Characterization of vancomycin resistance in Enterococcus faecium and Enterococcus faecalis.

Vancomycin resistance in Enterococcus faecium 180, a clinical isolate from England, was studied. Resistance to vancomycin was transferable by conjugation to other enterococci. Expression of resistance was inducible and coincided with the appearance of a new membrane protein.     

Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis

Resistance to linezolid has been associated with a G2576U mutation in domain V of the 23S rRNA. We analyzed nine clinical isolates of linezolid-resistant enterococci and showed a clear association between the number of 23S rRNA genes containing this mutation and the level of linezolid resistance expressed.     

氨苄西林预报粪肠球菌和屎肠球菌亚胺培南敏感性的可行性研究

目的 评价氨苄西林预报粪肠球菌和屎肠球菌亚胺培南敏感性的可行性。方法 收集23家医院的127株粪肠球菌和124株屎肠球菌非重复临床分离株。采用微量肉汤稀释法和纸片扩散法测定粪肠球菌和屎肠球菌对青霉素、氨苄西林、亚胺培南的敏感性。结果 纸片扩散法青霉素和氨苄西林均敏感或均耐药的粪肠球菌,微量肉汤稀释法氨苄西林-亚胺培南的分类一致率（CA）均为100.0%,未出现非常重大误差（VME）和重大误差（ME）;纸片扩散法青霉素和氨苄西林均耐药的粪肠球菌,微量肉汤稀释法氨苄西林-亚胺培南的CA为77.8%,ME占22.2%;青霉素耐药、氨苄西林敏感的粪肠球菌,微量肉汤稀释法氨苄西林-亚胺培南的CA为57.1%,纸片扩散法的CA为81.8%。结论 氨苄西林预报粪肠球菌和屎肠球菌亚胺培南敏感性的可靠性优于青霉素,但氨苄西林的敏感性不能用于预报青霉素耐药、氨苄西林敏感表型粪肠球菌和屎肠球菌的亚胺培南敏感性;青霉素敏感可预报粪肠球菌和屎肠球菌对氨苄西林敏感。     

Characterization of a vancomycin-resistant Enterococcus faecium outbreak caused by 2 genetically different clones at a neonatal intensive care unit

In July 2010, we identified an outbreak of vancomycin-resistant enterococci (VRE) in our 26-bed neonatal intensive care unit. We performed an epidemiological investigation after clinical cultures of 2 neonates were positive for VRE. Identification, susceptibility testing, and molecular characterization were performed. Cultures of 3 surveillance stool samples of inpatients and 5 environmental samples were positive for VRE. All isolates were identified as Enterococcus faecium containing the vanA gene. Two distinct clones were identified by performing pulsed-field gel electrophoresis. The 2 clones exhibited different pulsotypes, but they represented identical Tn1546 types. Two sequence types, ST18 and ST192, were identified among all of the isolates with multilocus sequence typing. Our investigation determined that the outbreak in the neonatal intensive care unit was caused by 2 genetically different clones. The outbreak may have occurred through clonal spread and horizontal transfer of the van gene.     

2015年重庆市粪肠球菌和屎肠球菌耐药性监测结果分析

目的 统计分析2015年重庆市细菌耐药监测网成员单位提交的粪肠球菌和屎肠球菌耐药性监测数据,为该市有效应用抗菌药物提供依据和参考.方法 各成员单位根据全国细菌耐药监测网技术方案要求,对目标细菌进行鉴定和药敏试验,并依据美国临床和实验室标准化协会(CLSI)2015版标准进行结果判读,使用WHONET5.6软件对药敏数据进行统计分析,耐药性差异采用SPSS21.0软件进行分析.结果 共分离到非重复粪肠球菌和屎肠球菌分别为1 811株和1 601株,共占所有阳性菌株的13.1%,两者对万古霉素的耐药性分别为0.5%和1.8%,对利奈唑胺的耐药率分别为2.5%和0.5%,均未发现对替加环素耐药的菌株.粪肠球菌对奎奴普丁/达福普汀的耐药性最高,为90.1%,对四环素的耐药率也高达78.8%,对高浓度庆大霉素的耐药率为43.0%,对青霉素、氨苄西林、呋喃妥因的耐药率均低于7.0%.除奎奴普丁/达福普汀和四环素外,屎肠球菌对其他药物的耐药率高于粪肠球菌(P<0.05).儿童和成人患者,以及重症监护病房(ICU)和非ICU患者分离株对部分药物的耐药率比较差异有统计学意义(P<0.05).结论 该市肠球菌感染主要病原菌是屎肠球菌和粪肠球菌,两者耐药性不同.做好耐药性监测有助于指导临床医生规范、有效使用抗菌药物.     

Mechanism of resistance to vancomycin in Enterococcus faecium D366 and Enterococcus faecalis A256.

