肺炎链球菌β-内酰胺类抗生素最小抑菌质量浓度与青霉素结合蛋白1A和2B变异的关系

Relationship between Minimal Inhibitory Concentrations of β-Lactams Against Streptococcus Pneumoniae Mutation of Substitutions in Penicillin-Binding Proteins 1A and 2B

目的探讨肺炎链球菌(Sp)β-内酰胺类抗生素最小抑菌质量浓度(MIC)与青霉素结合蛋白(PBP)1A和2B变异的关系。方法对55株Sp用E-test法进行7种β-内酰胺类抗生素进行药敏试验,并提取每株Sp的DNA,分别对其青霉素结合蛋白基因(pbp)1a和pbp2b中编码青霉素结合区域(PBD)进行套式PCR扩增和直接测序,并用Clustalx软件进行序列比对,分析PBP1A和PBP2BPBD保守序列的氨基酸置换。结果青霉素敏感株4株中发现PBP2B保守序列SSN后苏氨酸(Thr)445→丙氨酸(Ala),Thr451→Ala和谷氨酸(Glu)481→甘氨酸(Gly)置换,其余6株PSSP未发现PBP1A和PBP2B保守序列氨基酸置换。4株青霉素中介株(PISP)(MIC0.19～0.38mg/L)PBP2BThr445→Ala,Thr451→Ala和Glu481→Gly置换,其中1株发现PBP2B保守序列SVVK后第431-432位点处脯氨酸(Pro)的插入,余3株存在谷氨酰胺(Gln)432→亮氨酸(Leu)置换。另16株PISP(MIC0.5～1.0mg/L)在此基础上出现PBP1ASRN后Pro432→Thr,STMK中Thr371→丝氨酸(Ser)/Ala和KTG附近的短镶嵌序列Thr574→天冬氨酸(ASn)Ser575→Thr,Gln576→Gly,苯丙氨酸(Phe)577→酪氨酸(Tyr)置换,且后二种变异存在于所有青霉素和头孢类中介和耐药株中。20/25株青霉素耐药株存在PBP2B保守序列KTG附近Ala624→Gly,包含Ala624→GlySp组青霉素、头孢呋新、阿莫西林等抗生素平均MIC值明显高于不包含这一置换的Sp组。在青霉素MIC≥0.5mg/L的菌株中均同时存在PBP1A和PBP2B的变异。结论β-内酰胺类抗生素对肺炎链球菌临床分离株MIC值与PBP1A和PBP2B编码青霉素结合活性区域的保守序列中或附近的氨基酸置换有关。

Objective To explore the relationship between minimal inhibitory concentrations（MIC） of β-lactams against streptococcus pneumoniae（Sp） and mutation of penicillin-binding proteins（PBP）1A and 2B.Methods Fifty-five Sp isolates were tested 7 kinds β-Lactams sensitivity by E-test method.Fifty-five Sp isolates Chromosomal DNA was extracted and PBD of pbp1a,pbp2b were amplified by net polymerase chain reaction（nPCR） followed by DNA sequencing.Amino acid sequences of PBP1A and PBD2B in conserved motifs were deduced by Clustalx software program.Results There were mutations in PBP2B（Thr445→Ala,Thr451→Ala and Glu481→Gly） in 4 penicillin-sensitive streptococcus pneumoniae（PSSP） strains,while there was no mutation of PBP1A,PBP2B in the conserved motifs of the other 6 PSSP strains.Fore penicillin-intermediate streptococcus pneumoniae（PISP） strains（MIC 0.19-0.38 mg/L） had mutations in PBP2B（Thr445→Ala,Thr451→Ala and Glu481→Gly）.Proline was inserted between residues 431 and 432 in one strain,and Gln was changed to Leu at residue 432 in PBP2B of the other 3 strains.In addition,16 PISP（MIC 0.5-1.0 mg/L） strains had same changes seen in PISP strains,as well as PBP1A Thr371→Ser/Ala,Pro432→Thr and Thr574→Ala,Ser575→Thr,Gln576→Gly,Phe577→Tyr in PBP1A.The mutations at residue 371 and residues 574 to 577 of PBP1A were common to all penicilin-and cephalosporins-intermediate and-resis-tant isolates.Ala624→Gly in PBP2B was detected in 20/25 penicillin-resistant streptococcus pneumoniae strains.The unique change at Ala624→Gly of PBP2B was found in Sp strains with whose penicillin,amoxicillin and cefuroxime average MIC were obviously higher.For all isolates which penicilin MIC≥0.5 mg/L had alterations in both PBP1A and PBP2B.Conclusions These data reveal that the MIC for β-lactams against the Sp strains correlated with the amino acid alterations in or flanking the PBP conserved amino acid motifs.