Antibacterial effect of sophoraflavanone G by destroying the cell wall of Enterococcus faecium

Enterococcus faecium has appeared as an important opportunistic pathogen that can cause urinary tract infections,surgical site infections, bacteremia, and endocarditis. Therefore, it is imperative to develop alternative therapeuticmethods to treat enterococcal infections. Sophoraflavanone G (5,7,2′,4′-tetrahydroxy-8-lavandulylflavanone, SPF-G)exhibited the strongest antibacterial activity based on the minimum inhibitory concentration values on two E. faeciumstrains (6.25 and 12.5 μg/ml at 24 and 48 hours of treatment, resp.) in the broth microdilution assay among thetested compounds and a remarkable bactericidal effect with a 12.5 μg/ml minimum bactericidal concentration value.Sophoraflavanone G (12.0 ± 2.3 mm inhibition zone for Korean Agricultural Culture Collection, Korea (KACC)11954 and 11.0 ± 3.0mm for Culture Collection of Antimicrobial Resistant Microbes, Korea (CCARM) 5506) alsoexhibited the highest susceptibility based on the agar diffusion assay. Membrane-permeabilizing agents with a lowdose of sophoraflavanone G synergistically activated anti-E. faecium activity through a 67% reduction of E. faeciumgrowth, and E. faecium-derived peptidoglycans (PGN) blocked the antibacterial activity. These results indicate thatsophoraflavanone G could bind to the bacterial cell wall and induce E. faecium cell wall damage. Transmissionelectron microscopy (TEM) images of E. faecium treated with sophoraflavanone G also exhibited cell lysis, followedby leakage of intracellular components, confirming that sophoraflavanone G has anti-E. faecium activity by bindingto the PGN and disrupting the cell wall. This study showed the possible usage of sophoraflavanone G as an effectivenatural anti-E. faecium compound

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium Isolated from the Chungcheong Area / 대한임상미생물학회지

BACKGROUND: Enterococcus faecium has emerged as an important nosocomial pathogen worldwide, and this trend has been associated with the dissemination of a genetic lineage designated clonal complex 17 (CC17). In the present study, characterization of the glycopeptide resistance mechanism, genetic relatedness, and pathogenicity in isolates of vancomycin-resistant E. faecium in the Chungcheong area were investigated. METHODS: A total of 37 consecutive, non-duplicate, vancomycin-resistant E. faecium were isolated at three university hospitals in the Chungcheong area. The mechanism of glycopeptide resistance and pathogenicity factors were studied using PCR, and the genetic relatedness was determined via multilocus sequence type and esp repeat profile analysis. Additionally, the quinolone resistance-determining regions of parC and gyrA were sequenced to identify mutations involved in ciprofloxacin resistance. RESULTS: Two genotypes of VRE were confirmed: VanA-phenotype vanA genotype VRE (25 isolates) and VanB-phenotype vanA genotype VRE (12 isolates). MLST analysis revealed five sequence types. A significant result was that ST414 and CNS4 (4-1-1-1-1-1-1) were considered as belonging to CC17. The esp and hyl genes were found in 100% and 86.4% of the isolates, respectively. A total of 37 isolates showed genetic mutations in parC and gyrA. CONCLUSION: All isolated strains in the present study belonged to one of the CC17 genotypes including ST414 and CNS4 (4-1-1-1-1-1-1), which were not previously detected in Korea. The combination of MLST and the esp gene repeat profiles can be useful for genetic characterization of VREF isolates with regard to the evolutionary process and epidemiology of the clones.

Characterization of Vancomycin-Resistant Enterococci Isolated from Stools and Their Acquisition of Vancomycin Resistance

