258株肠球菌耐药性分析及耐万古霉素基因检测

目的研究肠球菌的耐药率及耐万古霉素肠球菌(VRE)的耐药表型和基因型。方法按照美国临床和实验室标准化研究所(CLSI)2009年推荐的微量稀释法进行临床分离肠球菌对各类药物的最小抑菌浓度(MIC)检测,VRE进一步用E-test药敏试验确认;PCR法检测VRE的耐药基因。结果 2010年7月至2011年11月沈阳军区总医院共检出粪肠球菌95株,屎肠球菌163株。粪肠球菌对万古霉素、替考拉宁保持较高敏感度,对氨苄西林、青霉素、呋喃妥因三种抗菌药物敏感度也在65%以上,对其他抗菌药物敏感度低,统计期内未检出耐万古霉素粪肠球菌菌株。屎肠球菌对多数抗菌药物表现为耐药,对氯霉素敏感率为70%,对万古霉素、替考拉宁敏感度下降,为90.7%。期间检出15株VRE,其耐药表型为多重耐药,PCR扩增结果显示,15株万古霉素耐药屎肠球菌VanA基因扩增均为阳性,产物长度在700～1000bp之间,约783bp,符合预期;VanB、VanC引物扩增均阴性。15株万古霉素耐药屎肠球菌对多数抗菌药物耐药,仅对氯霉素、四环素相对敏感,对万古霉素MIC>256mg/L,对替考拉宁也表现为耐药。结论屎肠球菌耐药性高于粪肠球菌,VRE多为多重耐药,给临床治疗带来困难,医院应加强对其预防监测。     

In vitro susceptibility of vancomycin-resistant enterococci (VRE) to fosfomycin

We evaluated the in vitro activity of fosfomycin against 75 clinical isolates of vancomycin-resistant enterococci (VRE). Using the NCCLS breakpoint for susceptibility of urinary tract isolates to fosfomycin (MIC > or = 256), 51 out of 52 Enterococcus faecium and all Enterococcus faecalis isolates tested were susceptible or intermediate to fosfomycin.     

Antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from North America and Europe: a report from the SENTRY antimicrobial surveillance program

Increases in prevalence of vancomycin-resistant enterococci (VRE) have been documented globally since its emergence in the 1980s. A SENTRY Antimicrobial Surveillance Program (2003) objective monitored VRE isolates with respect to antimicrobial susceptibility trends, geographic resistance variability, and clonal dissemination. In 2003, VRE isolates from North America (United States and Canada, n = 839, 26 sites) and Europe (n = 56, 10 sites) were susceptibility tested using Clinical and Laboratory Standards Institute (CLSI) reference methodologies. Based on resistance profiles, 155 isolates displayed similar multidrug-resistant (MDR) profiles and were temporally related; these were subsequently submitted for typing by pulsed-field gel electrophoresis (PFGE). Most of the submitted isolates were Enterococcus faecium (91.0%) and Enterococcus faecalis (7.8%). Among VRE, the VanA phenotype was more prevalent in North America (76%) than Europe (40%), and all isolates had elevated resistance rates to other antimicrobial classes including the following: 1) chloramphenicol resistance among E. faecalis being greater in North America than in Europe (28.6% versus 7.1%, respectively) but reversed among E. faecium (0.5% and 15.0%, the latter due to clonal occurrences); 2) ciprofloxacin resistance in North America >99% for both species and in Europe varying from 85.7% to 87.5%; 3) rare occurrences of linezolid resistance in North America (0.8% to 1.8%) due to G2576U ribosomal mutation; 4) higher quinupristin/dalfopristin resistance observed among European E. faecium strains (10.0% versus 0.6%); and 5) higher rifampin resistance rates among European E. faecalis (21.4% versus 5.4%). Thirty-five MDR epidemic clusters were identified by PFGE in 21 North American and 2 European medical centers including the following: 1) VanA (20 sites, 27 clonal occurrences) and VanB (1 site, 2 clonal occurrences); 2) elevated quinupristin/dalfopristin MIC results (not vatD/E, 3 sites); and 3) chloramphenicol resistance (chloramphenicol acetyltransferase-positive strains, 3 sites). The esp gene, part of the putative E. faecium pathogenicity island and a marker for the clonal complex-17 lineage, was detected in 76% of vancomycin-resistant E. faecium. Clonal spread appears to be a dominant factor of MDR VRE dissemination on both continents, and further monitoring is critical to assist in the control of these resistant pathogens.     

