Servicio de Microbiología and Unidad de Investigación, Instituto Universitario de Investigación en Ciencias de la Salud, Palma de Mallorca, Spain.
Antimicrob Agents Chemother. 2010 Sep;54(9):3557-63. doi: 10.1128/AAC.00385-10. Epub 2010 Jun 21.
AmpC hyperproduction is the most frequent mechanism of resistance to penicillins and cephalosporins in Pseudomonas aeruginosa and is driven by ampD mutations or the recently described inactivation of dacB, which encodes the nonessential penicillin-binding protein (PBP) PBP 4. Recent work showed that nagZ inactivation attenuates beta-lactam resistance in ampD mutants. Here we explored whether the same could be true for the dacB mutants with dacB mutations alone or in combination with ampD mutations. The inactivation of nagZ restored the wild-type beta-lactam MICs and ampC expression of PAO1 dacB and ampD mutants and dramatically reduced the MICs (for example, the MIC for ceftazidime dropped from 96 to 4 microg/ml) and the level of ampC expression (from ca. 1,000-fold to ca. 50-fold higher than that for PAO1) in the dacB-ampD double mutant. On the other hand, nagZ inactivation had little effect on the inducibility of AmpC. The NagZ inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate attenuated the beta-lactam resistance of the AmpC-hyperproducing strains, showing a greater effect on the dacB mutant (reducing the ceftazidime MICs from 24 to 6 microg/ml) than the ampD mutant (reducing the MICs from 8 to 4 microg/ml). Additionally, nagZ inactivation in the dacB mutant blocked the overexpression of creD (blrD), which is a marker of the activation of the CreBC (BlrAB) regulator involved in the resistance phenotype. Finally, through population analysis, we show that the inactivation of nagZ dramatically reduces the capacity of P. aeruginosa to develop ceftazidime resistance, since spontaneous mutants were not obtained at concentrations > or = 8 microg/ml (the susceptibility breakpoint) for the nagZ mutant but were obtained with wild-type PAO1. Therefore, NagZ is envisaged to be a candidate target for preventing and reverting beta-lactam resistance in P. aeruginosa.
AmpC 超表达是铜绿假单胞菌对青霉素类和头孢菌素类耐药的最常见机制，由 ampD 突变或最近描述的 dacB 失活驱动，dacB 编码非必需的青霉素结合蛋白（PBP）PBP4。最近的研究表明，nagZ 失活可减弱 ampD 突变体的β-内酰胺耐药性。在这里，我们探讨了 dacB 突变体（单独或与 ampD 突变体组合）是否也可以如此。nagZ 的失活恢复了 PAO1 dacB 和 ampD 突变体的野生型β-内酰胺 MIC 和 ampC 表达，并显著降低了 MIC（例如，头孢他啶的 MIC 从 96 降至 4μg/ml）和 ampC 表达水平（从 ca.1000 倍到 ca.50 倍高于 PAO1）在 dacB-ampD 双突变体中。另一方面，nagZ 失活对 AmpC 的诱导作用影响不大。NagZ 抑制剂 O-(2-乙酰氨基-2-脱氧-D-葡萄糖基亚氨基-N-苯基氨基甲酰基）对 AmpC 过度产生的菌株的β-内酰胺耐药性有减弱作用，对 dacB 突变体的效果更大（将头孢他啶的 MIC 从 24 降至 6μg/ml），而对 ampD 突变体的效果较小（将 MIC 从 8 降至 4μg/ml）。此外，dacB 突变体中 nagZ 的失活阻止了 creD（blrD）的过表达，creD 是参与耐药表型的 CreBC（BlrAB）调节剂激活的标志物。最后，通过群体分析，我们表明 nagZ 的失活显著降低了铜绿假单胞菌产生头孢他啶耐药性的能力，因为在浓度>或=8μg/ml（nagZ 突变体的药敏折点）时，没有获得自发突变体，但用野生型 PAO1 获得了突变体。因此，NagZ 有望成为预防和逆转铜绿假单胞菌β-内酰胺耐药性的候选靶标。
