目的 对2株临床分离的烟曲霉菌株进行病原学检测和分子鉴定,并对不同检测方法进行比较分析.方法 收集2例侵袭性肺曲霉菌病(IPA)患者痰液标本,采用直接压片、亚甲蓝染色和改良GMS染色,以及分离、培养等病原学检测,应用rodA、anxc4、β-tub、ITS、cyp51A和cyp51B基因进行PCR、环介导等温扩增(LAMP)、分子克隆和Sanger测序.结果 痰涂片镜检可见典型的烟曲霉鹿角样菌丝、分生孢子头和分生孢子;真菌显色培养基上菌落颜色由白色转为蓝绿色;rodA、anxc4、β-tub、ITS、cyp51A和cyp51B与Genbank数据库中烟曲霉相应序列的同源性均在99.7％以上;LAMP扩增产物肉眼可见白色沉淀,琼脂糖凝胶电泳呈典型梯度条带,HNB可视化检测结果呈淡蓝色,real-time LAMP呈"S"型扩增曲线.结论 临床疑似烟曲霉感染时,可通过LAMP进行快速分子鉴定,辅助临床提升诊断效率,为临床治疗提供依据.

目的 比较耶氏肺孢子菌(P.jirovecii)不同病原学染色形态特征,分析其在肺孢子菌肺炎(PJP)诊断中的价值.方法 收集1例PJ P患者的支气管肺泡灌洗液,制成涂片,运用不同病原学染色方法分别对其进行染色,分析、评价不同染色方法在PJ P诊断中的效果.结果 革兰染色和抗酸染色可见透明空泡样结构;真菌荧光染色可见发出蓝色荧光的P.jirovecii包囊;HE染色P.jirovecii包囊囊壁不着色,呈透明晕圈状,囊内可见4～8个囊内小体;Wright-Giemsa染色和改良Giemsa染色可见圆形或卵圆形P.jirovecii包囊,囊壁不着色但折光性强,呈新月形,囊内可见4～8个囊内小体;改良GMS染色P.j irovecii包囊囊壁为深褐色或黑色,呈特征性括弧样结构,囊内小体不着色,中间有1条黑色褶皱或中心点状深染.结论 HE染色、Wright-Giemsa染色、改良Giemsa染色、改良GMS染色均可用于确诊PJP,但HE染色、Wright-Giemsa染色、改良Giemsa染色对检验人员要求较高,而改良GMS染色的染色效果好,是临床确诊PJP的推荐病原学染色方法.

大肠埃希菌和肺炎克雷伯杆菌是医院感染常见的革兰阴性杆菌,第三代头孢菌素的广泛使用使细菌的耐药性越来越强, 产生超广谱β-内酰胺酶 (extended-spectrum β-lacta-mases,ESBLs) 是革兰阴性菌对第三代头孢菌素耐药的主要原因.自1983年首次报道发现ESBLs菌株以后,ESBLs的流行日益严重,世界各地均有报道,且数量和基因型不断增加.ESBLs通常为质粒介导, 同一质粒可同时携带多个耐药基因, 引起多重耐药. 最常见的ESBLs由TEM-1、TEM-2和SHV-1突变而来, 主要由克雷伯菌属和大肠埃希菌产生,大部分是头孢他啶酶,少部分是头孢噻肟酶. 其次还有不断报道的其他类型的耐药酶类, 头孢他啶酶PER、VEB、TLA-1、GES/IBC和头孢噻肟酶CTX-M(Toho)、SFO-1、BES-1等[1].本研究以肺炎克雷伯菌作为研究对象, 以3个常见的基因型TEM型、SHV型和CTX-M型为检测目标,鉴定细菌产ESBLs的情况,以及产ESBLs菌株的耐药特性,探讨耐药性与基因型的关系,对临床合理使用抗生素起到积极的指导作用.

