肺炎克雷伯杆菌生物被膜肺感染模型的建立及对抗生素的敏感性研究
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摘要
前言
肺炎克雷伯杆菌是存在于正常人肠道及呼吸道的重要的条件致病菌，它可粘附于机体腔道粘膜表面，分泌多糖基质，形成生物被膜(biofilm，BF)。细菌形成BF后，可逃逸机体免疫功能和抗菌药物的杀灭作用，常常使感染变得难治、易复发，使临床治疗极为棘手。肺炎克雷伯杆菌BF在慢性肺部感染性疾病中十分常见，它引起的BF相关感染已被临床广泛关注。
要对细菌BF进行研究，首先要建立一个理想的实验模型，体内模型与体外模型相比，更有利于综合考虑内环境因素对细菌生物被膜的影响。关于肺炎克雷伯杆菌BF的体内研究国外学者对此报道不多，国内则未见报道。本课题采用吸入法在豚鼠体内建立肺炎克雷伯杆菌生物被膜肺感染模型，并通过活菌计数、光学及电子显微镜探讨肺内肺炎克雷伯杆菌BF的发生发展变化。研究头孢噻肟单用及与罗红霉素合用对肺内生物被膜肺炎克雷伯杆菌的杀菌作用，为生物被膜菌相关感染的防治提供依据。
实验方法
1．实验分组：50只豚鼠随机分为四组：A组10只，接种灭菌生理盐水；B组30只，接种肺炎克雷伯杆菌菌悬液；C组5只，单独应用头孢噻肟(赫美罗赫斯特医药公司，0.5g／支，批号B011004)皮下注射；D组5只，头孢噻肟皮下注射与罗红霉素(法国罗素公司，150mg／片，批号731)口服合用。
2．肺炎克雷伯杆菌生物被膜肺感染模型的建立：A、B两组
动物均事先给予地塞米松皮下注射，剂量为3m才kgi日，每日一
次，连续七天，以抑制豚鼠免疫功能。实验开始后第8天，每小组
5只动物置于与喷雾器配套的塑料容器内，A上两组动物分别接
种灭菌生理盐水及6．25 X 10、FU／Inl的肺炎克雷伯杆菌菌悬液，
通过喷雾器以0．765k才cm’压力，喷雾40min接种。A组在接种
灭菌生理盐水后第3＊天下组在接种肺炎克雷伯杆菌菌悬液后
第3＊＊ ＊ ＊7在1天各取5只动物手术取出肺组织。
3．抗生素的干预：C对组动物与A在组动物同时以上述同
样方法应用地塞米松及接种肺炎克雷伯杆菌菌悬液，并于接种后
第7天开始用药，C组单独应用头抱＠M皮下注射，剂量为
100mg／kg／日，每日二次，连用三天。D组应用头抱＠M皮下注射
（剂量同 C组）与罗红霉素口服K 日，每日二次）联合应
用，连用三天。接种后第 10天，比较 B人山三组动物肺内活菌数
及病理变化。
4．体内感染肺组织活菌数的测定：取一侧肺叶，用灭菌生理
盐水行支气管肺泡灌洗，反复3次从去除浮游菌。无菌切取小块
肺组织，称取组织块的重量，置于组织捣碎器内，加人豆d灭菌生
理盐水制成组织悬液，然后用灭菌生理盐水做10倍系列稀释至
1000倍，分别取各稀释度标本10pl接种于血琼脂平板上，于37℃
培养过夜，并以最低可数的稀释倍数计算菌落数＊FU人然后换算
每克肺组织有多少CFU。
CFU／克肺组织一菌落数 X稀释倍数 X 100／称重肺组织重量
5．病理学检查：取材日，手术取出肺组织标本，分别用10％福
尔马林溶液及 2．5％戊二醛溶液固定lE及 PAS染色，普通光学
显微镜及扫描电镜（Scanning Elecbnic Micro％opy，SEM）观察。
6．统计分析
在接种细菌后第 10天对 B八月三组肺内活菌数取＊ 值进
行方差分析。
·2·
结 果
1．B组有2只动物于接种后第1天死亡，其余动物在取材日
之前未再死亡。
2．肺炎克雷伯杆菌生物被膜肺感染模型的建立与鉴定
细菌学检测：整个实验过程中o组动物肺组织内未培养出细
菌人组动物在吸人肺炎克雷伯杆菌3天以后肺内培养出的细菌
菌落较小、生长缓慢、革兰氏染色发现菌体变短，此时肺内细菌数
量为 10’－10’CFU／g。第7、10上今天略有增力，在 10’－10’CFU／
g之间。14天以后细菌部分被清除，体内细菌数量有所下降，第
21天时基本稳定在10‘－10”’CFU／g。
光学显微镜检查：接种肺炎克雷伯杆菌后第3天，靠近胸膜边
缘实变肺组织内可见炎性细胞多灶性聚集，以中性粒细胞及淋巴
细胞为主。第7天，中性粒细胞减少，以淋巴细胞为主的炎性细胞
聚集形成颗粒样结节，结节外周由成纤维细胞及类上皮细胞包绕，
形成肉芽肿。第10＊ 天，肉芽肿颗粒的形成明显增大。第门。
ZI天带有细菌集落的肉芽肿颗粒略有减小，形成稳定的BF菌慢
性感染。
SEM观察：在接种细菌后第7天，肺组织的颗粒样结构内可
见到多糖蛋白复合物包绕的细菌，细菌菌体之间相互以粘液丝相
连，可见到淋巴细胞嵌于其中。接种后第10天，细菌聚集成团，
多糖蛋白复合物明显增多。
3．抗生素的治疗效果：接种细菌后第 10天，乙D二组肺内活
菌数明显少于 B组人组与 C组人组与 D组比较 p＜0．05人组
与 D组比较 p＜0．01。C组经抗生素治疗后肺内的颗粒样结构减
小JF内的粘液样物质及细菌数量减少为F厚度变薄。D组较C
组更为明显，SEM显示BF稀疏，细菌数量及细菌分泌的粘液样物
·3·
Preface
Klebsiella pneumoniae is responsible for opportunistic infections, particularly of the respiratory tract and intestinal tract, in humans. It can adhere to interface and secrete glycocalyx to form biofilm. Biofilm acts as a barrier to protect the infecting cells from host defense system and antibiotics, which makes some infections intractable and relapsing. Klebsiella pneumoniae biofilm is very common in chronic airway infections, and the infection of biofilm has attracted the attention of clinical doctors.