European survey of vancomycin-resistant enterococci in at-risk hospital wards and in vitro susceptibility testing of ramoplanin against these isolates

A survey in eight European countries, including 13 hospitals, of vancomycin-resistant enterococci (VRE) in at-risk hospital wards (such as the ICU and the haematology ward) was performed in 2001, and the in vitro susceptibility of the isolates ramoplanin and other drugs was tested. A total of 1314 non-duplicate clinical enterococcal isolates were collected, and 38 (2.9%) were vancomycin resistant: 27 Enterococcus faecium and 11 Enterococcus faecalis; 35 VanA and three VanB phenotypes. Rates of VRE among clinical enterococcal isolates varied between 0 and 1.7% for the participating countries, except the UK (10.4%) and Italy (19.6%). One hundred and twenty-three (3.5%) VRE were found among 3499 stool samples tested for the presence of these organisms: 111 (3.2%) E. faecium and 12 (0.3%) E. faecalis; 114 (3.3%) VanA and nine (0.3%) VanB phenotypes. Rates of intestinal colonization with VRE varied between 0 and 1.2% for the participating countries, except Italy (7.5%) and the UK (32.6%). In vitro susceptibility testing showed that the Italian and UK VRE are multi-resistant (including resistance to ampicillin and high-level resistance to gentamicin and streptomycin), and that ramoplanin was active against all strains of VRE, with an MIC90 of 0.5 mg/L for clinical isolates. Pulsed-field gel electrophoresis showed that the high prevalence of VRE in the Italian and UK centres was related to the monoclonal emergence and spread of three centre-specific clones. This survey suggests that in some centres in Europe, a similar situation may be encountered to that in the USA (monoclonal spread of multi-resistant VRE in at-risk wards).     

VanD-type glycopeptide-resistant Enterococcus faecium BM4339.

Enterococcus faecium BM4339 was constitutively resistant to vancomycin (MIC, 64 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml). A 605-bp product obtained with the V1 and V2 primers for amplification of genes encoding D-Ala:D-Ala ligases and related glycopeptide resistance proteins was sequenced after cloning. The deduced amino acid sequence had 69% identity with VanA and VanB and 43% identity with VanC, consistent with the finding that BM4339 synthesized peptidoglycan precursors terminating in D-lactate. This new type of glycopeptide resistance phenotype was designated VanD.     

In vitro activity of RP59500, an injectable streptogramin antibiotic, against vancomycin-resistant gram-positive organisms.

The in vitro activity of RP59500, a streptogramin antibiotic, against 146 clinical isolates of vancomycin-resistant gram-positive bacteria was examined. Five strains of the species Enterococcus casseliflavus and Enterococcus gallinarum, for which the MIC of vancomycin was 8 micrograms/ml, were also studied. Twenty-eight vancomycin-susceptible strains of Enterococcus faecalis and Enterococcus faecium were included for comparison. The drug was highly active against Leuconostoc spp., Lactobacillus spp., and Pediococcus spp. (MICs, < or = 2 micrograms/ml). RP59500 was more active against vancomycin-susceptible strains of E. faecium than E. faecalis (MICs for 90% of the strains [MIC90s], 1.0 versus 32 micrograms/ml). Vancomycin-resistant strains of E. faecalis were as resistant to RP59500 as vancomycin-susceptible strains (MIC90, 32 micrograms/ml), but some vancomycin-resistant E. faecium strains were relatively more resistant to the new agent (MIC90, 16; MIC range, 0.5 to 32 micrograms/ml) than were vancomycin-susceptible organisms of this species.     

