肺炎链球菌pbp2b和pbp1a基因突变与青霉素耐药的相关性研究

Relationship between the mutation of pbp2b/pbp1a genes and the penicillin resistance of Streptococcus pneumoniae

目的探讨耐青霉素肺炎链球菌pbp2b和pbp1 a基因的突变与青霉素耐药的关系,为明了肺炎链球菌的耐药性变异机制,防治其感染提供实验依据。方法从呼吸道感染患儿痰标本中分离肺炎链球菌163株,液体培养基连续稀释法测定其对青霉素的最小抑菌浓度(M IC),套式聚合酶链反应(nPCR)扩增pbp2b和pbp1 a基因,扩增产物直接DNA测序,所测序列与青霉素敏感株(SPN R6)的基因序列进行比较,并分析其氨基酸结构的改变。结果 163株肺炎链球菌中检出青霉素敏感菌75株,中度敏感17株,青霉素耐药菌71株(44%)。耐药菌中58株存在pbp2b突变(81.7%),其中,56株为点突变,2株为CCT插入突变;在27株有pbp2b基因突变的B型和C型耐药菌中,21株出现了不同程度的pbp1 a基因突变。PBP2B氨基酸结构改变以苏氨酸变为丙氨酸、精氨酸变为赖氨酸为主,PBP1A以丙氨酸变为苏氨酸、谷氨酸变为天门冬氨酸为主。结论肺炎链球菌的pbp2b和pbp1 a基因突变与对青霉素的耐药性密切相关,PBP2b突变导致低水平耐药;PBP2b和PBP1A突变导致高水平耐药。

Objective To understand the mechanisms of the resistance mutation,and the relationship between the mutation of pbp2b/pbp1a genes and the penicillin resistance of S.pneumoniae,in order to provide evidence for the prevention and cure of S.pneumoniae infection.Method 163 strains of S.pneumoniae were isolated from the secretion of respiratory tracts of children with respiratory infection diseases.The minimal inhibition concentration（MIC） of penicillin to pneumococcal isolates was determined by the method of liquid media serial dilution.The genes of pbp2b and pbp1a in the isolates were detected by the method of nested PCR.The DNA sequences of PCR products were detected and contrasted with the DNA sequences of R6 strain sensitive to penicillin.The changes of amino acid structures in PBP2B and PBP1A were analyzed.Result In the 163 isolates of S.pneumoniae,75 strains were susceptible to penicillin;17 strains were moderately sensitive and 71 strains were resistant to penicillin（44%）;58（81.7%） strains had pbp2b mutation,56 of which were point mutation and 2 were CCT insert mutation.In 27 B and C type of pbp2b mutation strains,21 were found having pbp1a mutation.The changes of amino acids were threonine to alanine and arginine to lysine in PBP2B,and alanine to threonine and glutamic acid to aspartic acid in PBP1A.Conclusion There is a close correlation between the mutation of pbp1a/pbp2b genes and penicillin resistance of S.pneumoniae.The mutation of pbp2b results in low level resistance while the mutations of pbp2b and pbp1a result in high level resistance.