【摘要】：研究背景和目的目前鮑曼不動(dòng)桿菌已成為醫(yī)院內(nèi)感染的重要致病菌之一,而且在燒傷病房尤其突出,筆者所在科室燒傷重癥病房2011年1月至2014年12月收治的162例燒傷患者的血液標(biāo)本共計(jì)1658份,病原菌檢出339株,前三位分別是鮑曼不動(dòng)桿菌(Acinetobacter baumannii,ABA),金黃色葡萄球菌(Staphylococcus aureus,SAU)以及銅綠假單胞菌(Pseudomonas aeruginosa,PAE)。國(guó)內(nèi)大部分文獻(xiàn)報(bào)道鮑曼不動(dòng)桿菌的檢出率僅低于銅綠假單胞菌,位居第二。據(jù)國(guó)外文獻(xiàn)報(bào)道,1991年紐約首次暴發(fā)鮑曼不動(dòng)桿菌感染以來(lái),世界各地對(duì)鮑曼不動(dòng)桿菌耐藥現(xiàn)象的報(bào)道層出不窮,尤其是對(duì)碳青霉烯類(lèi)抗菌素耐藥現(xiàn)象的增多,使成功治療鮑曼不動(dòng)桿菌所致感染變得困難。加上開(kāi)發(fā)新的抗菌素周期長(zhǎng),耗費(fèi)資金巨大以及細(xì)菌耐藥變異迅速,亟需尋找新的治療方案。噬菌體是一種以細(xì)菌為宿主的病毒,噬菌體制劑經(jīng)過(guò)臨床驗(yàn)證能夠有效治療細(xì)菌感染。1958年,我國(guó)細(xì)菌學(xué)教授余賀,就利用噬菌體成功治療了銅綠假單胞菌所致燒傷患者感染。過(guò)去幾十年里,大量抗生素得以研發(fā)并廣泛應(yīng)用于臨床,相應(yīng)限制了噬菌體的使用。近年來(lái),隨著細(xì)菌耐藥情況越來(lái)越嚴(yán)重,歐美國(guó)家開(kāi)始重新重視噬菌體在細(xì)菌感染治療中的作用,國(guó)內(nèi)一些單位也開(kāi)始進(jìn)行相關(guān)研究。筆者單位在篩選分離噬菌體并建立噬菌體庫(kù)方面進(jìn)行探索。本研究通過(guò)篩選泛耐藥鮑曼不動(dòng)桿菌裂解性噬菌體,建立泛耐藥鮑曼不動(dòng)桿菌所致小鼠膿毒癥模型,并使用所篩選到的噬菌體進(jìn)行相關(guān)的動(dòng)物實(shí)驗(yàn)。通過(guò)分析噬菌體治療后小鼠存活情況、對(duì)白細(xì)胞計(jì)數(shù)的影響及其細(xì)菌清除情況,觀察和研究噬菌體對(duì)來(lái)源于燒傷患者的泛耐藥鮑曼不動(dòng)桿菌所致小鼠膿毒癥的治療效果。研究方法(1)泛耐藥鮑曼不動(dòng)桿菌裂解性噬菌體的分離與保存使用混合宿主菌擴(kuò)增噬菌體的方法,以泛耐藥鮑曼不動(dòng)桿菌為宿主菌,在筆者所在醫(yī)院未經(jīng)處理的污水池中取污水篩選得到泛耐藥鮑曼不動(dòng)桿菌裂解性噬菌體8株,并測(cè)定各株裂解性噬菌體的裂解譜,選取其裂解譜最寬、裂解效果最好的裂解性噬菌體備用,作為抗菌劑進(jìn)行后續(xù)動(dòng)物治療實(shí)驗(yàn)。(2)小鼠泛耐藥鮑曼不動(dòng)桿菌膿毒癥模型的建立將48只8~12周齡雄性健康BALB/c小鼠,隨機(jī)分為4組,每組12只,記為A,B,C,D四組。分別向A,B,C,D,四組小鼠腹腔注射泛耐藥鮑曼不動(dòng)桿菌5×108 CFU/mL,2.5×108 CFU/mL,1×108 CFU/mL,5×107 CFU/mL這四個(gè)濃度的菌液各1mL。注射成功后,將各組小鼠按正常飼養(yǎng),密切觀察各組小鼠生命體征,死亡小鼠數(shù)量,腹腔灌洗液細(xì)菌培養(yǎng)結(jié)果,血培養(yǎng)結(jié)果。(3)噬菌體對(duì)小鼠泛耐藥鮑曼不動(dòng)桿菌膿毒癥的治療效果Ⅰ.將60只BALB/c小鼠,按隨機(jī)數(shù)字表法分為空白對(duì)照組、膿毒癥對(duì)照組、抗生素治療組、噬菌體治療組、噬菌體對(duì)照組,每組12只。空白對(duì)照組小鼠腹腔(注射部位下同)注射生理鹽水1 mL;膿毒癥對(duì)照組、抗生素治療組、噬菌體治療組小鼠注射5×107 CFU/mL鮑曼不動(dòng)桿菌1 mL建立膿毒癥模型。2 h后,膿毒癥對(duì)照組注射生理鹽水1 mL,抗生素治療組注射1 mg/mL亞胺培南/西司他丁1 mL,噬菌體治療組腹腔注射1×108 PFU/mL噬菌體1 m L;噬菌體對(duì)照組注射1×108 PFU/mL噬菌體1 mL。每組均連續(xù)注射7 d,每天觀察記錄小鼠存活情況。Ⅱ.另取60只BALB/c小鼠,同Ⅰ分組處理。實(shí)驗(yàn)第2、4、6天當(dāng)天注射前,每組選1~3只存活小鼠,每只鼠取眶靜脈血20μL檢查白細(xì)胞計(jì)數(shù)。實(shí)驗(yàn)第2天取血小鼠另取1 mL眶靜脈血行細(xì)菌培養(yǎng);另取小鼠肺、肝、腎、脾組織,勻漿、稀釋后行菌落計(jì)數(shù)后計(jì)算細(xì)菌含量。對(duì)數(shù)據(jù)行Wilcoxon秩和檢驗(yàn)、單因素方差分析、LSD檢驗(yàn)及Kruskal-Wallis秩和檢驗(yàn)。結(jié)果(1)以22株泛耐藥鮑曼不動(dòng)桿菌為宿主菌,筆者所在醫(yī)院未經(jīng)消毒處理的污水池為噬菌體來(lái)源,共篩選得到8株裂解性噬菌體,通過(guò)噬菌體裂解譜測(cè)定,其中Bp201404072號(hào)噬菌體裂解譜59%,高于其他7株噬菌體,故選取該株噬菌體作為抗菌劑,用于后續(xù)動(dòng)物實(shí)驗(yàn),4℃冰箱凍存。(2)各組小鼠注射不同濃度鮑曼不動(dòng)桿菌后,四組小鼠均出現(xiàn)典型的膿毒癥相關(guān)癥狀,其中A,B兩組小鼠較C,D兩組急性感染癥狀明顯。實(shí)驗(yàn)第7天,A,B,C,D四組小鼠均死亡。