金黄色葡萄球菌万古霉素体外诱导中介株凝固酶基因序列改变

目的研究万古霉素敏感金黄色葡萄球菌经过体外诱导变成万古霉素中介耐药的金黄色葡萄球菌后凝固酶基因序列有无改变。方法利用酶切及SSCP技术检测凝固酶基因序列改变情况,并测序进行确证。结果检测到1株金黄色葡萄球菌凝固酶基因序列有改变。结论凝固酶基因酶切位点的改变及其他位点碱基的改变,可能是凝固酶活性降低的原因之一。     

血液中“金萄菌变异株”的鉴定

由于抗菌药物的广泛应用,导致金葡菌变异菌株增多。变异菌株失去了产生色素和合成血浆凝固酶的能力,如按传统的凝固酶试验进行鉴定,经常易将“金葡菌变异株”误诊为表皮葡萄球菌。作者自1985年以来共收集变异菌58株,经培养特性、甘露醇厌氧发酵、血浆-RF 胶乳-IgG 协同凝集试验及DNA 酶试验等多种方法鉴定,与初次分离菌株比较,确定为     

金黄色葡萄球菌临床分离株对万古霉素敏感性的监测

目的检测金黄色葡萄球菌（金葡菌）对万古霉素药物敏感情况,以监控对万古霉素敏感性降低金葡菌[vaneomyein-intermediate Staphylococcus aureus（S．aureus）,VISA]或耐万古霉素金葡菌（vancomycin-resistant S．aureus,VRSA）的出现,防止其暴发流行。方法对石家庄市区2家三级甲等医院分离自痰液、支气管肺泡灌洗液等临床标本的55株金葡菌应用苯唑西林纸片和头孢西丁纸片检测是否为耐甲氧西林的金葡菌（methieilin-resistant S．aureus,MRSA）;对检出的MRSA,再用胶乳凝集试验检测青霉素结合蛋白2a（PBp-2a）进一步确认。应用Kirb-Bauer纸片法和琼脂稀释法测定万古霉素对55株金葡菌的抑菌环大小和最低抑菌浓度（MIC值）,并比较MRSA与对甲氧西林敏感的金葡菌（methieilin-suseeptibility S．aureus,MSSA）的MIC差异。同时对2004～2005年河北医科大学第二医院分离金葡菌的耐药性状况进行了回顾分析。结果55株金葡菌的万古霉素抑菌环直径15～21mm,MIC≤1mg／L,且MRSA与MSSA两组之间差异无统计学意义（P〉0．05）。河北医科大学第二医院2004年、2005年MRSA的检出率分别为56．8％和64．5％。结论石家庄市区医院分离的55株金葡菌中未检出VISA或VRSA,但部分菌株已经接近耐药折点,应密切关注其发展动向;MRSA的耐药机制可能与金葡菌对糖肽类抗生素耐药无直接相关性。     

呼吸监护病区金黄色葡萄球菌耐药性监测(附21株资料分析)

目的：对呼吸监护病区（ＲＩＣＵ）分离的２１株金黄色葡萄球菌（金葡菌）进行耐药性监测，方法；药敏试验采用纸片扩散法，用色原头孢菌素（ｎｉｔｒｏｃｅｆｉｎ）纸片法检测内酰胺酶，结果：ＲＩＣＵ分离的金葡菌高于普通病房，检出耐甲氧西林金葡菌（ＭＲＳＡ）１７株，ＭＲＳＡ有１２株产内酰胺酶，结论：金葡菌产酶株与非产朱的耐药相比有差别，万古霉素是唯一对金葡菌完全敏感的抗菌药物。     

金黄色葡萄球菌高保守性fib基因的鉴定及其表达

金葡菌为全球性重要致病菌之一，它产生大量的细胞外蛋白，是重要的致病因子，包括凝固醇和纤维蛋白原结合蛋白（Fibrinogen－BindingFib）在内。后者可与纤维蛋白原结合，与金葡菌粘附于静脉内导管或内皮细胞有关，也可增加中性粒细胞的杀菌活性。Fib分子量为19kDa，它存在于金葡菌体内，但由于金葡菌分泌的水解酶的作用，不能100％检测出来，故Fib基因常用于金葡菌的鉴定。作者等曾纯化制备出Fib，它与凝固酶的C-末端区具有同源性。作者进一步从DNA水平，mRNA和蛋白质水平对Fib基因和Fib进行了研究。临床分离的葡萄球菌中，只有金…     

