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目的:探讨肺炎链球菌(Streptococcus pneumoniae,SP)临床菌株pbp2B、pbp2X和pbp1A基因编码的青霉素结合蛋白(PBPs)的青霉素结合区(PBD)中SVVK、SSN和KTG基序及其邻近序列的突变与β-内酰胺类抗生素耐药相关性。方法:采用二倍稀释法检测36株肺炎链球菌临床菌株对4种β-内酰胺类抗生素的最低抑菌浓度(minimal inhibitoryconcentration,MIC)。采用PCR扩增各菌株DNA中pbp2B、pbp2X和pbp1A基因片段并测序。根据药敏试验及测序结果,分析上述基因PBD或附近序列突变与β-内酰胺类抗生素耐药的相关性。结果:36株肺炎链球菌临床菌株中,10株为青霉素敏感株(penicillin-susceptible SP,PSSP)、10株为青霉素中介耐药株(penicillin-intermediate SP,PISP)、16株为青霉素耐药株(penicillin-resistance SP,PRSP)。4株PSSP、9株PISP及16株PRSP菌株PBP2B中SSN的C端T445A/S突变,其余6株PSSP和1株PISP菌株无突变。MIC≥0.5 mg/L的7株PISP及16株PRSP菌株PBP2X中STMK出现T338A突变、KSG的N端出现L546V突变,2株PISP菌株SSN的N端分别出现H394Y/L突变,另有2株高度耐药PRSP菌株STMK的C端发生M342I突变。所有36株菌株PBP1A的SRN和KTG、10株PSSP菌株STMK序列均未发生突变,所有MIC≥1 mg/L的PISP和PRSP菌株PBP1A的STMK均出现T371A/S突变、SRN的C端出现P432T突变。结论:本研究结果显示肺炎链球菌临床菌株对β-内酰胺类抗生素耐药性与PBP2B、PBP2X和PBPA的PBD中SVVK、SSN和KTG及其邻近序列突变密切相关。
OBJECTIVE: To investigate the mutations of the SVVK, SSN and KTG motifs and their adjacent sequences in the penicillin-binding region (PBD) of penicillin binding proteins (PBPs) encoded by the Streptococcus pneumoniae (SP) clinical isolates pbp2B, pbp2X and pbp1A β-lactam antibiotic resistance-related. Methods: The minimal inhibitory concentration (MIC) of 36 strains of Streptococcus pneumoniae strains against 4 kinds of β-lactam antibiotics was determined by double dilution method. The DNA fragments of pbp2B, pbp2X and pbp1A were amplified by PCR and sequenced. According to the results of drug sensitivity test and sequencing, the correlation between the mutation of PBD or its vicinity and the resistance of β-lactam antibiotics was analyzed. Results: Among 36 Streptococcus pneumoniae clinical isolates, 10 were penicillin-susceptible SP (PSSP), 10 were penicillin-intermediate SP (PISP) and 16 were penicillin resistant strains (penicillin-resistance SP, PRSP). 4 CSP, 9 PISP and 16 PRSP strains PBP2B SSN C-terminal T445A / S mutation, and the remaining 6 strains of PSSP and 1 strains of PISP strains without mutation. T338A mutation was found in STMK of MIC≥0.5 mg / L and in PBP2X of 16 PRSP strains, L546V mutation was observed in the N-terminal of KSG, and H394Y / L mutation was observed in the N-terminal of two strains of PISP SSN, respectively The M342I mutation occurred on the C-terminal of the resistant PRSP strain STMK. All 36 strains of PBP1A did not mutate in SRN and KTG, 10 strains of PSSP, and all of them showed the T371A / S mutation in the STMK of PISP with MIC≥1 mg / L and PRP strain PBP1A, and the P432T mutation in the C terminus of SRN . Conclusion: The results of this study showed that the antibiotic resistance of Streptococcus pneumoniae strains to β-lactam antibiotics was closely related to the mutation of SVVK, SSN and KTG in PBD of PBP2B, PBP2X and PBPA and its adjacent sequence.