Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance
Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance
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DOI：
10.1128/aac.43.11.2648
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发表时间：
1999-11-01
影响因子：
4.9
通讯作者：
Woods, DE
中科院分区：
文献类型：
--
作者：
Burtnick, MN;Woods, DE
鼻疽伯克霍尔德氏菌是一种革兰氏阴性细菌，可引起类鼻疽病。这种病原体是东南亚和澳大利亚北部的地方病，在泰国东北部尤为严重。此前有报道称，鼻疽伯克霍尔德氏菌对阳离子抗菌肽（包括人中性粒细胞肽、硫酸鱼精蛋白、聚 L-赖氨酸、magainin 和多粘菌素）的杀灭作用具有抗药性。最近，我们还发现强毒临床分离菌鼻疽伯克氏菌 1026b 能够在含有浓度 >100 mg/ml 的多粘菌素 B 的培养基中复制。为了鉴定与这种特定抗性表型相关的遗传位点，我们采用了 Tn5-OT182 诱变系统与复制平板筛选相配合来分离多粘菌素 B 敏感突变体。在通过这种方法筛选的 17,000 个 Tn5-OT182 突变体中，获得了 5 个多粘菌素 B 敏感突变体。其中三个突变体在基因位点内含有 Tn5-OT182 插入，表明与其他革兰氏阴性细菌中存在的 lytB 基因具有很强的同源性。其余两个突变体中，一个在参与脂多糖核心生物合成（waaF）的基因座中包含转座子插入，而另一个在与UDP-葡萄糖脱氢酶基因同源的开放阅读框中包含插入。还通过等位基因交换构建了同基因突变体，并用于互补分析研究，以进一步表征各个基因位点对于拟鼻疽杆菌 1026b 表现出的多粘菌素 B 抗性表型的相对重要性。
Burkholderia pseudomallei is a gram-negative bacterium that causes the disease known as melioidosis, This pathogen is endemic to Southeast Asia and northern Australia and is particularly problematic in northeastern Thailand, It has been previously reported that B. pseudomallei is resistant to the killing action of cationic antimicrobial peptides, including human neutrophil peptide, protamine sulfate, poly-L-lysine, magainins, and polymyxins. Recently, we have also found that the virulent clinical isolate B. pseudomallei 1026b is capable of replicating in media containing polymyxin B at concentrations of >100 mg/ml. In order to identify genetic loci that are associated with this particular resistance phenotype, we employed a Tn5-OT182 mutagenesis system in coordination with a replica plating screen to isolate polymyxin B-susceptible mutants. Of the 17,000 Tn5-OT182 mutants screened via this approach, five polymyxin B-susceptible mutants were obtained. Three of these mutants harbored Tn5-OT182 insertions within a genetic locus demonstrating strong homology to the lytB gene present in other gram-negative bacteria. Of the remaining two mutants, one contained a transposon insertion in a locus involved in lipopolysaccharide core biosynthesis (waaF), while the other contained an insertion in an open reading frame homologous to UDP-glucose dehydrogenase genes. Isogenic mutants were also constructed via allelic exchange and used in complementation analysis studies to further characterize the relative importance of each of the various genetic loci with respect to the polymyxin B resistance phenotype exhibited by B, pseudomallei 1026b.