本文選題：肺炎克雷伯桿菌 + 超廣譜β-內(nèi)酰胺酶　； 參考：《川北醫(yī)學(xué)院》2016年碩士論文

【摘要】：目的:檢測本院臨床分離的80株肺炎克雷伯桿菌(Klebsiella pneumoniae)對臨床常用的18種抗生素的藥物敏感性,并調(diào)查分析感染者的臨床特征;運用雙紙片增效法測定細(xì)菌產(chǎn)超廣譜β-內(nèi)酰胺酶(extended-spectrumβ-lactamase,ESBLs)的情況,并選擇ESBLs陽性菌,檢測其產(chǎn)ESBLs相關(guān)耐藥基因;對細(xì)菌來源的病人進行流行病學(xué)分析,并分析已檢出的產(chǎn)ESBLs相關(guān)基因與細(xì)菌耐藥性之間的關(guān)系,揭示本院肺炎克雷伯桿菌的耐藥特點,為臨床用藥提供理論參考。方法:本研究收集了川北醫(yī)學(xué)附屬醫(yī)學(xué)院檢驗科微生物室2014年9月至2015年9月臨床分離,非同一患者、非同一部位的肺炎克雷伯桿菌共80株。采用二倍瓊脂稀釋法檢測臨床常用18種抗菌藥物對80株肺炎克雷伯桿菌的最低抑菌濃度(minimal inhibitory concentration,MIC)。采用雙紙片增效法篩選出ESBLs陽性肺炎克雷伯桿菌,提取上述ESBLs陽性菌株總DNA,用PCR的擴增技術(shù)擴增8種與產(chǎn)ESBLs相關(guān)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)。同時收集細(xì)菌來源的患者的相關(guān)臨床資料,分析細(xì)菌的耐藥特點,以及與產(chǎn)ESBLs相關(guān)基因之間的關(guān)系。結(jié)果:80例患者病史資料顯示平均年齡為50.13歲,其中男性比例為51.25%。80株細(xì)菌對應(yīng)的患者所患疾病以肺部感染為主,占50%(40/80),其次是敗血癥12.5%,尿路感染10%,腹部感染10%,肛周膿腫10%,膽道感染6.25%,腦炎2.5%。標(biāo)本來源科室分布:普外科和小兒外科占22.5%(18/80),呼吸科和消化科占20%(16/80),肛腸科和胸外占12.5%(10/80),兒科和新生兒科占10%(8/80),泌尿外科和神經(jīng)外科占7.5%(6/80),其余科室散在分布。患者有基礎(chǔ)疾病的占16.25%(13/80)。入院前使用了抗菌藥物的患者比例為71.25%(57/80)。入院患者使用抗菌藥物情況:i聯(lián)用藥占28.75%(23/80),ii聯(lián)或ii聯(lián)以上用藥占71.25%(57/80)。平均住院天數(shù)為20.3天,患者的預(yù)后情況:好轉(zhuǎn)出院占78.75%(63/80)。80株細(xì)菌的藥敏結(jié)果為:對氨芐西林鈉耐藥率最高,耐藥率高達85%。對不含酶抑制劑的頭孢一、二代耐藥率分別為42.5%、35%,對頭孢他啶、頭孢曲松鈉耐藥率分別為26.25%、25%、對含酶抑制劑的頭孢三代耐藥率明顯降低,對頭孢哌酮他唑巴坦和頭孢哌酮舒巴坦耐藥率分別為15%、13.75%。對單環(huán)類抗生素耐藥率為3.25%,對氟喹諾酮類抗生素如環(huán)丙沙星、左氧氟沙星耐藥率分別為21.25%、20%,對氨基糖胺類抗生素如阿米卡星、慶大霉素耐藥率分別為22.5%、37.5%,對大環(huán)內(nèi)酯類抗生素如阿奇霉素耐藥率為31%。而肺炎克雷伯桿菌對磺胺類抗生素如復(fù)方新諾明敏感性高,耐藥率僅為21.25%。仍然出現(xiàn)了對碳青霉烯抗生素耐藥的菌株,對比阿培南耐藥率為3.75%。