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P683 Molecular characterisation of vancomycin-resistant Enterococcus isolates in clinical samples from Chile
S164 VREfm/VSEfm = 0.745, CI95: 0.58–0.96, p = 0.024), as were the cluster sizes (number of isolates belonging to one genotype) of VREfm and of VSEfm (mean: 6.47 vs. 4.77, median: 3 vs. 2, p = 0.028). MLVA-analysis of the most prominent PFGE-cluster revealed the involvement of the epidemic clonal complex-17 (MT 1, MT 3 and MT 7). Conclusion: The lower SD and the greater median cluster size of VREfm express a higher genetic relationship of VREfm in comparison to VSEfm. Considering the 36 unique genotypes and the occurrence of identical genotypes on different wards without epidemiological linkage, this is not explained by simple patient-to-patient transmission of VREfm, which accounted for approximately 30% of VREfm. Further analysis has to be done to clarify the causes of the increase in VREfm at MHH. P683 Molecular characterisation of vancomycin-resistant Enterococcus isolates in clinical samples from Chile G. Saavedra, J. Hormaz´abal, A. Maldonado, J. Mota, F. Baquero, J. Silva, R. del Campo (Panam´a, PA; Santiago de Chile, CL; Madrid, ES; Antofagasta, CL) Objectives: The aim of this work was to characterise a collection of vancomycin-resistant isolates recovered from clinical samples in Chile. Methods: A total of 70 vancomycin resistant enterococal strains recovered during 2003–2005 was included (10 E. faecalis and 60 E. faecium). Strains were recovered from different samples (34 faecal swabs, 15 urine, 6 exudates, 3 blood and 12 others) corresponding to unrelated patients attended in 16 different hospitals corresponding to four regions of Chile. All strains presented vancomycin-resistance and were sent to the National Reference Institute. Susceptibility to several antimicrobial agents was tested using the microdilution test. Clonal relationships were studied by PFGE-SmaI assays, and presence of the vanA or vanB genes were demonstrated by PCR experiments. Amplicons were purified and sequenced. Presence of ace, agg, cylA, esp, efaA and gelE genes was also investigated in the different clones detected. Results: All strains were resolved into 7 different clones (2 E. faecalis and 5 E. faecium) showing resistance to vancomycin, and susceptibility to teicoplanin. Resistance to macrolides, tetracycline, quinolones, b-lactams (only in E. faecium clones), and high level resistance to aminoglycosides was observed in all enterococcal clones. Positive amplification with the generic vanB primers was observed, and the sequences correspond to the previously described vanB2 variant. Presence of ace, agg, cylA, esp, efaA and gelE genes was only detected in the E. faecalis clones. Conclusions: vanB2-containing E. faecium and E. faecalis clones have been isolates dispersed in 16 different hospitals in Chile. P684 Molecular epidemiology of vancomycin-resistant enterococci isolated in 2003–2005 in a large teaching hospital in Warsaw W. Grzybowska, G. Mlynarczyk, A. Mlynarczyk, A. Mrowka, S. Tyski, M. Luczak (Warsaw, PL) Objectives: Enterococcal infections caused by multiresistant bacteria might constitute a severe problem for patients with serious diseases. Glycopeptides are the most frequently used antibiotics in such cases. In the investigated hospital, vancomycin resistant enterococci (VRE) did not occur frequently up to the 2003. A significant increase in the number of VRE isolated recently has suggested a possibility of an outbreak. The aim of this study was to perform epidemiological analysis of VRE strains isolated from clinical materials of hospitalised patients. Methods: All VRE strains originated from patients of the investigated hospital and were isolated in the years 2003–2005. Strains were initially identified in the Diagnostic Laboratory of Microbiology Department. Identification was performed by API ID32 Strep and the susceptibility was tested by ATB Entero. Vancomycin resistant or intermediate isolates were checked by E-tests. All resistant strains were analysed for the presence of vanA, van B, vanD or vanG ligase genes by PCR. The identification of the strains was proven by PCR with application of the ddl primers. Only one VRE isolate from one patient was taken to epidemiological analysis. Strains were compared on the basis of
17th ECCMID / 25th ICC, Posters differences in patterns obtained by PFGE of SmaI-restricted whole genomes analysis. Results: Although the frequency of enterococci isolation in our laboratory was usually about 1000 strains per year, until the 2003, VRE occurred sporadically. In 2003 they appeared in one of internal wards, and were isolated from urine (9 patients) and from blood (1 patient). In 2004 VRE were isolated from 9 patients of the same ward and appeared in The Intensive Therapy Unit (OIOT) (2 patients) and in surgery (2 patients). In 2005 VRE were isolated from 11 patients of internal ward, from one patient of OIOT and from 21 patients of surgery. All together 53 VRE isolates were available for PFGE examination (12 E. faecalis, 41 E. faecium). All examined strains possessed vanA determinant of resistance. PFGE analysis revealed that most E. faecium strains belonged to one of the 3 clusters. The same strains occurred in three wards of the hospital. The PFGE relationship analysis between isolated E. faecium and between E. faecalis strains showed their high similarity: 75% and 82%, respectively. Conclusion: The PFGE and SmaI restrictase turned out to be very useful for epidemiological analysis of enterococci. P685 Molecular characterisation of vancomycin-resistant Enterococcus faecium isolated from German hospitals between 1991 and 2006 reveals differences in emergence and spread G. Werner, I. Klare, W. Witte (Wernigerode, DE) Objectives: To investigate the molecular background of vanA-type glycopeptide resistance in 57 clinical vancomycin-resistant Enterococcus faecium (VREF) from 29 German hospitals of three separate periods (group I, 1991–1995; II, 1996–1999; III, 2004–2006). Methods: Isolates were characterised by multi-locus sequence typing (MLST), SmaI macrorestriction analysis in pulsed-field gel electrophoresis (PFGE), and multiple-locus variable-number tandem repeat analysis (MLVA). Phylogenetic relatedness between strains was identified using BioNumerics and eBURST software. Distribution of virulence genes esp and hylEfm was investigated by PCR. The structure of the vanA gene clusters was investigated by PCR, long PCR, sequencing and Southern hybridisations. Results: Group I isolates (n = 6) are quite diverse by different typing methods, lack mostly any of the investigated virulence genes, and possess all an identical vanA cluster type. Two and all group II and III isolates belong to the clonal complex of hospital-adapted strain types (MLST CC17, MLVA C1). Isolates of group II (n = 8) are identical by MLST (ST-117), PFGE and MLVA (MT-12), harbour all the esp but not the hylEfm gene and possess an identical or slightly modified vanA cluster type (insertion of ISEf1). Group III VREF (n = 43) are rather diverse constituting different strain types, different virulence markers and vanA clusters. Within this diversity we found supportive data for a dissemination of related VREF among various hospitals and spread of identical vanA gene clusters into different strain types within single hospitals. Conclusions: Our data suggest that VREF from the three periods investigated here have evolved differently. The increase in the rates of VREF among German hospital patients within the last two years might be rather complex and due to different molecular events and scenarios. P686 Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a Turkish University Hospital A. Ozcan, B. Ozhak Baysan, D. Ogunc, D. Inan, T. Naas, D. Colak, P. Nordmann (Antalya, TR; Kremlin-Bicetre, FR) Objectives: Vancomycin-resistant enterococci (VRE) are widespread worldwide. Despite growing concern about VRE as nosocomial pathogens, especially in the USA, they are rarely isolated in Turkish hospitals. In 2001, unrelated VRE isolates were first described in Turkey (Colak D, JAC, 2002, 50:397–401.). Here we report the emergence of a novel VRE clone responsible of a nosocomial outbreak at a Turkish University Hospital.
17th ECCMID / 25th ICC, Posters differences in patterns obtained by PFGE of SmaI-restricted whole genomes analysis. Results: Although the frequency of enterococci isolation in our laboratory was usually about 1000 strains per year, until the 2003, VRE occurred sporadically. In 2003 they appeared in one of internal wards, and were isolated from urine (9 patients) and from blood (1 patient). In 2004 VRE were isolated from 9 patients of the same ward and appeared in The Intensive Therapy Unit (OIOT) (2 patients) and in surgery (2 patients). In 2005 VRE were isolated from 11 patients of internal ward, from one patient of OIOT and from 21 patients of surgery. All together 53 VRE isolates were available for PFGE examination (12 E. faecalis, 41 E. faecium). All examined strains possessed vanA determinant of resistance. PFGE analysis revealed that most E. faecium strains belonged to one of the 3 clusters. The same strains occurred in three wards of the hospital. The PFGE relationship analysis between isolated E. faecium and between E. faecalis strains showed their high similarity: 75% and 82%, respectively. Conclusion: The PFGE and SmaI restrictase turned out to be very useful for epidemiological analysis of enterococci. P685 Molecular characterisation of vancomycin-resistant Enterococcus faecium isolated from German hospitals between 1991 and 2006 reveals differences in emergence and spread G. Werner, I. Klare, W. Witte (Wernigerode, DE) Objectives: To investigate the molecular background of vanA-type glycopeptide resistance in 57 clinical vancomycin-resistant Enterococcus faecium (VREF) from 29 German hospitals of three separate periods (group I, 1991–1995; II, 1996–1999; III, 2004–2006). Methods: Isolates were characterised by multi-locus sequence typing (MLST), SmaI macrorestriction analysis in pulsed-field gel electrophoresis (PFGE), and multiple-locus variable-number tandem repeat analysis (MLVA). Phylogenetic relatedness between strains was identified using BioNumerics and eBURST software. Distribution of virulence genes esp and hylEfm was investigated by PCR. The structure of the vanA gene clusters was investigated by PCR, long PCR, sequencing and Southern hybridisations. Results: Group I isolates (n = 6) are quite diverse by different typing methods, lack mostly any of the investigated virulence genes, and possess all an identical vanA cluster type. Two and all group II and III isolates belong to the clonal complex of hospital-adapted strain types (MLST CC17, MLVA C1). Isolates of group II (n = 8) are identical by MLST (ST-117), PFGE and MLVA (MT-12), harbour all the esp but not the hylEfm gene and possess an identical or slightly modified vanA cluster type (insertion of ISEf1). Group III VREF (n = 43) are rather diverse constituting different strain types, different virulence markers and vanA clusters. Within this diversity we found supportive data for a dissemination of related VREF among various hospitals and spread of identical vanA gene clusters into different strain types within single hospitals. Conclusions: Our data suggest that VREF from the three periods investigated here have evolved differently. The increase in the rates of VREF among German hospital patients within the last two years might be rather complex and due to different molecular events and scenarios. P686 Nosocomial outbreak of vancomycin-resistant Enterococcus faecium at a Turkish University Hospital A. Ozcan, B. Ozhak Baysan, D. Ogunc, D. Inan, T. Naas, D. Colak, P. Nordmann (Antalya, TR; Kremlin-Bicetre, FR) Objectives: Vancomycin-resistant enterococci (VRE) are widespread worldwide. Despite growing concern about VRE as nosocomial pathogens, especially in the USA, they are rarely isolated in Turkish hospitals. In 2001, unrelated VRE isolates were first described in Turkey (Colak D, JAC, 2002, 50:397–401.). Here we report the emergence of a novel VRE clone responsible of a nosocomial outbreak at a Turkish University Hospital.