耐甲氧西林金黄色葡萄球菌分型方法研究进展

金黄色葡萄球菌(金葡菌)是革兰阳性球菌,在一般人群中的携带率为25％～50％,但在某些情况下也可成为致病菌,尤其是耐甲氧西林金葡菌(MRSA)已经成为了医院和社区获得性感染重要病原之一[1].     

两种检测耐甲氧西林金黄色葡萄球菌的K-B法应用评价

目的评价两种分别使用MH琼脂(头孢西丁)和高盐琼脂(苯唑西林)纸片扩散法检测耐甲氧西林金黄色葡萄球菌的临床应用价值。方法用头孢西丁纸片在MH琼脂平板上进行纸片扩散法(K-B法)检测临床分离的89株耐甲氧西林金黄色葡萄球菌,并与苯唑西林纸片扩散法(K-B法)、mecA基因检测进行比较。结果在MH琼脂使用头孢西丁纸片检测MRSA优于高盐琼脂苯唑西林纸片扩散法,并与PCR检测mecA基因的方法高度一致。结论头孢西丁纸片扩散法是筛选和确认耐甲氧西林金黄色葡萄球菌(MRSA)一种简单、可靠的实验方法。     

Emergence of linezolid-resistant mutants in a susceptible-cell population of methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus with a MIC of linezolid of 4 μg/ml, isolated from a patient who had undergone unsuccessful linezolid therapy, yielded linezolid-resistant mutants in blood agar at 48 h of incubation. The resistant clones showed a MIC of linezolid ranging from 8 to 64 μg/ml and accumulated the T2500A mutation(s) of the rRNA genes. Emergence of these resistant clones appears to be facilitated by a cryptic mutation or mutations associated with chloramphenicol resistance.     

Development of a rapid diagnostic assay for methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococcus

We describe here a rapid assay for the detection of the tuf gene for the identification of Staphylococcus genus, the femB gene for the identification of Staphylococcus aureus species, and the mecA gene for the identification of methicillin resistance directly from BACTEC blood culture bottles showing Gram-positive cocci in clusters. The test, configured on a thin-film biosensor platform, allows for detection of genomic DNA from blood culture samples without the need for nucleic acid amplification. In an initial study to validate the technology, 107 consecutive positive blood cultures were tested on the thin-film biosensor, and the assay exhibited 100% concordance in comparison with standard microbiological methods for identifying methicillin-susceptible and methicillin-resistant S. aureus and for identifying methicillin-susceptible and methicillin-resistant coagulase-negative Staphylococcus. Results were obtained within 90 min directly from signal positive bottles with no instrumentation required.     

Evaluation of the Cathra inoculating device for susceptibility testing of methicillin-susceptible and methicillin-resistant Staphylococcus aureus strains.

We compared the antimicrobial susceptibility results obtained with the Cathra replicator and reference methods when Staphylococcus aureus strains were tested by using agar dilution techniques. The Cathra replicator and the 0.001-ml calibrated loop gave results that fell within +/- 1 log2 dilution for greater than or equal to 95% of isolates when methicillin and cefamandole were tested.     

Comparison of four methods for detection of teicoplanin resistance in methicillin-resistant Staphylococcus aureus

OBJECTIVES: To determine which method of determining the MIC of teicoplanin produces a result closely related to outcome in the critically ill patient. METHODS: Four methods of teicoplanin susceptibility testing-disc diffusion, Etest, VITEK (Legacy and VITEK 2) and agar incorporation-were compared for 47 methicillin-resistant Staphylococcus aureus (MRSA) isolates from invasive intensive care unit (ICU) infections and 83 isolates from ICU patients colonized with the organism. Clinical outcome was recorded prospectively for all the patients. Another 13 reference laboratory strains of MRSA with reduced susceptibility to teicoplanin were tested. RESULTS: Both VITEK systems failed to demonstrate resistance in the three isolates identified as resistant by Etest or agar incorporation, and disc testing detected only one resistant isolate. A higher MIC, as found by Etest or agar incorporation, was associated with lower survival (n = 130, 95% CI -0.082 to -0.006, P = 0.023, Etest; n = 130, 95% CI -0.156 to -0.020, P = 0.011, agar). The findings for the 13 reference strains were similar, with a > or = 4-fold reduction in MIC between agar incorporation or Etest and VITEK2 for six isolates. CONCLUSIONS: Neither disc diffusion nor the VITEK systems are reliable for detection of teicoplanin resistance in MRSA. Etest and agar incorporation remain the methods of choice.     

