Chromosomal antioxidant genes have metal ion-specific roles as determinants of bacterial metal tolerance.

Microbiological metal toxicity involves redox reactions between metal species and cellular molecules, and therefore, we hypothesized that antioxidant systems might be chromosomal determinants affecting the susceptibility of bacteria to metal toxicity. Here, survival was quantified in metal ion-exposed planktonic cultures of several Escherichia coli strains, each bearing a mutation in a gene important for redox homeostasis. This characterized approximately 250 gene-metal combinations and identified that sodA, sodB, gor, trxA, gshA, grxA and marR have distinct roles in safeguarding or sensitizing cells to different toxic metal ions (Cr(2)O(7)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), AsO(2)(-), SeO(3)(2-) or TeO(3)(2-)). To shed light on these observations, fluorescent sensors for reactive oxygen species (ROS) and reduced thiol (RSH) quantification were used to ascertain that different metal ions exert oxidative toxicity through disparate modes-of-action. These oxidative mechanisms of metal toxicity were categorized as involving ROS and thiol-disulfide chemistry together (AsO(2)(-), SeO(3)(2-)), ROS predominantly (Cu(2+), Cr(2)O(7)(2-)) or thiol-disulfide chemistry predominantly (Ag(+), Co(2+), Zn(2+), TeO(3)(2-)). Corresponding to this, promoter-luxCDABE fusions showed that toxic doses of different metal ions up- or downregulate the transcription of gene sets marking distinct pathways of cellular oxidative stress. Altogether, our findings suggest that different metal ions are lethal to cells through discrete pathways of oxidative biochemistry, and moreover, indicate that chromosomally encoded antioxidant systems may have metal ion-specific physiological roles as determinants of bacterial metal tolerance.

Copper and quaternary ammonium cations exert synergistic bactericidal and antibiofilm activity against Pseudomonas aeruginosa.

Biofilms are slimy aggregates of microbes that are likely responsible for many chronic infections as well as for contamination of clinical and industrial environments. Pseudomonas aeruginosa is a prevalent hospital pathogen that is well known for its ability to form biofilms that are recalcitrant to many different antimicrobial treatments. We have devised a high-throughput method for testing combinations of antimicrobials for synergistic activity against biofilms, including those formed by P. aeruginosa. This approach was used to look for changes in biofilm susceptibility to various biocides when these agents were combined with metal ions. This process identified that Cu(2+) works synergistically with quaternary ammonium compounds (QACs; specifically benzalkonium chloride, cetalkonium chloride, cetylpyridinium chloride, myristalkonium chloride, and Polycide) to kill P. aeruginosa biofilms. In some cases, adding Cu(2+) to QACs resulted in a 128-fold decrease in the biofilm minimum bactericidal concentration compared to that for single-agent treatments. In combination, these agents retained broad-spectrum antimicrobial activity that also eradicated biofilms of Escherichia coli, Staphylococcus aureus, Salmonella enterica serovar Cholerasuis, and Pseudomonas fluorescens. To investigate the mechanism of action, isothermal titration calorimetry was used to show that Cu(2+) and QACs do not interact in aqueous solutions, suggesting that each agent exerts microbiological toxicity through independent biochemical routes. Additionally, Cu(2+) and QACs, both alone and in combination, reduced the activity of nitrate reductases, which are enzymes that are important for normal biofilm growth. Collectively, the results of this study indicate that Cu(2+) and QACs are effective combinations of antimicrobials that may be used to kill bacterial biofilms.