广州市五所医院铜绿假单胞菌耐药现状和危险因素及其分子特征研究

发布时间：2018-01-06 00:21

  本文关键词：广州市五所医院铜绿假单胞菌耐药现状和危险因素及其分子特征研究　出处：《广东药学院》2013年硕士论文　论文类型：学位论文

  更多相关文章： 铜绿假单胞菌 多重耐药 泛耐药 脉冲场凝胶电泳 危险因素 基因

【摘要】：目的了解广州市部分医院铜绿假单胞菌不同耐药程度流行情况及其危险因素；探究不同耐药程度铜绿假单胞菌β-内酰胺酶、基因的分布和基因分型状况及其可能的耐药机制。 方法收集2008年7月到2012年12月在广州某五所医院院内感染患者的铜绿假单胞菌348株，经鉴定后，采用回顾性查阅病例方法收集病史资料。348株PA进行药敏试验后，筛选敏感菌株、1-2类耐药菌株、多重耐药菌株及泛耐药菌株，分析其耐药现况及通过单因素和多因素采用无序多分类logistic回归分析1-2类耐药菌株、多重耐药菌株及泛耐药菌株感染的危险因素。 运用双纸片复合法和双纸片协同法筛选PA产β-内酰胺酶（超广谱β-内酰胺酶、金属酶、AmpC酶）的情况，同时运用PCR扩增β-内酰胺酶的编码基因（NDM-1、TEM、PER、OXA-10、IMP、VIM）、氨基糖苷类钝化酶编码基因（ant(3″)-Ⅰ与aac(6′)-Ⅱ）、外膜孔蛋白Oprd2，整合子及gyrA、parC管家基因，然后运用PCR-RFLP的方法对整合子进行分型及检测gyrA、parC管家基因的QRDR片段的突变情况。最后分析五所医院感染菌株的β-内酰胺酶、耐药基因及管家基因突变的情况，并运用卡方检验分析β-内酰胺酶产生、耐药基因或管家基因突变与耐药菌株（包括1-2类耐药组、多重耐药组、泛耐药组菌株）的关系。 采用脉冲场凝胶电泳方法对铜绿假单胞菌菌株进行分子分型，并运用Bionumerie6.0软件进行聚类分析，分析菌株间的亲缘关系及鉴定散发或暴发流行趋势。 结果2008年-2012年间共收集348株铜绿假单胞菌，其中多重耐药组中MDRPA149例，，多重耐药率为42.8%(149/348)，39株PDRPA，泛耐药率为11.2%（39/348）。MDRPA感染的患者中男性占63.3%(114/180)，中位数是73岁；PDRPA感染男性占59.0%（23/39）,中位数是74岁。PDRPA主要分布在重症加强护理病房（12/39），呼吸内科（11/39）和神经内科（8/39），MDRPA分布最多的亦为以上三个科室。 MDRPA与PDRPA的标本来源大部分来自痰,分别达81.1%(146/180)、82.1%（32/39）。PA对抗菌药物耐药性最高的前三位是β-内酰胺类的TIC44.8%、PIP39.7%、ATM34.8%，敏感性最高的是MEN74.1%，13种抗菌药物中除了LEV、ATM外，耐药率随着时间变化而呈现上升的趋势。 单因素分析发现差异具有统计学意义的因素包括：手术、检出前入住ICU、使用碳青霉烯类抗菌药物、使用二代头孢抗菌药物、抗菌药物使用总天数、感染前住院天数、使用抗菌药物≥3种、混合感染、吸痰、机械通气、导尿管插管、鼻饲胃管插管。多因素分析发现（以敏感组为对照组）：1-2类耐药组的危险因素有吸痰(调整后OR=2.79,95%CI=1.09-7.12)；多重耐药组危险因素有曾使用碳青霉烯类抗菌药物（调整后OR=2.29,95%CI=1.02-5.15），检出前入住ICU（调整后OR=2.90，95%CI=1.22-6.91）；泛耐药组的危险因素有曾使用碳青霉烯类抗菌药物（调整后OR=5.94，95%CI=1.97-17.85）和机械通气（调整后OR=11.78，95%CI=3.14-44.20）。 β-内酰胺酶的产生及其编码基因检测发现：348株菌产MBL酶占11.2%（39/348），产AmpC酶占12.9%（45/348）, ESBLs酶占12.1%（42/348）；各种产酶率在四组分布的差异具有统计学意义（P0.05）。β-内酰胺酶编码基因NDM-1暂未检出，而TEM达43.4%（151/348），PER达22.7%（79/348），OXA-10达23.3%（81/348），IMP达15.2%（53/348），VIM达4.6%（16/348）；除了PER与IMP基因外，其余耐药基因在泛耐药组（PDRPA）和敏感组检出率差异都具有统计学意义。 