头孢噻利对铜绿假单胞菌和甲氧西林耐药金葡菌的体外抗菌作用研究

目的探讨头孢噻利与阿米卡星对铜绿假单胞菌及头孢噻利与万古霉素对MRSA的体外联合抗菌作用,为联合抗菌治疗提供依据。方法收集30株不同耐药表型的铜绿假单胞菌和30株耐甲氧西林的金葡菌(MRSA),采用棋盘法测定头孢噻利与阿米卡星、头孢噻利与万古霉素的联合抗菌作用。结果头孢噻利与阿米卡星联合对铜绿假单胞菌具有协同、相加和无关作用者分别占26.7%、40.0%和33.3%。头孢噻利与万古霉素联合对MRSA主要是相加作用(63.3%),其余为无关作用(36.7%)。两组细菌中均未发现拮抗现象。结论头孢噻利与阿米卡星对铜绿假单胞菌及头孢噻利与万古霉素对MRSA在体外均表现出较好的联合抗菌作用。     

耐甲氧西林金黄色葡萄球菌MecA基因的实时荧光定量PCR方法学评价

目的评估实时荧光定量聚合酶链反应(FQ-PCR)检测临床标本中耐甲氧西林金黄色葡萄球菌(MRSA)MecA耐药基因的价值。方法选择该院2013年6~12月125份临床标本,包括血液40份,痰液52份,创面分泌物33份进行FQ-PCR检测和细菌鉴定,分析结果符合率,以检测FQ-PCR的灵敏度及特异性。结果FQ-PCR的灵敏度为0.159pg/μL,检测经细菌培养鉴定的菌株,特异性达到100%,与临床标本结果符合率为97.6%。结论 FQ-PCR检测MRSA的MecA基因更加方便、快速,且灵敏度和特异性等性能指标均比较理想,与细菌鉴定结果符合率较高,适合临床广泛应用。     

Improvement of water-soluble cephalosporin derivatives having antibacterial activity against methicillin-resistant Staphylococcus aureus

We evaluated a series of novel cephem antibiotics, N-alkylpyridinium (alkyl group), N-carboxyethylpyridinium (carboxylic group), N-sulfoethylpyridinium (sulfonic group) and N-alkylquaternary ammonium salts (ammonioethyl group), N-alkyl-aromatic-quaternary ammonium salts and N-alkyl-heterocyclic quaternary ammonium salts (cyclic group) as vinylthio pyridinium derivatives at the C-3 position and hydroxyiminoaminothiazol at the C-7 position, for their activity against methicillin-resistant Staphylococcus aureus (MRSA) and their solubility, by measuring the minimum inhibitory concentrations (MICs) and the dissolving test in phosphate buffer. All tested compounds, except for the alkyl group, showed good solubility (>10%) in 1/15 M phosphate buffer (pH 7.2). The concentrations required to inhibit 80% of the bacterial strains (MIC80s) of the alkyl group, carboxylic group, sulfonic group, ammonioethyl group and cyclic group against MRSA were 1.56, 12.5-25, 6.25, 1.56 and 1.56 microg/ml, respectively. These results indicated that the ammonioethyl and cyclic groups yield the maximum anti-MRSA and anti-Enterococcus faecalis activity, and also good water solubility.     

Current antimicrobial resistance profiles among methicillin-resistant Staphylococcus aureus encountered in the outpatient setting

