【作者】 胡立芬；

【导师】 李旭；

【作者基本信息】 安徽医科大学 ， 内科学， 2007， 硕士

【摘要】 目的了解本省志贺菌耐药情况,以指导合理用药;了解安徽地区临床分离志贺菌中产超广谱β-内酰胺酶(ESBLs)和质粒介导AmpC型β-内酰胺酶情况;探讨临床分离志贺菌螺旋酶A亚单位gyrA基因及拓扑异构酶ⅣC亚单位parC基因突变与志贺菌对氟喹诺酮类药物的耐药表型的关系;研究临床分离志贺菌中主动外排系统acrAB-tolC结构基因的分布和acrAB基因表达水平。材料与方法菌株来源收集我省35所医院2005年9月份临床标本中分离的志贺菌91株,并常规保存于-70℃。药敏试验质控菌大肠埃希菌ATCC25922。方法采用平皿琼脂对倍稀释法检测我省35所医院2005年9月份收集的91株志贺菌对多种抗菌药物的敏感性。用煮沸法提取细菌总DNA,碱裂解法提取质粒。用聚合酶链反应(PCR)方法扩增CTX-M组、OXA组、blaTEM-1、blaSHV-1、ampC基因。PCR产物纯化测序法明确β-内酰胺酶基因型。用细菌总DNA为摸板,PCR技术扩增gyrA和parC基因的喹诺酮耐药决定区(QRDR)相关序列和acrAB-tolC基因,用DNA直接测序的方法对26株(其中19株对环丙沙星耐药,7株对环丙沙星敏感)志贺菌的gyrA及parC基因序列进行测序分析,结果至GenBank进行比对。用RT-PCR测定15株多重耐药株和13株敏感株的acrAB基因mRNA表达水平。结果91株志贺菌中,有福氏志贺菌57株、宋内志贺菌31株、鲍氏志贺菌3株;91株志贺菌对头孢哌酮/舒巴坦和哌拉西林/他唑巴坦的耐药率明显低于其他头孢类抗菌药物,对碳青霉烯类敏感率达100%,对喹诺酮类环丙沙星及帕珠沙星的耐药率分别是27.47%、32.97%。91株志贺菌产β-内酰胺酶的检出率为58株( 63.74%),产β-内酰胺酶菌中blaTEM-1、blaCTX-M-1、blaCTX-M-13、blaOXA-1和blaampc扩增阳性率分别为62.07%(36/58)、17.24%(10/58)、24.14%(14/58)、36.21%(21/58)和10.34%(6/58),产Ampc中全为EBC型;携带2种耐药基因的菌株占20.88%,携带3种耐药基因的为5.49%,未测出CTX-M-2、CTX-M-8、CTX-M-25、OXA-2、OXA-10、SHV-1型。20株发生gyrA第83位氨基酸改变,7株发生gyrA第87位改变;9株发生parC第80位氨基酸改变(Ser80→Ile),3株发生parC第57位改变(Ser57→Thr),3株parC第64位氨基酸突变(Ala64→Cys, Ala64→Asp)。其中志贺菌gyrA83(Ser→Ile),parC57(Ser→Thr)及parC64(Ala→Cys, Ala→Asp)的改变在国内外尚属首次报道,这些测序结果已递交GenBank,获得的序列号为DQ898178、DQ898179、DQ898180、DQ898305。同时具gyrA和parC突变菌株的MIC值是仅具gyrA突变菌株MIC值的2～8倍,gyrA突变合并parC64位突变菌株比gyrA突变合并parC57或parC80位突变菌株的耐药更严重。多重耐药株acrAB基因mRNA表达水平高于敏感野生株(P<0.05)。结论本地区志贺菌对喹诺酮类和第三代头孢类抗菌药物有一定耐药性,应加强耐药性监测和防治。本地区志贺菌β-内酰胺酶的检出率较高,以TEM-1型, OXA-1型和CTX-M型基因为主。在研究耐氟喹诺酮类药物志贺菌螺旋酶A亚单位gyrA基因及拓扑异构酶ⅣC亚单位parC基因方面检测到可引起氨基酸改变的四个新基因型已递交GenBank,获得的序列号为DQ898178、DQ898179、DQ898180、DQ898305。gyrA83、87位突变参与志贺菌对氟喹诺酮类药物耐药,合并parC57、64、80位突变后会导致高度耐药。acrAB基因高表达与志贺菌多重耐药有关。更多还原

