本文關(guān)鍵詞：廣州市五所醫(yī)院銅綠假單胞菌耐藥現(xiàn)狀和危險(xiǎn)因素及其分子特征研究　出處：《廣東藥學(xué)院》2013年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 銅綠假單胞菌 多重耐藥 泛耐藥 脈沖場(chǎng)凝膠電泳 危險(xiǎn)因素 基因

【摘要】：目的了解廣州市部分醫(yī)院銅綠假單胞菌不同耐藥程度流行情況及其危險(xiǎn)因素；探究不同耐藥程度銅綠假單胞菌β-內(nèi)酰胺酶、基因的分布和基因分型狀況及其可能的耐藥機(jī)制。 方法收集2008年7月到2012年12月在廣州某五所醫(yī)院院內(nèi)感染患者的銅綠假單胞菌348株，經(jīng)鑒定后，采用回顧性查閱病例方法收集病史資料。348株P(guān)A進(jìn)行藥敏試驗(yàn)后，篩選敏感菌株、1-2類耐藥菌株、多重耐藥菌株及泛耐藥菌株，分析其耐藥現(xiàn)況及通過(guò)單因素和多因素采用無(wú)序多分類logistic回歸分析1-2類耐藥菌株、多重耐藥菌株及泛耐藥菌株感染的危險(xiǎn)因素。 運(yùn)用雙紙片復(fù)合法和雙紙片協(xié)同法篩選PA產(chǎn)β-內(nèi)酰胺酶（超廣譜β-內(nèi)酰胺酶、金屬酶、AmpC酶）的情況，同時(shí)運(yùn)用PCR擴(kuò)增β-內(nèi)酰胺酶的編碼基因（NDM-1、TEM、PER、OXA-10、IMP、VIM）、氨基糖苷類鈍化酶編碼基因（ant(3″)-Ⅰ與aac(6′)-Ⅱ）、外膜孔蛋白Oprd2，整合子及gyrA、parC管家基因，然后運(yùn)用PCR-RFLP的方法對(duì)整合子進(jìn)行分型及檢測(cè)gyrA、parC管家基因的QRDR片段的突變情況。最后分析五所醫(yī)院感染菌株的β-內(nèi)酰胺酶、耐藥基因及管家基因突變的情況，并運(yùn)用卡方檢驗(yàn)分析β-內(nèi)酰胺酶產(chǎn)生、耐藥基因或管家基因突變與耐藥菌株（包括1-2類耐藥組、多重耐藥組、泛耐藥組菌株）的關(guān)系。 采用脈沖場(chǎng)凝膠電泳方法對(duì)銅綠假單胞菌菌株進(jìn)行分子分型，并運(yùn)用Bionumerie6.0軟件進(jìn)行聚類分析，分析菌株間的親緣關(guān)系及鑒定散發(fā)或暴發(fā)流行趨勢(shì)。 結(jié)果2008年-2012年間共收集348株銅綠假單胞菌，其中多重耐藥組中MDRPA149例，，多重耐藥率為42.8%(149/348)，39株P(guān)DRPA，泛耐藥率為11.2%（39/348）。MDRPA感染的患者中男性占63.3%(114/180)，中位數(shù)是73歲；PDRPA感染男性占59.0%（23/39）,中位數(shù)是74歲。PDRPA主要分布在重癥加強(qiáng)護(hù)理病房（12/39），呼吸內(nèi)科（11/39）和神經(jīng)內(nèi)科（8/39），MDRPA分布最多的亦為以上三個(gè)科室。 MDRPA與PDRPA的標(biāo)本來(lái)源大部分來(lái)自痰,分別達(dá)81.1%(146/180)、82.1%（32/39）。PA對(duì)抗菌藥物耐藥性最高的前三位是β-內(nèi)酰胺類的TIC44.8%、PIP39.7%、ATM34.8%，敏感性最高的是MEN74.1%，13種抗菌藥物中除了LEV、ATM外，耐藥率隨著時(shí)間變化而呈現(xiàn)上升的趨勢(shì)。 單因素分析發(fā)現(xiàn)差異具有統(tǒng)計(jì)學(xué)意義的因素包括：手術(shù)、檢出前入住ICU、使用碳青霉烯類抗菌藥物、使用二代頭孢抗菌藥物、抗菌藥物使用總天數(shù)、感染前住院天數(shù)、使用抗菌藥物≥3種、混合感染、吸痰、機(jī)械通氣、導(dǎo)尿管插管、鼻飼胃管插管。