We must establish an ideal model to study the biofilm bacteria. Compared with models in vitro, an in vivo model is beneficial to consider the influence of internal environment. We are to establish the Klebsiella biofilm model of pulmonary infection in guinea pigs in an inhalation method, then we will investigate the evolution of Klebsiella pneumoniae biofilm in vivo by quantifying bacteria and observing through light and electron microscopy firstly. Secondly, we are to e-valuate the efficacies of cefotaxime( CTX) and the combination of CTX and roxithromycin( RXM) , which would be beneficial to the treatment
of Klebsiella pneumoniae biofilm - associated infections.
Methods
1. The animals were divided into four groups. 10 animals in group A were inoculated with saline, 30 animals in group B were inoculated with the suspension of Klebsiella pneumoniae, 5 animals in group C were treated with CTX and 5 animals in group D were treated with the combination of CTX and RXM after inoculation by Klebsiella pneumoniae.
2. The guinea pigs were subcutaneously treated with dexamethson at a dose of 3mg/kg/day for seven consecutive days before inoculation. Five animals in each group were placed in a plastic chamber e-quipped with a nebulizer. All animals were inoculated by physiological saline solution or the bacterial suspension of which the concentration was adjusted to 6. 25 x 108 colony forming unit( CFU/ml) . Eight mil-liliters of bacterial suspension was dispersed through the nebulizer for 40 minutes at an atomospheric pressure of about 0.765kg/cm2.
3. On day 7 after inoculation, animals in group C were subcutaneously treated with CTX at a dose of 50mg/kg twice a day for 3 consecutive days. Animals in group D were treated with oral administration of RXM at a dose of 2.5mg/kg twice a day and subcutaneous injection of CTX at the same dose.
4. All animals were sacrificed by cardiac puncture on the sampled days. The lung tissues were aseptically removed and weighted, then homogenized with 1ml of saline. Serial tenfold dilutions were made with saline, and 10ul of each dilution was cultured on blood agar plate overnight at 37 C. Colonies grown on blood agar plate were
identified routinely and counted, the colony forming unit (CFU)/g -lung was quantified.
