社区呼吸道感染肺炎链球菌对头孢妥仑的耐药机制研究

摘要

Objective To study the mechanisms of cefditoren-resistance in the clintcal strains of Streptococcus pneumoniae. Methods Thirty-seven strains of S. pneumoniae with cefditoren MICs of ≥ 1 mg/L were isolated from community-acquired respiratory tract infections in 10 teaching hospitals from January 2009 to January 2010. Six strains of S. pneumoniae with cefditoren MICs of ≤0. 5 mg/L were collected as control group. Broth microdilution method was used to determine the MICs of penicillin, amoxicillirrclavulanic acid, cefuroxime, cefaclor, cefditoren, ceftriaxone, azithromycin, clarithromycin, levofloxacin, moxifloxacin and vancomycin, Multiplex-PCR was used to determine the serotypes of all the test isolates, PCR was used to amplify the genes of pbp2b, pbpa, pbp2x and murM in 43 clinical isolates. PCR products were digested by restriction enzyme to analyze the DNA fingerprints. One to five FCR products exhibiting the same DNA fingerprint pattern were sequenced. Amino acid sequences were then compared to the sequence of S. pneumoniae strain R6 to analyze the mutations in the conserved motifs of PBP genes, especially for SXXK box, SXN box and KT(S)G box. Results Isolates were divided into three groups according to the susceptibility profile: Cl group included 3 strains which were susceptible to penicillin and with cefditoren MIC of≤0. 5 mg/L; C2 group included 3 strains which were intermediate to penicillin and with cefditoren MIC of≤O, 5 mg/L; R group included 37 strains which were intermediate to penicillin and with cefditoren MIC of 1-4 mg/L Isolates from group R were all susceptible to levofloxacin, moxifloxacin and vancomycin, and resistant to cefuroxime, cefaclor, azithromycin and clarithromycin. Only 8.2% and 5.4% of the isolates were susceptible to amoxicillin-clavulanic acid and ceftriaxone, separately. The serotyping results indicated that 37 isolates of group R included 35 isolates with type 19F, 1 with type 14 and 1 with type 19A. The conserved motifs of PBPs in Cl group had no mutations when compared with R6. However, the conserved motifs of PBPs in C2 group isolates had following common mutations; Thr445→Ala in PBP2B, Thr371→Ser/Ala and Pro432→Thr in PBPlA, Thr338→Ala and Leu546→Val in PBP2X. For group R, additional mutations of Ala618→Gly in PBP2B, Met339→Phe and Tyr595→Phein PBP2X were found in addition to the mutations in group Cl. Conclusions The mutations of PBP2B, PBPlA and PBP2X have close relationship to cefditoren-resistance in S, pneumoniae. Ala618→Gly in PBP2B, Met339→Phe and Tyr595→Phe in PBP2X may be highly related to the increasing cefditoren MICs.%目的 研究头孢妥仑敏感性降低肺炎链球菌的耐药机制.方法 收集2009年1月至2010年1月来自全国各地10所教学医院的37株头孢妥仑最低抑菌浓度(MIC)≥1 mg/L的社区呼吸道感染肺炎链球菌作为研究组,并取6株头孢妥仑MIC≤0.5 mg/L的社区呼吸道感染肺炎链球菌作为对照组.采用微量肉汤稀释法测定菌株对11种抗菌药物的MIC.采用多重PCR法对所有菌株进行血清分型.采用PCR法扩增43株菌的pbp2b、pbp1a、pbp2x和murM基因.用限制性内切酶进行pbp2b、pbp1a、pbp2x的指纹图谱分析.根据酶切试验分型结果,同一型的菌株挑选1～5个PCR产物送测序,测序结果与肺炎链球菌标准菌株R6比较以分析耐药菌株的青霉素结合蛋白(PBP)保守基序,特别是SXXK盒、SXN盒、KT(S)G盒的突变情况.结果 根据药敏试验结果将菌株分为3组:3株对青霉素敏感、头孢妥仑MIC值≤0.5 mg/L作为C1组；3株对青霉素中介、头孢妥仑MIC值≤0.5 mg/L作为C2组；37株对青霉素中介,头孢妥仑MIC为1～4mg/L的为R组.R组的肺炎链球菌对左氧氟沙星、莫西沙星和万古霉素均敏感,对头孢呋辛、头孢克洛、阿奇霉素和克拉霉素均耐药,仅有8.2％和5.4％的菌株分别对阿莫西林-克拉维酸和头孢曲松敏感.37株R组菌株血清学分型结果35株为19F型,1株为14型,1株为19A型.C1组菌的PBP氨基酸保守基序与野生株R6相比无突变.C2组菌PBP氨基酸保守基序与R6株相比在PBP2B、PBP2X和PBP1A中均发生3点突变.R组菌株PBP氨基酸保守基序与R6株相比则在C2组突变的基础上增加了2～3个活性位点的突变.结论 PBP2B、PBP1A和PBP2X的突变与肺炎链球菌的头孢妥仑耐药性密切相关,特别是PBP2B的Ala618→Gly,PBP2X的Met339→Phe和Tyr595→Phe可能与头孢妥仑MIC升高相关.