Epidemiology and Molecular Characterization of Vancomycin-Resistant Enterococcus faecalis / 대한임상미생물학회지

BACKGROUND: Vancomycin-resistant Enterococci (VRE) infections are caused by Enterococcus faecium in about 90% of the cases but can also be caused by Enterococcus faecalis. Thus, this study investigates factors that cause a low isolation rate of vancomycin-resistant E. faecalis (VREfs). To this end, the authors study the clinical traits, resistant gene structure, genomic classification, and molecular characteristics of the virulent factor. METHODS: From January 2001 through September 2011, 17 vanA-containing E. faecalis isolates were collected from hospitalized patients at Ajou University Hospital in Korea. Identification, antimicrobial susceptibility testing, and PCR of van and esp genes were performed. Pulsed-field gel electrophoresis (PFGE) was used for strain typing. PCR and sequencing of the internal regions of Tn1546 were performed for structural analysis of the van gene. RESULTS: Of 4,235 VRE infections, 3,918 (92.5%) were caused by E. faecium, and 95 (2.2%) were caused by E. faecalis. In 67% of VREfs infections, there was a preceding occurrence of E. faecium infection. All isolates were of genotype vanA. Our isolates were divided into three types according to the distribution of IS elements integrated into Tn1546 (types I to IIb). The PFGE results showed no clonal relatedness among isolates. CONCLUSION: Our study found that VREfs infections affect patients who have experienced vancomycin-resistant E. faecium. (VREfm) infection or undergo invasive procedures. The VREfs seems to involve the horizontal transfer of Tn1546 transposon from VREfm.

Comparison of Tn1546-like Elements in vanA Genotype Enterococcus gallinarum and E. faecalis or E. faecium / 대한임상미생물학회지

BACKGROUND: Enterococcus gallinarum, an organism often found in the intestine, is intrinsically resistant to low level vancomycin, but some of them are highly resistant to vancomycin due to acquisition of vanA gene. We occasionally detected both vanA carrying E. gallinarum and E. faecalis or E. faecium in the same patients, suggesting transfer of the resistance gene from the latter. In this study, the structures of Tn1546-like elements in E. gallinarum, and E. faecalis or E. faecium from the same patients were compared to determine the clinical significance of the vanA genotype E. gallinarum isolates. METHODS: Six pairs of vanA genotype E. gallinarum and E. faecalis or E. faecium were isolated at a tertiary- care hospital in Korea during 2000 to 2004. Species identification was performed by conventional methods. and the vancomycin-resistance genotypes were determined by PCR. For structural analysis of Tn1546-like elements, overlapping PCR amplification and sequencing of the internal regions were performed. RESULTS: All isolates were positive for vanA genes by PCR. The analysis of Tn1546-like elements showed structurally related three types of distribution of IS elements integrated: Type I had insertion of an IS1542 in the orf2-vanR intergenic region, and an IS1216V in the vanX-vanY intergenic region. Type II was similar with Type I but accompanied with the partial and complete deletions of orf1 and orf2. Type III had an IS1216V and an IS1542 in the orf2-vanR intergenic region, and an IS1216V in the vanX-vanY intergenic region. Two of the six vanA clusters in E. gallinarum isolates had structures identical to those in E. faecalis or E. faecium strains isolated from the same patients. However, in some isolate pairs, the origin of Tn1546 cannot be determined, because the structures were not identical, and colonization of multiple clones was supposed. CONCLUSION: vanA clusters in some E. gallinarum, and in E. faecalis or E. faecium isolates from the same patients had an identical structure, indicating their transfer from the latter. It is assumed that vanA type E. gallinarum can act as a reservoir of vanA gene for interspecies spread.

