脉冲场凝胶电泳在铜绿假单胞菌分子流行病学研究中的应用
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摘要
目的：
建立脉冲场凝胶电泳(Pulsed-Field Gel Electrophoresis, PFGE)基因分型方法,对天津市南开医院和天津市儿童医院临床分离的铜绿假单胞菌(Pseudomonas aeruginosa, PA)进行基因分型,进而对PA进行流行病学分析。
方法：
(1)临床送检标本按《全国临床检验操作规程》鉴定为PA,质控菌株为PAATCC27853。
(2)采用K-B纸片扩散法对PA进行药敏试验,测试其对不同抗菌药物的敏感性,药敏结果按美国CLSI标准判读。南开医院PA的药敏试验检测14种抗菌药物,儿童医院PA的药敏试验检测15种抗菌药物。
(3)对两家医院临床分离得到的128株PA进行PFGE基因分型和流行病学分析。
(4)对临床分离出PA宿主的临床病例进行回顾性分析,判断菌株分离时段是否处在感染状态。
结果：
(1)感染和定植菌株的判断：来自于南开医院的72株PA中,有57株(79%)菌可能为感染菌株；而来自于儿童医院的56株PA中,有39株(70%)可能为感染菌株。两家医院的定植菌株均来自于呼吸道。
(2)菌株的临床来源和分布：两家医院的PA菌株主要分离于痰标本,南开医院有51株,占71%；儿童医院为51株,占90%；其他菌株分离于胆汁、腹腔液、脓性分泌物、血液和尿液标本。南开医院的PA菌株主要来自于中西结合脑病科和呼吸科,而儿童医院菌株主要分离于呼吸科和内分泌科。
(3)菌株的药物敏感性：南开医院的PA菌株对14种抗菌药物的耐药率在30%-70%之间；但儿童医院的PA菌株对抗生素的敏感性与南开医院菌株有所不同,耐药率在0%-100%之间,儿童医院菌株均对氨苄西林耐药,对丁胺卡那霉素、环丙沙星敏感。
(4) PFGE分型：南开医院的72株PA被分为48个PFGE型,Ⅰ型5株；Ⅱ和Ⅲ型各4株；有14种PFGE谱各由2株菌共有,其余31株菌各表现为独特的PFGE谱。儿童医院56株PA被分为18个型别,A型26株；B型12株；C型3株：其余15株各成独特的PFGE谱。两家医院PA菌株中未见相同或相近的PFGE谱菌株。
(5)儿童医院PFGE优势型和耐药性的关系：儿童医院的A和B型菌株几乎均对4种抗菌药物耐药和3种抗菌药物敏感。但对其余8种药物两型表现了不同的耐药率,其中4种药物耐药率有明显差别。
(6)追溯两家医院主要PFGE型别菌株的时间和空间分布：南开医院Ⅰ型菌株是同一患者不同时间分离得到的菌株,Ⅱ型菌株和Ⅲ型菌株由不同科室患者在不同时间分离得到；儿童医院A型菌株来自于呼吸科的不同患儿,而B型菌株主要来自内分泌科的不同患儿。
结论：
(1)新建立的PFGE方法能够明确地对PA进行基因分型,可以作为有效的技术手段用于分子流行病学的研究和临床实践。
(2)不同医院的临床分离得到的PA菌株抗生素耐药谱不同。
(3)南开医院的PA菌株呈散发感染的特征,而儿童医院PA菌株表现为科室间流行趋势；两家医院PA菌株无相同基因型,故未见院间传播；PA菌株进化来源具有遗传学多元性。
(4) PFGE在基因水平上对PA进行分析,可分析PA的遗传学特征、感染来源、传播途径及分布规律,对感染流行的监测提供可靠依据。
Objectives:
To establish the genotyping method of pulsed field gel electrophoresis (PFGE) for Pseudomonas aeruginosa (PA), and investigate the molecular epidemiological characteristics of PA clinical isolates collected from Tianjin Nankai Hospital and Tianjin Children's Hospital.
Methods:
(1) The bacterial strains isolated from clinical samples were identified according to the procedure described in The Clinical Inspection SOP. The ATCC27853 was used as reference strain.
(2) According to CLSI, K-B disk diffusion method was employed to examine the antimictobial susceptibility of PA. The 14 and 15 kinds of antibiotics were respectively used in susceptibiliyi tests for PA isolates from Tianjin Nankai Hospital and Tianjin Children's Hospital.
(3) The genotypes of the 128 strains were characterized by PFGE analysis.
