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• W1533590911 abstract "Objective To examine the clonal diversity of vancomycin-resistant enterococci (VRE). Methods A total of 900 clinical isolates of enterococci were obtained, and VRE isolates were subjected to antimicrobial susceptibility tests, biochemical fingerprinting with the PhPlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE) typing. Results Forty-nine of all enterococcal isolates were resistant to high levels of vancomycin (MIC ≥ 128) and identified as Enterococcus faecium. Biochemical fingerprinting with PhP showed that the VRE isolates were highly diverse (diversity index, Di = 0.93) and belonged to 24 PhP-types. The VRE could be separated into 34 and 27 types with PFGE and ribotyping, giving diversity indices of 0.98 and 0.97, respectively. The PFGE method was more discriminatory than ribotyping and PhP system for E. faecium isolates. A combination of either of the two typing methods resulted in at least 44 types. Furthermore, sequencing analysis of vanS of Tn1546 showed one nucleotide mutation (C→A) at position 5727 in comparison with the prototype BM4147, which was found to be unique in all Iranian VRE isolates. Conclusion The isolated clinical VRE strains were highly diverse in Tehran. Objectif Examiner la diversité clonale des entérocoques résistants à la vancomycine (ERV). Méthodes Un total de 900 isolats cliniques d’entérocoques a été obtenu et les isolats ERV ont été soumis à des tests de sensibilité aux antimicrobiens, au typage biochimique avec le système PhPlate (PhP) et au ribotypage par la méthode du Pulse Field Gel Electrophoresis (PFGE). Résultats 49 de tous les isolats d’entérocoques étaient résistants avec des niveaux de résistance élevés pour la vancomycine (CMI ≥ 128) et étaient identifiés comme Enterococcus faecium. Le typage biochimiques avec le PhP a révélé que les isolats ERV étaient très divers (Di = 0,93) et appartenaient à 24 types PhP. Les ERV pourraient être classifiés en 34 et 27 types par le PFGE et le ribotypage, donnant des indices de diversité de 0,98 et 0,97, respectivement. La méthode du PFGE est plus discriminative que le ribotypage et le système PhP pour les isolats de E. faecium. La combinaison de deux des méthodes de typage mène à au moins 44 types. En outre, l’analyse du séquençage de vanS de Tn1546 a révélé une mutation nucléotidique (C → A), à la position 5727 en comparaison avec le prototype BM4174, qui s’est avéréêtre unique dans tous les isolats iraniens ERV. Conclusion Les isolats cliniques ERV sont très diversifiés à Téhéran. Objetivo Examinar la diversidad clonal de enterococos vancomicina resistentes (EVR). Métodos Se obtuvo un total de 900 aislados clínicos de enterococo, y los aislados EVR fueron sometidos a pruebas de susceptibilidad antibíótica, análisis de “fingerprinting” bioquímico con el sistema PhPlate (PhP), ribotipaje y electroforesis de campo pulsado (PFGE). Resultados 49 del total de aislados de enterococo eran resistentes a altos niveles de vancomicina (CIM ≥ 128) y fueron identificados como Enterococcus faecium. El “fingerprinting” bioquímico con PhP mostró que los aislados EVR eran altamente diversos (Di = 0.93) y pertenecían a 24 tipos de PhP. Los EVR podían separarse en 34 y 27 tipos mediante PFGE y ribotipaje, dando índices de diversidad de 0.98 y 0.97, respectivamente. El método PFGE era más discriminatorio que el ribotipaje o el sistema de PhP para aislados de E. faecium. Combinaciones de cualquiera de los dos métodos de tipaje resultaba en al menos 44 tipos. Más aún, el análisis de la secuencia vanS de Tn1546 mostró una mutación de un nucleótido (C→A) en la posición 5727, en comparación con el prototipo BM4174, el cual se encontró que era único en todos los aislados EVR iraníes. Conclusión Los aislados clínicos de cepas de EVR eran altamente diversos en Teherán. Enterococci, an important cause of clinical infections, are often recovered from patients with urinary tract infections (UTI) (Alangaden et al. 2006), wounds (Berger et al. 2006), bacteraemia (Von Baum et al. 2005), endocarditis (Hill et al. 2007) and meningitis (Guardado et al. 2006). Genetically, enterococci have a remarkable capacity to transfer the antibiotic-resistant genetic elements