耐甲氧西林金黄色葡萄球菌（MRSA）是引起医院和社区感染的重要病原菌之一［1］。近年来随着广谱及超广谱β-内酰胺类抗菌药物在临床上的广泛使用，MRSA的检出率不断增加［2］。MRSA感染有逐年增高的趋势，且具有多重耐药的特点。对MRSA耐药机制进行研究，寻找药物作用的新靶点，是目前全世界都密切关注的问题之一。由于MRSA在不同国家、不同地区的流行菌株、耐药情况、多重耐药模式、基因型别等均存在着很大差异，因此，有必要探明本地区一些大型医院MRSA的流行状况和耐药模式，为明确耐药机制、致病机制、寻找药物作用的靶点及研发新药奠定基础，同时还可以指导临床治疗MRSA感染，控制耐药谱迅速扩大。

耐甲氧西林金黄色葡萄球菌(MRSA)是携带mecA基因的金黄色葡萄球菌或苯唑西林最小抑菌浓度(MIC)≥4 mg/L的金黄色葡萄球菌.大部分MRSA菌株携带mecA基因,但少数MRSA不携带mecA基因,存在其他的耐药机制,如青霉素结合蛋白(PBP)改变或高产β-内酰胺酶等. l MRSA的检测 1.1纸片扩散法:制备0.5麦氏单位菌悬液涂布在水解酪蛋白琼脂(MHA)平皿上,贴30 μg头孢西丁纸片或者1μg苯唑西林纸片,33～35℃(35℃以上可能检测不到MRSA)孵育24 h判读结果,头孢西丁可作为苯唑西林耐药性检测的替代物,应根据头孢西丁结果报告苯唑西林敏感或耐药.如果2个药物同时用于金黄色葡萄球菌,任何一种药物耐药,报告该菌株对苯唑西林耐药.

Objective To study the resistance characteristics and the tendency of AmpC β-lactamase in acinetobacter baumannii by analyzing their resistant phenotype and genotype in Shanxi province.Methods Totally 63 strains of multidrug resistant acinetobacter baumannii were collected by clinical dissociation from two comprehensive hospitals of Shanxi province from January to October of 2010.Drug sensitivity test was performed by agar diffusion method.Polymerase chain reaction(PCR) was used to detect AmpC enzyme.Results Sixty-one strains out of the 63 were resistant to cefoxitin,with a resistant rate of 97%,and the resistant rates to other commonly used antibiotics were also rather high,all above 90% except for imipenem,meropenem,minocycline,amikacin,cefoperazone /sulbactam,ticarcillin /clavulanic acid.Multiple drug resistance was noted,and the sensitivity rate to minocycline was the top rate,at 56%,that to cefoperazone /sulbactam ranked the second,at 41%.AmpC enzyme positive rate was 67% in all the 63 strains of acinetobacter baumannii.Conclusion The situation of acinetobater baumannii resistance is very serious in Shanxi province,and multiple drug resistance is spreading.A strain resisting all current antibiotics is noted.AmpC enzyme plays an important role in acinetobater baumanii drug-resistant mechanism,the carrying rate of which is 67%.Minocycline and cefoperazone /sulbactam are presently the most effective antibiotics in Shanxi.

目的 分析产超广谱β-内酰胺酶(extended spectrum β lactamases,ESBLs)及AmpC酶的阴沟肠杆菌的分布情况及耐药特征.方法 选择49株阴沟肠杆菌,采用纸片扩散法进行药物敏感试验,按美国临床实验室标准化协会推荐的确证试验检测产ESBLs阳性菌株；头孢西丁纸片扩散法初步筛选产AmpC酶阳性菌株,采用PCR检测AmpC酶基因.结果 抗生素敏感试验结果显示阴沟肠杆菌对头孢西丁、替卡西林/克拉维酸、头孢曲松、氨曲南的耐药率均达79.6％以上,对美罗培南敏感率较高,达91.8％.49株阴沟肠杆菌中对头孢西丁耐药的菌株46株(93.9％)；产ESBLs菌株29株(59.2％),PCR 扩增产AmpC酶的阴沟肠杆菌36株(73.5％),同时产两种酶的菌株为13株(26.5％).结论 产ESBLs 及AmpC酶阴沟肠杆菌普遍且耐药现象严重.