2010年中国CHINET葡萄球菌属细菌耐药性监测和分析

目的 了解国内不同地区医院葡萄球菌属细菌临床分离株对抗菌药物的耐药性.方法 中国14所综合性医院,按统一方案进行葡萄球菌属细菌的耐药性监测和分析.结果 7 530株葡萄球菌属细菌临床分离株中,金葡菌占59.1％;凝固酶阴性葡萄球菌占40.9％(均分离自血液和无菌体液).后者中有表皮葡萄球菌(21.6％)、人型葡萄球菌(7.3％)和溶血葡萄球菌(4.5％)等.金葡菌和凝固酶阴性葡萄球菌中耐甲氧西林菌株分别占51.7％(11.5％～77.6％)和74.8％ (62.7％00～95.5％).60％以上的甲氧西林耐药金葡菌(MRSA)对磷霉素和甲氧苄啶-磺胺甲(啞)唑敏感.甲氧西林耐药凝固酶阴性葡萄球菌(MRCNS)中分别有68.7％和87.8％菌株对磷霉素和利福平敏感.重症监护病房患者中,MRSA和MRCNS检出率最高,尤其在某些医院中的某些科室有相对集中趋势.未发现对糖肽类抗生素和利奈唑胺耐药的菌株.结论 国内临床上甲氧西林耐药葡萄球菌属的检出率较高,细菌对抗菌药物的耐药率不同医院和地区间存在较大的差异,须重视对异质性万古霉素耐药金葡菌(hVISA)和万古霉素耐药金葡菌(VRSA)的检测.     

多种金葡菌分型法间的关系

金黄色葡萄球菌引起人类多种严重感染,多重耐药的金葡菌导致院内感染逐年增加,流行病学调查需要将金葡菌分型.常用的金葡菌分型方法有:噬菌体分型、荚膜多糖血清型及酶分型、核酸分型和质粒分型.作者等利用SmaI消化金葡菌染色体DNA然后进行脉冲场凝胶电泳,依酶切图形对流行病学无关的两个来源的69株金葡菌分型(36株来自法国.33株来自德国的囊性纤维化病人的4周夏令营地),并与噬菌体、荚膜和酶分型法比较.     

金黄色葡萄球菌耐消毒剂基因qacA的流行病学研究

目的 了解重庆医科大学附一院近年临床分离的金葡菌中,耐消毒剂基因qacA的流行情况,从而指导临床抗菌药物、消毒剂的合理使用.方法 选取有代表性的1价和2价化合物,检测126株金葡菌的MIC,阳性株用PCR法检测qacA基因型,采用CLSI推荐的30 μg头孢西丁纸片法进行MRSA鉴定.结果 临床分离的126株金葡菌中,12株qacA基因型呈阳性(阳性率为9.5%),52株为MRSA(其中qacA基因阳性率为17.3%).结论 临床分离的金葡菌中,qacA基因携带率较高.     

铜陵地区2003年度细菌耐药性监测

目的：了解安徽省铜陵地区临床分离菌株耐药状况。方法：2003年1—12月铜陵地区临床分离菌株用Kitby-Bauer法进行药敏试验。结果：918株细菌中革兰阳性菌393株占42．8％，革兰阴性菌525株占57．2％。耐甲氧西林金黄色葡萄球菌(金葡菌)和耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)分别占金葡菌和凝固酶阴性葡萄球菌(CNS)的35．4％和85．6％。金葡菌和CNS对青霉素、氨苄西林及庆大霉素等均高度耐药，对利福平、磷霉素及氯霉素的耐药率均较低；未见耐万古霉素葡萄球菌。粪肠球菌对青霉素、氨苄西林的耐药率较低，对利福平、磷霉素、万古霉素和替考拉宁无耐药；屎肠球菌对万古霉素和替考拉宁也无耐药。大肠埃希菌和克雷伯菌属中产超广谱β内酰胺酶(ESBLs)株分别占32．7％和33．9％，产ESBLs株对16种抗菌药物的耐药率均较不产ESBLs株高，对亚胺培南均无耐药。结论：细菌耐药有一定的地区性，定期对本地区细菌耐药性进行监测，对合理使用抗菌药物、减少耐药菌株的产生和流行有重要临床指导价值。     