多因素分析發(fā)現(xiàn)（以敏感組為對(duì)照組）：1-2類耐藥組的危險(xiǎn)因素有吸痰(調(diào)整后OR=2.79,95%CI=1.09-7.12)；多重耐藥組危險(xiǎn)因素有曾使用碳青霉烯類抗菌藥物（調(diào)整后OR=2.29,95%CI=1.02-5.15），檢出前入住ICU（調(diào)整后OR=2.90，95%CI=1.22-6.91）；泛耐藥組的危險(xiǎn)因素有曾使用碳青霉烯類抗菌藥物（調(diào)整后OR=5.94，95%CI=1.97-17.85）和機(jī)械通氣（調(diào)整后OR=11.78，95%CI=3.14-44.20）。 β-內(nèi)酰胺酶的產(chǎn)生及其編碼基因檢測(cè)發(fā)現(xiàn)：348株菌產(chǎn)MBL酶占11.2%（39/348），產(chǎn)AmpC酶占12.9%（45/348）, ESBLs酶占12.1%（42/348）；各種產(chǎn)酶率在四組分布的差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。β-內(nèi)酰胺酶編碼基因NDM-1暫未檢出，而TEM達(dá)43.4%（151/348），PER達(dá)22.7%（79/348），OXA-10達(dá)23.3%（81/348），IMP達(dá)15.2%（53/348），VIM達(dá)4.6%（16/348）；除了PER與IMP基因外，其余耐藥基因在泛耐藥組（PDRPA）和敏感組檢出率差異都具有統(tǒng)計(jì)學(xué)意義。 外膜通透性調(diào)控基因檢測(cè)發(fā)現(xiàn)：OprD2的總的缺失率達(dá)19.5%（68/348）；1-2類耐藥組、多重耐藥組和泛耐藥組的OprD2基因的缺失率與敏感組比較，差異具有統(tǒng)計(jì)學(xué)意義，其OR值分別為：1-2類耐藥組（OR=12.57,95%CI=1.65-111.37），多重耐藥組（OR=38.53,95%CI=5.21-285.19），泛耐藥組（OR=66.08,95%CI=8.33-524.24）。 氨基糖苷類鈍化酶編碼基因檢測(cè)發(fā)現(xiàn)：aac(6＇)-Ⅱ基因攜帶率達(dá)24.7%（86/348），ant(3”)-Ⅰ達(dá)32.2%（112/348）；aac(6＇)-Ⅱ、ant(3”)-Ⅰ的基因的檢出率在多重耐藥組和泛耐藥組中分別與敏感組比較，得多重耐藥組中aac(6＇)-Ⅱ（OR=3.45,95%CI=1.68-7.07）、ant(3”)-Ⅰ（OR=3.13,95%CI=1.69-5.81），泛耐藥組中aac(6＇)-Ⅱ（OR=11.11,95%CI=4.54-27.19）、ant(3”)-Ⅰ（OR=3.23,95%CI=1.42-7.38）,且具差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 整合子檢測(cè)結(jié)果：Ⅰ類整合子檢出172株，占49.4%（172/348），未檢出Ⅱ、Ⅲ類整合子；其中MDRPA的Ⅰ類整合子達(dá)53.7%（80/149）。PDRPA中Ⅰ類整合子達(dá)84.6%（33/39）；慶大霉素、妥布霉素、阿米卡星、哌拉西林、替卡西林、亞胺培南、美羅培南、左氧氟沙星、環(huán)丙沙星、氨曲南、頭孢他啶、頭孢吡肟12種抗菌藥物在整合子陽(yáng)性菌株中的耐藥率明顯高于整合子陰性組，差別具有統(tǒng)計(jì)學(xué)意義（P0.05)。