(4) The clinical data of the PA hosts were retrospectively analyzed to evaluate the infectious status during the period of bacteria isolated.
Results:
(1) Colonization and infection:In 72 strains collected from Tianjin Nankai Hospital,57 strains were likely-pathogens; Among 56 strains isolated from Tianjin Children's Hospital,39 were likely-pathogens.
(2) Infection sites:The most of PAs were isolated from sputum, which were 51 (71%) in Nankai Hospital isolates and 51 (91%) in Children's Hospital strains. Other strains were collected from the samples of sanies, ascite or peritoneal drainage fluid, blood or urine.
(3) Antimicrobial susceptiblity:The PAs from the two hospitals showed different patterns. For those from Nankai Hospital, the resistant rates for different antibiotics were 30% to 70%; the strains from Children's Hospital, the rates were 0% to 100% and the stains were identically resistant to ampicillin, but sensitive to amikacin and ciprofloxacin.
(4) PFGE analysis:the 72 stains from Nankai Hospital were classified into 48 types; 5 strains for typeⅠ,4 for each of typeⅡandⅢ,2 for each of 14 types, remaining 31 for the other 31 types. The 56 strains from Children's Hospital were divided into 18 types. More than half of the strains belonged to type A and B,3 to type C, others to unique type.
(5) Relationship between PFGE and antimicrobial susceptibility:The strains of typeA and B were resistant to 6 kinds of antibiotics, but sensitive to 3 kinds of antibiotics. There was obviously different in 4 kinds of antibiotics between the two types.
(6) Based upon the location and time distribution of PA stains in the two hospitals, the occurrence and spread of PAs could be clearly identified and tracked according to the genetic characteristics of PA.
Conclusion:
(1) The procedure of PFGE for PA genotyping was well established and was shown to be an effective meothod for the molecular epidemiological investigation of clinical PA isolates.
(2) The PA strains grouped on the hospitals showed different in antimicrobial resistant patterns.
(3) By PFGE analysis, it was found that the feature of sporadic infection was identified in Nankai Hospital and a trend of epidemic spreading existed in Children's Hospital. Nevertheless, there is no cross-infection between the two hospitals.
(4) In this study, PA strains were characterized by PFGE genotyping. Therefore the source, spread route and distribution of the clinical PA strains could be readily determined and tracked. The reliable evidence could be provided for surveillance and control of infections by PFGE genotyping.
建立脉冲场凝胶电泳(Pulsed-Field Gel Electrophoresis, PFGE)基因分型方法,对天津市南开医院和天津市儿童医院临床分离的铜绿假单胞菌(Pseudomonas aeruginosa, PA)进行基因分型,进而对PA进行流行病学分析。
方法：
(1)临床送检标本按《全国临床检验操作规程》鉴定为PA,质控菌株为PAATCC27853。
(2)采用K-B纸片扩散法对PA进行药敏试验,测试其对不同抗菌药物的敏感性,药敏结果按美国CLSI标准判读。南开医院PA的药敏试验检测14种抗菌药物,儿童医院PA的药敏试验检测15种抗菌药物。
(3)对两家医院临床分离得到的128株PA进行PFGE基因分型和流行病学分析。
(4)对临床分离出PA宿主的临床病例进行回顾性分析,判断菌株分离时段是否处在感染状态。
结果：
(1)感染和定植菌株的判断：来自于南开医院的72株PA中,有57株(79%)菌可能为感染菌株；而来自于儿童医院的56株PA中,有39株(70%)可能为感染菌株。两家医院的定植菌株均来自于呼吸道。
(2)菌株的临床来源和分布：两家医院的PA菌株主要分离于痰标本,南开医院有51株,占71%；儿童医院为51株,占90%；其他菌株分离于胆汁、腹腔液、脓性分泌物、血液和尿液标本。南开医院的PA菌株主要来自于中西结合脑病科和呼吸科,而儿童医院菌株主要分离于呼吸科和内分泌科。
(3)菌株的药物敏感性：南开医院的PA菌株对14种抗菌药物的耐药率在30%-70%之间；但儿童医院的PA菌株对抗生素的敏感性与南开医院菌株有所不同,耐药率在0%-100%之间,儿童医院菌株均对氨苄西林耐药,对丁胺卡那霉素、环丙沙星敏感。
(4) PFGE分型：南开医院的72株PA被分为48个PFGE型,Ⅰ型5株；Ⅱ和Ⅲ型各4株；有14种PFGE谱各由2株菌共有,其余31株菌各表现为独特的PFGE谱。儿童医院56株PA被分为18个型别,A型26株；B型12株；C型3株：其余15株各成独特的PFGE谱。两家医院PA菌株中未见相同或相近的PFGE谱菌株。
(5)儿童医院PFGE优势型和耐药性的关系：儿童医院的A和B型菌株几乎均对4种抗菌药物耐药和3种抗菌药物敏感。但对其余8种药物两型表现了不同的耐药率,其中4种药物耐药率有明显差别。
(6)追溯两家医院主要PFGE型别菌株的时间和空间分布：南开医院Ⅰ型菌株是同一患者不同时间分离得到的菌株,Ⅱ型菌株和Ⅲ型菌株由不同科室患者在不同时间分离得到；儿童医院A型菌株来自于呼吸科的不同患儿,而B型菌株主要来自内分泌科的不同患儿。
结论：