horizontally within the enterococci populations or other genera such as Staphylococcus aureus (Nallapareddy et al. 2005). As a result, a high level of resistance to glycopeptides, ampicillin and aminoglycosides by enterococci has been reported worldwide, which is of great clinical importance (Simonsen et al. 2003). The spread of vancomycin-resistant enterococci (VRE) represents an immediate threat to public health where combined antimicrobial therapy is used (Torell et al. 2003). Identification of the genetic lineage of the globally spread VRE is, therefore, essential for the implementation of control measures (Bedendo & Pignatari 2000). A variety of typing methods have been used to examine clonal relatedness among human VRE isolates. Among the phenotypic methods, biochemical fingerprinting using the PhPlate system (PhP) is a simple and highly discriminatory method (Kühn et al. 2003). Among the genotyping methods, pulsed-field gel electrophoresis (PFGE) is considered the gold standard (Brisse et al. 2002). Ribotyping is also reproducible in analyses of the enterococcal conserved rDNA (Price et al. 2002). It has been suggested that the combination of different typing methods would yield a higher discrimination and more valuable information about the diversity, clonality and dissemination of the isolated strains (Cetinkaya et al. 2000). Over a period of 1 year, we obtained a large number of clinical isolates of enterococci, 49 of which were resistant to vancomycin. In the absence of any in-depth report on the genetic relatedness of nosocomial VRE strains in Tehran, Iran, we undertook this study to characterize these isolates using the above phenotypic and genotypic methods. Between September 2005 and June 2006, we collected 900 enterococcal clinical isolates in Tehran. The clinical samples were from urine (759), wounds (50), blood (41), body fluids (20), respiratory tract (19) and abscess (11). The presumed enterococci strains were identified to the species level by biochemical characterization using Facklam’s recommendations (Facklam & Collins 1989). Species identification was also confirmed by PCR using species-specific primer as described previously (Kariyama et al. 2000). Antimicrobial susceptibility tests were performed and interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS 2000). The following antimicrobial disks were used: vancomycin (30 μg), ampicillin (10 μg), gentamicin (120 μg), erythromycin (15 μg), ciprofloxacin (5 μg)(BD BBL, Sparks, MD, USA). Dalfopristin-quinupristin (15 μg) and linezolid (30 μg) were purchased from Mast Diagnostics Ltd. (Bootle, Mersey Side, UK), and teicoplanin (30 μg) from Bio-Rad (Hercules, CA, USA). VRE were also tested with the E-test method (AB Biodisk, Solna, Sweden) for vancomycin and teicoplanin resistance according to the manufacturer’s specification. For each VRE isolate, van genotypes (vanA and vanB) were identified by PCR as described by Kariyama et al. (2000). E. faecium BM4147 (vanA-positive) and E. faecalis V583 (vanB-positive) were used as positive controls. All VRE strains were typed using PhP-RF plates (PhPlate Microplates Techniques AB, Sweden) according to the manufacturer’s instructions. The inoculated microplates were incubated at 37 °C, and the absorbance value (A620) of each well was read at 16, 40 and 64 h. Biochemical fingerprints of the isolates were compared pairwise and the similarity between strains was calculated as a correlation coefficient. Isolates showing correlation coefficients higher than identity level were assigned to the same biochemical type (common type: CT). The diversity of bacterial isolates was calculated using Simpson’s diversity index (Di). Optical readings, calculation of correlation coefficients and diversity indexes were performed using PhPWin software (PhPlate Microplates Techniques AB) (Kühn et al. 2003). The isolates were grown in brain heart infusion (BHI) broth overnight at 37 °C with shaking. The cultures were centrifuged and cells resuspended in 5 ml of TE buffer [10 mm Tris, 0.1 mM EDTA (pH 8.0], mixed with an equal volume of 2% low melting agarose (Bio-Rad) in distilled water and poured into a plug mold. The plugs were treated overnight at 37 °C with lysis buffer containing lysozyme (1 mg/ml) and 