耐万古霉素金黄色葡萄球菌耐药机制及检测方法研究进展

我国是一个耐苯唑西林金黄色葡萄球菌（MRSA）菌株发生率极高的国家。万古霉素保持了对MRSA很高的活性，目前国内有关耐万古霉素金黄色葡萄球菌（简称金葡菌）分离株的报道较少。但是某些MRSA菌株由于继续突变或基因转移出现耐万古霉素金葡菌，这将是未来必然要面对的一个严重的临床问题。因此，了解国内外耐万古霉素金葡菌感染现状、耐药机制及实验室检测方法很有必要。     

Evaluation of latex agglutination and microtube coagulase tests for detection of Staphylococcus aureus

In a blind study, a latex agglutination test (Serostat Staphylococcus, Scott Laboratories) and a microtube coagulase test (Staphase, API) were evaluated for their ability to detect Staphylococcus aureus. Of 289 isolates of catalase-positive, gram-positive cocci, 122 were identified as S. aureus based on positive reactions in at least three of the following tests: tube coagulase, slide coagulase, DNase production, or anaerobic fermentation of mannitol. The latex agglutination test gave positive reactions for all S. aureus isolates and 10 (6%) non-S. aureus isolates. The slide coagulase test was positive for 121 S. aureus isolates and three (2%) non-S. aureus isolates. The microtube coagulase test detected 53, 90, and 98% of the S. aureus isolates after 2, 4, and 24 hr, respectively. In contrast, the conventional tube coagulase test detected 97% of the S. aureus isolates after 2 hr, and 98% after 4 and 24 hr. Two isolates of S. aureus gave negative tube coagulase reactions at 37 degrees C, but positive reactions at room temperature after 24 hr. The combination of tube and slide coagulase tests provided the most reliable results. The slide and tube coagulase tests gave more reliable results than the latex agglutination and microtube coagulase tests, respectively.     

In-vitro activity of coumermycin against methicillin-resistant staphylococci: a comparison with six other agents

The in-vitro activity of coumermycin was compared with that of vancomycin, rifampicin, fusidic acid, trimethoprim-sulphamethoxazole, norfloxacin and cefamandole against seven isolates of methicillin-resistant Staphylococcus aureus and 97 isolates of methicillin-resistant coagulase negative staphylococci. Apart from one strain of methicillin-resistant S. aureus all isolates were inhibited by less than or equal to 0.06 mg/l of coumermycin. Cefamandole was more active against strains of S. epidermidis than against other coagulase negative staphylococci.     

Effects of short interfering RNA against methicillin-resistant Staphylococcus aureus coagulase in vitro and in vivo

OBJECTIVES: The emergence of antibiotic-resistant bacteria such as Staphylococcus aureus calls for inventive research and development strategies. Inhibition of bacterial pathogenesis may be a promising therapeutic approach in this regard. The gene-silencing effect of short interfering RNA (siRNA) is useful for this strategy. We investigated the efficacy of siRNA on the expression of coagulase because it is the one of the most important enzymes in the pathogenesis of methicillin-resistant S. aureus (MRSA) infection. METHODS: We designed and synthesized 21 bp siRNA duplexes against staphylococcal coagulase. RT-PCR was performed to determine whether the siRNAs inhibit the expression of the coagulase mRNA and radio-labelled siRNA was used to confirm transfection to bacteria in vitro. The efficacy of siRNA was determined in a murine model of haematogenous pulmonary infection. RESULTS: RT-PCR showed that siRNAs significantly inhibited the expression of the coagulase mRNA. The coagulase titres in the siRNA and control groups were 8 and 32, respectively. Measurement of incorporated radioactivity indicated that the siRNAs were delivered into the bacteria. In the murine infection model, in control and siRNA groups, 7.64 +/- 0.42 and 6.29 +/- 0.23 log cfu/mL (mean +/- SEM) MRSA were detected, respectively, showing that there was a significant decrease in the number of viable bacteria in the siRNA group (P < 0.05). CONCLUSIONS: The results show that siRNA inhibited both mRNA expression and the activity of MRSA coagulase in vitro. The in vivo results revealed that the siRNA was effective in reducing the bacterial load in a murine model of haematogenous pulmonary infection. Targeting of coagulase with siRNA appears to be a novel strategy for treating MRSA infections.     

