Adrian P V, Klugman K P
Pneumococcal Diseases Research Unit of South African Institute for Medical Research, University of the Witwatersrand, and Medical Research Council, Johannesburg.
Antimicrob Agents Chemother. 1997 Nov;41(11):2406-13. doi: 10.1128/AAC.41.11.2406.
Streptococcus pneumoniae isolates resistant to several antimicrobial agent classes including trimethoprim-sulfamethoxazole have been reported with increasing frequency throughout the world. The MICs of trimethoprim, sulfamethoxazole, and trimethoprim-sulfamethoxazole (1:19) for 259 clinical isolates from South Africa were determined, and 166 of these 259 (64%) isolates were resistant to trimethoprim-sulfamethoxazole (MICs > or =20 mg/liter). Trimethoprim resistance was found to be more strongly correlated with trimethoprim-sulfamethoxazole resistance (correlation coefficient, 0.744) than was sulfamethoxazole resistance (correlation coefficient, 0.441). The dihydrofolate reductase genes from 11 trimethoprim-resistant (MICs, 64 to 512 microg/ml) clinical isolates of Streptococcus pneumoniae were amplified by PCR, and the nucleotide sequences were determined. Two main groups of mutations to the dihydrofolate reductase gene were found. Both groups shared six amino acid changes (Glu20-Asp, Pro70-Ser, Gln81-His, Asp92-Ala, Ile100-Leu, and Leu135-Phe). The first group included two extra changes (Lys60-Gln and Pro111-Ser), and the second group was characterized by six additional amino acid changes (Glu14-Asp, Ile74-Leu, Gln91-His, Glu94-Asp, Phe147-Ser, and Ala149-Thr). Chromosomal DNA from resistant isolates and cloned PCR products of the genes encoding resistant dihydrofolate reductases were capable of transforming a susceptible strain of S. pneumoniae to trimethoprim resistance. The inhibitor profiles of recombinant dihydrofolate reductase from resistant and susceptible isolates revealed that the dihydrofolate reductase from trimethoprim-resistant isolates was 50-fold more resistant (50% inhibitory doses [ID50s], 3.9 to 7.3 microM) than that from susceptible strains (ID50s, 0.15 microM). Site-directed mutagenesis experiments revealed that one mutation, Ile100-Leu, resulted in a 50-fold increase in the ID50 of trimethoprim. The resistant dihydrofolate reductases were characterized by highly conserved redundant changes in the nucleotide sequence, suggesting that the genes encoding resistant dihydrofolate reductases may have evolved as a result of inter- or intraspecies recombination by transformation.
据报道，对包括甲氧苄啶 - 磺胺甲恶唑在内的多种抗菌药物耐药的肺炎链球菌分离株在全球范围内的出现频率日益增加。测定了来自南非的259株临床分离株对甲氧苄啶、磺胺甲恶唑以及甲氧苄啶 - 磺胺甲恶唑（1:19）的最低抑菌浓度（MIC），这259株分离株中有166株（64%）对甲氧苄啶 - 磺胺甲恶唑耐药（MIC≥20mg/L）。结果发现，甲氧苄啶耐药与甲氧苄啶 - 磺胺甲恶唑耐药的相关性更强（相关系数为0.744），而与磺胺甲恶唑耐药的相关性较弱（相关系数为0.441）。通过聚合酶链反应（PCR）扩增了11株对甲氧苄啶耐药（MIC为64至512μg/ml）的肺炎链球菌临床分离株的二氢叶酸还原酶基因，并测定了核苷酸序列。发现二氢叶酸还原酶基因的突变主要有两组。两组都共有六个氨基酸变化（Glu20 - Asp、Pro70 - Ser、Gln81 - His、Asp92 - Ala、Ile100 - Leu和Leu135 - Phe）。第一组还包括另外两个变化（Lys60 - Gln和Pro111 - Ser），第二组的特征是还有六个氨基酸变化（Glu14 - Asp、Ile74 - Leu、Gln91 - His、Glu94 - Asp、Phe147 - Ser和Ala149 - Thr）。耐药分离株的染色体DNA以及编码耐药二氢叶酸还原酶的基因的克隆PCR产物能够将肺炎链球菌的敏感菌株转化为对甲氧苄啶耐药。来自耐药和敏感分离株的重组二氢叶酸还原酶的抑制剂谱显示，来自对甲氧苄啶耐药分离株的二氢叶酸还原酶的耐药性比对敏感菌株的二氢叶酸还原酶高50倍（50%抑制剂量[ID50]，3.9至7.3μM对0.15μM）。定点诱变实验表明，一个突变Ile100 - Leu导致甲氧苄啶的ID50增加了50倍。耐药的二氢叶酸还原酶的特征是核苷酸序列中存在高度保守的冗余变化，这表明编码耐药二氢叶酸还原酶的基因可能是通过转化在种间或种内重组而进化的。