5. After sampled, lung tissues were fixed with 10% formalin and 2.5% glutaraldehyde respectively, then observed with light microscopy and scanning electron microscopy ( SEM) .
6. We used analysis of variance as the statistic method to deal with these data in group B, C, D on day 10.
Result
1. Almost all the animals survived to the sampled days after inoculation except for two died animals in group B on day 1.
2. No organisms were detected from the lung tissues in group A during the whole experiment. The organisms in group B after the 3rd day were smaller in colony, slower in growth, shorter in length than those before inoculation. Under light microscopy, Klebsiella pneu-moniae biofilm existed in the form of tubercles which consisted of poly-morphonuclear cells (PMNs) , lymphatic cells, mononuclear cells and so on. The outer layer of granulation tissues consisted of fibroblasts and epithelioid cells. Under SEM, on day 7 after inoculation, the bacteria were seen enclosed by glycocalix in the tubercles and aggregated to each other through fibrous structure, and lymphatic cells could be seen in the biofilm. on day 10, bacteria clustered , and glycocalix was more than that on day 7.
3. On day 10 after
肺炎克雷伯杆菌是存在于正常人肠道及呼吸道的重要的条件致病菌，它可粘附于机体腔道粘膜表面，分泌多糖基质，形成生物被膜(biofilm，BF)。细菌形成BF后，可逃逸机体免疫功能和抗菌药物的杀灭作用，常常使感染变得难治、易复发，使临床治疗极为棘手。肺炎克雷伯杆菌BF在慢性肺部感染性疾病中十分常见，它引起的BF相关感染已被临床广泛关注。
要对细菌BF进行研究，首先要建立一个理想的实验模型，体内模型与体外模型相比，更有利于综合考虑内环境因素对细菌生物被膜的影响。关于肺炎克雷伯杆菌BF的体内研究国外学者对此报道不多，国内则未见报道。本课题采用吸入法在豚鼠体内建立肺炎克雷伯杆菌生物被膜肺感染模型，并通过活菌计数、光学及电子显微镜探讨肺内肺炎克雷伯杆菌BF的发生发展变化。研究头孢噻肟单用及与罗红霉素合用对肺内生物被膜肺炎克雷伯杆菌的杀菌作用，为生物被膜菌相关感染的防治提供依据。
实验方法
1．实验分组：50只豚鼠随机分为四组：A组10只，接种灭菌生理盐水；B组30只，接种肺炎克雷伯杆菌菌悬液；C组5只，单独应用头孢噻肟(赫美罗赫斯特医药公司，0.5g／支，批号B011004)皮下注射；D组5只，头孢噻肟皮下注射与罗红霉素(法国罗素公司，150mg／片，批号731)口服合用。
2．肺炎克雷伯杆菌生物被膜肺感染模型的建立：A、B两组
动物均事先给予地塞米松皮下注射，剂量为3m才kgi日，每日一
次，连续七天，以抑制豚鼠免疫功能。实验开始后第8天，每小组
5只动物置于与喷雾器配套的塑料容器内，A上两组动物分别接
种灭菌生理盐水及6．25 X 10、FU／Inl的肺炎克雷伯杆菌菌悬液，
通过喷雾器以0．765k才cm’压力，喷雾40min接种。A组在接种
灭菌生理盐水后第3＊天下组在接种肺炎克雷伯杆菌菌悬液后
第3＊＊ ＊ ＊7在1天各取5只动物手术取出肺组织。
3．抗生素的干预：C对组动物与A在组动物同时以上述同
样方法应用地塞米松及接种肺炎克雷伯杆菌菌悬液，并于接种后
第7天开始用药，C组单独应用头抱＠M皮下注射，剂量为
100mg／kg／日，每日二次，连用三天。D组应用头抱＠M皮下注射
（剂量同 C组）与罗红霉素口服K 日，每日二次）联合应
用，连用三天。接种后第 10天，比较 B人山三组动物肺内活菌数
及病理变化。
4．体内感染肺组织活菌数的测定：取一侧肺叶，用灭菌生理
盐水行支气管肺泡灌洗，反复3次从去除浮游菌。无菌切取小块
肺组织，称取组织块的重量，置于组织捣碎器内，加人豆d灭菌生
理盐水制成组织悬液，然后用灭菌生理盐水做10倍系列稀释至
1000倍，分别取各稀释度标本10pl接种于血琼脂平板上，于37℃
培养过夜，并以最低可数的稀释倍数计算菌落数＊FU人然后换算
每克肺组织有多少CFU。
CFU／克肺组织一菌落数 X稀释倍数 X 100／称重肺组织重量