A蛋白与凝固酶凝集试验在金黄色葡萄球菌快速鉴定中的应用

目的探讨金黄色葡萄球菌快速检测的可能性。方法用凝固酶(CA)(试管法和玻片法)和葡萄球菌A蛋白(SPA)两种方法检测667株葡萄球菌。结果378株金黄色葡萄球菌中二者同时呈阳性的有303株,阳性率为80.2%,特异性、准确性均为100%;CA与SPA试验联合使用的阳性率之和高至98.9%,而其他289株葡萄球菌CA与SPA试验的阳性率之和仅为12.4%。结论CA与SPA试验联合运用对金黄色葡萄球菌的鉴定具有快速、准确、简便易行及特异性强等优点。     

Prosthetic joint infection due to Staphylococcus lugdunensis

Staphylococcus lugdunensis, a coagulase-negative staphylococcus, is being increasingly recognized as the cause of serious infections. We report 2 cases of total knee arthroplasty infection caused by S lugdunensis. S lugdunensis frequently produces a clumping factor that can result in a positive slide (short) coagulase test result. If the microbiology laboratory does not use the tube coagulase (long) test to confirm the slide coagulase test result, the organism may be misidentified as Staphylococcus aureus. S lugdunensis is more virulent than other coagulase-negative staphylococci and in many clinical situations behaves like S aureus, further increasing the confusion. However, S lugdunensis differs from S aureus in that it is susceptible to most antibiotics. This fact may alert the microbiology laboratory or the clinician that the isolate is likely not S aureus and prompt further testing of a specific isolate. Accurate identification of S lugdunensis isolates facilitates studies to define the epidemiology and pathogenesis of prosthetic joint infection due to S lugdunensis and delineates optimal medical and surgical therapies.     

多重聚合酶链反应检测耐甲氧西林葡萄球菌

目的应用一种敏感快速的多重聚合酶链反应（ＰＣＲ）,检测耐甲氧西林的金黄色葡萄球菌（ＭＲＳＡ）和凝固酶阴性葡萄球菌（ＭＲＣＮＳ）。方法临床分离的北京地区金黄色葡萄球菌１２３株,ＭＲＣＮＳ１２２株,用溶壁素和蛋白酶Ｋ制备模板ＤＮＡ,设计葡萄球菌甲氧西林耐药的决定基因,金黄色葡萄球菌独有的一个辅助基因和细菌中均有的１６ＳｒＲＮＡ基因引物,通过多重ＰＣＲ技术对标本进行扩增。结果１２３株金黄色葡萄球菌的ｆｅｍＡ基因１００％（１２３／１２３）阳性,ｍｅｃＡ基因阳性的占１８．７％（２３／１２３）,１２２株ＭＲＣＮＳ的ｆｅｍＡ１００％（１２２／１２２）阴性,ｍｅｃＡ阳性的占２４．６％（３０／１２２）。１６ＳｒＲＮＡ基因片断在多重ＰＣＲ中作为内部参照避免了假阴性结果的出现。结论建立的多重ＰＣＲ技术检测ＭＲＳＡ和ＭＲＣＮＳ具有敏感、快速、特异的特点,是一种可靠的实验诊断手段。     

Development and validation of microarray-based assay for epidemiological study of MRSA