Nitrofurantoin is active against vancomycin-resistant enterococci

The activity of nitrofurantoin was tested against 300 isolates of Enterococcus faecium, Enterococcus faecalis, and Enterococcus gallinarum. No isolates tested were resistant to nitrofurantoin (MIC, >/=128 microg/ml), including vancomycin-resistant E. faecium isolates with vanA- and vanB-positive genotypes and vancomycin-resistant E. gallinarum isolates. We conclude that nitrofurantoin may provide effective treatment of urinary tract infections caused by vancomycin-resistant enterococci.     

Glycopeptide susceptibility among Danish Enterococcus faecium and Enterococcus faecalis isolates of animal and human origin and PCR identification of genes within the VanA cluster.

The MICs of vancomycin and avoparcin were determined for isolates of Enterococcus faecium and isolates of Enterococcus faecalis recovered from the feces of humans and animals in Denmark. Two hundred twenty-one of 376 (59%) isolates of E. faecium and 2 of 133 (1.5%) isolates of E. faecalis were resistant to vancomycin (MICs, 128 to > or = 256 micrograms/ml), and all vancomycin-resistant isolates were resistant to avoparcin (MICs, 64 to > or = 256 micrograms/ml). All vancomycin-resistant isolates examined carried the vanA, vanX, and vanR genes, suggesting that a gene cluster similar to that of the transposon Tn1546 was responsible for the resistance.     

Vancomycin-resistant enterococci (VRE) colonization of high-risk patients in tertiary care Canadian hospitals. Canadian VRE Surveillance Group.

We isolated 1487 Enterococcus species from 1200 stool specimens collected from high-risk patients in 12 Canadian tertiary care hospitals between October 1995 and November 1996. The composition of the 1487 isolates was 601 vancomycin-sensitive Enterococcus faecalis (40.4%), 667 vancomycin-sensitive Enterococcus faecium (44.9%), 18 vancomycin-resistant (nine isolates MIC 8-16 micrograms/mL; nine isolates MIC > or = 32 micrograms/mL) E. faecium (VREF) (1.2%), 95 vancomycin-sensitive Enterococcus gallinarum (6.4%), 29 vancomycin-resistant (all MICs 8-16 micrograms/mL) E. gallinarum (2.0%), and 77 vancomycin-sensitive Enterococcus casseliflavus (5.2%). Nine of the 18 VREF isolates collected possessed the vanA genotype and were from three patients at one hospital. Two other VREF isolates, of the vanB genotype, were from a single patient at a second hospital, and the remaining seven isolates, also all of the vanB genotype, were from five patients at a third hospital. All VREF were ampicillin resistant (MIC > or = 16 micrograms/mL), streptomycin resistant (MIC > 1000 micrograms/mL), and ciprofloxacin resistant (MIC > or = 4 micrograms/mL). Ten of the 18 VREF were also resistant to gentamicin (MIC > 500 micrograms/mL), while all 18 isolates had quinupristin/dalfopristin MICs < or = 0.5 microgram/mL. In conclusion, high-risk patients in tertiary care Canadian hospitals are rarely colonized (9/1200 patients, 0.75%) with VREF in their lower gastrointestinal tract. These findings correlate well with the lack of reported VREF infection in high-risk patients in Canadian hospitals. Quinupristin/dalfopristin demonstrated excellent in vitro activity against VREF and other non-faecalis species of Enterococcus, many of which also possessed high-level ampicillin, and/or high-level aminoglycoside, and/or ciprofloxacin resistance.     

