流感嗜血杆菌表型和脉冲场凝胶电泳分型研究

Study on characteristics of Haemophilus influenzae strains by phenotyping and pulsed-field gel electrophoresis genotyping

目的应用生物学分型、血清学分型和脉冲场凝胶电泳技术（PFGE）分析临床分离流感嗜血杆菌的表型和分子分型特征，了解流感嗜血杆菌的分子流行病学规律。方法1988年和2004--2007年期间，成都市儿童医院从患者的下呼吸道分泌物分离培养273株流感嗜血杆菌，菌株的分离培养与鉴定参照美国《临床微生物手册》。菌株的生物学分型和血清学分型分别参照Kilian和Pittman分类法。应用PCR方法对所有的菌株进行荚膜鉴定和血清学分型鉴定。随机选择100株流感嗜血杆菌菌株，应用PFGE技术进行分子分型研究。结果273株流感嗜血杆菌可分为8个生物型。17．6％（48／273）的菌株为生物型Ⅰ型，43．6％（119／273）为生物型Ⅱ型，22．7％（62／273）为生物型Ⅲ型，7．3％（20／273）为生物型Ⅳ型，5．9％（16／273）为生物型Ⅴ型，0．4％（1／273）为生物型Ⅵ型，1．8％（5／273）为生物型Ⅶ型，0．7％（2／273）为生物型Ⅷ型。99．6％（272／273）的菌株为无荚膜的、血清学不可分型的流感嗜血杆菌，1株为血清型f型。100株流感嗜血杆菌经PFGE分析，可分为96个PFGE基因型，93株菌株各自代表一个PFGE基因型。基因型的分布与菌株的分离时间无明显的相关性。结论引起儿童下呼吸道感染的流感嗜血杆菌主要是无荚膜的、不可分型流感嗜血杆菌菌株，生物型主要是Ⅰ、Ⅱ和Ⅲ型。血清学不可分型流感嗜血杆菌菌株PFGE基因型呈现明显的多态性，菌株的分子多态性可能是流感嗜血杆菌感染在成都市没有形成暴发与流行的分子流行病学原因；PFGE基因分型与生物学分型和血清学分型比较，具有很强的菌株分辨能力，是流感嗜血杆菌感染流行病学研究适用的分析方法。

Objective To investigate the epidemiological and molecular typing features of the pathogenic Haemophilus influenzae （H. influenzae） by biotyping, serotyping and pulsed-field gel eleetropho- resis （PFGE）. Methods A total of 273 invasive isolates of H. influenzae were collected from the pediatric patients with pneumonia at Chengdu Children Hospital of Sichuan province from 1988 and 2004 to 2007. The identification of H. influertzae strains were done according to the laboratory standard methodology described by Manual of Clinical Microbiology （American）. All strains were biotyped according to Kilian＇s classifica- tion with the API~ NH system. And serotyped by a slide agglutination assay with type a to f specific antise- rum as described by Pittman. PCR method for identification of H. influenzae were performed as described by Falla. One hundred of 273 strains were analyzed by PFGE as described by Saito with some modifications. The results of PFGE were analyzed by Bionumerics soft （ Version 4.0, Applied Maths BVBA, Belium）. Results 78.2% of 273 cases occurred under 1 years old. Eight biotypes were found among the 273 H. influenzae isolates. 17.6% （48/273） of all isolates belonged to biotype I , 43.6% （ 119/273 ） were biotype Ⅱ, 22.7% （62/273） were biotype Ⅲ, 7.3% （20/273） were biotype Ⅳ, 5.9% （ 16/273 ） were biotype Ⅴ, 0.4% （ 1/273 ） were biotype Ⅵ, 1.8% （5/273） were biotype Ⅶ and 0.7% （2/273） were biotype Ⅷ, re- spectively. 99.6% of all 273 isolates were nontypeable. There was only one isolate was serotype f. Ninty-six PFGE genotypes were obtained in this study. One hundred strains demonstrated a variety of genomic patterns by PFGE. The most isolates of the same PFGE genotype （ type 35） was 3 isolates. Each of 93 PFGE geno- types was represented by only a single isolate. The genotypes distribution didn＇t correlate with the time dis- tribution of the strains were isolated. Conclusion Nontypeable H. influenzae primarily caused acute pneumonia in children under 1 years old. They mostly belonged to biotype Ⅰ , Ⅱ and m biotypes. The nontypeable H. influenzae strains appeared to more heterogeneous patterns by PFGE genotyping. Genotyping may help understand the molecular characteristics of outbreak and endemicity according to the results of PFGE. PFGE genotyping proved to have a much stronger discriminatory power than either serotyping or biotyping. Our findings suggest that PFGE analysis is useful for the epidemiological study of H. influenzae infections.