The role of the glycopeptide-inducible proteins of Enterococcus faecium D366 (39.5 kilodaltons) and Enterococcus faecalis A256 (39 kilodaltons) in the mechanism of resistance to vancomycin and teicoplanin was examined. Crude cell walls from noninduced cells or from induced cells treated with sodium dodecyl sulfate to remove the inducible proteins were shown to bind vancomycin, in contrast to cell walls containing the cytoplasmic membrane-associated induced proteins, which did not bind vancomycin. Cytoplasmic membranes from vancomycin-induced cells did not inactivate (bind) vancomycin or teicoplanin, but they could protect the glycopeptides from being bound to the synthetic pentapeptide. This protection could be competitively abolished by D-alanyl-D-alanine. A decrease in glycopeptide binding to the pentapeptide was observed in a time-dependent fashion after treatment of the pentapeptide with the cytoplasmic membranes from induced cells. We hypothesize that the inducible proteins are responsible for glycopeptide resistance due to the binding to, and subsequent enzymatic modification of, the pentapeptide precursor of peptidoglycan, which is considered to be the natural target of glycopeptides.     

Phenotypic Distinction in Enterococcus faecium and Enterococcus faecalis Strains between Susceptibility and Resistance to Growth-Enhancing Antibiotics

Susceptibility of Enterococcus faecium and Enterococcus faecalis strains from animals and foods to growth-promoting antibiotics used in animal feed was tested by the agar dilution technique. Acquired resistance to bacitracin, narasin, tylosin, and virginiamycin was seen for both species, and for E. faecium, resistance to avilamycin and avoparcin was also seen. Drawing the distinction between susceptibility and resistance based on frequency distributions of MICs was easy with avoparcin, avilamycin, and tylosin but difficult with virginiamycin and to some extent also with bacitracin and narasin.     

Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis

OBJECTIVES: The enterococcal surface protein gene, esp, is a major putative pathogenicity marker in clinical isolates of Enterococcus faecium and Enterococcus faecalis. This study demonstrates in vitro conjugative transfer of the esp gene among E. faecium and E. faecalis. MATERIALS AND METHODS: Enterococcal isolates from clinical samples, positive for esp, were mated on filters with enterococcal recipients. Transconjugants were checked for transfer of antibiotic resistance determinants and co-mobilization of the esp gene. They were also characterized by PCR and plasmid profiling/PFGE typing including Southern hybridizations with labelled esp probes. Transfer as triggered by excision was tested using Taqman PCR. RESULTS: Two of five E. faecalis and five of nine E. faecium transferred antibiotic resistance determinants into a recipient. Of the transconjugants analysed by PCR for acquisition of esp, only isolates from two E. faecalis and a single E. faecium mating were positive. In the donor strains, the esp gene was located on the chromosome. Molecular analysis revealed a plasmid localization of esp in the E. faecium transconjugant and chromosome-to-chromosome transfer in E. faecalis. CONCLUSION: The esp gene is transferable by conjugation among enterococcal isolates.     

Differences in antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium strains isolated from farm and pet animals

The prevalence of acquired resistance in 146 Enterococcus faecium and 166 Enterococcus faecalis strains from farm and pet animals, isolated in 1998 and 1999 in Belgium, against antibiotics used for growth promotion and for therapy was determined. Acquired resistance against flavomycin and monensin, two antibiotics used solely for growth promotion, was not detected. Avoparcin (glycopeptide) resistance was found sporadically in E. faecium only. Avilamycin resistance was almost exclusively seen in strains from farm animals. Resistance rates were higher in E. faecium strains from broiler chickens than in strains from other animal groups with tylosin and virginiamycin and in E. faecalis as well as in E. faecium strains with narasin and bacitracin. Resistance against ampicillin was mainly found among E. faecium strains from pets and was absent in E. faecalis. Tetracycline resistance occurred most often in strains from farm animals, while enrofloxacin resistance, only found in E. faecalis, occurred equally among strains from all origins. Resistance against gentamicin was very rare in broiler strains, whereas resistance rates were high in strains from other origins. It can be concluded that resistance against antibiotics used solely for growth promotion was more prevalent in E. faecium strains than in E. faecalis strains. With few exceptions, resistance against the different categories of antibiotics was more prevalent in strains from farm animals than in those from pets.     