2010年中国CHINET肠球菌属细菌耐药性监测

目的了解2010年中国主要地区临床分离肠球菌属细菌对各类抗菌药物的耐药性。方法国内主要地区14所教学医院(12所综合性医院,2所儿童医院)按统一方案、采用统一的材料、方法(K-B法)和判断标准(CLSI 2010年版)进行肠球菌属细菌的耐药性监测。结果共分离到4 046株非重复肠球菌属细菌,最常见菌种为粪肠球菌1 829株(45.2%)、屎肠球菌1 817株(44.9%)、鹑鸡肠球菌78株(1.9%)、鸟肠球菌54株(1.3%)、铅黄肠球菌49株(1.2%)。肠球菌属对利奈唑胺、万古霉素、替考拉宁仍极敏感,耐药率<4%,万古霉素耐药粪肠球菌和屎肠球菌检出率分别为0.6%、3.6%。粪肠球菌对呋喃妥因、磷霉素和氨苄西林耐药率较低,分别为3.2%、5.7%和11.3%,对高浓度庆大霉素耐药率为44.0%;屎肠球菌耐药性明显高于粪肠球菌,对氨苄西林耐药率接近90%,对高浓度庆大霉素耐药率接近70%,对氯霉素耐药率仅为7.3%,儿童屎肠球菌分离株对磷霉素、呋喃妥因耐药率<10%。不同医院分离的肠球菌属细菌对抗菌药物的耐药率有一定差异。结论屎肠球菌的分离率有增加趋势,肠球菌属细菌对利奈唑胺、万古霉素、替考拉宁依然保持极高的敏感性。     

Comparison of phenotypic methods to identify enterococci intrinsically resistant to vancomycin (VanC VRE)

Clinical laboratories must be able to differentiate between enterococci possessing acquired resistance to vancomycin (vanA and vanB genotypes) from those that are inherently resistant (vanC1 and vanC2/C3 genotypes). We compared several routine phenotypic tests to determine the species identity of clinical isolates of enterococci and a PCR assay for the van ligase genes was used to confirm identification of VanC VRE.The Vitek Gram Positive Identification card identified 53/60 (88%) Enterococcus faecalis and E. faecium isolates and 81/141 (57%) VanC VRE without additional testing. Another 32 of the VanC VRE required additional testing (e.g., motility and pigmentation) for correct identification. However, 7 of these 32 VanC VRE were nonmotile. The rapid ID 32 STREP strips identified 50/60 (83%) E. faecalis and E. faecium isolates and 102/141 (72%) VanC VRE. All E. faecalis and E. faecium isolates were nonmotile and did not acidify 1% methyl-alpha-D-glucopyranoside (MGP). Only 93/115 (81%) E. gallinarum and 21/26 (81%) E. casseliflavus/E. flavescens were motile but all 141 VanC VRE acidified MGP. MGP acidification can accurately differentiate VanC VRE from E. faecalis and E. faecium. Because some VanC VRE isolates are nonmotile, MGP acidification is preferred as a simple and less costly test for identification of these isolates.     

Antibiotic activity against urinary tract infection (UTI) isolates of vancomycin-resistant enterococci (VRE): results from the 2002 North American Vancomycin Resistant Enterococci Susceptibility Study (NAVRESS)

BACKGROUND: The purpose of this study was to assess the prevalence of vancomycin-resistant enterococci (VRE) in urinary isolates in North America, and the activity of various antibiotics against VRE. MATERIALS AND METHODS: Twenty-eight medical centres in the United States and 10 centres in Canada assessed the prevalence of VRE in urinary isolates in 2002. Each study site was asked to collect up to a maximum of 50 consecutive VRE (Enterococcus faecium, Enterococcus faecalis only) urinary isolates. Susceptibility was determined by NCCLS broth microdilution. The prevalence of vanA and vanB resistance genotypes was determined by multiplex PCR. RESULTS: From the 28 US medical centres, a total of 697 VRE (616 [88.4%] E. faecium and 81 [11.6%] E. faecalis) were received. Approximately 75% of all VRE (E. faecium and E. faecalis) isolates demonstrated a VanA phenotype (resistance to both vancomycin and teicoplanin). PCR detection of vanA and vanB resistance determinants showed that the vanA genotype was present in 584 of 697 (83.8%) VRE isolates, whereas 113 (16.2%) isolates possessed the vanB gene. The most active agents were linezolid, nitrofurantoin and chloramphenicol, with 0.3%, 0.6% and 2.4% resistance, respectively. The majority (77.8%) of vancomycin-resistant E. faecium isolates displayed the VanA phenotype, and 538 of these 616 (87.3%) isolates were PCR-positive for vanA; the vanB genotype was detected in 78 (12.7%) isolates. Resistance was lowest with linezolid, chloramphenicol and nitrofurantoin at 0.3%, 0.3% and 0.5%, respectively. Only three genetically indistinguishable vanA-positive E. faecium were isolated from the 10 Canadian medical centres. CONCLUSION: VRE urinary isolates are common in the United States, are primarily of the vanA genotype and are very susceptible to linezolid, nitrofurantoin and chloramphenicol. In Canada, VRE urinary isolates remain uncommon.     