Diversity of Tn1546 Elements in Vancomycin-Resistant Enterococci Isolated from Korea / 대한진단검사의학회지

BACKGROUND: The vanA gene cluster of vancomycin-resistant enterococci (VRE) is carried as a part of Tn1546-like elements. In this study we characterized the structure of Tn1546-like elements in Enterococcus. faecium isolated from patients in Korea. The isolates were also typed by pulsed-field gel electrophoresis (PFGE). METHODS: During 2000, 21 clinical isolates of vanA-containing E. faecium were collected from ten university hospitals in Korea. E. faecium BM4147 was used as a control. PFGE was performed on a CHEF-DR III apparatus. For structural analysis of Tn1546, the overlapping PCR amplification of internal regions of Tn1546 was performed. The purified PCR products were directly sequenced by using ABI Prism 3100 DNA SEQUENCER. RESULTS: All isolates were divided into 3 types according to the distribution of insertion sequences (IS elements) integrated Tn1546 elements. Type I and II were characterized by an IS1542 insertion in the orf2-vanR intergenic region and an IS1216V insertion in the vanX-vanY intergenic region. Type III represented two copies of IS1216V at the orf1 and in the vanX-vanY intergenic region as well as IS1542 in the orf2-vanR intergenic region. No isolates were identical to the prototype, which was identical to the predicted pattern for the published sequence of Tn1546. The PFGE results revealed that all strains except A13, C1, A2 and A9 were genetically unrelated. CONCLUSIONS: The distribution of IS in Tn1546-like elements of the Korean isolates is similar to that of the European VREs. Considering the results of PFGF and Tn1546 typing, the horizontal transfer of vanA resistance gene may be occurring among genetically diverse strains of E. faecium in Korea.

Molecular Characterization of Vancomycin-Resistant Enterococci Isolated from Poultry in Korea / 대한임상미생물학회지

BACKGROUND: Vancomycin-resistant enterococci (VRE) have been increasingly isolated worldwide as a nosocomial pathogen. In Korea, because avoparcin has been used as a growth promoter in animal feed, vanA-containing enterococci have been found in animals. The aim of this study is to understand the epidimiology of VRE isolated from chicken of diverse geographic areas. METHODS: Thirty eight isolates of VanA VRE from chicken of diverse geographic areas were investigated. Multiplex PCR was used to confirm the genotype of VRE. Long PCR and restriction fragment length polymorphism (long PCR-RFLP) were performed to analyze the structure of Tn1546. If the RFLP pattern was different from the prototype, PCR amplification of internal regions of Tn1546 and subsequent sequencing analysis was performed. RESULTS: All 38 isolates harbored vanA gene and divided into 3 types by long PCR-RFLP. Thirty five isolates (92%) were classified as type I. Two isolates and one isolate were belonging to type II and type III, respectively. Type II revealed an insertion of IS1216V in vanX-vanY intergenic region. IS element integrated type III did not match any previously reqorted seguence. CONCLUSION: Structural analysis of vanA gene cluster from chicken revealed that the majority of isolates (type I) have indistinguishable structure to E. fecium BM4147. This results indicated horizontal spread of resistance gene among isolates from chicken. Genetic rearrangement of Tn1546 was infrequently detected in Korea.

Molecular Characterization of vanA-containing Enterococcus faecium Isolated from a Teaching Hospital / 대한임상미생물학회지

BACKGROUND: The widespread dissemination of Tn1546 has been attributed to transposition into plasmid or transferable elements. The transposition has been achieved through the activity of insertion sequences. Genetic diversity in Tn1546 includes integration of IS elements such as IS1216V, IS1251, IS1476 and IS1542. We investigated molecular typing and the distribution of insertion sequences in vanA-containing Enterococcus faecium isolated from patients in a teaching hospital. METHODS: Sixteen strains of vanA-containing E. faecium isolated from Ajou university hospital were analyzed. PCR amplification of internal regions of Tn1546 was performed and PCR amplicons were directly sequenced on both DNA strands by the dideoxy termination method. RESULTS: For all 16 isolates, IS1216V sequences were located within Tn1546. IS1542 sequences were detected in the genome of 9 isolates. One isolate contained IS1216V in the vanS intragenic region. The structural analysis of Tn1546 in VRE isolates produced three different groups by the presence of the insertion sequences. Group I was characterized by deletions of orf1 and orf2 and IS1216V insertion in the vanXY intergenic region. Group II represented IS1542 in the orf2-vanR and IS1216V in the vanXY intergenic region with deletion of orf1 region. Group III represented IS1542 in the orf2-vanR and IS1216V in the vanXY intergenic region without any deletion. CONCLUSIONS: Most of Korean isolates contained IS1216V and IS1542 sequences. Transposon typing of isolates in this study revealed a similarity to the European's. The identification of insertion sequence within vanA gene cluster can be a useful tool in epidemiological investigations.

