezolid. Since the G2576T mutation was not detected in the heterozygous isolate by PCR and sequencing, the real-time PCR must be the method of choice for the characterisation of this emergent resistance mechanism. P890 Linezolid resistance in a Staphylococcus haemolyticus strain isolated in an intensive care unit A. Mazzariol, G. Lo Cascio, R. Koncan, L. Maccacaro, R. Fontana, G. Cornaglia (Verona, IT) Objectives: Although even recent reports confirm resistance to linezolid to be minimal among coagulase-negative staphylococci, and virtually confined to a few cases of Staphylococcus epidermidis encountered in the USA, a number of documented cases have occurred in South America and Europe, too. In a recent report from intensive care units (ICUs) in Madrid, Spain, variable resistance percentages were found in Staphylococcus epidermidis (0.81%), Staphylococcus hominis (0.71%), and most noticeably Staphylococcus haemolyticus (6.84%). A 65-year-old male patient was admitted to the ICU of the Verona University Hospital on 1 August 2006, after extensive surgery for acute pancreatitis. Empirical therapy with teicoplanin (600 mg) was started on admission. On the 16th day of therapy, a strain of S. haemolyticus was isolated from the blood culture. The strain proved intermediate to teicoplanin (MIC, 16 mg/L), which was immediately suspended and replaced with linezolid (MIC, 0.25 mg/L). After a further 8 days, a new blood culture showed that the same strain had become resistant to linezolid (MIC, 32 mg/L). Methods: Antimicrobial susceptibility testing was routinely performed by diffusion test according to the latest CLSI documents. The MICs to all antibiotics were determined by means of the E-test and confirmed by dilution tests. PFGE was carried out by standard procedures.