白念珠菌ERG11基因突变与氟康唑耐药性的关系

探讨白念珠菌氟康唑作用的靶酶编码基因 (ERG11)突变与耐药性的关系。分离抽提分别对氟康唑敏感和耐药的白念珠菌基因组 DNA,根据 ERG11编码序列设计一对上下游引物 ,对靶酶活性中心血色素结合区和疏水端螺旋区的基因表达序列进行 PCR扩增 ,经 DNA测序比较敏感和耐药菌株的碱基差异。结果 :6株氟康唑耐药白念珠菌此段序列有 5个位点发生突变 ,其中 5株第 1344 bp位点 G→A的突变致所编码的第 44 8个氨基酸由甘氨酸 (G)变为谷氨酸 (E)。结论 :ERG11基因突变致所编码的氨基酸改变与氟康唑耐药有关。     

特比萘芬与氟康唑或伊曲康唑体外联合抗白念珠菌的作用

目的探讨特比萘芬与氟康唑或伊曲康唑体外联合抗白念珠菌的作用。方法采用微量液体稀释法测定特比萘芬、氟康唑、伊曲康唑对生殖器部位分离的36株白念珠菌药敏试验，应用棋盘微量稀释法测定特比萘芬与氟康唑或伊曲康唑对36株白念珠菌的联合药敏试验。结果白念珠菌菌株对氟康唑、伊曲康唑、特比萘芬耐药株分别为3株（8．33％）、4株（11．11％）、6株（16．67％）。特比萘芬和氟康唑联用MIC几何平均数较其单独应用均显著降低（t=3．590，P〈0．05；t=3．252，P〈0．05），15株有协同作用（42％），18株有相加作用（50％），3株无关作用（8％）；特比萘芬和伊曲康唑联用后的MIC几何平均数较其单独应用均显著降低（t=3．849，P〈0．001；t=2．409，P〈0．05），表现为协同作用有14株（39％），相加作用的有17株（47％），无关作用的有5株（14％）；均未发现拮抗作用。结论特比萘芬与氟康唑或伊曲康唑联合应用对大部分白念珠菌能产生协同作用和相加作用，提示特比萘芬与氟康唑或伊曲康唑联合应用能增强抗白念珠菌的作用。     

念珠菌性包皮龟头炎55例致病菌药敏分析

目的 对念珠菌感染包皮龟头炎的55例患者,所分离出的55株念珠菌菌株进行分类鉴定及药敏试验,以了解念珠菌性包皮龟头炎的致病菌种感染及耐药情况.方法 通过科玛嘉显色培养基、菌株显微镜下形态观察、对包皮龟头炎分离出念珠菌进行培养、菌种鉴定及药敏试验.结果 1)念珠菌性包皮龟头炎分离出的阳性标本经鉴定,51株为白念珠菌、3株为近平滑念珠菌、1株为近平滑念珠菌及白念珠菌复合感染株.2)药敏试验对制霉菌素、氟康唑、伊曲康唑和特比萘芬的敏感率分别为96.4％、94,5％、85.5％及14.5％.结论 福建地区男性包皮龟头炎念珠菌感染以白念珠菌为主;念珠菌的药敏情况对制霉菌素敏感性最高,其次为氟康唑及伊曲康唑,特比萘芬最低.     

应用比色微量稀释药敏试验方法检测念珠菌临床分离株对特比萘芬的敏感性

目的检测特比萘芬对念珠菌临床分离株的最低抑菌浓度（MIC），了解念珠菌对特比萘芬的体外敏感性，为临床合理用药提供参考依据。方法参照美国国家临床试验室标准化委员会（NCCLS／CLSI）推荐的M27-A药敏试验方案的微量稀释法，并应用氧化还原指示剂Alamar blue通过比色判定MIC值，检测了129株念珠菌属对特比萘芬的体外敏感性。结果129株临床分离的念珠菌属对特比萘芬的MIC值范围为：白念珠菌0．5～16μg／ml，MIC50为4μg／ml，MIC90为8μg／ml；热带念珠菌2～16μg／ml，MIC50为8μg／ml，MIC90为16μg／ml；季也蒙念珠菌1～4μg／ml，MIC50为2μ／ml，MIC90为4μg／ml。结论比色微量稀释药敏试验方法有较好的可重复性和稳定性，且简单省时，特比萘芬对念珠菌属有良好的体外抑菌作用。     

白念珠菌ERG11基因突变及高表达对耐药的影响

目的 探讨白念珠菌ERG11基因突变、高表达及二者的相互作用在氟康唑耐药中的作用.方法 采用PCR法扩增实验菌株的ERG11基因,并进行测序及生物信息学分析,筛查突变位点;抽提实验菌株总RNA,并逆转录合成cDNA,采用实时荧光定量PCR（FQ-RT-PCR）法检测ERG11基因的表达水平.结果 24株临床菌株中,共检测出ERG 11基因存在24个同义突变位点和5个错义突变位点,其中,E174A、T123I、V234F为新发位点;白念珠菌ERG11基因在耐药组中的表达量高于敏感组,差异具有统计学意义未发生错义突变的白念珠菌耐药株ERG11基因的表达量高于发生错义突变的,差异具有统计学意义.结论 白念珠菌ERG 11基因的5个错义突变A114V、E174A、T123I、Y132H、V234F可能与氟康唑耐药形成有关;ERG11基因的高表达可能与氟康唑耐药有关;ERG11基因的错义突变可能对该基因的高表达存在抑制作用.     