多重耐藥組、泛耐藥組的整合子的檢出率分別與敏感組比較差異，得多重耐藥組（OR=2.43,95%CI=1.43-4.15），泛耐藥組（OR=11.53,95%CI=4.37-30.40），且差異均具有統(tǒng)計(jì)學(xué)意義（P0.05)。 喹諾酮耐藥決定區(qū)突變情況：在162株喹諾酮耐藥的銅綠假單胞菌，gyrA基因突變占40.7%(66/162)，parC突變占17.3%（28/162）； gyrA基因的QRDR片段突變主要發(fā)生在第83位氨基酸密碼子的ACC→ATC，其編碼的氨基酸由The→Ⅱe，與此同時(shí)，耐藥菌株在132位氨基酸密碼子(CAC→CAT)有一靜止突變，該突變未引起氨基酸的改變，而parC測(cè)序結(jié)果表明QRDR片段突變主要位于第79位氨基酸密碼子發(fā)生TCC→GCC的突變，該位點(diǎn)突變引起氨基酸Ser→Ala的改變；泛耐藥組中g(shù)yrA與parC的QRDR片段的突變率與1-2類耐藥組的菌株比較，得出gyrA （OR=3.93，95%CI=1.13-13.62）， parC （OR=1.36，95%CI=1.11-1.16），且差異均具有統(tǒng)計(jì)學(xué)差異（P0.05）。 PFGE分子分型結(jié)果：348株菌Dice系數(shù)為100%的PA一共有8對(duì)，分別為A、B、D、C、E、F、G、H型， PFGE為A、C、D、E、F型中同一型里兩或三位患者均來(lái)自同一家醫(yī)院，不但住院時(shí)間日期有重疊且同一個(gè)科室，加之耐藥譜相似，容易導(dǎo)致患者間互相感染的發(fā)生，因此上述5型中的同源菌株考慮科室內(nèi)交叉感染而產(chǎn)生。而B、G、H型的菌株同一型的患者均來(lái)自同一家醫(yī)院，但檢出科室不同，有發(fā)生科室間交叉感染的可能，感染途徑還有待追究。泛耐藥株的PFGE分型Dice系數(shù)在44.3%至97.1%之間， Dice系數(shù)在80%或以上的同源株有5組，同一型的菌株均來(lái)自同一家醫(yī)院，共11株P(guān)DRPA,其余的28株均為散發(fā)。 結(jié)論廣州市院內(nèi)感染PA菌株的耐藥發(fā)生率較為普遍，除了LEV和ATM外，耐藥率隨著時(shí)間呈現(xiàn)上升的趨勢(shì)。 吸痰為1-2類耐藥PA的危險(xiǎn)因素；曾使用碳青霉烯類抗菌藥物、檢出前入住ICU為MDRPA的危險(xiǎn)因素；機(jī)械通氣、曾使用碳青霉烯類抗菌藥物為PDRPA的危險(xiǎn)因素。 β-內(nèi)酰胺酶及其基因（TEM、OXA-10、VIM）、氨基糖苷類鈍化酶編碼基因aac(6＇)-Ⅱ、OprD2基因的缺失、整合子、gyrA和ParC基因的喹諾酮耐藥決定區(qū)突變?yōu)镻DRPA產(chǎn)生耐藥的主要機(jī)制。 未發(fā)現(xiàn)PA大范圍單克隆流行情況,大部分菌株呈散發(fā)流行趨勢(shì)，但在同醫(yī)院或病區(qū)存在輕微的克隆播散現(xiàn)象，ICU與呼吸內(nèi)科出現(xiàn)頻率較高，也有不同科室間出現(xiàn)交叉感染。
[Abstract]:Objective to understand the prevalence and risk factors of Pseudomonas aeruginosa in different hospitals in Guangzhou, and to explore the distribution and genotype of Pseudomonas aeruginosa, and the possible mechanism of resistance.
