Analysis of penicillin-binding protein lb and 2a genes from Streptococcus pneumoniae.
Analysis of penicillin-binding protein lb and 2a genes from Streptococcus pneumoniae.
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DOI：
10.1089/107662900419438
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发表时间：
2000
影响因子：
2.6
通讯作者：
M. du Plessis;A. M. Smith;K. Klugman
中科院分区：
文献类型：
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作者：
M. du Plessis;A. M. Smith;K. Klugman
从南非各地的医院随机选择50株肺炎链球菌临床分离株（青霉素MIC，0.03-8 μ g/mL），以及在匈牙利分离的7株菌株（青霉素MIC，16-32 μ g/mL）。通过PCR扩增青霉素结合蛋白（pbp）1b和2a基因，并将纯化的DNA用HinfI、StyI和MseI + DdeI限制性内切酶消化。将片段进行放射性末端标记并在聚丙烯酰胺凝胶上分离，并在放射自显影后观察DNA指纹。进一步选择分离物的集合用于pbp 1b和2a的序列分析。DNA指纹分析显示，这两个基因的所有菌株之间的统一配置文件。所有分离株在其pbp 1b基因中最多只有7个核苷酸替换，导致PBP 1B中最多3个氨基酸替换。在pbp 2a基因的情况下，观察到多达13个核苷酸取代随机分布在青霉素敏感和耐药菌株，揭示了最多的5个氨基酸取代的PBP 2A。未发现所有青霉素耐药分离株中常见的氨基酸取代。用从两种耐药菌株（MIC，4和16微克/毫升）分离的pbp 1b和2a基因进行的转化实验未能将肺炎球菌菌株转化为青霉素耐药性水平增加的菌株。这些结果表明，这里检查的pbp 1b和2a基因不显示在青霉素耐药肺炎球菌的pbp 2x、2b和1a基因中观察到的典型嵌合基因模式。此外，转化研究表明PBPs 1B和2A可能在某些肺炎球菌青霉素耐药性的发展中不起作用。
Fifty clinical isolates (penicillin MICs, 0.03-8 microg/mL) of Streptococcus pneumoniae were randomly selected from hospitals throughout South Africa, together with seven strains isolated in Hungary (penicillin MICs, 16-32 microg/mL). Penicillin-binding protein (pbp) 1b and 2a genes were amplified by PCR, and the purified DNA was digested with HinfI, StyI, and MseI + DdeI restriction enzymes. The fragments were radioactively end-labeled and separated on polyacrylamide gels, and the DNA fingerprints were visualized following autoradiography. A collection of isolates was further selected for sequence analysis of pbp1b and 2a. DNA fingerprint analysis revealed a uniform profile amongst all isolates for both genes. All isolates revealed a maximum of only seven nucleotide substitutions in their pbp1b genes, resulting in a maximum of three amino acid substitutions in PBP 1B. In the case of the pbp2a gene, up to 13 nucleotide substitutions were observed randomly distributed amongst penicillin-susceptible and resistant isolates, revealing a maximum of five amino acid substitutions in PBP 2A. No amino acid substitutions were found to be common amongst all penicillin-resistant isolates. Transformation experiments with pbp1b and 2a genes isolated from two resistant strains (MICs, 4 and 16 microg/mL) failed to transform pneumococcal strains to increased levels of penicillin resistance. These results show that the pbp1b and 2a genes examined here do not display the typical mosaic gene patterns observed in the pbp2x, 2b, and 1a genes of penicillin-resistant pneumococci. In addition, the transformation studies suggest that PBPs 1B and 2A may not play a role in the development of penicillin resistance in some pneumococci.