The acquisition of the mec gene complex by methicillin-susceptible Staphylococcus aureus in the community and the increased spread of methicillin-resistant Staphylococcus aureus (MRSA) from the health care setting to the community underscore a need to monitor the resistance phenotypes likely to be encountered among outpatient MRSA. Data from the LEADER 2004 surveillance program were analyzed to evaluate current resistance profiles among outpatient MRSA. Outpatient MRSA exhibited 26 different resistance phenotypes; the 4 most common were resistance to erythromycin only (40.8%), multidrug resistance to erythromycin, clindamycin, and levofloxacin (21.5%), double drug resistance to erythromycin and levofloxacin (11.3%), and double drug resistance to clindamycin and erythromycin (5.1%). These phenotypes were also the most common among inpatient MRSA (n = 946), but multidrug resistance to erythromycin, clindamycin, and levofloxacin (43.7%) was most common. Fifty percent (256) of the outpatient MRSA were resistant to 2 or more agents, whereas resistance to either vancomycin or linezolid was not encountered. The extensive similarities in resistance profiles between inpatient and outpatient MRSA have important implications for establishing outpatient management and treatment guidelines for staphylococcal infections.     

Susceptibility testing of methicillin-resistant Staphylococcus aureus with three commercial microdilution systems

The antimicrobial susceptibilities of 100 methicillin-resistant Staphylococcus aureus isolates were determined concurrently by API Uniscept KB, Micro-Media, MicroScan, standardized disk diffusion, and reference broth microdilution to evaluate whether these commercial microdilution systems would reliably defect methicillin-resistant S. aureus. The methicillin minimal inhibitory concentration for all isolates was greater than or equal to 16 micrograms/ml as determined by the reference minimal inhibitory concentration panels containing 2% NaCl supplemented Mueller-Hinton broth. Using the breakpoints established by the National Committee for Clinical Laboratory Standards for reporting susceptible and resistant methicillin results, there was 100% agreement between the reference methods and API Uniscept KB at 24 hr. The Micro-Media and MicroScan systems had 47% and 8% very major discrepancies at 24 hr, respectively. At 48 hr, these two systems exhibited 15% and 0% very major discrepancies. Micro-Media and MicroScan were in agreement with the reference microdilution method (+/- 1 log2 dilution) for 62% and 68% of the strains at 24 hr, respectively and 88% and 85% of the isolates at 48 hr, respectively. The results of this study indicate that API Uniscept KB would provide a practical and reliable method for the detection of methicillin-resistant S. aureus.     

Evaluation of once-daily vancomycin against methicillin-resistant Staphylococcus aureus in a hollow-fiber infection model

For methicillin-resistant Staphylococcus aureus (MRSA) infections, data suggest that the clinical response is significantly better if the total vancomycin area under the concentration-time curve (AUC)/MIC ratio is ≥400. While the AUC/MIC ratio is the accepted pharmacokinetic/pharmacodynamic (PK/PD) index for vancomycin, this target has been achieved using multiple daily doses. We are unaware of a systematically designed dose fractionation study to compare the bactericidal activity of once-daily administration to that of traditional twice-daily administration. A dose fractionation study was performed with vancomycin in an in vitro hollow-fiber infection model against an MRSA USA300 strain (MIC of 0.75 μg/ml) using an inoculum of ～10(6) CFU/ml. The three vancomycin regimens evaluated for 168 h were 2 g every 24 h (q24h) as a 1-h infusion, 1 g q12h as a 1-h infusion, and 2 g q24h as a continuous infusion. Free steady-state concentrations (assuming 45% binding) for a total daily AUC/MIC ratio of ≥400 were simulated for all regimens. A validated liquid chromatography-tandem mass spectrometry method was used to determine vancomycin concentrations. Although once-daily and twice-daily dosage regimens exhibited total trough concentrations of <15 μg/ml, all regimens achieved similar bactericidal activities between 24 and 168 h and suppressed the amplification of nonsusceptible subpopulations. No colonies were found on agar plates with 3× MIC for any of the treatment arms. Overall, the results suggest that once-daily vancomycin administration is feasible from a PK/PD perspective and merits further inquiry in the clinical arena.     

Methicillin-resistant Staphylococcus aureus in dermatology]

Staphylococcus aureus (SA) is among the most important causes of skin infections. The incidence of Methicillin-resistant SA (MRSA) strains isolated from skin and skin structure infections was about 20-40%. In deep-seated pyoderma such as furuncle and furunculosis, MRSA was more frequently isolated than in other type of infectious diseases of the skin. But the incidence was gradually increasing. As to coagulase typing, type IV was most frequently isolated in MRSA. The damaged skin is easily colonized by high numbers of SA on its surface and within hair follicles. Through the indwelling catheters or decubitus SA on the skin could cause easily severe systemic MRSA infections such as sepsis or endocarditis of in-patients.     