Objective:To probe the relationship between active efflux pump MexAB-OprM in clinical isolated Pseudomonas aeruginosa strains and the carbopenem resistance,and study its molecular mechanism of drug resistance.Methods:Antimicrobial susceptibili of Pseudomonas aeruginosa clinical isolates were tested by Kirby-Bauer method against inipenem and meropenem.The strains were divided into Carbapenems-resistant group and Carbapenems-sensitive group according to carbopenems sensitivity.RT-PCR method was used to study the level of mRNA expression of oprM which was the structural gene in active efflux pump MexAB-OprM.PCR method was used to amplify the mexR gene of these two group strains,and analyze the PCR products with DNA sequencing.Results:The expression level of mRNA of oprM in the strains of the Carbapenems-resistant group was significantly higher than those in Carbapenems-sensitive group(P0.05).One strains had the amino acid substitu-tionin in MexR protein,Val(GTG)→Glu(GAG) at the position of amino acid 126.Two strains had the amino acid substitutionin of Arg(CGG)→Gln(CAG) at the position of amino acid 70.Conclusion:In The First Affiliated Hospital of Shanxi Medical University,active efflux pumps in clinical isolated Pseudomonas aeruginosa mediate the resistance to carbopenem.High level expression of MexAB-OprM is related to mexR gene mutation and other mutant type.

Objective:To identify and investigate if the variant OmpC being expressed by the clinical strains was playing a role in their antibiotic resistance.Methods:A low copy number plasmid containing the wild type E.coli K-12 ompC gene was transformed into the two different clinical isolates.Increased expression of OmpC,indicating expression of the chromosomal copy and the plasmid copy of the gene,was confirmed by carrying out outer membrane preparations and running SDS-PAGE gels.Results:To In the isolate 1 there were no significant changes in MIC with or without the wild type OmpC The isolate 7 however did show significant decreases in MICs for the carbapenem antibiotics,meropenem and imipenem,when wild type OmpC was expressed.It appears that the expression of wild type OmpC in the last isolate at least partially restores permeability allowing antibiotics into the cell and reducing the level of antibiotic resistance.This result indicates that at least one of the amino acid changes present in the last isolate 7.Conclusion:Prliminary data suggest that changes in the amino acid sequence of the contribute to antibiotic resistance via the modulation of ompC active.

Objective: To investigate the drug-resistance phenotype of Escherichia coli which produces extended-spectrum β-lactamases(ESBLs) and AmpC enzymes genotype.Methods: Escherichia coli producing both ESBLs and AmpC strains were screened and detected by double-disc diffusion and E-test.And the AmpC enzymes drug-resistance genotype was confirmed by PCR gene amplification method.Results: There was 1 AmpC strain among 68 strains producing extended-spectrum β-lactamases(ESBLs) Escherichia coli.Conclusion:Escherichia coli is a common bacterium which produces ESBLs and produces strains of AmpC enzymes at the same time to make the drug-resistance increased.It is important to detect and monitor the Escherichia coli in the laboratory.

Objective To investigate the methods of detecting Escherichia coli producing AmpC β-lactamase.Methods A total of 10 clinical isolates of non-repeated Escherichia coli were collected from different hospitals in Shanxi province.AmpC β-lactamase producing isolates were identified by cefoxitin three-dimensional test and antibiotic susceptibility was identified by agar dilution test.Plasmid extraction and PCR amplification of corresponding group was performed,followed by sequencing.Results The positive rate of cefoxitin three-dimensional test was 10%.The susceptibility test showed that all strains were resistant to cephamycins and piperacillin,and susceptible to imipenem.One strain of Escherichia coli was confirmed producing resistance to fourth-generation cephalosporin.Conclusion Escherichia coli which produces both ESBL,AmpC and clavulanate may induce hyper production of the AmpC β-lactamase leading to hydrolysis of the third generation cephalosporin thus masking any synergy arising from inhibition of the ESBL.So the Escherichia coli isolates of which disk diffusion screening test are positive should be confirmed by the PCR.Carbapenems could be the first choice for the treatment of infection caused by AmpC-lactamase producers.

随着抗生索在临床应用的不断增多,革兰阴性杆菌引起的多重耐药问题已成为全球关注的焦点,其中尤以β内酰胺类抗生素耐药为主~([1]).我国是世界上滥用抗生素最为严重的国家之一,耐药菌引起的医院感染人数,已占到住院感染患者总人数的30%左右~([2])。

