临床分离80株肺炎克雷伯杆菌的药敏检测及超广谱β-内酰胺酶相关耐药基因的检测

发布时间：2018-06-20 15:19

本文选题：肺炎克雷伯杆菌 + 超广谱β-内酰胺酶　；参考：《川北医学院》2016年硕士论文

【摘要】：目的:检测本院临床分离的80株肺炎克雷伯杆菌(Klebsiella pneumoniae)对临床常用的18种抗生素的药物敏感性,并调查分析感染者的临床特征;运用双纸片增效法测定细菌产超广谱β-内酰胺酶(extended-spectrumβ-lactamase,ESBLs)的情况,并选择ESBLs阳性菌,检测其产ESBLs相关耐药基因;对细菌来源的病人进行流行病学分析,并分析已检出的产ESBLs相关基因与细菌耐药性之间的关系,揭示本院肺炎克雷伯杆菌的耐药特点,为临床用药提供理论参考。方法:本研究收集了川北医学附属医学院检验科微生物室2014年9月至2015年9月临床分离,非同一患者、非同一部位的肺炎克雷伯杆菌共80株。采用二倍琼脂稀释法检测临床常用18种抗菌药物对80株肺炎克雷伯杆菌的最低抑菌浓度(minimal inhibitory concentration,MIC)。采用双纸片增效法筛选出ESBLs阳性肺炎克雷伯杆菌,提取上述ESBLs阳性菌株总DNA,用PCR的扩增技术扩增8种与产ESBLs相关的基因(CTX-M-1、2、7、9、15、16型ESBLs基因、TEM型ESBLs基因、SHV型ESBLs基因)。同时收集细菌来源的患者的相关临床资料,分析细菌的耐药特点,以及与产ESBLs相关基因之间的关系。结果:80例患者病史资料显示平均年龄为50.13岁,其中男性比例为51.25%。80株细菌对应的患者所患疾病以肺部感染为主,占50%(40/80),其次是败血症12.5%,尿路感染10%,腹部感染10%,肛周脓肿10%,胆道感染6.25%,脑炎2.5%。标本来源科室分布:普外科和小儿外科占22.5%(18/80),呼吸科和消化科占20%(16/80),肛肠科和胸外占12.5%(10/80),儿科和新生儿科占10%(8/80),泌尿外科和神经外科占7.5%(6/80),其余科室散在分布。患者有基础疾病的占16.25%(13/80)。入院前使用了抗菌药物的患者比例为71.25%(57/80)。入院患者使用抗菌药物情况:i联用药占28.75%(23/80),ii联或ii联以上用药占71.25%(57/80)。平均住院天数为20.3天,患者的预后情况:好转出院占78.75%(63/80)。80株细菌的药敏结果为:对氨苄西林钠耐药率最高,耐药率高达85%。对不含酶抑制剂的头孢一、二代耐药率分别为42.5%、35%,对头孢他啶、头孢曲松钠耐药率分别为26.25%、25%、对含酶抑制剂的头孢三代耐药率明显降低,对头孢哌酮他唑巴坦和头孢哌酮舒巴坦耐药率分别为15%、13.75%。对单环类抗生素耐药率为3.25%,对氟喹诺酮类抗生素如环丙沙星、左氧氟沙星耐药率分别为21.25%、20%,对氨基糖胺类抗生素如阿米卡星、庆大霉素耐药率分别为22.5%、37.5%,对大环内酯类抗生素如阿奇霉素耐药率为31%。而肺炎克雷伯杆菌对磺胺类抗生素如复方新诺明敏感性高,耐药率仅为21.25%。仍然出现了对碳青霉烯抗生素耐药的菌株,对比阿培南耐药率为3.75%。通过检测筛选出产esbls肺炎克雷伯杆菌20株,而这些esbls阳性菌株的相关耐药基因检出率最高的shv基因,比例是65%;其次为ctx-m-9基因,比例为60%;ctx-m-1为35%,tem为25%,ctx-m-2、ctx-m-7及ctx-m-16均为20%,检出率最低的基因为ctx-m-15基因,比例为10%。20株产esbls肺炎克雷伯杆菌对18种常用抗菌药物的耐药率分别为:耐药率较高的是头孢唑啉(90%)、氨苄西林(70%)、头孢呋辛(65%)、复方新诺明(55%)、头孢他啶(50%)、庆大霉素(45%)、阿奇霉素(45%)、氨曲南(45%)、头孢曲松(40%)、环丙沙星(30%)以及左氧氟沙星(30%),耐药率较低的是哌拉西林舒巴坦(15%)、美洛西林舒巴坦(15%)、阿米卡星(15%)、头孢哌酮他唑巴坦(5%)、头孢哌酮舒巴坦(5%)、亚胺培南(5%),全部敏感的是比阿培南(0%)。20株产esbls菌中多重耐药菌占90%(18/20),对一种抗生素耐药占5%(1/20),对两种抗生素耐药占5%(1/20)。18株esbls阳性多重耐药菌对18种抗生素耐药率较高的是头孢唑啉(94.44%)、氨苄西林(72.22%)、头孢呋辛(66.67%)、复方新诺明(61.11%)、头孢他啶(55.56%)、庆大霉素(50%)、氨曲南(50%)、头孢曲松(44.44%)以及阿奇霉素(44.44%),耐药率较低的是头孢哌酮他唑巴坦(5.56%)、头孢哌酮舒巴坦(5.56%)、亚胺培南(5.56%)、哌拉西林舒巴坦(16.67%)、美洛西林舒巴坦(16.67%)以及阿米卡星(16.67%),全部敏感的是比阿培南。18株多重耐药菌中各基因亚型分布情况为:shv占66.67%(12/18),ctx-m-9占50%(9/18),ctx-m-1占33.33%(6/18),tem占27.78%(5/18),ctx-m-2占22.22%(4/18),ctx-m-7、ctx-m-15、ctx-m-16均占11.11%(2/18)。13株产shv-esbls菌对18种抗生素的耐药率分别为:氨苄西林(61.54%),哌拉西林舒巴坦(15.38%),美洛西林舒巴坦(23.08%),头孢唑啉(100%),头孢呋辛(61.54%),头孢他啶(53.85%),头孢曲松(53.85%),头孢哌酮他唑巴坦(7.69%),头孢哌酮舒巴坦(7.69%),亚胺培南(7.69%),比阿培南(7.69%),环丙沙星(23.08%),左氧氟沙星(15.38%),庆大霉素(53.85%),阿米卡星(0.00%),阿奇霉素(38.46%),复方新诺明(61.54%),氨曲南(46.15%)。12株产ctx-m-9-esbls菌对18种抗生素的耐药率分别为:氨苄西林(66.67%),哌拉西林舒巴坦(8.33%),美洛西林舒巴坦(8.33%),头孢唑啉(91.67%),头孢呋辛(75.00%),头孢他啶(41.67%),头孢曲松(41.67%),头孢哌酮他唑巴坦(0.00%),头孢哌酮舒巴坦(0.00%),亚胺培南(0.00%),比阿培南(0.00%),环丙沙星(16.67%),左氧氟沙星(25.00%),庆大霉素(41.67%),阿米卡星(8.33%),阿奇霉素(50.00%),复方新诺明(58.33%),氨曲南(33.33%)。其余亚型(ctx-m-1、ctx-m-2、ctx-m-7、ctx-m-15、ctx-m-16及tem)产esbls菌亦表现出不同的耐药率差异。