大肠埃希菌临床菌株优势β-内酰胺酶基因型及其诱导表达与抑制的研究

谈潘莉; 汪浙炯; 孙爱华; 严杰; 赵金方

Abstract

谈潘莉,汪浙炯,孙爱华,严杰,赵金方.大肠埃希菌临床菌株优势β-内酰胺酶基因型及其诱导表达与抑制的研究[J].Chinese journal of Epidemiology,2015,36(5):484-490

大肠埃希菌临床菌株优势β-内酰胺酶基因型及其诱导表达与抑制的研究

Predominant β-lactamase genotypes of Escherichia coli isolates and induction and inhibition mechanisms of β-lactamase gene expression

Received:November 29, 2014

DOI：10.3760/cma.j.issn.0254-6450.2015.05.016

KeyWord: 大肠埃希菌 β-内酰胺类抗生素耐药性 β-内酰胺酶/基因型/表达组氨酸激酶/抑制剂

English Key Word: Escherichia coli β-lactam antibiotics Resistance β-lactamase/genotype/expression Histidine kinase/inhibitor

FundProject:国家自然科学基金(81271893); 浙江省自然科学基金(LY12H19002); 浙江省卫生厅科研项目(2011KYA005)

Author Name	Affiliation	E-mail
Tan Panli	Department of Laboratory Medicine, the First Affiliated Hospital of Zhejiang Traditional Chinese Medicine University, Hangzhou 310006, China
Wang Zhejiong	Department of Laboratory Medicine, the First Affiliated Hospital of Zhejiang Traditional Chinese Medicine University, Hangzhou 310006, China
Sun Aihua	Zhejiang Medical College
Yan Jie	Department of Medical Microbiology and Parasitology, Medical School of Zhejiang University
Zhao Jinfang	Department of Laboratory Medicine, the First Affiliated Hospital of Zhejiang Traditional Chinese Medicine University, Hangzhou 310006, China	xinqiuzh@163.com

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Abstract:

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。

English Abstract:

Objective To understand the predominant β-lactamase genotypes and their carrying modes of Escherichia coli isolates in Zhejiang province, and the effects of β-lactam antibiotics on inducing or histidine kinase inhibitor closantel (CLO) on inhibiting the expression of β-lactamase 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-lactam 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-lactamase 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-lactamase genes (68.8%) was also significantly higher than that of single β-lactamase 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.

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