Objective To study the mechanism of the Carbapenems-tolerance in Pseudomona aeruginosa ( P.aeruginosa ). Method Thirty-one strains of P. aeruginosa isolated from November 2008 to April 2009 in our hospital were analyzed. According to the results of drug sensitivity test, the thirty-one strains of P. aeruginosa were divided into Carbapenems-tolerance group(21 strains) and Carbapenems-sensitive group( 10 strains). Meanwhile, a standard strain of P.aeruginosa ATCC 27853 was also studied and an imipenem-EDTA inhibition test was conducted to detect metallo-beta-lactamases. PCR method was used to detect theoprD2 gene in P. aeruginosa,so as to study the mechanism of the Carbapenemstolerance inP. aeruginosa. Result 7 of 21 strains of Carbapenems-tolerance P. aeruginosa produced metallo-beta-lactamases; 15 of 21 strains of Carbapenems-tolerance P. aeruginosa were shown to be oprD2 gene negative, while the other 6 were positive. In contrast,all of the 10 strains of the Carbapenems-sensitive P. aeruginosa were found to be oprD2 gene positive,and so was the standard strain ATCC 27853. The results of the Chi-square test revealed that the difference between the positive rates of the oprD2 gene in the Carbapenems-tolerance and sensitive P. aeruginosa was statistically highly significant.Conclusion Loss of the oprD2 gene and metallo-beta-lactamases-producing ability were the important mechnisms of the Carbapenems-tolerance in P. aeruginosa of our hospital.

目的:为了解临床葡萄球菌感染的状况及对常用抗生素耐药性特点。方法:采用常规方法鉴定菌种,用K-B法及微量稀释法作15种抗生素体外抗菌活性分析。结果:285株葡萄球菌中凝固酶阳性耐甲氧西林金黄色葡萄球菌(MRSA)占51.79%,耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)占49.14%。对15种抗生素耐药性试验都显示出较高的耐药率,但MRSA耐药率高于凝固酶阳性对甲氧西林敏感的金黄色葡萄球菌(MSSA)和MRCNS。MRSA只对万古霉素有100%的抗菌活性;MSSA对苯唑西林、万古霉素、利福平及新生霉素100%敏感,耐药率小于20%有阿米卡星、头孢唑林、头孢呋汀(头孢呋肟);CNS对万古霉素、利福平、亚胺培南100%敏感,耐药率小于20%的有头孢唑林和头孢呋汀。结论:G+菌感染近年来日渐增多,MSSA、MRCNS、MSCNS与MRSA一样成为医院感染的重要病原菌,但对MRSA的感染更需引起足够的重视。切实加强实验室监测,对指导临床合理用药和制定有效治疗方案等具有十分重要的意义。

OBJECTIVE To identify and analyze whether the mutations in porins ompC and ompF result in a decrease in outer membrane permeability,and resistence to a number of chemically unrelated antibiotics.METHODS The ompC and ompF genes were sequenced using primers ompC6F,ompC2R,ompC8F and ompC4R.Primers were designed to correspond to the protein regions conserved between K-12(U00096) and ompC from the isolates,which were identified by the mass spectrometry analysis of tryptic fragments derived from ompC protein separated by SDS-PAGE of outer membrane extracts.RESULTS Analysis of outer membrane proteins by SDS-PAGE and peptide mass fingerprinting of bands reveals the presence of ompA and ompC,but not ompF.PCR analysis confirmed the presence of the ompF gene,but no evidence for its expression was obtained.It was notable that exhibited reduced levels of ompC and ompA in the last two isolates(6 and 7).Analysis of the PCR products showed that the fhuA PCR product was shorter for the clinical isolates than for Escherichia coli K-12(54bp).CONCLUSIONS Preliminary data suggest that changes in the amino acid sequence contribute to antibiotic resistance via the modulation of ompC activity.

OBJECTIVE To study the antibiotic sensitivities and characterization of β-lacamase activity and the antibiotic resistance mechanisms.METHODS The extended-spectrum β-lactamases were detected.Agar dilution method was used to determine the minimum inhibitory concentration(MIC)of severel antibiotics.The plasmids of clinical isolates were cloned into competent cells(K-12).RESULTS Isoelectric focusing(IEF):The last 5 clinical isolates have all got an activity at a high pH 9 which could be presumptively identified as AmpC.The last two clinical isolates had a weak activity with pH of about 8.0,which belonged to SHV type β-lactamases.All the isolates had a group of activities at the lower pH range of the gel(pH5).These could be presumptively identified as TEM β-lactamases.CONCLUSIONS The data suggest that each isolate clearly originate from the same population but possess mutations that leads to antibiotic resistance.The combinations of mutations in same isolates suggest that an earlier strain have infected other compartments and become the founder for later antibiotic resistant variants.

