Schurek Kristen N, Sampaio Jorge L M, Kiffer Carlos R V, Sinto Sumiko, Mendes Caio M F, Hancock Robert E W
Centre for Microbial Diseases & Immunity Research, University of British Columbia, 2259 Lower Mall, Vancouver, British Columbia, Canada.
Antimicrob Agents Chemother. 2009 Oct;53(10):4345-51. doi: 10.1128/AAC.01267-08. Epub 2009 Jul 27.
During investigation of susceptibility testing methods for polymyxins, 24 multidrug-resistant clinical isolates of Pseudomonas aeruginosa were observed to have a distinct, reproducible phenotype in which skipped wells were observed during broth microdilution testing for polymyxin B. Possible mechanisms underlying this phenotype were investigated. The effects of various concentrations of polymyxin B on growth, the expression of resistance genes, and outer-membrane permeability were observed. Real-time PCR was performed to compare the expression, in response to selected concentrations of polymyxin B, of genes related to the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems in polymyxin B-susceptible isolate PAO1, polymyxin B-resistant isolate 9BR, and two isolates (19BR and 213BR) exhibiting the skipped-well phenotype. 19BR and 213BR appeared to have similar basal levels of expression compared to that of PAO1 for phoQ, arnB, and PA4773 (from the pmrAB operon), and in contrast, 9BR had 52- and 280-fold higher expression of arnB and PA4773, respectively. The expression of arnB and PA4773 increased in response to polymyxin B in a concentration-dependent manner for 9BR but not for 19BR and 213BR. For these isolates, expression was significantly increased for arnB and PA4773, as well as phoQ, only upon exposure to 2 mug/ml polymyxin B but not at a lower concentration of 0.125 microg/ml. The sequencing of the pmrAB and phoPQ operons for all three isolates revealed a number of unique mutations compared to that for PAO1. 1-N-phenylnaphthylamine (NPN) was used to study the effect of preincubation with polymyxin B on the self-promoted uptake of polymyxin B across the outer membrane. The preincubation of cells with 2 microg/ml polymyxin B affected baseline membrane permeability in 19BR and 213BR and also resulted in a reduced rate of NPN uptake in these isolates and in PAO1 but not in 9BR. The results presented here suggest that the skipped-well isolates have the ability to adapt to specific concentrations of polymyxin B, inducing known polymyxin B resistance genes involved in generating alterations in the outer membrane.
在对多粘菌素药敏试验方法的研究过程中，观察到24株耐多药铜绿假单胞菌临床分离株具有一种独特的、可重复的表型，即在多粘菌素B肉汤微量稀释试验中出现跳孔现象。对该表型潜在的可能机制进行了研究。观察了不同浓度多粘菌素B对生长、耐药基因表达及外膜通透性的影响。进行实时聚合酶链反应（PCR），以比较在选定浓度的多粘菌素B作用下，多粘菌素B敏感菌株PAO1、多粘菌素B耐药菌株9BR以及两株表现出跳孔表型的菌株（19BR和213BR）中，与PhoP-PhoQ和PmrA-PmrB双组分调节系统相关基因的表达情况。与PAO1相比，19BR和213BR的phoQ、arnB和PA4773（来自pmrAB操纵子）的基础表达水平似乎相似；相反，9BR的arnB和PA4773的表达分别高出52倍和280倍。对于9BR，arnB和PA4773的表达随多粘菌素B浓度的增加呈浓度依赖性增加，而19BR和213BR则不然。对于这些分离株，仅在暴露于2μg/ml多粘菌素B时，arnB、PA4773以及phoQ的表达才显著增加，而在较低浓度0.125μg/ml时则未增加。对所有三株分离株的pmrAB和phoPQ操纵子进行测序，发现与PAO1相比存在一些独特的突变。使用1-N-苯基萘胺（NPN）研究预先用多粘菌素B孵育对多粘菌素B跨外膜自促进摄取的影响。用2μg/ml多粘菌素B对细胞进行预先孵育，影响了19BR和213BR的基线膜通透性，并且还导致这些分离株以及PAO1中NPN摄取速率降低，但9BR中未出现这种情况。此处呈现的结果表明，跳孔分离株有能力适应特定浓度的多粘菌素B，诱导参与外膜改变的已知多粘菌素B耐药基因。