结论:1、本院肺炎克雷伯杆菌标本来源以普外科、小儿外科、肛肠外科、胸外科为主,疾病来源以肺部感染多见,其次是败血症、尿路感染、腹部感染、肛周脓肿、胆道感染以及脑炎,从实验结果可以看出,南充地区肺炎克雷伯杆菌对青霉素类、头孢一二代抗生素的耐药率较高,尽管碳青霉烯类抗生素是目前治疗肺炎克雷伯杆菌最有效的抗菌药物,但仍然出现了对碳青霉烯抗生素耐药菌株,如对比阿培南的耐药率为3.75%,而对单环类抗生素氨曲南的耐药率为仅为3.25%,因此在治疗上应尽快根据药敏试验结果选择敏感抗生素,避免多重耐药菌的产生。2、本院临床分离的80株肺炎克雷伯杆菌中,产esbls型菌株占25%(20/80),20株产esbls菌对18种常用抗生素的耐药率最高的是头孢唑啉(90%),其次为氨苄西林(70%),耐药率较低的是含β内酰胺酶抑制剂的头孢三代以及碳青霉烯类;而20株产esbls菌中又以shv型和ctx-m-9型多见,tem型产esbls菌占到一定比例(25%,5/20),且tem型对含酶抑制剂的青霉素类抗菌药具有一定的耐药率(25%),可能与细菌在抗菌药物长期压力下发生基因组水平的变异有关。3、本院20株产esbls菌中,多重耐药菌占90%(18/20),表明多重耐药性的获得与esbls的产生密切相关,而18株产esbls多重耐药菌耐药率最高的是头孢唑啉、氨苄西林、头孢呋辛,耐药率较低的头孢哌酮舒巴坦、头孢哌酮他唑巴坦,全部敏感的是比阿培南,但对亚胺培南的耐药率达到5.56%。基因亚型分布以shv、ctx-m-9、ctx-m-1多见,分别占66.67%,50.00%及33.33%,tem型亦占一定比例(27.78%)。新基因型的出现可能导致了产esbls菌耐药性差异的多样化趋势。4、各亚型产esbls菌的耐药率不尽相同,产shv-esbls型、ctx-m-9型及ctx-m-1型耐药率较高的是氨苄西林、头孢唑啉、头孢呋辛、头孢他啶、头孢曲松以及复方新诺明,而耐药率较低的是哌拉西林舒巴坦、美洛西林舒巴坦、头孢哌酮他唑巴坦、头孢哌酮舒巴坦以及亚胺培南,但shv型对亚胺培南有7.69%的耐药率;ctx-m-2、ctx-m-7型对头孢他啶、左氧氟沙星、庆大霉素、氨曲南等耐药率较低,ctx-m-16型esbls菌对氨曲南、庆大霉素的耐药率均达到50%,而ctx-m-15型esbls菌对氨曲南和庆大霉素全部耐药;tem型esbls菌对含β-内酰胺酶抑制剂的哌拉西林舒巴坦、美洛西林舒巴坦以及氨基糖苷类阿米卡星均表现出20%的耐药率。临床上可根据不同亚型产esbls菌耐药特点灵活选用敏感的抗菌药物,再根据细菌的产酶特点的变化、药敏实验结果以及疗效决定是否调整抗菌药物种类。5、为预防耐药菌的暴发流行或者多重耐药菌株的出现,应对本地区esbls阳性肺炎克雷伯杆菌进行临床危险因素的调查,并采取有效的医源性控制措施;同时通过分子生物学的检验方法,对肺炎克雷伯杆菌的基因型进行分析,根据不同基因型的耐药性或产酶特征,针对性选择敏感的抗菌药物,并进一步探索病原菌的耐药机理、耐药基因的传播渠道和流行发展趋势,对减少和预防新耐药菌及多重耐药菌的产生和传播具有积极意义。
[Abstract]:Objective: to detect the sensitivity of 80 clinical isolates of Klebsiella pneumoniae (Klebsiella pneumoniae) to 18 commonly used antibiotics, and to investigate the clinical characteristics of the infected people, and to determine the condition of extended-spectrum beta lactamase (extended-spectrum beta -lactamase, ESBLs) by double paper synergistic method and select ESBLs. The positive bacteria were used to detect the ESBLs related resistance genes, the epidemiological analysis of the patients with bacterial origin, and the relationship between the detected ESBLs related genes and the bacterial resistance were analyzed, and the drug resistance characteristics of Klebsiella pneumoniae were revealed. The laboratory department of microbiology was isolated from September 2014 to September 2015, and 80 strains of Klebsiella pneumoniae were isolated from the same area. The minimum inhibitory concentration (minimal inhibitory concentration, MIC) for 80 strains of Klebsiella pneumoniae (Klebsiella pneumoniae) was detected by two times agar dilution method. ESBLs positive Klebsiella pneumoniae was screened and the total DNA of the ESBLs positive strain was extracted. 8 ESBLs related genes (CTX-M-1,2,7,9,15,16 ESBLs gene, TEM type ESBLs gene, SHV ESBLs gene) were amplified by PCR amplification. The clinical data of the patients with bacterial origin were collected and the characteristics of bacterial resistance were analyzed. Results: the relationship between ESBLs related genes. Results: the average age of 80 patients was 50.13 years old, and the male proportion of 51.25%.80 strain was mainly pulmonary infection, 50% (40/80), followed by septicemia 12.5%, urinary tract infection 10%, abdominal infection 10%, perianal abscess 10%, biliary tract infection 6.25%, brain