通過檢測篩選出產(chǎn)esbls肺炎克雷伯桿菌20株,而這些esbls陽性菌株的相關(guān)耐藥基因檢出率最高的shv基因,比例是65%;其次為ctx-m-9基因,比例為60%;ctx-m-1為35%,tem為25%,ctx-m-2、ctx-m-7及ctx-m-16均為20%,檢出率最低的基因為ctx-m-15基因,比例為10%。20株產(chǎn)esbls肺炎克雷伯桿菌對18種常用抗菌藥物的耐藥率分別為:耐藥率較高的是頭孢唑啉(90%)、氨芐西林(70%)、頭孢呋辛(65%)、復(fù)方新諾明(55%)、頭孢他啶(50%)、慶大霉素(45%)、阿奇霉素(45%)、氨曲南(45%)、頭孢曲松(40%)、環(huán)丙沙星(30%)以及左氧氟沙星(30%),耐藥率較低的是哌拉西林舒巴坦(15%)、美洛西林舒巴坦(15%)、阿米卡星(15%)、頭孢哌酮他唑巴坦(5%)、頭孢哌酮舒巴坦(5%)、亞胺培南(5%),全部敏感的是比阿培南(0%)。20株產(chǎn)esbls菌中多重耐藥菌占90%(18/20),對一種抗生素耐藥占5%(1/20),對兩種抗生素耐藥占5%(1/20)。18株esbls陽性多重耐藥菌對18種抗生素耐藥率較高的是頭孢唑啉(94.44%)、氨芐西林(72.22%)、頭孢呋辛(66.67%)、復(fù)方新諾明(61.11%)、頭孢他啶(55.56%)、慶大霉素(50%)、氨曲南(50%)、頭孢曲松(44.44%)以及阿奇霉素(44.44%),耐藥率較低的是頭孢哌酮他唑巴坦(5.56%)、頭孢哌酮舒巴坦(5.56%)、亞胺培南(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株多重耐藥菌中各基因亞型分布情況為: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株產(chǎn)shv-esbls菌對18種抗生素的耐藥率分別為:氨芐西林(61.54%),哌拉西林舒巴坦(15.38%),美洛西林舒巴坦(23.08%),頭孢唑啉(100%),頭孢呋辛(61.54%),頭孢他啶(53.85%),頭孢曲松(53.85%),頭孢哌酮他唑巴坦(7.69%),頭孢哌酮舒巴坦(7.69%),亞胺培南(7.69%),比阿培南(7.69%),環(huán)丙沙星(23.08%),左氧氟沙星(15.38%),慶大霉素(53.85%),阿米卡星(0.00%),阿奇霉素(38.46%),復(fù)方新諾明(61.54%),氨曲南(46.15%)。12株產(chǎn)ctx-m-9-esbls菌對18種抗生素的耐藥率分別為:氨芐西林(66.67%),哌拉西林舒巴坦(8.33%),美洛西林舒巴坦(8.33%),頭孢唑啉(91.67%),頭孢呋辛(75.00%),頭孢他啶(41.67%),頭孢曲松(41.67%),頭孢哌酮他唑巴坦(0.00%),頭孢哌酮舒巴坦(0.00%),亞胺培南(0.00%),比阿培南(0.00%),環(huán)丙沙星(16.67%),左氧氟沙星(25.00%),慶大霉素(41.67%),阿米卡星(8.33%),阿奇霉素(50.00%),復(fù)方新諾明(58.33%),氨曲南(33.33%)。其余亞型(ctx-m-1、ctx-m-2、ctx-m-7、ctx-m-15、ctx-m-16及tem)產(chǎn)esbls菌亦表現(xiàn)出不同的耐藥率差異。結(jié)論:1、本院肺炎克雷伯桿菌標(biāo)本來源以普外科、小兒外科、肛腸外科、胸外科為主,疾病來源以肺部感染多見,其次是敗血癥、尿路感染、腹部感染、肛周膿腫、膽道感染以及腦炎,從實驗結(jié)果可以看出,南充地區(qū)肺炎克雷伯桿菌對青霉素類、頭孢一二代抗生素的耐藥率較高,盡管碳青霉烯類抗生素是目前治療肺炎克雷伯桿菌最有效的抗菌藥物,但仍然出現(xiàn)了對碳青霉烯抗生素耐藥菌株,如對比阿培南的耐藥率為3.75%,而對單環(huán)類抗生素氨曲南的耐藥率為僅為3.25%,因此在治療上應(yīng)盡快根據(jù)藥敏試驗結(jié)果選擇敏感抗生素,避免多重耐藥菌的產(chǎn)生。