CAJ | 학술논문

目的了解浙江地区大肠埃希菌临床菌株优势β-内酰胺酶基因型及其携带模式、β-内酰胺类抗生素诱导β-内酰胺酶基因表达及组氨酸激酶抑制剂氯氰碘柳胺(CLO)抑制其表达的作用.方法采用微量稀释法和E-test检测大肠埃希菌临床菌株对β-内酰胺类抗生素耐药率和最低抑菌浓度(MIC).采用PCR及其产物测序法检测大肠埃希菌耐药菌株β-内酰胺酶基因型及携带模式.采用实时荧光定量RT-PCR和β-内酰胺酶确证试验分别检测1/4 MIC头孢噻肟或青霉素及CLO对大肠埃希菌耐药菌株β-内酰胺酶基因转录和表达的影响.结果浙江地区61.7％(285/462)大肠埃希菌对青霉素、氨苄青霉素、头孢西丁、头孢噻肟和头孢他啶耐药.285株耐药菌株中,TEM和CTX-M基因检出率(83.2％和75.1％)显著高于KPC、SHV和OXA基因(1.4％～10.2％)(P＜0.01),两种以上β-内酰胺酶基因携带率(68.8％)显著高于单基因(31.2％)(P＜0.01),其中61.4％菌株携带TEM+ CTX-M基因(P＜0.01).除KPC基因外,1/4 MIC头孢噻肟和青霉素能诱导89株β-内酰胺酶单基因菌株TEM、CTX-M、SHV和OXA mRNA水平迅速升高(P＜0.01),但可被50～ 500 μg/ml CLO所抑制(P＜0.01).100 μg/ml CLO预处理后,82.8％～ 85.6％耐药菌株对上述抗生素敏感(P＜0.01),β-内酰胺酶检出率也从95.1％下降至16.1％ (P＜0.01).结论 TEM和CTX-M是浙江地区大肠埃希菌临床菌株优势β-内酰胺酶基因型,并以TEM-1+CTX-M为优势携带模式.低浓度头孢噻肟和青霉素可经细菌二元信号系统上调β-内酰胺酶基因表达,但可被组氨酸激酶抑制剂CLO所抑制.
목적 료해절강지구대장애희균림상균주우세β-내선알매기인형급기휴대모식、β-내선알류항생소유도β-내선알매기인표체급조안산격매억제제록청전류알(CLO)억제기표체적작용.방법 채용미량희석법화E-test검측대장애희균림상균주대β-내선알류항생소내약솔화최저억균농도(MIC).채용PCR급기산물측서법검측대장애희균내약균주β-내선알매기인형급휴대모식.채용실시형광정량RT-PCR화β-내선알매학증시험분별검측1/4 MIC두포새우혹청매소급CLO대대장애희균내약균주β-내선알매기인전록화표체적영향.결과 절강지구61.7％(285/462)대장애희균대청매소、안변청매소、두포서정、두포새우화두포타정내약.285주내약균주중,TEM화CTX-M기인검출솔(83.2％화75.1％)현저고우KPC、SHV화OXA기인(1.4％～10.2％)(P＜0.01),량충이상β-내선알매기인휴대솔(68.8％)현저고우단기인(31.2％)(P＜0.01),기중61.4％균주휴대TEM+ CTX-M기인(P＜0.01).제KPC기인외,1/4 MIC두포새우화청매소능유도89주β-내선알매단기인균주TEM、CTX-M、SHV화OXA mRNA수평신속승고(P＜0.01),단가피50～ 500 μg/ml CLO소억제(P＜0.01).100 μg/ml CLO예처리후,82.8％～ 85.6％내약균주대상술항생소민감(P＜0.01),β-내선알매검출솔야종95.1％하강지16.1％ (P＜0.01).결론 TEM화CTX-M시절강지구대장애희균림상균주우세β-내선알매기인형,병이TEM-1+CTX-M위우세휴대모식.저농도두포새우화청매소가경세균이원신호계통상조β-내선알매기인표체,단가피조안산격매억제제CLO소억제.
Objective To understand the predominant β-lactamase genotypes and their carrying modes ofEscherichia coli isolates in Zhejiang province,and the effects of β-1actam antibiotics on inducing or histidine kinase inhibitor closantel (CLO) on inhibiting the expression of β-1actamase genes.Methods Micro-dilution method and E-test were applied to measure the resistant rate and minimal inhibitory concentration (MIC) in E.coli isolates against β-1actam antibiotics.PCR and sequence analysis of PCR products were conducted to detect the β-lactamase genotypes and their carrying modes.Real-time fluorescent quantitative RT-PCR and β-lactamase confirmation test were performed to determine the influence of 1/4 MIC penicillin and cefotaxime,and CLO on the transcription and expression of β-lactamase genes in the resistant E.coli isolates.Results Among the 462 E.coli strains isolated in Zhejiang,285 (61.7％) were resistant to penicillin,ampicillin,cefoxitin,cefotaxim and ceftazidime.In the 285 resistant isolates,the detection rate of TEM or CTX-M β-1actamase gene (83.2％ or 75.1％) was significantly higher than that of KPC,SHV or OXA β-lactamase gene (1.4％-10.2％) (P＜0.01) and the carrying rate of two or more β-1actamase genes (68.8％) was also significantly higher than that of single β-1actamase gene (31.2％) (P＜0.01),and 61.4％ of the resistant isolates carried TEM + CTX-M genes (P＜0.01).Except KPC gene,1/4 MIC of cefotaxim and penicillin induced a rapid increase of TEM-mRNA,CTX-M-mRNA,SHV-mRNA or OXA-mRNA levels (P＜0.01),but 50-500 μg/ml CLO inhibited these levels (P＜0.01).After pre-treatment with 100 μg/ml CLO,82.8％-85.6％ of the resistant isolates became sensitive to β-lactam antibiotics (P＜0.01),while the detection rate of β-lactamases was also decreased from 95.1％ to 16.1％ (P＜0.01).Conclusion TEM and CTX-M are the predominant β-lactamase genotypes in E.coli isolates in Zhejiang and TEM+CTX-M is the predominant carrying mode of β-lactamase genes.Low concentrations of β-lactam antibiotics can up-regulate the expression levels of β-lactamase genes in E.coli through bacterial two-component signaling systems,but this effect can be inhibited by CLO,a histidine kinase inhibitor.