其中A組小鼠實(shí)驗(yàn)第3天均死亡,B組小鼠于實(shí)驗(yàn)第4天全部死亡,C組小鼠于實(shí)驗(yàn)第5天全部死亡,D組小鼠于實(shí)驗(yàn)第7天均死亡(其中一只小鼠存活時(shí)予以處死,取眶靜脈血做細(xì)菌培養(yǎng))。各組小鼠生存天數(shù)采用log-rank test,與D組相比,其他各組存活天數(shù)較短(P=0.002,有統(tǒng)計(jì)學(xué)差異)。A,B,C,D四組小鼠腹腔灌洗液細(xì)菌培養(yǎng)可見(jiàn)明顯菌落形成。D組存活小鼠眶靜脈血培養(yǎng)結(jié)果提示陽(yáng)性,細(xì)菌學(xué)鑒定結(jié)果提示為鮑曼不動(dòng)桿菌。對(duì)比各組實(shí)驗(yàn)結(jié)果,D組注射菌液濃度(5×107CFU/mL)符合后續(xù)動(dòng)物實(shí)驗(yàn)要求。故后續(xù)小鼠泛耐藥鮑曼不動(dòng)桿菌膿毒癥模型菌液濃度定為5×107 CFU/mL。(3)Ⅰ.實(shí)驗(yàn)第7天,空白對(duì)照組、膿毒癥對(duì)照組、抗生素治療組、噬菌體治療組、噬菌體對(duì)照組小鼠存活數(shù)分別為12、0、8、10、12只。與膿毒癥對(duì)照組比較,其余4組小鼠存活比均明顯升高(Z值為55.635~106.593,P值均小于0.05);噬菌體治療組小鼠存活率稍高于抗生素治療組,但差異無(wú)統(tǒng)計(jì)學(xué)意義(Z=2.797,P0.05)。Ⅱ.膿毒癥對(duì)照組、抗生素治療組、噬菌體治療組小鼠實(shí)驗(yàn)第2天分別死亡3、2、1只,實(shí)驗(yàn)第4天分別死亡10、7、6只,實(shí)驗(yàn)第6天分別死亡12、11、8只。實(shí)驗(yàn)第2天,空白對(duì)照組、噬菌體治療組、噬菌體對(duì)照組小鼠白細(xì)胞計(jì)數(shù)相近,3組均明顯低于膿毒癥對(duì)照組的(P0.05),且后2組明顯低于抗生素治療組的(P值均小于0.05)。實(shí)驗(yàn)第4天,抗生素治療組、噬菌體治療組、噬菌體對(duì)照組小鼠白細(xì)胞計(jì)數(shù)相近,明顯低于空白對(duì)照組(P0.05),前4組白細(xì)胞計(jì)數(shù)均明顯低于膿毒癥對(duì)照組(P0.01)。實(shí)驗(yàn)第6天,空白對(duì)照組、抗生素治療組、噬菌體治療組、噬菌體對(duì)照組小鼠白細(xì)胞計(jì)數(shù)差異無(wú)統(tǒng)計(jì)學(xué)意義(χ2=4.128,P0.05)。實(shí)驗(yàn)第2天,空白對(duì)照組、膿毒癥對(duì)照組、抗生素治療組、噬菌體治療組、噬菌體對(duì)照組分別有0、12、7、2、0只小鼠血細(xì)菌培養(yǎng)結(jié)果為陽(yáng)性�？瞻讓�(duì)照組、噬菌體治療組、噬菌體對(duì)照組小鼠血細(xì)菌培養(yǎng)陽(yáng)性比顯著低于膿毒癥對(duì)照組(χ2值為-30.0~30.0,P值均小于0.01)�？股刂委熃M小鼠血細(xì)菌培養(yǎng)陽(yáng)性比顯著高于空白對(duì)照組和噬菌體對(duì)照組(χ2值分別為17.5、-17.5,P值均小于0.05)。實(shí)驗(yàn)第2天,除噬菌體治療組小鼠腎組織外,空白對(duì)照組、噬菌體治療組、噬菌體對(duì)照組小鼠各臟器組織細(xì)菌含量均為0,均顯著低于膿毒癥對(duì)照組(χ2值為-9.0~9.0,P值均小于0.01);抗生素治療組小鼠腎組織細(xì)菌含量顯著高于空白對(duì)照組和噬菌體對(duì)照組(χ2值分別為-7.5、7.5,P值均小于0.05)。結(jié)論通過(guò)篩選得到的泛耐藥鮑曼不動(dòng)桿菌裂解性噬菌體作為抗菌劑,用于噬菌體治療小鼠泛耐藥鮑曼不動(dòng)桿菌所致膿毒癥,能明顯提高膿毒癥小鼠生存率,控制炎癥反應(yīng),證明了泛耐藥鮑曼不動(dòng)桿菌噬菌體運(yùn)用于臨床治療相關(guān)細(xì)菌感染的可行性。
[Abstract]:BACKGROUND AND OBJECTIVE Acinetobacter baumannii has become one of the most important pathogens of nosocomial infections, especially in burn wards. 162 blood samples from 162 burn patients admitted to our department from January 2011 to December 2014 were collected. 339 strains of pathogens were detected. The first three strains of Acinetobacter baumannii were immobile. Acinetobacter baumannii (ABA), Staphylococcus aureus (SAU) and Pseudomonas aeruginosa (PAE). Most domestic literatures reported that the detection rate of Acinetobacter baumannii was only lower than Pseudomonas aeruginosa, ranking second. According to foreign literature reports, the first outbreak of Acinetobacter baumannii in New York in 1991. Since the infection of Acinetobacter baumannii, reports of drug resistance to Acinetobacter baumannii have