Inconsistent bactericidal activity of triple-combination therapy with vancomycin, ampicillin, and gentamicin against vancomycin-resistant, highly ampicillin-resistant Enterococcus faecium.

Combination therapy with ampicillin, vancomycin, and gentamicin in vitro against several clinical isolates of vancomycin-resistant, highly ampicillin-resistant Enterococcus faecium, including VanA and VanB strains, was evaluated. The MICs of ampicillin were not significantly decreased by induction with vancomycin, and the combination of ampicillin and vancomycin was not inhibitory for any strain. Triple-combination therapy was least active against highly resistant VanA isolates, achieving a reduction of less than 1 log CFU at 24 h, but demonstrated slightly more activity against VanB strains.     

Susceptibility to quinupristin/dalfopristin and other antibiotics of vancomycin-resistant enterococci from the UK, 1997 to mid-1999

Susceptibility to quinupristin/dalfopristin and other antibiotics was studied for clinical isolates of vancomycin-resistant enterococci (VRE) referred by UK hospitals between January 1997 and June 1999. Single isolates of VRE from 858 patients in 136 hospitals were received, of which 76% were Enterococcus faecium and 21% were Enterococcus faecalis, the remainder comprising minor species. Most isolates were multi-resistant. After allowing for the effect of blood, which raised the MICs of quinupristin/dalfopristin four-fold, 98.3% of E. faecalis isolates and all the Enterococcus avium, Enterococcus casseliflavus and Enterococcus gallinarum appeared resistant to quinupristin/dalfopristin, whereas 98.8% of the E. faecium isolates and the single Enterococcus raffinosus isolate were susceptible.     

Time-kill and synergism studies of ceftobiprole against Enterococcus faecalis, including beta-lactamase-producing and vancomycin-resistant isolates

Ceftobiprole (BAL9141) is an investigational cephalosporin with broad in vitro activity against gram-positive cocci, including enterococci. Ceftobiprole MICs were determined for 93 isolates of Enterococcus faecalis (including 16 beta-lactamase [Bla] producers and 17 vancomycin-resistant isolates) by an agar dilution method following the Clinical and Laboratory Standards Institute recommendations. Ceftobiprole MICs were also determined with a high inoculum concentration (10(7) CFU/ml) for a subset of five Bla producers belonging to different previously characterized clones by a broth dilution method. Time-kill and synergism studies (with either streptomycin or gentamicin) were performed with two beta-lactamase-producing isolates (TX0630 and TX5070) and two vancomycin-resistant isolates (TX2484 [VanB] and TX2784 [VanA]). The MICs of ceftobiprole for 50 and 90% of the isolates tested were 0.25 and 1 microg/ml, respectively. All Bla producers and vancomycin-resistant isolates were inhibited by concentrations of 相似文献   

Synergistic killing of vancomycin-resistant enterococci of classes A, B, and C by combinations of vancomycin, penicillin, and gentamicin

Using both high and low inocula for time-kill curves, we examined the antibiotic killing of clinical isolates of glycopeptide-resistant enterococci (Enterococcus faecium, E. faecalis, and E. gallinarum) belonging to phenotypic resistance classes A, B, and C. None were resistant to high levels (greater than 500 mg/liter) of gentamicin. Vancomycin-penicillin-gentamicin resulted in 2 or more logs of killing above that of the most effective two-antibiotic combination for all strains except two of three E. gallinarum (VanC) strains and a constitutive mutant of a VanB strain. This strategy may be useful clinically.     