Ciprofloxacin for methicillin-resistant Staphylococcus aureus infections.

Thirty-seven patients with methicillin-resistant Staphylococcus aureus infections and/or colonization were treated with oral ciprofloxacin (750 mg twice a day). Clinical cure or improvement of infections occurred in 91% of the patients, and bacteriologic cure occurred in 60%. Ciprofloxacin therapy suppressed methicillin-resistant S. aureus colonization in 55% of the patients. Ciprofloxacin-resistant strains emerged in 6 of the 37 patients.     

Comparative analysis of methicillin-resistant Staphylococcus aureus isolated from outpatients of dermatology unit in hospitals and clinics

Community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) is a causative agent of intractable skin infections. In general, clinical symptoms of hospital outpatients with skin infections are severer than those of clinic patients. Hence, molecular epidemiological features of the CO-MRSA strains from hospital outpatients are predicted to be different from those of clinic patients. Here, we conducted a comparative analysis for CO-MRSA isolates from outpatients with impetigo in hospitals and clinics located in the same district of Tokyo, Japan. Incidence of MRSA infection was higher in hospital outpatients (21.5%, 20/93 isolates) than in clinic patients (14.5%, 121/845 isolates). The resistance rate to clindamycin, which is a common topical antimicrobial agent in dermatology, in the isolates from hospital outpatients (60.0%) was higher than those from clinic patients (31.4%). Proportion of the staphylococcal cassette chromosome (SCC) mec type II, which is a representative type of hospital-acquired MRSA in Japan, in the isolates from hospital outpatients (65.0%) was significantly higher than those from clinic patients (30.6%) (P < 0.01). Multilocus sequence typing showed that the clonal complex 89-SCCmec type II (CC89-II) clone, which exhibits clindamycin resistance, was the most predominant (55.0%) in the isolates from hospital outpatients. On the other hand, all CC8-IV, CC121-V, and CC89-V clones accounted for 60% in clinic patients were susceptible to clindamycin. Our findings suggested that the clindamycin-resistant CC89-II CO-MRSA clone might be more related to skin infections in hospital outpatients than clinic patients.     

Management of healthcare-associated methicillin-resistant Staphylococcus aureus

Healthcare-associated methicillin-resistant Staphylococcus aureus is a major cause of nosocomial infections worldwide, with significant attributable morbidity and mortality in addition to pronounced healthcare costs. Treatment results with vancomycin--the current recommended antibiotic for serious methicillin-resistant S. aureus infections--have not been impressive. The recent availability of effective antimicrobial agents other than glycopeptides, such as linezolid and daptomycin, as well as the anticipated approval of newer agents with diverse mechanisms of action, has somewhat ameliorated the threat posed by this organism. However, these drugs are expensive, and there is still no overall satisfactory strategy for reducing the incidence of healthcare-associated methicillin-resistant S. aureus in endemic regions. Although early results with the Society for Healthcare Epidemiology of America guidelines give cause for cautious optimism, long-term experience is lacking, and it is likely that these guidelines will have to be adapted according to local conditions and resources before implementation. Trends to keep in mind when considering the problem of healthcare-associated methicillin-resistant S. aureus include the advent of community-associated methicillin-resistant S. aureus, and the propensity of S. aureus to evolve and acquire resistance determinants over time. This was last vividly demonstrated by the handful of vancomycin-resistant S. aureus isolated recently, which had acquired the vancomycin resistance gene from vancomycin-resistant enterococci.     

Evidenced-based practice for control of methicillin-resistant Staphylococcus aureus

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THE INCREASING PREVALENCE of methicillin-resistant Staphylococcus aureus (MRSA) has become a global issue and affects nursing practice in many clinical areas. This article explores methods for effective control of MRSA in hospital settings.

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BASED ON INFECTION CONTROL guidelines provided by the Centers for Disease Control and Prevention, the College of Nurses of Ontario, AORN, the World Health Organization, and several evidence-based studies, strategies for MRSA infection control measures include hand hygiene, contact isolation, and hospital environment hygiene. AORN J 81 (February 2005) 361-372.