浅谈抗真菌药特比萘芬的研究及临床应用

本文对近几年特比萘芬的研究及临床应用进行了综述。烯丙胺类抗真菌药特比萘芬,抗菌谱广,杀菌力强,对临床上常见皮肤真菌病疗效显著。作用机制主要为选择性抑制真菌膜上的角鲨烯环氧化酶,而产生抑杀作用,对细胞色素P450无任何影响,因而无严重肝、肾不良反应。     

热休克蛋白90抑制剂格尔德霉素对白色念珠菌耐药性的影响

目的探讨热休克蛋白90(HSP90)抑制剂格尔德霉素(GA)对白色念珠菌体外诱导耐药菌株耐药性的影响,以期证实HSP90抑制剂的体外抗真菌作用。方法用HSP90抑制剂GA处理对特比萘芬片具有稳定耐药性的白色念珠菌体外诱导耐药菌株AC2013112-R,参照美国国家临床试验室标准化委员会(NCCLS)推荐的M27VA药敏试验方案的微量稀释法,应用氧化还原指示剂Alamarblue通过比色判定最低抑菌浓度(MIC)值,分别检测格尔德霉素处理前后白色念珠菌ATCC90028-R菌株对特比萘芬的体外敏感性。结果经过格尔德霉素处理后,对特比萘芬片具有稳定耐药性的白色念珠菌体外诱导耐药菌株AC2013112-R的对特比萘芬的MIC值从≥512μg/mL降低为64μg/mL。结论 HSP90的抑制剂GA具有体外抗念珠菌活性,这种抑菌作用可能与GA的药理作用及白色念珠菌菌株产生耐药性的方式有关。     

临床白念珠菌耐药性与ERG3基因突变及表达的关系

目的明确白念珠菌ERG3基因突变及高表达对抗真菌药物耐药的调控作用。方法用微量稀释法测得36株临床分离白念珠菌的最小抑菌浓度（MIC）;提取基因组DNA,PCR基因扩增,将扩增后的产物纯化测序,并与genbank中已知的标准序列（AF069572）进行BLAST比对分析;采用实时定量PCR（RT—PCR）方法,对白念珠菌ERG3基因表达的mRNA进行相对定量分析。结果36株白念珠菌ERG3基因序列共发现6个突变位点,其中19株发生同义突变,突变位点为T51C、T432C、T381C、C438T、T1044C;2株发生错义突变,突变位点为C1052T,并产生耐药;白念珠菌对氟康唑耐药组ERG3高表达的发生率高于敏感组（P〈0．05）。结论ERG3基因突变及高表达增加了抗真菌药物的耐药性。同时有多种调节机制共同参与白念珠菌耐药性的形成。     

抗真菌药布替萘芬的合成

布替萘芬（ｂｕｔｅｎａｆｉｎｅ,１）,商品名为Ｍｅｎｔａｘ,化学名为Ｎ－４－叔丁基苄基－Ｎ－甲基－１－萘甲胺盐酸盐［１］,由Ｋａｋｅｎ公司开发,１９９２年在日本上市。１与萘替芬和特比萘芬同属于烯丙胺类抗真菌药,通过抑制角鲨烯环氧化酶,阻断真菌细胞膜麦...     

2011年深圳地区解脲脲原体药敏及耐药机制的研究

目的了解泌尿生殖道解脲脲原体(Uu)对抗菌药物的敏感性,以指导临床合理用药;探讨解脲脲原体对氟喹诺酮类药物的耐药状况与gyrA基因突变之间的关系。方法 2011年深圳地区采用药敏试剂盒对Uu进行了药敏分析;随机抽取4株耐喹诺酮菌株,聚合酶链反应(PCR)扩增gyrA基因并测序,采用Blast比对法分析临床菌株与标准菌株的序列差异。结果在4株解脲脲原体菌株中,经测序分析发现gyrA基因1种突变形式(302位碱基C-T)。结论解脲脲原体对氟喹诺酮类药物的耐药可能是由于gyrA基因突变导致相应氨基酸改变所介导。     

Resistance mechanisms in fluconazole-resistant Candida albicans isolates from vaginal candidiasis