emerged all over the world. Especially, the increasing resistance to carbapenems makes it difficult to successfully treat Acinetobacter baumannii infection. Phage is a bacterial-hosted virus that has been proved to be effective in the treatment of bacterial infections. In 1958, Yu He, a professor of bacteriology in China, successfully treated burn patients with Pseudomonas aeruginosa infection by using phages. In the past few decades, a large number of antibiotics have been developed and widely used. In recent years, with the bacterial drug resistance becoming more and more serious, European and American countries began to attach importance to the role of phages in the treatment of bacterial infections, and some domestic units began to carry out relevant research. To establish a model of sepsis in mice induced by pan-resistant Acinetobacter baumannii by screening the lysing phages of pan-resistant Acinetobacter baumannii and to carry out related animal experiments with the selected phages. Methods (1) Isolation and preservation of lysis phages of pan-resistant Acinetobacter baumannii using mixed host bacteria to amplify phages. Pan-resistant Acinetobacter baumannii was used as host bacteria and was not found anywhere in our hospital. Eight strains of pan-resistant Acinetobacter baumannii lysing phages were screened out from the sewage in a rational sewage tank, and the lysing spectra of each strain were determined. The lysing phages with the broadest lysing spectra and the best lysing effect were selected as reserve for subsequent animal treatment as antimicrobial agents. (2) Pan-resistant Acinetobacter baumannii sepsis in mice Forty-eight healthy male BALB/c mice aged from 8 to 12 weeks were randomly divided into four groups, 12 mice in each group, which were divided into four groups: A, B, C and D. The mice in each group were injected intraperitoneally with pan-drug-resistant Acinetobacter baumannii 5 *108 CFU/mL, 2.5 *108 CFU/mL, 1 *108 CFU/mL and 5 *107 CFU/mL, respectively. The vital signs, the number of dead mice, the results of bacterial culture in peritoneal lavage fluid and blood culture were observed. (3) The therapeutic effect of bacteriophage on Pan-drug-resistant Acinetobacter baumannii sepsis in mice I. 60 BALB/c mice were divided into blank control group, sepsis control group and antibiotic treatment group according to random number table. The mice in blank control group were injected with 1 mL normal saline, the mice in sepsis control group, the mice in antibiotic treatment group, and the mice in phage treatment group were injected with 5 *107 CFU/mL Acinetobacter baumannii to establish sepsis model. The treatment group was injected with 1 mg/mL imipenem/cilastatin 1 mL, the phage treatment group was injected with 1 x 108 PFU/mL phage 1 mL, and the phage control group was injected with 1 x 108 PFU/mL phage 1 mL. Before irradiation, 1-3 mice in each group were selected, and 20 mu L of orbital vein blood was taken from each mouse to check the white blood cell count.On the second day of the experiment, another 1 mL of orbital vein blood was taken from the blood of the mice for bacterial culture.The lung, liver, kidney, spleen tissue, homogenate of the mice were taken and the bacterial content was counted after dilution.The data were tested by Wilcoxon rank sum test, one-way ANOVA and LSD. Kruskal-Wallis rank sum test was performed. Results (1) A total of 8 lysing phages were obtained from 22 pan-resistant Acinetobacter baumannii strains as host bacteria and non-disinfected sewage pools in our hospital. The lysis spectrum of phage Bp2014072 was 59%, which was higher than that of the other 7 strains. The bacteriophage was used as an antimicrobial in the following animal experiments and frozen at 4 C for storage. (2) After injecting different concentrations of Acinetobacter baumannii into each group of mice, typical sepsis-related symptoms were found in the four groups. The acute infection symptoms of A, B, C and D mice were more obvious than those of C and D mice. All the mice in group B died on the 3rd day, all the mice in group B died on the 4th day, all the mice in group C died on the 5th day, and all the mice in group D died on the 7th day of the experiment (one of the mice was killed while surviving, and the orbital vein blood was taken for bacterial culture). The bacterial culture of peritoneal lavage fluid of mice in groups A, B, C and D showed obvious colony formation. The blood culture of orbital vein of surviving mice in group D was positive, and the result of bacteriological identification was Acinetobacter baumannii. (3) On the 7th day of the experiment, there were 12, 0, 8, 10 and 12 mice in the blank control group, sepsis control group, antibiotic treatment group, bacteriophage treatment group and bacteriophage control group, respectively. The survival rate of mice in the phage treatment group was slightly higher than that in the antibiotic treatment group, but there was no significant difference (Z = 2.797, P 0.05). II. Sepsis control group, antibiotic treatment group and phage treatment group died 3, 2, 1 mice on the second day, 10, 7, 6 mice on the fourth day, respectively. On the