5．病理学检查：取材日，手术取出肺组织标本，分别用10％福
尔马林溶液及 2．5％戊二醛溶液固定lE及 PAS染色，普通光学
显微镜及扫描电镜（Scanning Elecbnic Micro％opy，SEM）观察。
6．统计分析
在接种细菌后第 10天对 B八月三组肺内活菌数取＊ 值进
行方差分析。
·2·
结 果
1．B组有2只动物于接种后第1天死亡，其余动物在取材日
之前未再死亡。
2．肺炎克雷伯杆菌生物被膜肺感染模型的建立与鉴定
细菌学检测：整个实验过程中o组动物肺组织内未培养出细
菌人组动物在吸人肺炎克雷伯杆菌3天以后肺内培养出的细菌
菌落较小、生长缓慢、革兰氏染色发现菌体变短，此时肺内细菌数
量为 10’－10’CFU／g。第7、10上今天略有增力，在 10’－10’CFU／
g之间。14天以后细菌部分被清除，体内细菌数量有所下降，第
21天时基本稳定在10‘－10”’CFU／g。
光学显微镜检查：接种肺炎克雷伯杆菌后第3天，靠近胸膜边
缘实变肺组织内可见炎性细胞多灶性聚集，以中性粒细胞及淋巴
细胞为主。第7天，中性粒细胞减少，以淋巴细胞为主的炎性细胞
聚集形成颗粒样结节，结节外周由成纤维细胞及类上皮细胞包绕，
形成肉芽肿。第10＊ 天，肉芽肿颗粒的形成明显增大。第门。
ZI天带有细菌集落的肉芽肿颗粒略有减小，形成稳定的BF菌慢
性感染。
SEM观察：在接种细菌后第7天，肺组织的颗粒样结构内可
见到多糖蛋白复合物包绕的细菌，细菌菌体之间相互以粘液丝相
连，可见到淋巴细胞嵌于其中。接种后第10天，细菌聚集成团，
多糖蛋白复合物明显增多。
3．抗生素的治疗效果：接种细菌后第 10天，乙D二组肺内活
菌数明显少于 B组人组与 C组人组与 D组比较 p＜0．05人组
与 D组比较 p＜0．01。C组经抗生素治疗后肺内的颗粒样结构减
小JF内的粘液样物质及细菌数量减少为F厚度变薄。D组较C
组更为明显，SEM显示BF稀疏，细菌数量及细菌分泌的粘液样物
·3·
Preface
Klebsiella pneumoniae is responsible for opportunistic infections, particularly of the respiratory tract and intestinal tract, in humans. It can adhere to interface and secrete glycocalyx to form biofilm. Biofilm acts as a barrier to protect the infecting cells from host defense system and antibiotics, which makes some infections intractable and relapsing. Klebsiella pneumoniae biofilm is very common in chronic airway infections, and the infection of biofilm has attracted the attention of clinical doctors.
We must establish an ideal model to study the biofilm bacteria. Compared with models in vitro, an in vivo model is beneficial to consider the influence of internal environment. We are to establish the Klebsiella biofilm model of pulmonary infection in guinea pigs in an inhalation method, then we will investigate the evolution of Klebsiella pneumoniae biofilm in vivo by quantifying bacteria and observing through light and electron microscopy firstly. Secondly, we are to e-valuate the efficacies of cefotaxime( CTX) and the combination of CTX and roxithromycin( RXM) , which would be beneficial to the treatment
of Klebsiella pneumoniae biofilm - associated infections.
Methods
1. The animals were divided into four groups. 10 animals in group A were inoculated with saline, 30 animals in group B were inoculated with the suspension of Klebsiella pneumoniae, 5 animals in group C were treated with CTX and 5 animals in group D were treated with the combination of CTX and RXM after inoculation by Klebsiella pneumoniae.