【Abstract】 Objective:To investigate the resistance of Shigella isolates against 16 antimicrobial agents in Anhui province and offer the resistance data to clinical therapy.To investigate the production of extended-spectrumβ-lactamases (ESBLs) and plasmid-mediated AmpCβ-lactamases in the clinical Shigella isolates in Anhui province.To investigate the correlation between the alterations of DNA gyrase gyrA and TopoisomeraseⅣparC genes and fluoroquinolone resistance in Shigella isolates.To detect the distribution of the energy-dependent multidrug efflux pump gene acrAB-tolC and expression level of acrAB gene in Shigella isolates.Materials and Methods:Isolates:From September 1 to September 30, 2005, a total of 91 Shigella isolates were collected in 31 hospitals in Anhui of China.Method:The MICs of the following agents were determined by the agar dilution method recommended by CLSI (Clinical Laboratory Standard Institute), 2005. Total DNA was extracted by suspending a few overnight colonies in 0.5 mL of double-distilled water and heating the mixture at 100°C for 10 min. Plasmid DNA was extracted and purified by Qiagen plasmid Mini Kit. The extracted DNA was subjected to amplification by PCR for the quinolone resistance-determining regions of gyrA, parC on 26 Shigella isolates (19strains showing ciprofloxacin≥4 ug/mL, and 7 strains selected showing ciprofloxacin < 4 ug/mL), the multidrug efflux pump acrAB-tolC genes on 91 strains were also identified by PCR. PCR analysis was also used to investigate the distribution ofβ-lactamases and Qnr present in Shigella strain. Reverse transcription PCR (RT-PCR) of acrAB genes were used to confirm the presence of acrAB.Results:There were 57 strains of S. flexnery, 31 strains S. sonnei and 2 strains S. boydii among 91 strains of Shigella isolates. The resistance rates of Shigella isolates to cefoperazone-sulbactam and piperacillin-tazobactam were remarkably lower than other third generation cephalosporins. The susceptible rates to carbopenems were 100%. The resistance rates to ciprofloxacin and pazufloxacin were 27.47% and 32.97%, respectively. 14 of 91 isolates were susceptible to all tested antimicrobial agents in this study. In this study we did not found the qnr genes.β-lactamases-producing Shigella isolates were 63.74% (58/91), the genes of blaTEM-1, blaCTX-M-1, blaCTX-M-13, blaOXA-1 group and ampC were detected in 62.07%(36/91), 17.24%(10/91), 24.14%(14/91), 36.21%(21/91) and 10.24% (6/91) among 91 isolates. 20 strains had a mutation at codon 83 of gyrA, 7 strains had a mutation at codon 87 of gyrA. 9 strains had a mutation at codon 80 (Ser80→Ile) of parC. Three of the ciprofloxacin-resistance isolates had a novel mutation at codon 64 (Ala64→Cys, Ala64→Asp) of parC, the mutation at codon 57 (Ser57→Thr) of parC and the mutations at codon 83 of gyrA (Ser→Ile) were firstly reported in Shigella isolates. The partial sequences of gyrA and parC genes reported in this article have been deposited in the GenBank database and assigned accession no. DQ898178, DQ898179, DQ898180, DQ898305. The strains with both gyrA and parC mutations were two to eight times more resistant than the strains which had only gyrA mutations. Isolates with a single gyrA mutation were less resistant to fluoroquinolones than those with an additional parC mutation, the novel mutation at codon 64 plus one other gyrA mutation conferred higher level resistance to fluoroquinolones than the mutation at codon 57 or 80 of parC together with one other gyrA mutation. The levels of mRNA expression of acrAB in multidrug-resistant strains were significantly higher than those in wild susceptible strains.Conclusion:There was a certain resistance rate of the Shigella isolates to fluoroquinolones and the third generation cephalosporins. More attention should be paid to the surveillance and control of such resistance. The prevalence ofβ-lactamases among clinical Shigella isolates was high, theβ-lactamase-genes possibly were the TEM-1, OXA-1 and CTX-M types in Shigella isolates in our locality.The mutations of gyrA at codon 83 and 87 were related to fluroquinolone resistance,and they woμLd make higher resistance together with additional parC mutation. The partial sequences of gyrA and parC genes reported in this article have been deposited in the GenBank database and assigned accession no. DQ898178, DQ898179, DQ898180, DQ898305.Overexpression of the acrAB genes was responsible for resistance to fluoroquinolones in Shigella isolates.更多还原