Methods from July 2008 to December 2012 in Guangzhou some five of nosocomial infection of Pseudomonas aeruginosa in 348 strains was identified, using a retrospective review of cases collected history data.348 PA strains were drug sensitive test, screening of sensitive strains, 1-2 resistant strains, multiple resistant strains and pan resistant strains, analysis the current situation and drug resistance by using univariate and multivariate logistic regression analysis of 1-2 kinds of resistant strains of multi resistant strains and pan resistant strains of the infection risk factors.
The use of double disk composite method and double disc synergy method for screening PA beta lactamases (ESBLs, metal enzyme, AmpC enzyme) conditions, while the use of PCR encoding gene amplification of beta lactamase (NDM-1, TEM, PER, OXA-10, IMP, VIM), aminoglycosides inactivation of genes encoding enzymes (amino ant (3 ") - 1 and AAC (6 ') - II), outer membrane protein Oprd2, parC and gyrA integron, housekeeping genes, and then use the PCR-RFLP method to integrate the sub classification and detection of the gyrA mutation, parC housekeeping gene QRDR fragment. The final analysis strain infection in five hospitals of the beta lactamase resistance gene and the housekeeping gene mutation, and the use of the production of beta lactamase analyzed by chi square test, resistance gene or housekeeping gene mutation and drug resistant strain (including 1-2 resistance group, multidrug resistance group, group of Pan resistant strains) relationship.
The Pseudomonas aeruginosa strains were genotyped by pulsed field gel electrophoresis, and cluster analysis was performed by Bionumerie6.0 software. The phylogenetic relationships among strains were identified, and the epidemic trend of sporadic or outbreaks was identified.
Results during the period of 2008 -2012, a total of 348 strains of Pseudomonas aeruginosa, the multipleresistant group in MDRPA149 cases, multiple drug resistance rate was 42.8% (149/348), 39 strains of PDRPA, pan drug resistant rate was 11.2% (39/348).MDRPA infection in male patients accounted for 63.3% (114/180), the median is 73 years old; male PDRPA infection accounted for 59% (23/39), the median is 74 year old.PDRPA was mainly distributed in the intensive intensive care unit (12/39), Department of respiratory medicine (11/39) and Department of Neurology (8/39) MDRPA, the largest distribution is also above three sections. MDRPA and PDRPA were mostly from sputum were 81.1% (146/180), 82.1% (32/39.PA) on the drug resistance of the top three is a beta lactam TIC44.8%, PIP39.7%, ATM34.8%, the sensitivity of MEN74.1% is the highest, 13 kinds of antibacterial drugs in addition to LEV, ATM, the resistance rate of change with time and showed an upward trend.
Single factor analysis showed that the factors including: the difference was statistically significant in ICU before surgery, detection, the use of carbapenem antibiotics, the use of the two generation cephalosporin antibiotics, antibiotic use days before infection, hospitalization, use of antibiotics was 3, mixed infection, sputum aspiration, mechanical ventilation, catheter intubation nasogastric tube, intubation. Multivariate analysis (in sensitive group) the risk factors of:1-2 class resistance group sputum (adjusted OR=2.79,95%CI=1.09-7.12); multidrug resistance group risk factors had the use of carbapenem antibiotics (adjusted OR=2.29,95%CI=1.02-5.15), detected before ICU admission (adjusted OR=2.90,95%CI=1.22-6.91); risk pan resistant factors group had used carbopenems (adjusted OR=5.94,95%CI=1.97-17.85) and mechanical ventilation (adjusted OR= 11.78,95%CI=3.14-44.20).