2、本院臨床分離的80株肺炎克雷伯桿菌中,產(chǎn)esbls型菌株占25%(20/80),20株產(chǎn)esbls菌對18種常用抗生素的耐藥率最高的是頭孢唑啉(90%),其次為氨芐西林(70%),耐藥率較低的是含β內(nèi)酰胺酶抑制劑的頭孢三代以及碳青霉烯類;而20株產(chǎn)esbls菌中又以shv型和ctx-m-9型多見,tem型產(chǎn)esbls菌占到一定比例(25%,5/20),且tem型對含酶抑制劑的青霉素類抗菌藥具有一定的耐藥率(25%),可能與細(xì)菌在抗菌藥物長期壓力下發(fā)生基因組水平的變異有關(guān)。3、本院20株產(chǎn)esbls菌中,多重耐藥菌占90%(18/20),表明多重耐藥性的獲得與esbls的產(chǎn)生密切相關(guān),而18株產(chǎn)esbls多重耐藥菌耐藥率最高的是頭孢唑啉、氨芐西林、頭孢呋辛,耐藥率較低的頭孢哌酮舒巴坦、頭孢哌酮他唑巴坦,全部敏感的是比阿培南,但對亞胺培南的耐藥率達到5.56%�；騺喰头植家詓hv、ctx-m-9、ctx-m-1多見,分別占66.67%,50.00%及33.33%,tem型亦占一定比例(27.78%)。新基因型的出現(xiàn)可能導(dǎo)致了產(chǎn)esbls菌耐藥性差異的多樣化趨勢。4、各亞型產(chǎn)esbls菌的耐藥率不盡相同,產(chǎn)shv-esbls型、ctx-m-9型及ctx-m-1型耐藥率較高的是氨芐西林、頭孢唑啉、頭孢呋辛、頭孢他啶、頭孢曲松以及復(fù)方新諾明,而耐藥率較低的是哌拉西林舒巴坦、美洛西林舒巴坦、頭孢哌酮他唑巴坦、頭孢哌酮舒巴坦以及亞胺培南,但shv型對亞胺培南有7.69%的耐藥率;ctx-m-2、ctx-m-7型對頭孢他啶、左氧氟沙星、慶大霉素、氨曲南等耐藥率較低,ctx-m-16型esbls菌對氨曲南、慶大霉素的耐藥率均達到50%,而ctx-m-15型esbls菌對氨曲南和慶大霉素全部耐藥;tem型esbls菌對含β-內(nèi)酰胺酶抑制劑的哌拉西林舒巴坦、美洛西林舒巴坦以及氨基糖苷類阿米卡星均表現(xiàn)出20%的耐藥率。臨床上可根據(jù)不同亞型產(chǎn)esbls菌耐藥特點靈活選用敏感的抗菌藥物,再根據(jù)細(xì)菌的產(chǎn)酶特點的變化、藥敏實驗結(jié)果以及療效決定是否調(diào)整抗菌藥物種類。5、為預(yù)防耐藥菌的暴發(fā)流行或者多重耐藥菌株的出現(xiàn),應(yīng)對本地區(qū)esbls陽性肺炎克雷伯桿菌進行臨床危險因素的調(diào)查,并采取有效的醫(yī)源性控制措施;同時通過分子生物學(xué)的檢驗方法,對肺炎克雷伯桿菌的基因型進行分析,根據(jù)不同基因型的耐藥性或產(chǎn)酶特征,針對性選擇敏感的抗菌藥物,并進一步探索病原菌的耐藥機理、耐藥基因的傳播渠道和流行發(fā)展趨勢,對減少和預(yù)防新耐藥菌及多重耐藥菌的產(chǎn)生和傳播具有積極意義。
[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.
【學(xué)位授予單位】：川北醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R446.5

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