外膜通透性调控基因检测发现：OprD2的总的缺失率达19.5%（68/348）；1-2类耐药组、多重耐药组和泛耐药组的OprD2基因的缺失率与敏感组比较，差异具有统计学意义，其OR值分别为：1-2类耐药组（OR=12.57,95%CI=1.65-111.37），多重耐药组（OR=38.53,95%CI=5.21-285.19），泛耐药组（OR=66.08,95%CI=8.33-524.24）。 氨基糖苷类钝化酶编码基因检测发现：aac(6＇)-Ⅱ基因携带率达24.7%（86/348），ant(3”)-Ⅰ达32.2%（112/348）；aac(6＇)-Ⅱ、ant(3”)-Ⅰ的基因的检出率在多重耐药组和泛耐药组中分别与敏感组比较，得多重耐药组中aac(6＇)-Ⅱ（OR=3.45,95%CI=1.68-7.07）、ant(3”)-Ⅰ（OR=3.13,95%CI=1.69-5.81），泛耐药组中aac(6＇)-Ⅱ（OR=11.11,95%CI=4.54-27.19）、ant(3”)-Ⅰ（OR=3.23,95%CI=1.42-7.38）,且具差异有统计学意义（P0.05）。 整合子检测结果：Ⅰ类整合子检出172株，占49.4%（172/348），未检出Ⅱ、Ⅲ类整合子；其中MDRPA的Ⅰ类整合子达53.7%（80/149）。PDRPA中Ⅰ类整合子达84.6%（33/39）；庆大霉素、妥布霉素、阿米卡星、哌拉西林、替卡西林、亚胺培南、美罗培南、左氧氟沙星、环丙沙星、氨曲南、头孢他啶、头孢吡肟12种抗菌药物在整合子阳性菌株中的耐药率明显高于整合子阴性组，差别具有统计学意义（P0.05)。多重耐药组、泛耐药组的整合子的检出率分别与敏感组比较差异，得多重耐药组（OR=2.43,95%CI=1.43-4.15），泛耐药组（OR=11.53,95%CI=4.37-30.40），且差异均具有统计学意义（P0.05)。 喹诺酮耐药决定区突变情况：在162株喹诺酮耐药的铜绿假单胞菌，gyrA基因突变占40.7%(66/162)，parC突变占17.3%（28/162）； gyrA基因的QRDR片段突变主要发生在第83位氨基酸密码子的ACC→ATC，其编码的氨基酸由The→Ⅱe，与此同时，耐药菌株在132位氨基酸密码子(CAC→CAT)有一静止突变，该突变未引起氨基酸的改变，而parC测序结果表明QRDR片段突变主要位于第79位氨基酸密码子发生TCC→GCC的突变，该位点突变引起氨基酸Ser→Ala的改变；泛耐药组中gyrA与parC的QRDR片段的突变率与1-2类耐药组的菌株比较，得出gyrA （OR=3.93，95%CI=1.13-13.62）， parC （OR=1.36，95%CI=1.11-1.16），且差异均具有统计学差异（P0.05）。 PFGE分子分型结果：348株菌Dice系数为100%的PA一共有8对，分别为A、B、D、C、E、F、G、H型， PFGE为A、C、D、E、F型中同一型里两或三位患者均来自同一家医院，不但住院时间日期有重叠且同一个科室，加之耐药谱相似，容易导致患者间互相感染的发生，因此上述5型中的同源菌株考虑科室内交叉感染而产生。而B、G、H型的菌株同一型的患者均来自同一家医院，但检出科室不同，有发生科室间交叉感染的可能，感染途径还有待追究。泛耐药株的PFGE分型Dice系数在44.3%至97.1%之间， Dice系数在80%或以上的同源株有5组，同一型的菌株均来自同一家医院，共11株PDRPA,其余的28株均为散发。 结论广州市院内感染PA菌株的耐药发生率较为普遍，除了LEV和ATM外，耐药率随着时间呈现上升的趋势。 吸痰为1-2类耐药PA的危险因素；曾使用碳青霉烯类抗菌药物、检出前入住ICU为MDRPA的危险因素；机械通气、曾使用碳青霉烯类抗菌药物为PDRPA的危险因素。 β-内酰胺酶及其基因（TEM、OXA-10、VIM）、氨基糖苷类钝化酶编码基因aac(6＇)-Ⅱ、OprD2基因的缺失、整合子、gyrA和ParC基因的喹诺酮耐药决定区突变为PDRPA产生耐药的主要机制。 未发现PA大范围单克隆流行情况,大部分菌株呈散发流行趋势，但在同医院或病区存在轻微的克隆播散现象，ICU与呼吸内科出现频率较高，也有不同科室间出现交叉感染。
[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鍒嗗瓙鍒嗗瀷缁撴灉锛

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