Antimicrobial studies with the Pseudomonas aeruginosa two-allele library require caution.
Antimicrobial studies with the Pseudomonas aeruginosa two-allele library require caution.
复制标题
铜绿假单胞菌双等位基因库的抗菌研究需要谨慎。
DOI：
10.1128/aac.00419-08
复制
发表时间：
2008
影响因子：
4.9
通讯作者：
Zhao,Xilin
中科院分区：
文献类型：
--
作者：
Wu,Xiangli;Wang,Hexiang;Zhao,Xilin
抗菌素耐药性日益普遍，需要不断努力寻找新的抗菌素。多种微生物基因组序列的可用性促进了用于系统基因功能分析的全基因组、单基因敲除文库的发展。这些文库，如大肠杆菌庆应义塾文库(1)和华盛顿大学铜绿假单胞菌双等位基因文库(3)，可用于高通量表型阵列研究(7)；因此，它们似乎非常适合于识别新的抗菌靶点。然而，我们最近在铜绿假单胞菌双等位基因突变文库中注意到，内在的、未知的因素可能会影响许多随机选择的突变体对某些抗菌素的敏感性，从而模糊了特定候选突变体的作用。在大肠杆菌中，phoU敲除突变使其对多种抗菌素和其他应激产生超敏反应(5)。为了确定P. aeruginosa中phoU同源物的失活是否会赋予类似的表型，我们从华盛顿大学的双等位基因文库中获得了一个phoU突变体和亲本野生型菌株（PAO1，缺乏转座子插入）。当测试喹诺酮类药物的敏感性时，观察到环丙沙星和氧喹啉酸的MIC降低了4- 16倍（表1），正如预期的(5)。然而，当测试10个不相关的突变体时，每个突变体也表现出对喹诺酮类药物的超敏感（表1）。此外，PCR分析未能证实转位在phoU等位基因内。因此，phoU突变体的易感性增加不能特别归因于phoU突变。大多数突变体的菌落颜色比野生型菌株深（蓝绿色），表明突变体产生更多的非那嗪色素。由于苯那嗪，如pyocyanin，可能参与氧化应激，从而影响抗菌药物的敏感性(2)，我们测试了另外两种不产生pyocyanin的突变体（phzS和phzM突变体）(6)。观察到与其他突变体相似的mic降低。因此，花青素产生的变异不是降低喹诺酮类药物敏感性的原因。由于测试的突变体在各种染色体位置都有转座子插入，其中一些远远超出开放阅读框，转座子介导的极性也不太可能解释MIC降低。当对其他抗菌药物进行测试时，观察到氯霉素的超敏反应（4- 64倍），而妥布霉素、美罗培南或头孢他啶的超敏反应则没有（表1）。因此，被测试的突变体对某些人普遍敏感，但是
The increasing prevalence of antimicrobial resistance requires a continuous effort to find new antimicrobials. The availability of genome sequences for a variety of microbes has facilitated the development of whole-genome, single-gene knockout libraries for systematic gene function analysis. These libraries, such as the Keio library of Escherichia coli (1) and the University of Washington two-allele library of Pseudomonas aeruginosa (3), can be used in high-throughput phenotypic array studies (7); thus, they seem well suited for identifying new antimicrobial targets. However, we recently noticed with the P. aeruginosa two-allele mutant library that intrinsic, unidentified factors may affect susceptibility for some antimicrobials with many randomly picked mutants, thereby obscuring effects attributed to specific candidate mutants. In E. coli, a phoU knockout mutation confers hypersensitivity to a variety of antimicrobials and other stresses (5). To determine whether inactivation of a phoU homologue in P. aeruginosa confers a similar phenotype, we obtained a phoU mutant and the parental wild-type strain (PAO1, lacking a transposon insertion) from the University of Washington twoallele library. When quinolone susceptibility was tested, a 4-to 16-fold decrease in MIC was observed for both ciproffoxacin and oxolinic acid (Table 1), as expected (5). However, when 10 unrelated mutants were tested, each of those mutants also exhibited hypersusceptibility to quinolones (Table 1). Moreover, PCR analysis failed to confirm transposition being inside the phoU allele. Thus, increased susceptibility of the phoU mutant could not be specifically attributed to the phoU mutation.Colony color with most of these mutants was darker (bluish green) than with the wild-type strain, indicating production of more phenazine pigments in the mutants. Since phenazines, such as pyocyanin, may be involved in oxidative stress and thereby affect antimicrobial susceptibility (2), we tested two additional mutants (the phzS and phzM mutants) that do not produce pyocyanin (6). Reduced MICs, similar to those seen with the other mutants, were observed. Thus, variation in pyocyanin production was not responsible for reduced quinolone susceptibility. Since the mutants tested had transposon insertions at a variety of chromosomal locations, some of which were far outside open reading frames, transposon-mediated polarity is also an unlikely explanation for reduced MIC. When other antimicrobial classes were tested, hypersusceptibility was observed with chloramphenicol (4-to 64-fold) but not with tobramycin, meropenem, or ceftazidime (Table 1). Thus, the mutants tested were universally hypersusceptible to some, but