儿童肺炎链球菌耐药性与pbp2b基因RFLP和PFGE图型分析

目的　监测肺炎链球菌对青霉素和其他抗生素的耐药情况 ;分析pbp2b基因扩增产物图谱与青霉素耐药性的相关性 ;对 3 1株青霉素耐药菌株进行血清分型 ;分析血清型 2 3F和血清型 6的青霉素耐药菌株的脉冲电场凝胶电泳 (PFGE)图型 ,初步了解北京地区耐药菌株分子流行病学上的特点。方法　 ( 1)抗生素药物敏感试验 ;( 2 )用聚合酶链反应 (PCR)和限制性内切酶片段长度多态性分析 (RFLP)方法 ,分析pbp2b基因扩增产物图谱与青霉素耐药性的相关性 ;( 3 )使用乳胶凝集的方法 ,对 3 1株青霉素耐药菌株进行血清分型 ;( 4 )使用PFGE图型方法分析 6株血清型 2 3F和 3株血清型 6的青霉素耐药菌株流行病学上的基本特性。结果　 ( 1)对青霉素的耐药性由 1997年 9 9%上升到 2 0 0 0年 14 6% (P >0 0 5)。对红霉素、复方磺胺甲基异唑和氯霉素耐药性分别由 1997年76 8%、74 7%和 2 2 6%上升到 2 0 0 0年的 87 4%、88 3 %和 40 8% (P <0 0 5) ;( 2 )通过PCR和RFLP方法分析青霉素耐药菌株和青霉素敏感菌株表明 ,pbp2b基因扩增图谱与青霉素耐药性之间有较好的相关性 ;( 3 )对 3 1株青霉素不敏感肺炎链球菌 (PRSP)做了血清型 2 3F和 6分型。前者为 6株 ( 19% ) ;后者为 3株 ( 9% )。 6株血清型 2 3F肺炎链球菌均为低水平

Analysis of antimicrobial resistance of Streptococcus pneumoniae with restriction fragment length polymorphism of pbp2b gene and pulsed-field gel electrophoresis profiles among children

Streptococcus pneumoniae is a common cause of potentially life-threatening infections such as meningitis, bacteraemia, pneumonia worldwide, for which children of preschool age are at particularly high risk. Since the late 1970s and 1980s, antibiotic resistance among pneumococci has become an emerging problem. Several multidrug-resistant clones have rapidly spread throughout the world. OBJECTIVE: (1) To investigate the prevalence of penicillin and other antibiotics nonsusceptibility among pneumococci. (2) To analyze the correlation of pbp2b amplicon profiles with penicillin resistance. (3) To serotype 31 isolates of penicillin-resistant pneumococci by latex agglutination. (4) To analyze the chromosomal relatedness of serotype 23F and 6 isolates of penicillin-resistant pneumococci by using pulsed-field gel electrophoresis (PFGE) and characterize these isolates in molecular epidemiology. METHODS: (1) Susceptibility was determined by using broth microdilution, E-test, and K-B disk. (2) The correlation of pbp2b amplicon profiles with penicillin resistance was assessed by restriction fragment length polymorphism (RFLP). (3) Serotyping of penicillin-resistant pneumococcal isolates was performed by using latex agglutination. (4) The properties of serotype 23F and 6 isolates of penicillin-resistant pneumococci were assessed by PFGE. RESULTS: S. pneumoniae with increased nonsusceptibility (including intermediate strains and resistant strains) to penicillin G was 9.9% in 1997, 12.6% in 1998, 14.6% in 2000; to cefuroxime 4.2%, 1.5%, 8.2%; to cefotaxime 0.0%, 1.7%, 1.0% respectively. There were no statistically significant differences (P > 0.05). While resistance to erythromycin, trimethoprim-sulfamethoxazole and chloramphenicol increased significantly from 76.8% in 1997 to 87.4% in 2000, from 74.7% to 88.3%, and from 22.6% to 40.8%, respectively (P < 0.05). RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. Of the 31 strains of penicillin-resistant pneumococci (MICs 0.12 - 2.0 micro g/ml) studied, 6 (19.4%) strains (MICs 0.12 - 0.19 micro g/ml) were serotype 23F and 3 (9.7%) strains (MICs 0.5 - 1.5 micro g/ml) were serotype 6. There were nearly identical susceptibility to antibiotics and identical PFGE patterns in the former, and there were different susceptibility to antibiotics and different PFGE patterns in the latter. Three serotype 6 strains had different susceptibility to antibiotics and different PFGE patterns, which suggested that those strains may be scattered. CONCLUSION: Generally beta-lactams retained their activity against S. pneumoniae in Beijing. Resistance to erythromycin, trimethoprim-sulfamethoxazole, and chloramphenicol increased drastically. RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. In 6 strains of serotype 23 F there were nearly identical susceptibility to antibiotics and identical PFGE patterns, which suggested the probability that there was a spread of serotype 23F isolates with low-level penicillin resistance in local area.