The distribution of the source section of inflammatory 2.5%. specimens: Department of general surgery and pediatric surgery accounted for 22.5% (18/80), Department of respiration and digestive department accounted for 20% (16/80), anorectal department and outside of the chest accounted for 12.5% (10/80), pediatrics and newborn children accounted for 10% (8/80), 7.5% (6/80), the remaining sections were scattered. Patients with basic diseases accounted for 16.25% (13/80). Before admission to hospital use. The proportion of the patients with antibiotics was 71.25% (57/80). The use of antibiotics in hospitalized patients was 28.75% (23/80) and 71.25% (57/80) with II or II. The average hospital days were 20.3 days, and the prognosis of the patients was 78.75% (63/80) and 78.75% (63/80). The drug sensitivity of ampicillin sodium was the highest, The resistance rate was as high as 85%. for ceftazidin and two generation, 42.5% and 35%, respectively. The resistance rate of ceftriaxone and ceftriaxone sodium was 26.25%, 25% respectively. The resistance rate of ceftriaxone with ceftriaxone and cefoperazone and cefoperazone and sulbactam was 15% for cefoperazone and cefoperazone and 13.75%. to mono ring resistance, respectively. The rate of antibiotic resistance was 3.25%, and the resistance rates of fluoroquinolones such as ciprofloxacin and levofloxacin were 21.25% and 20% respectively. The resistance rate of aminoglycan amines such as Amikacin and gentamicin was 22.5% and 37.5% respectively. The drug resistance rate of macrolides, such as azithromycin, was 31%. and Klebsiella pneumoniae against sulfonamides. In the case of high sensibility of compound sennox, the resistance rate was only 21.25%., which was still resistant to carbapenems, 20 strains of Klebsiella pneumoniae were produced by 3.75%., and the SHV gene with the highest detection rate of the related resistance genes of these ESBLs positive strains was 65%, followed by ctx-m. The ratio of -9 was 60%, ctx-m-1 was 35%, TEM was 25%, ctx-m-2, ctx-m-7 and ctx-m-16 were 20%, and the gene for the lowest detection rate was ctx-m-15 gene. The ratio of ESBLs pneumonia by Klebsiella pneumoniae to 18 commonly used antibiotics was the high rate of cefazolin (90%), ampicillin (70%), cefuroxime (65%). Fang Xin NOMIN (55%), ceftazidime (50%), azithromycin (45%), amamethoni (45%), ceftriaxone (40%), ciprofloxacin (30%) and levofloxacin (30%), piperacillin sulbactam (15%), meloxicine sulbactam (15%), Amikacin (15%), cefoperazone (5%), cefoperazazol Batan (5%), cefoperazone Shu Batan (5%), Imipenem (5%) was all sensitive to 90% (18/20) of multidrug-resistant bacteria producing ESBLs strains of.20 strain of apenem (0%), 5% (1/20) against one antibiotic, 5% (1/20).18 strain of two antibiotics and higher rates of resistance to 18 antibiotics were cefazolin (94.44%), ampicillin (72.22%), and cefuroxime (66. 