second day of the experiment, the white blood cell counts of the blank control group, the phage treatment group and the phage control group were similar. The white blood cell counts of the three groups were significantly lower than those of the sepsis control group (P 0.05), and the latter two groups were significantly lower than those of the antibiotic treatment group (P < 0.05). The white blood cell count of the control group was similar to that of the blank control group (P 0.05). The white blood cell count of the first four groups was significantly lower than that of the sepsis control group (P 0.01). On the sixth day of the experiment, the white blood cell count of the blank control group, the antibiotic treatment group, the phage treatment group and the phage control group had no significant difference (2=4.128, P 0.05). There were 0,12,7,2,0 mice in the blank control group, sepsis control group, antibiotic treatment group, phage treatment group and bacteriophage control group respectively. The positive rate of blood bacterial culture in the blank control group, phage treatment group and bacteriophage control group was significantly lower than that in the sepsis control group (_2 value - 30.0-30.0, P value was less than 0. The positive rate of blood bacterial culture in antibiotic treatment group was significantly higher than that in blank control group and bacteriophage control group (_2 value was 17.5, -17.5, P value was less than 0.05 respectively). On the second day of experiment, the bacterial content in all organs of mice except the kidney tissue of bacteriophage treatment group, blank control group, bacteriophage treatment group and bacteriophage control group were all 0. It was significantly lower than the sepsis control group (_2 value was - 9.0-9.0, P value was less than 0.01); the bacterial content of kidney tissue in the antibiotic treatment group was significantly higher than that in the blank control group and the phage control group (_2 value was - 7.5, 7.5, P value was less than 0.05). Conclusion The pan-drug-resistant Acinetobacter baumannii lysis phages were screened as antibiotics and used as antibacterial agents. Phage therapy for sepsis caused by pan-drug-resistant Acinetobacter baumannii in mice can significantly improve the survival rate of septic mice and control inflammatory reaction, which proves the feasibility of pan-drug-resistant Acinetobacter baumannii phage in clinical treatment of related bacterial infections.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R644

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