Molecular Epidemiologic Analysis of vanA-Containing Vancomycin-Resistant Enterococci / 대한진단검사의학회지

BACKGROUND: Vancomycin-resistant enterococci (VRE) have been increasingly isolated world-wide as a nosocomial pathogen. To target infection control, epidemiologic investigations of VRE should include analysis of the resistance gene in addition to typing of strains. We performed molec-ular characterization of the vanA resistance gene to evaluate the inter or intraconstitutional spread. METHODS: Twenty isolates of VanA VRE from the Centers for Disease Control and Prevention (CDC) and 17 from Ajou University Hospital (AUH) were investigated. Minimum inhibitory concen-trations of vancomycin, teicoplanin, and ampicillin were tested by the agar dilution method. Pulsed-field gel electrophoresis (PFGE) and long PCR-restriction fragment length polymorphism (long PCR-RFLP) were performed. The long PCR negative strains were typed by ORF1-, vanS-vanH-, vanX-, vanY-vanZ-, vanZ-, and IS1216-specific PCRs. Filter matings were performed by using rifampin-resistant, fusidic acid-resistant E. faecalis J H2-2 as the recipient. RESULTS: The PFGE from the VRE of the CDC showed 15 patterns including 4 clusters and PFGE from isolates of AUH revealed 6 patterns including 3 clusters. Tn1546 amplicons were detected in 18 of 20 (90%) CDC strains and 16 of 17 (94%) AUH strains. RFLP of Tn1546 amplicons revealed 5 different patterns in the VRE of the CDC strains, and 2 patterns in the VRE of the AUH strains. The mean transfer efficiency of the CDC and the AUH strains are 3.0 X 10(-8)and 4.9 X 10(-5)transconju-gant/ donor, respectively. CONCLUSIONS: Molecular typing of isolates from the CDC suggests the horizontal spread of vanA genes among genetically diverse strains. Analysis of the VRE from the AUH shows a mixed pattern with clonal dissemination of strains and horizontal transfer of vanA.

Molecular Relationship of vanA Glycopeptide Resistance Gene in Enterococci from Hospitalized Patients and Poultry in Korea / 감염

BACKGROUND: Vancomycin-resistant enterococci (VRE) with vanA gene have been reported as a significant nosocomial pathogen. The vanA gene cluster (Tn1546) located on mobile DNA elements is known to be transferable from VRE to other enterococci. The purpose of this study was to investigate the genetic relationship between the vanA VRE strains isolated from hospitalizd patients and poultry. METHODS: Total 145 isolates, including 58 E. faecium, 12 E. faecalis, 3 E. casseliflavus, and 4 E. gallinarum from humans and 68 E. faecium from poultry, were studied. Antimicrobial susceptibility tests were done by disk diffusion or agar dilution methods and molecular epidemiological analysis was performed by pulsed-field gel electrophoresis (PFGE). The internal and structural regions of vanA gene cluster were analyzed by PCR fragment length polymorphism, restriction enzyme, and sequencing of Orf2D region and vanXY intergenic region. The point mutation at Tn1546 nucleotide position 8234 (G->T) within the vanX gene was screened with DdeI restriction enzyme. RESULTS: The antibiotic resistance patterns of human isolates were different from those of poultry. PFGE patterns revealed high heterogeneity. Three PCR fragment length patterns in the vanA gene cluster were found : (I) PCR amplicon of the same size as prototype (E. faecium BM4147) in 17% of human isolates and 100% of poultry ones; (II) PCR amplicon for vanXY intergenic region due to an insertion between vanX and vanY genes in 2.5% of human isolates; (III) the insertions in vanX-vanY intergenic and Orf2 regions in 81% of human isolates. The T type in vanX gene of human and poultry isolates was not found. CONCLUSION: Despite the diverse PFGE patterns, 81% of human and all of poultry isolates belonged to vanA gene cluster type III and I, respectively. These results indicate that the horizontal spread of vanA gene is occurring among genetically diverse strains of VRE in Korea.