67%), compound novamoxin (61.11%), ceftazidime (55.56%), gentamicin (50%), amamethoni (50%), ceftriaxone (44.44%) and azithromycin (44.44%), and the lower drug resistance rate was cefoperazone tazobactam (5.56%), cefoperazone Shubatan (5.56%), imipenem (5.56%), piperacillin sulbactam (16.67%), and mercillin sulbactam (16.67%). Amikacin (16.67%) was all sensitive to the distribution of the subtypes of gene subtypes in the multidrug-resistant bacteria of.18 plant, SHV, ctx-m-9 50% (9/18), ctx-m-1 33.33% (6/18), TEM 27.78% (5/18), ctx-m-2 22.22% (4/18), ctx-m-7, and 11.11% (11.11%) resistant to 18 antibiotics The drug rates were ampicillin (61.54%), piperacillin sulbactam (15.38%), meloxicine sulbactam (23.08%), cefazolin (100%), ceffuroxime (61.54%), ceftazidime (53.85%), ceftriaxone (53.85%), cefoperazone (7.69%), cefoperazone (7.69%), imipenem (7.69%), amperan (7.69%), ciprofloxacin (23.08%). The resistance rates of levofloxacin (15.38%), gentamicin (53.85%), Amikacin (0%), azithromycin (38.46%), compound sulfamethoxazole (61.54%), amamethoni (46.15%).12 strains of ctx-m-9-esbls strains to 18 antibiotics were ampicillin (66.67%), Pi La Shilling Shug Batan (8.33%), meloxicine Shubatan (8.33%), cefazolin (91.67%), cephalosporin Furoxime (75%), ceftazidime (41.67%), ceftriaxone (41.67%), cefoperazone (0%), cefoperazone (0%), cefoperazone (0%), imipenem (0%), ampere (0%), ciprofloxacin (16.67%), levofloxacin (25%), gentamicin (41.67%), Amikacin (8.33%), azithromycin (50%), compound Novamin (58.33%), amamennan (41.67%). 33.33%). The other subtypes (ctx-m-1, ctx-m-2, ctx-m-7, ctx-m-15, ctx-m-16 and TEM) also showed different resistance rates. Conclusion: 1, the source of Klebsiella pneumoniae is mainly from Department of general surgery, pediatric surgery, Department of anus & intestine surgery, and the disease comes from pulmonary infection, followed by sepsis, urinary tract infection and abdominal infection. Perianal abscess, biliary tract infection, and encephalitis showed that the resistance rate of Klebsiella pneumoniae to penicillins and one or two generation cephalosporins was higher in Nanchong area. Although carbapenems were the most effective antiseptic in the treatment of Klebsiella pneumoniae, the resistance to carbapenems still occurred. The resistance rate of the strains, such as the comparison of asinan, was 3.75%, and the drug resistance rate of mono antibiotic was only 3.25%. Therefore, the sensitive antibiotics should be selected as soon as possible according to the results of drug sensitivity test to avoid the production of.2. Of the 80 strains of Klebsiella pneumoniae, 25% (20/80) and 20 strains of ESBLs strains were produced in our hospital. The highest resistance rate of ESBLs bacteria to 18 common antibiotics was cefazolin (90%), followed by ampicillin (70%), the lower drug resistance rate was the three generation of cephalosporin containing beta lactamase inhibitor and carbapenems, while the 20 ESBLs producing strains were more SHV and ctx-m-9, TEM producing ESBLs bacteria accounted for a certain proportion (25%, 5/20), and TEM type pairs The