Simões Manuel, Simões Lúcia C, Cleto Sara, Machado Idalina, Pereira Maria O, Vieira Maria J
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological Engineering, Universidade do Minho, Campus de Gualtar, Braga, Portugal.
J Basic Microbiol. 2007 Jun;47(3):230-42. doi: 10.1002/jobm.200610280.
Biocides generally have multiple biochemical targets. Such a feature easily entangles the analysis of the mechanisms of antimicrobial action. In this study, the action of the dialdehyde biocide ortho-phtalaldehyde (OPA), on bacteria, was investigated using the Gram-negative Pseudomonas fluorescens. The targets of the biocide action were studied using different bacterial physiological indices. The respiratory activity, membrane permeabilization, physico-chemical characterization of the bacterial surfaces, outer membrane proteins (OMP) expression, concomitant influence of pH, contact time and presence of bovine serum albumin (BSA) on respiratory activity, morphological changes and OPA-DNA interactions were assessed for different OPA concentrations. With the process conditions used, the minimum inhibitory concentration was 1500 mg/l, the concentration to promote total loss of bacterial culturability was 65 mg/l and the concentration needed to inactivate respiratory activity was 80 mg/l. These data are evidence that culturability and respiratory activity were markedly affected by the biocide. OPA lead, moreover, to a significant change in cell surface hydrophobicity and induced propidium iodide uptake. Such results suggest cytoplasmic membrane damage, although no release of ATP was detected. At pH 5, the bactericidal action of OPA was stronger, though not influenced by BSA presence. Nevertheless, at pH 9, BSA noticeably (p < 0.05) impaired biocide action. A time-dependent effect in OPA action was evident when contemplating respiratory activity variation, mainly for the lower exposure times. Scanning electron microscopy allowed to detect bacterial morphological changes, translated on cellular elongation, for OPA concentrations higher than 100 mg/l. Interferences at DNA level were, however, restricted to extreme biocide concentrations. The overall bactericidal events occurred without detectable OMP expression changes. In conclusion, the results indicated a sequence of events responsible for the antimicrobial action of OPA: it binds to membrane receptors due to cross-linkage; impairs the membrane functions allowing the biocide to enter through the permeabilized membrane; it interacts with intracellular reactive molecules, such as RNA, compromising the growth cycle of the cells and, at last, with DNA.
杀生剂通常有多个生化靶点。这一特性很容易使对抗菌作用机制的分析变得复杂。在本研究中，使用革兰氏阴性荧光假单胞菌研究了双醛杀生剂邻苯二甲醛（OPA）对细菌的作用。利用不同的细菌生理指标研究了杀生剂作用的靶点。针对不同的OPA浓度，评估了呼吸活性、膜通透性、细菌表面的物理化学特性、外膜蛋白（OMP）表达、pH值、接触时间和牛血清白蛋白（BSA）的存在对呼吸活性的伴随影响、形态变化以及OPA与DNA的相互作用。在所采用的工艺条件下，最低抑菌浓度为1500 mg/l，促进细菌培养能力完全丧失的浓度为65 mg/l，使呼吸活性失活所需的浓度为80 mg/l。这些数据证明杀生剂对细菌的培养能力和呼吸活性有显著影响。此外，OPA导致细胞表面疏水性发生显著变化，并诱导碘化丙啶摄取。这些结果表明细胞质膜受到损伤，尽管未检测到ATP的释放。在pH值为5时，OPA的杀菌作用更强，不过不受BSA存在的影响。然而，在pH值为9时，BSA显著（p < 0.05）削弱了杀生剂的作用。考虑呼吸活性变化时，OPA作用存在时间依赖性效应，主要在较低暴露时间时明显。扫描电子显微镜能够检测到细菌形态变化，对于OPA浓度高于100 mg/l的情况，表现为细胞伸长。然而，在DNA水平的干扰仅限于极高的杀生剂浓度。总体杀菌过程中未检测到OMP表达变化。总之，结果表明了一系列导致OPA抗菌作用的事件：它通过交联作用与膜受体结合；损害膜功能，使杀生剂能够通过通透的膜进入；它与细胞内的反应性分子如RNA相互作用，破坏细胞的生长周期，最后与DNA相互作用。