Resistance to Linezolid: Characterization of Mutations in rRNA and Comparison of Their Occurrences in Vancomycin-Resistant Enterococci

To assess the potential for emergence of resistance during the use of linezolid, we tested 10 clinical isolates of vancomycin-resistant enterococci (VRE) (four Enterococcus faecalis, five Enterococcus faecium, and one Enterococcus gallinarum) as well as a vancomycin-susceptible control (ATCC 29212) strain of E. faecalis. The enterococci were exposed to doubling dilutions of linezolid for 12 passes. After the final passage, the linezolid plate growing VRE contained a higher drug concentration with E. faecalis than with E. faecium. DNA sequencing of the 23S rRNA genes revealed that linezolid resistance in three E. faecalis isolates was associated with a guanine to uracil transversion at bp 2576, while the one E. faecium isolate for which the MIC was 16 microg/ml contained a guanine to adenine transition at bp 2505.     

Real-time PCR for the rapid detection of vanA and vanB genes

A real-time PCR assay suitable for use on the Roche LightCycler platform was developed to replace an existing gel-based PCR assay for the simultaneous detection of the vanA & vanB genes in enterococcal isolates. Novel Fluorescence Resonance Energy Transfer (FRET) hybridization probes were designed. The multiplex real-time PCR assay and the existing gel-based assay were 100% concordant and both correctly detected the vanA or vanB genes in 4/4 VanA E. faecium and 25/25 VanB E. faecium. Additionally, 1/1 VanC1 E. gallinarum, 1/1 VanC2 E. casseliflavus and 47/47 vancomycin susceptible enterococci were negative for the vanA and vanB genes in both PCR assays. Results were available within 1.5 h for the real-time PCR assay compared to up to 5.5 h for the conventional PCR assay.     

In vitro activity of daptomycin against vancomycin-resistant enterococci of various Van types and comparison of susceptibility testing methods

The increasing prevalence of vancomycin-resistant enterococcal (VRE) infections and the limited number of antimicrobial agents for their treatment emphasize a need for new, more effective agents. In this study, the in vitro activity of daptomycin was determined against a collection of 156 VRE from seven different institutions. Van types were characterized by PCR, and pulsed-field gel electrophoresis was performed to exclude isolates with >85% relatedness by dendrogram. Included were 126 Enterococcus faecium (109 vanA, 17 vanB) isolates, 5 Enterococcus faecalis (3 vanA, 2 vanB) isolates, 2 Enterococcus avium (vanA) isolates, 1 Enterococcus durans (vanA) isolate, 10 Enterococcus gallinarum (vanC1) isolates, and 12 Enterococcus casseliflavus (vanC2) isolates. MICs of daptomycin and five additional agents were determined by the NCCLS broth microdilution method with Mueller-Hinton (MH) broth containing supplemental calcium. MICs were also determined using two investigational E-test strip formulations, and disk diffusion testing was performed by the standard NCCLS method. The MIC of daptomycin at which 50% of the isolates tested were inhibited for this isolate collection was 4 microg/ml, and the MIC at which 90% of the isolates tested were inhibited was 8 microg/ml. Two isolates of vanA E. faecium were resistant to linezolid, and one isolate was resistant to quinupristin-dalfopristin. MICs of daptomycin determined by the E test with and without added calcium varied by 8- to 16-fold, and disk diffusion zones varied by 3 to 6 mm according to the calcium content of the commercial MH agar lots used in the study. This study has shown daptomycin to have good activity against a diverse collection of contemporary VRE isolates. However, improved standardization of the calcium content of MH agar will be important for reliable testing of daptomycin by clinical laboratories using either the E test or disk diffusion methods.     

Activity of tigecycline (GAR-936), a novel glycylcycline,against Enterococci in the mouse peritonitis model

A novel glycylcycline agent, tigecycline (GAR-936), was evaluated in vivo in the mouse model of peritonitis against three Enterococcus faecalis and four Enterococcus faecium isolates with different susceptibilities to vancomycin and tetracyclines, all of which were inhibited by 相似文献