penicillins containing enzyme inhibitors have a certain rate of resistance (25%), which may be related to the variation of the genomic level of the bacteria under the long-term stress of the antimicrobial agents.3. Among the 20 strains of ESBLs producing bacteria in our hospital, 90% (18/20), indicating that the acquisition of multiple resistance is closely related to the production of ESBLs, and 18 strains of ESBLs multidrug-resistant bacteria. The highest drug resistance rate was cefazolin, ampicillin, cefuroxime, cefoperazone sulbactam, cefoperazone and cefoperazolobactam, which were all sensitive to amenenan, but the resistance rate of imipenem reached 5.56%. gene subtypes in SHV, ctx-m-9, and ctx-m-1, accounting for 66.67%, 50% and 33.33% respectively, and TEM type accounted for a certain ratio. 27.78%. 27.78%. The emergence of the new genotypes may lead to the diversity trend of the diversity of drug resistance of ESBLs bacteria.4. The resistance rates of ESBLs producing strains in each subtype are not the same. The high rate of shv-esbls, ctx-m-9 and ctx-m-1 is ampicillin, cefazolin, cefuroxime, ceftazidime, ceftriaxone, and compound Sulfamethoxine, and the drug resistance rate The lower is piperacillin sulbactam, meroxicillin sulbactam, cefoperazone tazobactam, cefoperazone sulbactam and imipenem, but SHV has a 7.69% resistance rate to imipenem; ctx-m-2, ctx-m-7 has low resistance to ceftazidime, levofloxacin, gentamicin, amamennan, and ctx-m-16 type ESBLs bacteria to amamethanone and mildew The resistance rates of the elements were all up to 50%, while the ctx-m-15 type ESBLs bacteria were all resistant to amamanin and gentamicin, and the resistance rate of TEM type ESBLs bacteria to piperacillin sulbactam, meloxilin sulbactam and aminoglycoside type Amikacin containing beta lactamase inhibitors was 20%. The drug resistance of ESBLs bacteria in different subtypes could be based on the drug resistance of different subtypes. The clinical risk factors of ESBLs positive Klebsiella pneumoniae in the local area were investigated and the clinical risk factors were investigated by using sensitive antibiotics, the changes in the characteristics of the bacteria producing enzymes, the results of the drug sensitivity test and the effect of the effect of the.5. Effective iatrogenic control measures; at the same time, the genotypes of Klebsiella pneumoniae were analyzed by molecular biology, and sensitive antimicrobial agents were selected according to the resistance of different genotypes or characteristics of enzyme production, and the mechanism of drug resistance of pathogenic bacteria was further explored, the transmission channels of resistance genes and the trend of epidemic development were further explored. It is of great significance to reduce and prevent the emergence and spread of new drug-resistant bacteria and multidrug-resistant bacteria.
【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.5

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