Defining Daptomycin Resistance Prevention Exposures in Vancomycin-Resistant Enterococcus faecium and E. faecalis

Daptomycin is used off-label for enterococcal infections; however, dosing targets for resistance prevention remain undefined. Doses of 4 to 6 mg/kg of body weight/day approved for staphylococci are likely inadequate against enterococci due to reduced susceptibility. We modeled daptomycin regimens in vitro to determine the minimum exposure to prevent daptomycin resistance (Dap^r) in enterococci. Daptomycin simulations of 4 to 12 mg/kg/day (maximum concentration of drug in serum [C_max] of 57.8, 93.9, 123.3, 141.1, and 183.7 mg/liter; half-life [t_1/2] of 8 h) were tested against one Enterococcus faecium strain (S447) and one Enterococcus faecalis strain (S613) in a simulated endocardial vegetation pharmacokinetic/pharmacodynamic model over 14 days. Samples were plated on media containing 3× the MIC of daptomycin to detect Dap^r. Mutations in genes encoding proteins associated with cell envelope homeostasis (yycFG and liaFSR) and phospholipid metabolism (cardiolipin synthase [cls] and cyclopropane fatty acid synthetase [cfa]) were investigated in Dap^r derivatives. Dap^r derivatives were assessed for changes in susceptibility, surface charge, membrane depolarization, cell wall thickness (CWT), and growth rate. Strains S447 and S613 developed Dap^r after simulations of 4 to 8 mg/kg/day but not 10 to 12 mg/kg/day. MICs for Dap^r strains ranged from 8 to 256 mg/liter. Some S613 derivatives developed mutations in liaF or cls. S447 derivatives lacked mutations in these genes. Dap^r derivatives from both strains exhibited lowered growth rates, up to a 72% reduction in daptomycin-induced depolarization and up to 6-nm increases in CWT (P < 0.01). Peak/MIC and AUC_0–24/MIC ratios (AUC_0–24 is the area under the concentration-time curve from 0 to 24 h) associated with Dap^r prevention were 72.1 and 780 for S447 and 144 and 1561 for S613, respectively. Daptomycin doses of 10 mg/kg/day may be required to prevent Dap^r in serious enterococcal infections.     

Analysis of daptomycin efficacy and breakpoint standards in a murine model of Enterococcus faecalis and Enterococcus faecium renal infection

Daptomycin efficacy against clinical isolates of Enterococcus faecalis, Enterococcus faecium, and a lab-derived daptomycin-resistant isolate of E. faecalis was investigated in a mouse model of renal infection. The daptomycin MICs against these enterococci ranged from 0.5 to 50 micro g/ml. The objective of this study was to determine the relationship between the MICs of drugs against E. faecalis and E. faecium and the level of daptomycin exposure needed to evaluate the drug's efficacy. Correlating the required therapeutic exposures of mice with the exposures achieved clinically allowed us to project enterococcal breakpoint values. Mice pretreated with carrageenan were infected intravenously with 3 x 10(8) to 4 x 10(8) CFU of E. faecalis or E. faecium. Daptomycin (5 to 50 mg of drug/kg of body weight) or saline control was administered 4 h postinfection and continued once daily for 2 days (three total doses). On day 4, infected kidneys were harvested, homogenized, and dilution plated. Efficacy was defined as a > or = 2-log(10) (99%) reduction in bacterial burden in infected kidneys. At clinically relevant dosages and exposures (area under the curve, 400 to 600 microg.hr/ml), daptomycin demonstrated similar and marked efficacy against all clinical enterococcal isolates tested. Daptomycin achieved efficacy with comparable doses against both vancomycin-sensitive (MIC, < or = 4 microg/ml) and -resistant enterococcal strains tested. Efficacy was also established against the lab-derived daptomycin-resistant E. faecalis isolate. In this murine renal infection model, clinically relevant exposures of daptomycin were effective against E. faecalis and E. faecium strains for which MICs were < or = 8 microg/ml. These murine efficacy data for daptomycin, along with surveillance data and human pharmacokinetic exposures achieved, suggest a breakpoint concentration value of < or = 8 microg/ml (susceptible) and > or = 16 microg/ml (resistant) for daptomycin against E. faecium and E. faecalis.     

Identical genes confer high-level resistance to gentamicin upon Enterococcus faecalis, Enterococcus faecium, and Streptococcus agalactiae.

The structural gene coding for the bifunctional aminoglycoside-modifying 6'-acetyltransferase-2'-phosphotransferase (6'AAC-2'APH) enzyme was specifically amplified by the polymerase chain reaction using template DNA of clinical isolates of enterococci (both Enterococcus faecalis and Enterococcus faecium) and the single high-level gentamicin-resistant Streptococcus agalactiae strain identified thus far. The results of the present study demonstrated that the genes encoding this antibiotic resistance trait are highly homologous in these species. In dot blot hybridization assays using nonradioactively labeled oligonucleotide probes, strains with and without high-level gentamicin resistance could be discerned unequivocally. The gene segment encoding 6'-acetylating activity and the gene segment encoding 2'-phosphorylating activity were simultaneously present in all isolates exhibiting high-level gentamicin resistance.