2. The guinea pigs were subcutaneously treated with dexamethson at a dose of 3mg/kg/day for seven consecutive days before inoculation. Five animals in each group were placed in a plastic chamber e-quipped with a nebulizer. All animals were inoculated by physiological saline solution or the bacterial suspension of which the concentration was adjusted to 6. 25 x 108 colony forming unit( CFU/ml) . Eight mil-liliters of bacterial suspension was dispersed through the nebulizer for 40 minutes at an atomospheric pressure of about 0.765kg/cm2.
3. On day 7 after inoculation, animals in group C were subcutaneously treated with CTX at a dose of 50mg/kg twice a day for 3 consecutive days. Animals in group D were treated with oral administration of RXM at a dose of 2.5mg/kg twice a day and subcutaneous injection of CTX at the same dose.
4. All animals were sacrificed by cardiac puncture on the sampled days. The lung tissues were aseptically removed and weighted, then homogenized with 1ml of saline. Serial tenfold dilutions were made with saline, and 10ul of each dilution was cultured on blood agar plate overnight at 37 C. Colonies grown on blood agar plate were
identified routinely and counted, the colony forming unit (CFU)/g -lung was quantified.
5. After sampled, lung tissues were fixed with 10% formalin and 2.5% glutaraldehyde respectively, then observed with light microscopy and scanning electron microscopy ( SEM) .
6. We used analysis of variance as the statistic method to deal with these data in group B, C, D on day 10.
Result
1. Almost all the animals survived to the sampled days after inoculation except for two died animals in group B on day 1.
2. No organisms were detected from the lung tissues in group A during the whole experiment. The organisms in group B after the 3rd day were smaller in colony, slower in growth, shorter in length than those before inoculation. Under light microscopy, Klebsiella pneu-moniae biofilm existed in the form of tubercles which consisted of poly-morphonuclear cells (PMNs) , lymphatic cells, mononuclear cells and so on. The outer layer of granulation tissues consisted of fibroblasts and epithelioid cells. Under SEM, on day 7 after inoculation, the bacteria were seen enclosed by glycocalix in the tubercles and aggregated to each other through fibrous structure, and lymphatic cells could be seen in the biofilm. on day 10, bacteria clustered , and glycocalix was more than that on day 7.
3. On day 10 after
引文
1．于润江．呼吸道的生物被膜病．中华内科杂志，1993；32：291-292
2. Costerton JW, Lewandowski Z, Caldwell DE, et al. Microbiol biofilms. Annu Rev Microbiol, 1995;49:711-45
3. Kobayashi H. Biofilm disease: its clinical manifestation and therapeutic possibilities of macrolides. Am J med, 1995; 99(6A):26S-28S
4. Otani T, Katami K, Une T, et al. Nonbactemic Pseudomonas pneumonia in immunosuppressed guinea pigs. Microbiol Immunol, 1982; 26:67-76
5．李影林．临床微生物学及检验．人民卫生出版社，1995；673
6. Ishlda Y. Experimental pneumonia with Pseudomonas aeruginosa in immunosuppressed guinea pigs as a model for biofilm-associated infection. Kansenshogaku Zasshi, 1995; 69(5):572-81
7. Ohgaki N. Bacterial biofilm in chronic airway infection. Kansenshogaku Zasshi, 1994;68(1): 138-51
8. Costerton JW, Chen KJ, Gessey GG, et al. Bacteria biofilm in nature and disease. Annu. Rev Microbiol, 1987;41:435-64
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and adherent Escherichia coli determines drug efficacy in device-related infections. Anfimierobial Agents and Chemocherapy, 1991;4:741-6
12. Brown MRW, Gilbert P. Sensitivity of biofilms to antimicrobial agents. J Appl Bacteriol, 1993;74, Suppl: 87-97
13. Anwar H, Strap JL, Chen K. Dynamic interactions of biofilms of mucoid Pesudomonas aemginosa with tobramycin. Antimicrob Agents Chemother, 1992; 36:1208-11
14. Labro MT, el Benna J, Babin-Chevaye C. Comparison of the in-vitro effect of several macrolides on the oxidative burst of human neutrophils. J Antimicrob Chemother, 1989; 24(4): 561-72
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