5 mg of RNAse per millilitre to lysis buffer [6 mm Tris (pH 7.5), 1 m NaCl, 100 mm EDTA (pH 7.5), 0.5% Brij-58, 0.2% sodium deoxycholate and 1% sodium lauryl sarcosine]. This solution was replaced by ES solution [0.5 m EDTA (pH 9.5), 1% sarcosine] and ESP solution [0.5 m EDTA (pH 9.5), 1% sarcosine and 0.5 mg of proteinase K/ml], and the plugs were incubated 48 h at 50 °C. The plugs were washed with TE buffer and stored at 4 °C. After digestion with 20 U SmaI (Roche Diagnostic GmbH, Mannheim, Germany), the plugs were placed in the wells of 1% agarose in 0.5% × TBE and electrophoresed with switch times ramped from 5 to 35 s at 6 V with a run time for 27 h at 16 °C in a Bio-Rad CHEF-DRII system. DNA from Salmonella choleraesuis serotype Branderup H9812 (Pulsenet, http://www.cdc.gov/pulsenet) was included as the molecular size marker (Turabelidze et al. 2000). Similarities between the PFGE bands, and hence between the isolates, were interpreted using the guidelines set out by Tenover et al. (1995). Extracted DNA from VRE isolates was cleaved by EcoRI restriction endonucleases (Roche Diagnostic GmbH). The fragments were separated by electrophoresis in agarose (0.8%) gel in Tris-borate buffer (89 mm Tris, 89 mm borate, 2 mm EDTA, pH 8.3) for 16 h at 1.5 V/cm. The DNA fragments were then transferred to nylon membrane, and hybridization was performed with the probes labelled with digoxigenin-11-dUTP (DIG). The membranes were then visualized by the addition of alkaline phosphate-conjugated digoxigenin antibody (Roche Diagnostic GmbH) and 5-bromo-4-chloro-3-indolyl phosphate substrate and nitroblue tetrazolium (Regnault et al. 1997). The vanS gene of Tn1546 was sequenced by Macrogen Research (Korea, Seoul) and compared with the prototype strain BM4147, which can be found in GenBank under accession number M97297.1. Of the 900 enterococcal isolates tested, 49 (5.4%) were identified as VRE. All of the VRE isolates were identified as E. faecium by conventional and PCR methods. Of the 49 VRE isolates, 40 (82%) were obtained from the UTI, 3 (6%) from wounds, 2 (4%) from abscess and 1 isolate each (2%) from CSF, blood, ascites and trachea samples. All the 49 VRE isolates were resistant to ampicillin, and 47 (96%) of them were also resistant to gentamicin (120 μg) (Table 1). This was followed by erythromycin (92%), ciprofloxacin (92%) and teicoplanin (90%). All isolates were susceptible to linezolid and dalfopristin-quinupristin. For 44 (90%) VRE isolates, the MIC to vancomycin and teicoplanin were ≥128 and ≥24 μg/ml, respectively. PCR was performed and the vanA gene found in all VRE isolates. All the 49 VR E. faecium isolates were typed with the PhPlate system, revealing a diversity index of 0.93. Clustering analysis of the PhP types showed that 17 (35%) isolates were single types and the remaining 32 isolates belonged to seven common types (CTs) (Figure 1). CT2 with 10 isolates (21%) was the dominant PhP type (Figure 1). The results of ribotyping showed the presence of 27 types. Six common ribotypes (with >2 isolates) accounted for 51% of the total isolates examined. PFGE identified 34 types. The dominating cluster contained five isolates with DNA fragments ranging in size from 80 to 245 kb. The diversity analysis by Simpson’s index showed PFGE with Di = 0.98, ribotyping with Di = 0.97 and PhP typing with Di = 0.93. Dendrogram cluster analysis of PFGE data for 49 vanA positive enterococci. Site of specimen collection, ribotype and PhP patterns are indicated. The abbreviations are: Gm, gentamicin; Cip, ciprofloxacin; Tip, teicoplanin; E, erythromycin; Am, ampicillin; CT, common type based on PhP typing. Since the first isolation of VRE in 1988 (Utdey et al. 1988), enterococci have been the object of many investigations worldwide. Data on the prevalence of these strains are scarce in Iran, and to the best of our knowledge there is no published information concerning the genetic relatedness of VRE isolates. In 2004, the rate of VRE in Iranian hospitals was around 2% (Feizabadi et al. 2004); 16% of enterococcal isolates in Iran are resistant to at least three antibiotics (Feizabadi et al. 2002). Comparison of our results with the findings reported by investigators from other countries in this region suggests that