We have isolated 6 vancomycin resistant (VR) Enterococcus faecium and 5 VR-E. gallinarum. Vancomycin resistant enterococcus (VRE) isolates were resistant to multi-drugs, but susceptible to linezolid and quinupristin/dalfopristin. VRE isolates showed 10 VanA phenotypes and 1 VanB phenotype (E. gallinarum). However, all of them showed vanA genotype. vanA gene was detected on both genomic and plasmid DNA from all VRE isolates. Almost of VR-E. faecium had IS1216V which is worldwide type and almost of VR-E. gallinarum had IS1542 which is European type. IS1216V and IS1542 genes were not related with antibiotic types of VRE. Copy numbers of vanA were decreased in VRE with IS1216V or IS1542 but not in VRE with both ISs in broth without vancomycin. The copy numbers of vanA were significantly decreased in VanB phenotype of VRE with IS1542 in broth without vancomycin. Copy numbers of vanA were recovered in the presence of vancomycin. Growth time of reference E. faecium is faster than that of reference E. faecalis when cultured in the broth containing vancomycin. Reference strains cultured in the broth containing vancomycin showed intermediate resistance or resistance to antibiotics without acquisition of van genes. Naturally, multidrug-resistant E. faecium might be fast adapted in the presence of vancomycin compared to E. faecalis. Taken together, VanA phenotype E. gallinarum as well as E. feacium have been increasing in nosocomial infection and showed acquired inducible resistance. E. faecium and E. faecalis showed intermediate resistance in long exposure of vancomycin without acquisition of vanA.

Molecular characterization of vancomycin-resistant Enterococci strains eight years apart from its first isolation in São Paulo, Brazil / Caracterização molecular de espécies de Enterococci resistentes à vancomicina oito anos após seu primeiro isolamento em São Paulo, Brasil

E. faecium was the first reported VRE species, carrying the vanA gene in Brazil. In spite of this, vancomycin-resistant E. faecalis has become the predominant species in Brazilian hospitals. The aim of this study was to evaluate the genetic relatedness of VREs isolated in a Brazilian teaching hospital eight years apart from its first isolation. We analyzed 38 VRE strains obtained from 81 surveillance cultures of patients admitted to the four largest intensive care units in Hospital São Paulo in February, 2006. Presence of the vanA gene was assayed by PCR and PFGE analysis was used for molecular characterization. All VRE strains carried the vanA gene. Two distinct clonal groups were observed among vancomycin-resistant E. faecalis. Vancomycin-resistant E. faecium belonged to five distinct clones were demonstrated by molecular typing. All of these clones were different from the first vancomycin-resistant enterococci clone isolated eight years ago in our hospital.

E. faecium contendo o gene vanA foi a primeira espécie de VRE descrita, no Brasil. Apesar disto, E. faecalis resistente a vancomicina tem se tornado a espécie predominante nos hospitais brasileiros.O objetivo desse estudo foi avaliar a relação genética de VREs isolados em um hospital de ensino brasileiro após oito anos de seu primeiro isolamento. Analisamos 37 isolados de VRE obtidos de 81 culturas de vigilância de pacientes admitidos nas quatro maiores Unidades de Tratamento Intensivo em Fevereiro de 2006. A presença do gene vanA foi analisada por PCR e a caracterização molecular por PFGE. Todas as amostras VRE carreavam o gene vanA. Entre os E. faecalis vancomicina-resistentes, dois distintos grupos clonais foram observados. E. faecium resistente a vancomicina pertencentes a cinco clones distintos foram demonstrados por tipagem molecular. Todos esses clones foram diferentes do primeiro clone de enterococo resistente a vancomicina isolado oito anos atrás em nosso hospital.

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium Isolated from Clinical Specimens

A total of 58 vancomycin-resistant E. faecium (VREF) was isolated from 3 hospitals located in Daegu, Korea. The VREF isolates were evaluated for the antimicrobial susceptibility pattern and resistance determinants against vancomcin, aminoglycosides, and macrolides. The multilocus sequence types (MLST) were determined to characterize the clonal diversity of the VREF isolates. The VREF isolates were highly resistance to teicoplanin, erythromycin, ciprofloxacin, gentamicin, and streptomycin, whereas quinupristin-dalfopristin and linezolid were the most susceptible drugs. All isolates carried the vanA gene. The aac6'-aph2" (n=53) and aadE (n=27) genes were detected in the high-level aminoglycoside resistant (HLAR) isolates. The aac6'-aph2" gene was located in the conjugally transferable plasmids. The ermB and ermA genes were detected in the 54 and 3 VREF isolates, respectively. The VREF isolates showed 11 different sequence types (ST). The VREF isolates belonging to ST192 was the most prevalent (n=19), but detected in one hospital, whereas the isolates belonging to ST203 (n=11) were detected in 3 hospitals. These results suggest that the VREF isolates resistant to aminoglycosides and erythromycin are originated from different clones and specific VREF clones are spread in the study hospitals.