Candida albicans is the most frequently identified yeast species causing mycotic vaginitis. A significant number of vaginal yeast isolates are resistant to azole antifungal agents in vitro. Here we investigated the molecular mechanisms of resistance in 22 randomly selected fluconazole-resistant vaginal C. albicans isolates. Twelve isolates in this collection were found to be cross-resistant to itraconazole and 15 to voriconazole. Most of them also displayed decreased susceptibility to terbinafine. Northern blot analyses revealed overexpression of the MDR1 gene in all isolates, which in some isolates was accompanied by elevated levels of CDR1/CDR2 and ERG11 expression. Sequence analysis of the polymerase chain reaction-amplified ERG11 gene of selected azole-resistant isolates identified D116E and V488I amino acid alterations in Erg11p that are known to be conserved in fluconazole-resistant strains. The results demonstrate that decreased susceptibilities of vaginal yeast isolates to clinically used azole derivatives are the result of a combination of several molecular mechanisms involving drug efflux and alterations in the structure or cellular amount of 14-alpha-lanosterol demethylase.     

Complete nucleotide sequence of the gyrA gene of Helicobacter pullorum and identification of a point mutation leading to ciprofloxacin resistance in poultry isolates

To assess the molecular basis of nalidixic acid and ciprofloxacin resistance in Helicobacter pullorum, the gyrA gene of H. pullorum CIP 104787T was sequenced. In addition, 9 isolates (2 susceptible to ciprofloxacin and resistant to nalidixic acid, 3 susceptible and 4 resistant to both antibiotics) were selected from 44 poultry isolates and the nucleotide sequences of their quinolone resistance-determining regions (QRDRs) were compared. The 2490 bp gyrA gene showed an open reading frame encoding a polypeptide of 829 amino acids. The deduced amino acid sequence of gyrA showed>or=72% identity to Helicobacter hepaticus, Helicobacter pylori and Wolinella succinogenes. Moreover, >or=98% amino acid sequence identity was found comparing the QRDR of the H. pullorum type strain with the QRDRs of the aforementioned bacterial species. All ciprofloxacin-resistant poultry isolates showed an ACA-->ATA (Thr-->Ile) substitution at codon 84 of gyrA, corresponding to codons 86, 87 and 83 of Campylobacter jejuni, H. pylori and Escherichia coli gyrA genes, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin-resistant phenotype of poultry isolates. This is the first report describing the complete 2490 bp nucleotide sequence of H. pullorum gyrA and confirming the involvement of the Thr84Ile substitution of GyrA in ciprofloxacin resistance of H. pullorum.     

耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变研究

目的　研究临床分离的耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变情况。方法　测定临床分离的 5 5株铜绿假单胞菌MIC值 ,从中筛选出 1株敏感菌和 8株耐药菌 ,以标准敏感菌株ATCC2 785 3作为质控菌株。用聚合酶链反应 (PCR)扩增gyrA及parC基因的喹诺酮耐药决定区 (QR DR) ,扩增产物片段长度分别为 35 1bp、397bp。用限制性内切酶SacⅡ消化gyrAPCR产物 ,同时对上述 10株菌的gyrA及parC基因的喹诺酮决定区 (QRDR)进行PCR DNA直接测序分析。结果　有 8株耐菌株的gyrA基因在 83位 (ACC→ATC)有突变 ,导致氨基酸Thr→Ile的改变 ;有 3株高度耐药菌gyrA基因同时在 87位 (GAC→GGC)有突变 ,导致氨基酸Asp→Gly的改变 ;有 4株耐药菌株的parC基因在 87位有TCG→TTG突变 ,导致氨基酸由Ser→Leu的改变。同时具gy rA和parC突变MIC值是仅具gyrA突变菌株MIC值的 2～ 16倍。未发现parC突变单独存在。另外 ,有 6株耐药菌gyrA的 132位有CAC→CAT的突变 ;所有耐药菌株parC基因 115位有GCT→GCG的突变 ,该突变未引起氨基酸的改变。结论　gyrA83、87位突变及parC基因 87位突变都可引起铜绿假单胞菌对氟喹诺酮类药物产生耐药 ,但以gyrA基因 83位突变为主 ,合并gyrA基因 87位及parC基因 87位突变可增加耐药程度。     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical strains isolated in the Philippines

The prevalence of mutations in the katG, inhA and oxyR-ahpC genes of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in the Philippines were determined. Of 306 M. tuberculosis isolates studied, 81 (26.5%) exhibited INH-resistance. Forty-four strains (54.3%) had mutations in the katG gene, eighteen strains (22.2%) had mutations in the putative inhA locus region, seven had mutations in both regions and five strains had mutations in the oxyR-ahpC operon. Only seven strains had no mutations. A total of 71 of the 81 (87.6%) resistant strains and 65 of the 72 (90.3%) INH sensitive randomly selected strains showed amino acid substitution in codon 463 (Arg to Leu) (88.9%). This fact supports the hypothesis that mutations at codon 463 are independent of INH-resistance and are linked to the geographical origins of the strains.     