the incidence of VRE in Iran is higher (Khan et al. 2002; Kacmaz & Aksoy 2005). On the contrary, our study indicates that the prevalence of VRE clinical isolates in Tehran is 5.4%, less than that reported in North America (up to 18%) (Biedenbach et al. 2004) and some countries in Europe (from 1% in France to 59% in Portugal) (Bouchillon et al. 2004). In our collection, all VRE isolates were E. faecium, suggesting the importance of these bacteria in clinical infections in Tehran. Worldwide, up to 90% of the clinical enterococci isolates are E. faecalis, accounting for up to 10% of enterococcal infections (Yeh et al. 2002). However, E. faecium is more commonly associated with vancomycin resistance than other enterococcal species (Treitman et al. 2005). In our study, all VRE isolates harboured the vanA gene and were highly resistant to vancomycin (100%) and teicoplanin (90%), a typical characteristic of the VanA phenotype. It was interesting to see that 10% of our teicoplanin-susceptible isolates also harboured vanA instead of vanB gene. Such an inconsistency may be due to mutations in the vanS regulatory gene of the vanA gene cluster which, in turn, could result in impaired resistance to teicoplanin causing VanB phenotype (Hashimoto et al. 2000). Sequence analysis of the vanS gene of transposon (Tn1546) of our VRE isolates was performed. A single nucleotide change (C→A) at position 5727 in the vanS gene was found in all of the isolates regardless of being sensitive or resistant to teicoplanin. The vanS showed high sequence identity (99.7% amino acid level) as compared to the prototype BM4147, suggesting that vanS were not responsible for the reduced MIC in teicoplanin-sensitive VRE isolates. A single nucleotide variation in the vanS in our E. faecium isolates may reflect a unique characteristic of the VRE in Iran, which has not been reported before. There is still controversy concerning the typing applicability of ribotyping for Enterococcus spp. Some investigators have reported that ribotyping is less discriminatory than PFGE (Price et al. 2002). Others have suggested riboprinting as a preferred method because of its rapidity (Brisse et al. 2002). Of the two genotyping methods used, PFGE was the preferred method showing more discrimination (34 types) than ribotyping (27 types) with a Simpson index of 0.98 and 0.97 for PFGE and ribotyping, respectively. The combination of ribotyping and PFGE resulted in the identification of 45 genotypes out of 49 isolates. Although simultaneous performance of two genotyping methods may be time-consuming and cumbersome, it provides significant discriminatory power. We suggest combining both methods for similar genotyping studies. It has also been suggested that enterococci consist of a metabolically diverse group of species, and therefore they could be considered as a suitable candidate for phenotypic analysis by the PhP system (Lund et al. 2002). Analysis of our isolates with this system revealed 24 PhP types with clusters containing up to 10 isolates (i.e. CT2). Combination of PFGE and PhP typing methods divided the isolates into 44 types similar to that of PFGE and ribotyping. This finding suggests that combination of PFGE/PhP typing can be used instead of the PFGE/ribotyping in epidemiological studies considering the ease of use and less time involved. Combining typing methods used in this study showed that our VRE isolates were highly heterogeneous, suggesting no clonal dissemination in Tehran. The fact that almost all of these isolates were highly resistant to five major antibiotics tested suggests that the acquisition of antibiotic-resistant genes among E. faecium clinical isolates in Tehran is not through a vertical transmission but is happening independently and most likely under the selective antibiotic pressures used in these hospitals. This work was supported in part by Swedish International Development Cooperation Agency (Sida) grant no. 6342 and Ministry of Health of Iran, Undersecretary of Research." @default.
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• W1533590911 title "Clonal heterogeneity of clinical isolates of vancomycin-resistant Enterococcus faecium with unique vanS" @default.
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