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.

False Susceptibility to Imipenem by Vitek GPS Card in Enterococcus faecium / 임상검사와정도관리

BACKGROUND: Enterococcus faecium (E. faecium) is potential pathogens of mixed infections for which a broad-spectrum antimicrobial agents such as imipenem has a therapeutic role. But controversy continues concerning testing imipenem versus enterococci. The purpose of this study were 1) to investigate the ability of penicillin and ampicillin minimum inhibitory concentration (MIC) to predict in vitro susceptibility of E. faecium versus imipenem. and 2) to compare MICs of ampicillin, penicillin and imipenem by the Vitek system with those by agar dilution method. METHODS: Fifty-two isolates of E. faecium between April 2002 and May 2002 were tested. Each isolate was tested versus penicillin, ampicillin and imipenem. MICs were determined by Vitek system and agar dilution method according to NCCLS guidelines. Imipenem MIC determinations were repeated by E-test. RESULTS: MIC of Vitek system tends to be lower than that of agar dilution method, but there was good concordance between MICs of penicillin and ampicillin by Vitek system and agar dilution method. But for imipenem, the MICs by the agar dilution method did not correspond with the Vitek results. Of the 52 E. faecium isolates tested, in vitro activity of penicillin and ampicillin accurately predicts that of imipenem. CONCLUSIONS: MICs of ampicillin and penicillin are reliable, but imipenem MIC is not reliable for E. faecium by Vitek system. In vitro activity of penicillin and ampicillin versus E. faecium accurately predicts that of imipenem.

A Case of Vancomycin-resistant E. faecium Peritonitis in Patient with Continuous Ambulatory Peritoneal Dialysis / 감염

Enterococcus is a normal flora of the gastrointestinal or genitourinary tract. With the increased use of vancomycin and third generation cephalosporins, vancomycin-resistant enterococci (VRE) have become one of the major nosocomial pathogens in USA and Europe since 1986. In Korea, patients with VRE infection or colonization were increasingly reported recently and VRE may become a serious nosocomial pathogen in the near future. So we report a case of vancomycin-resistant E. faecium peritonitis in a patient on continuous ambulatory peritoneal dialysis.

Antimicrobial Susceptibility of Beta-Lactam Antibiotics on Enterococcus / 대한임상미생물학회지

BACKGROUND: Enterococci exhibit intrinsic resistance or high-level minimum inhibitory concentration (MIC) to beta-lactams than other streptococci. This appears to be due to low affinity of penicillin-binding proteins and rarely production of beta-lactamase, which gives the reason of testing beta-lactamase for blood and cerebrospinal fluid isolates. Ampicillin is more effective than penicillin in vitro, and MIC of ampicillin is generally 1 dilution lower than that of penicillin. The purpose of this study is to detect beta-lactamase producing enterococci an6 to compare MICs of ampicillin and penicillin by Vitek system (bioMerieux, Hazelwood, MO, USA) with those by agar dilution method. METHODS: We collected 110 isolates of Enterococcus faecalis and 51 isolates of E. faecium from clinical specimens in 1998. MICs of antibiotics were determined by agar dilution method and Vitek system. We also performed beta-lactamase test by the Cefinase (Becton Dickinson, USA) for 512 isolates of E. faecalis and 189 isolates of E. faecium collected in 1998. RESULTS: The most common sites of isolates were blood, bile, surgical/traumatic wounds, closed and open pus and urine. MICs of ampicillin were 1 to 2 dilution lower than those of penicillin for E. faecalis (P=0.03). But there were no significant differences in MICs for E. faecium (P=0.19). Five isolates (4 E. faecalis and 1 E. faecium) were susceptible to ampicillin but resistant to penicillin. There were no beta-lactamase producing enterococci among 701 isolates tested. CONCLUSIONS: MIC by Vitek system tends to be 1 to 2 dilution lower than MIC by agar dilution method to beta-lactams, and MIC of ampicillin is 1 to 2 dilution lower than MIC of penicillin, which could result in discrepancy in interpretation of susceptibilty tests. A beta-lactamase test for enterococci is not recommeneded for routine test in Korea.