We have developed a microarray-based assay for the genotyping of Staphylococcus aureus strains. A DNA microarray consisting of 221 genes with 390 oligonucleotide probes was designed to identify characteristic genes or gene alleles of S. aureus. The 221 genes were chosen on the basis of the following criteria: (i) genes used as control for the microarray system, (ii) virulence genes, (iii) resistance genes and their regulators, and (iv) genes constituting genomic islands, e.g., SCCmec. The microarray system was established by determining the method to prepare targets by random-primer labeling with chromosomal DNA and the conditions for hybridization. We verified the system by using DNAs of seven strains, the genome of which has been fully sequenced. Furthermore, the presence of 32 genes and the types of SCCmec elements and coagulase genes carried by another 27 strains were examined and compared with the results of PCR. As a result, the presence or absence of 182 genes out of the 221 genes was verified. Our data showed the usefulness of the oligonucleotide microarray based assay in identifying important marker sets, such as toxin genes, resistance genes, SCCmec elements, and coagulase genes, for the molecular epidemiology of S. aureus.     

Virulence factor expression by Gram-positive cocci exposed to subinhibitory concentrations of linezolid

Linezolid is a new oxazolidinone with potent antibacterial activity against Gram-positive cocci; it uniquely inhibits bacterial translation through inhibition of 70S initiation complex formation. The effects of sub-growth-inhibitory concentrations of linezolid on the expression of various structural and soluble virulence factors of Staphylococcus aureus and Streptococcus pyogenes were examined. For S. aureus, strains Wood 46 and Cowan 1 (NCTC 8532) were used to measure protein A, coagulase, alpha-haemolysin (hla) and delta-haemolysin (hld). For S. pyogenes, strain NCTC 9994 was used to measure M protein, streptolysin O (SLO) and DNase. Coagulase was assayed by clotting of citrated rabbit plasma, and hla, hld and SLO by lysis of rabbit, human and horse erythrocytes, respectively. Protein A and M protein were measured indirectly using bacterial susceptibility to phagocytic ingestion of radiolabelled bacteria by human neutrophils. When S. aureus was grown in 1/2, 1/4 and 1/8 MIC, linezolid, coagulase, hla and hld production were impaired. Susceptibility to phagocytosis was changed by growth in the presence of 1/2 MIC linezolid compared with that in its absence (50.8 +/- 4.1% versus 38.9 +/- 2.9%; P 相似文献   

Attenuated virulence and biofilm formation in Staphylococcus aureus following sublethal exposure to triclosan

Subeffective exposure of Staphylococcus aureus to the biocide triclosan can reportedly induce a small-colony variant (SCV) phenotype. S. aureus SCVs are characterized by low growth rates, reduced pigmentation, and lowered antimicrobial susceptibility. While they may exhibit enhanced intracellular survival, there are conflicting reports regarding their pathogenicity. The current study reports the characteristics of an SCV-like strain of S. aureus created by repeated passage on sublethal triclosan concentrations. S. aureus ATCC 6538 (the passage 0 [P0] strain) was serially exposed 10 times to concentration gradients of triclosan to generate strain P10. This strain was then further passaged 10 times on triclosan-free medium (designated strain ×10). The MICs and minimum bactericidal concentrations of triclosan for P0, P10, and ×10 were determined, and growth rates in biofilm and planktonic cultures were measured. Hemolysin, DNase, and coagulase activities were measured, and virulence was determined using a Galleria mellonella pathogenicity model. Strain P10 exhibited decreased susceptibility to triclosan and characteristics of an SCV phenotype, including a considerably reduced growth rate and the formation of pinpoint colonies. However, this strain also had delayed coagulase production, had impaired hemolysis (P < 0.01), was defective in biofilm formation and DNase activity, and displayed significantly attenuated virulence. Colony size, hemolysis, coagulase activity, and virulence were only partially restored in strain ×10, whereas the planktonic growth rate was fully restored. However, ×10 was at least as defective in biofilm formation and DNase production as P10. These data suggest that although repeated exposure to triclosan may result in an SCV-like phenotype, this is not necessarily associated with increased virulence and adapted bacteria may exhibit other functional deficiencies.