Objective To analyze the distributive characteristics,antimicrobial sensitivity and antimicrobial resistance of bacterium isolates from patients with AECOPD.Methods Five hundred and twenty-one clinical phlegm specimens were isolated from 2004 to 2006 and bacterial susceptibility test was carried out by Kirby-Bauer method according to NCCLS standards.Results Major pathogens in wards of respiratory disease include Pneumococcus(28.8%),Pseudomonas aeruginosa(9.8%),Coagulase-negative staphylococci(8.1%),Acinetobacter baumannii(6.1%),Escherichia colil(3.3%),Stenotrophomonas maltophilia(2.3%)and Staphylococcus aureus(1.3%).The result of bacterial susceptibility test showed that drug resistance rate of Pneumococcus,Staphylococcus aureus and Coagulase-negative staphylococci to Penicillin is 100%,the rate of sentivity to Vancomycine and Teicoplanin is 100%.Pseudomonas aeruginosa,Acinetobacter baumannii and Escherichia coli for ampicillin and Compound Sulfamethoxazole is 100%,but the sensitivity rate for Imipenem is more than 83.3%.The sensitive rate of Escherichia coli,Stenotrophomonas maltophilia and Klebsiella pneumoniae to Imipenem is 100%.Conclusion The drug resistance of bacterium isolated form patients with AECOPD is serious.It is suggested that there is an urgent need to enhance drug sensitive test and use antibiotics reasonable in order to prevent the creation and diffusion of drug resistant strain.

Objective To study the effect of 1,25(OH)2VitD3 and DDP on growth of HO-8910 human ovarian cells.Methods Cell proliferation was detected by MTT method,distribution of cell cycle and rate of apoptosis were determined by flow cytometry,and the expression of Bax protein was detected by immunohistochemistry.Results The proliferation of HO-8910 cells was inhibited,the cell cycle was arrested in G0/G1 phase and rate of apoptosis was inducted by either 1,25(OH)2VitD3 or DDP alone(P0.01).Combined use of 1,25(OH)2VitD3 and DDP could increase the inhibitory effect and apoptosis rate(P0.01).1,25(OH)2VitD3 or DDP alone could up-regulate the Bax protein expression (P0.01).Combined use of 1,25(OH)2VitD3 and DDP could Bax protein expression(P0.01).Conclusion 1,25(OH)2VitD3 and DDP synergistically inhibit proliferation and induce apoptosis in ovarian cancer cells.

Objective To analyze the distribution characteristics,antimicrobial sensitivity and resistance of bacterial isolates from wards of respiratory diseases.Methods Between 2004 and 2006,1599 strains of pathogenic bacteria were isolated from sputum specimens collected in our wards of respiratory diseases.These strains were assessed for antibiotic susceptibility using Kirby-Bauer method according to NCCLS standards.Results The prevalent pathogens in wards of respiratory diseases included Streptococcus pneumoniae(28.3%),Pseudomonas aeruginosa(9.3%),Acinetobacter baumanni(7.8%),Escherichia coli(3.5%),Coagulase-negative Staphylococci(6.4%) and Staphylococcus aureus(1.4%).The results of bacterial susceptibility test showed: Streptococcus pneumoniae,Pseudomonas aeruginosa,Acinetobacter baumanni and Escherichia coli were 100% resistant to Ampicillin and Compound Sulfamethoxazole;Escherichia coli and Klebsiella pneumoniae were 100% and Acimetobacter baumanni over 86.7% sensitive to Imipenem;Pseudomonas aeruginosa showed varying degrees of resistance to common antibiotics,with highest susceptibility to Amikacin(>76.2%),followed by Imipenem and Meropenem(>30%).Streptococcus pneumoniae,Staphylococcus aureus and Coagulase-negative staphylococci were resistant to Pennicillin and sensitive to Vancomycin in 100% of time.Conclusion Nosocomial Gram-negative bacilli were the most common pathogens in wards of respiratory diseases.Antibiotic resistance has become an increasing concern.Proper use of antibiotics and surveillance on antibiotic resistance appear crucial to prevent selection and wide spreading of drug-resistant strains.