Reversal of antifungal resistance mediated by ABC efflux pumps from Candida albicans functionally expressed in yeast

The enhanced efflux of antifungal drugs through ATP-binding cassette (ABC) transporters constitutes a major cause of clinical multidrug resistance (MDR). The inhibition of drug efflux pumps by specific compounds is considered to be a feasible strategy to overcome clinical antifungal resistance. Therefore, several blockers of mammalian and yeast ABC drug pumps, including FK506, propafenones, as well as the antifungal drug terbinafine were tested for their capacity to reverse CDR-mediated azole resistance in bakers yeast and in clinical isolates of Candida albicans. We have functionally expressed the C. albicans Cdr1p and Cdr2p transporters in hypersensitive Saccharomyces cerevisiae recipient strains lacking several endogenous ABC pumps. Cdr1p and Cdr2p were functional in yeast, as they conferred pronounced drug resistance to known antifungal drugs, including azoles and terbinafine. We employ two functional assays to demonstrate that ABC pump inhibitors reverse CDR-mediated antifungal resistance, thereby restoring drug susceptibility of yeast cells and resistant clinical isolates. Our results suggest that reversal of antifungal resistance can be achieved through ABC pump-dependent and independent mechanisms.     

金铁锁鲨烯合酶cDNA的克隆和功能鉴定

濒危药用植物金铁锁(Psammosilene tunicoides W.C.Wu et C.Y.Wu.)的有效成分三萜总皂苷有显著的药理活性。为克隆和鉴定金铁锁三萜皂苷生物合成途径中的关键酶基因——鲨烯合酶的全长cDNA，本研究采用同源兼并引物PCR和cDNA末端快速扩增(RACE)等方法克隆了其全长cDNA；结合大肠杆菌异源表达、体外酶促反应及针对产物化学结构的GC和GC-MS分析等方法鉴定了其功能。研究结果表明：金铁锁鲨烯合酶cDNA全长为1 663 bp，含有1 245 bp的开放阅读框(ORF)，编码414个氨基酸(计算分子质量为47.69 kD)，5′非编码区(UTR)和3′UTR分别为260 bp和158 bp，GenBank注册号为EF585250，与三七、人参和甘草的鲨烯合酶的氨基酸序列有较高的同源性，分别为83%、 82%和82%，而与裂变酵母、白色念珠菌和人的氨基酸序列的同源性分别只有35%、 39%和47%；表达产物具有催化两分子法呢烯焦磷酸连接成鲨烯的活性。本研究克隆和鉴定了金铁锁鲨烯合酶的全长cDNA，为金铁锁次生代谢工程研究提供了重要基础。     

Differential effects of the combination of caspofungin and terbinafine against Candida albicans, Candida dubliniensis and Candida kefyr

The activity of caspofungin (CSP) combined with terbinafine (TRB) against Candida dubliniensis, Candida kefyr and azole-resistant Candida albicans was evaluated in vitro by checkerboard analysis. The combination of CSP with TRB resulted in positive interactive effects in vitro against C. albicans and C. kefyr but not against C. dubliniensis. Moreover, true synergism was observed only against TRB resistant strains which became susceptible to this drug in the presence of CSP. In contrast, indifference was observed against strains that were already sensitive to TRB indicating that CSP may inhibit resistance to TRB.     

铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系研究

目的:研究铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系,并对聚合酶联反应(PCR)-限制性片段长度多态性(RFLP)-DNA单链构象多态性(SSCP)分析铜绿假单胞菌临床分离株gyrA基因突变的可行性进行评估。方法:以铜绿假单胞菌临床分离株gyrA基因序列为靶序列,用PCR、PCR-RFLP、PCR-SSCP、DNA测序等方法对铜绿假单胞菌ATCC27853及16株临床分离株gyrA基因突变进行对比研究。结果:在8株耐环丙沙星铜绿假单胞菌中,有6株gyrA基因的83位表现出单点突变,其突变方式全为ACC→ATC,导致氨基酸苏氨酸→异亮氨酸的改变;gyrA基因的PCR扩增产物SacⅡ酶切片段与测序结果一致;SSCP分析结果显示,16株细菌中仅2株gyrA带型与ATCC27853相同,其它菌株gyrA带型与ATCC27853均不同。结论:临床分离的铜绿假单胞菌对喹诺酮类药物耐药的分子机制主要表现为gyrA基因83位氨基酸密码子突变,应用PCR-RFLP-SSCP系统可快速、准确地检测耐喹诺酮类药物的铜绿假单胞菌gyrA中碱基的变异。