Beta lactamase gene encoding and detection: 348 strains producing MBL enzyme accounted for 11.2% (39/348), AmpC enzyme production accounted for 12.9% (45/348), ESBLs enzyme accounted for 12.1% (42/348); enzyme production rate with statistical significance in the difference between the four groups (P0.05). The distribution of beta lactamase the coding gene NDM-1 not detected, and TEM was 43.4% (151/348), PER 22.7% (79/348), OXA-10 23.3% (81/348), IMP 15.2% (53/348), VIM 4.6% (16/348); in addition to the PER and IMP gene, the resistance gene in Pan drug resistant group (PDRPA) and sensitive group. The rate difference was statistically significant.
Detection of outer membrane permeability regulation gene: OprD2 of the total loss rate of 19.5% (68/348); 1-2 resistance group, compared with the sensitive group of multi drug resistant group and the group of Pan drug resistant OprD2 gene deletion rate, the difference was statistically significant, the OR values were: 1-2 group (OR= 12.57,95%CI=1.65-111.37), multidrug resistance multidrug resistance group (OR=38.53,95%CI=5.21-285.19), pan drug resistant (OR=66.08,95%CI=8.33-524.24) group.
Detection of aminoglycoside modifying enzyme gene encoding amino AAC (6 ') - II gene carrying rate was 24.7% (86/348), ant (3) - 1 32.2% (112/348); AAC (6') - 2, ant (3) - 1 gene in the detection rate of multi drug resistant group and pan drug resistant group respectively and sensitive group compared to multi drug resistance in the group of AAC (6 ') - II (OR=3.45,95%CI=1.68-7.07), ant (3) - 1 (OR=3.13,95%CI=1.69-5.81) in AAC group, pan drug resistant (6') - II (OR=11.11,95%CI=4.54-27.19), ant (3) - 1 (OR=3.23,95%CI=1.42-7.38). And the difference was statistically significant (P0.05).
Results: the detection of integron class 1 integron were detected in 172 strains, accounting for 49.4% (172/348), were not detected in II, class III integron; the MDRPA class I integron was 53.7% (80/149).PDRPA class I integron was 84.6% (33/39); gentamicin, tobramycin, Amikacin, and Rasilin, for Kasilin. Imipenem, meropenem, levofloxacin, ciprofloxacin, aztreonam, ceftazidime, cefepime 12 antimicrobial drug resistance in integron positive strains was significantly higher than that of the integron negative group, the difference was statistically significant (P0.05). Multidrug resistance group, respectively with the sensitive difference detection of integron pan the resistance group, have multiple drug resistance group (OR=2.43,95%CI=1.43-4.15), (OR=11.53,95%CI=4.37-30.40), pan drug resistant group and the differences were statistically significant (P0.05).
QRDR mutations in 162 strains of Pseudomonas aeruginosa quinolone resistance, gyrA gene mutation accounted for 40.7% (66/162), parC mutation accounted for 17.3% (28/162); gyrA gene mutation mainly occurred in the QRDR fragment at codon eighty-third of the ACC, ATC, amino acid encoding by The II and E. At the same time, drug resistant strains in 132 amino acid codons (CAC, CAT) having a stationary mutation, the mutation did not cause the change of amino acid, and parC sequencing results showed that the QRDR fragment mutations were located at codon seventy-ninth mutation of TCC to GCC, the Ser and Ala mutations caused amino acid change; mutation rate gyrA and parC resistance group QRDR fragment and 1-2 pan resistant strains of the group, the gyrA (OR=3.93,95%CI=1.13-13.62), parC (OR=1.36,95%CI=1.11-1.16), and the differences were statistically different (P0.05).
PFGE鍒嗗瓙鍒嗗瀷緇撴灉錛

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