(1)新建立的PFGE方法能够明确地对PA进行基因分型,可以作为有效的技术手段用于分子流行病学的研究和临床实践。
(2)不同医院的临床分离得到的PA菌株抗生素耐药谱不同。
(3)南开医院的PA菌株呈散发感染的特征,而儿童医院PA菌株表现为科室间流行趋势；两家医院PA菌株无相同基因型,故未见院间传播；PA菌株进化来源具有遗传学多元性。
(4) PFGE在基因水平上对PA进行分析,可分析PA的遗传学特征、感染来源、传播途径及分布规律,对感染流行的监测提供可靠依据。
Objectives:
To establish the genotyping method of pulsed field gel electrophoresis (PFGE) for Pseudomonas aeruginosa (PA), and investigate the molecular epidemiological characteristics of PA clinical isolates collected from Tianjin Nankai Hospital and Tianjin Children's Hospital.
Methods:
(1) The bacterial strains isolated from clinical samples were identified according to the procedure described in The Clinical Inspection SOP. The ATCC27853 was used as reference strain.
(2) According to CLSI, K-B disk diffusion method was employed to examine the antimictobial susceptibility of PA. The 14 and 15 kinds of antibiotics were respectively used in susceptibiliyi tests for PA isolates from Tianjin Nankai Hospital and Tianjin Children's Hospital.
(3) The genotypes of the 128 strains were characterized by PFGE analysis.
(4) The clinical data of the PA hosts were retrospectively analyzed to evaluate the infectious status during the period of bacteria isolated.
Results:
(1) Colonization and infection:In 72 strains collected from Tianjin Nankai Hospital,57 strains were likely-pathogens; Among 56 strains isolated from Tianjin Children's Hospital,39 were likely-pathogens.
(2) Infection sites:The most of PAs were isolated from sputum, which were 51 (71%) in Nankai Hospital isolates and 51 (91%) in Children's Hospital strains. Other strains were collected from the samples of sanies, ascite or peritoneal drainage fluid, blood or urine.
(3) Antimicrobial susceptiblity:The PAs from the two hospitals showed different patterns. For those from Nankai Hospital, the resistant rates for different antibiotics were 30% to 70%; the strains from Children's Hospital, the rates were 0% to 100% and the stains were identically resistant to ampicillin, but sensitive to amikacin and ciprofloxacin.
(4) PFGE analysis:the 72 stains from Nankai Hospital were classified into 48 types; 5 strains for typeⅠ,4 for each of typeⅡandⅢ,2 for each of 14 types, remaining 31 for the other 31 types. The 56 strains from Children's Hospital were divided into 18 types. More than half of the strains belonged to type A and B,3 to type C, others to unique type.
(5) Relationship between PFGE and antimicrobial susceptibility:The strains of typeA and B were resistant to 6 kinds of antibiotics, but sensitive to 3 kinds of antibiotics. There was obviously different in 4 kinds of antibiotics between the two types.
(6) Based upon the location and time distribution of PA stains in the two hospitals, the occurrence and spread of PAs could be clearly identified and tracked according to the genetic characteristics of PA.
Conclusion:
(1) The procedure of PFGE for PA genotyping was well established and was shown to be an effective meothod for the molecular epidemiological investigation of clinical PA isolates.
(2) The PA strains grouped on the hospitals showed different in antimicrobial resistant patterns.
(3) By PFGE analysis, it was found that the feature of sporadic infection was identified in Nankai Hospital and a trend of epidemic spreading existed in Children's Hospital. Nevertheless, there is no cross-infection between the two hospitals.
(4) In this study, PA strains were characterized by PFGE genotyping. Therefore the source, spread route and distribution of the clinical PA strains could be readily determined and tracked. The reliable evidence could be provided for surveillance and control of infections by PFGE genotyping.
引文
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