Effects of Carbon Monoxide and Carbon Monoxide–Releasing Molecules (CORMs) as Adjuvants to Antibiotics on Escherichia coli

Antibiotic-resistant pathogenic bacteria pose a major, growing public health risk. Antibiotics affect specific targets so that bacteria may develop cognate resistance to the target site. To cope with antibiotic resistance, combinatorial therapies involving two (or more) antimicrobial agents may be useful for minimizing antibiotic resistance phenomena. It is, therefore, necessary to investigate other antimicrobial agents. Carbon monoxide-releasing molecules (CORMs) have been shown to exert antimicrobial actions on several bacterial species both in the laboratory and in animal models. The aims of this thesis were to investigate the antibacterial effects of CORMs on the bacterial growth and viability for both strains of E. coli wild type (MG1655) and multidrug-resistant uropathogenic E. coli (EC958) and more importantly to test their interactions with antibiotics. The results revealed that CORMs have antimicrobial effects against both strains of E. coli. Importantly, a combination of sub-inhibitory concentrations of CORMs and antibiotics showed a significant potency on the action of conventional antibiotics with reduction of bacterial growth and viability. CO gas showed only slight antibacterial effects and there was no effect of CO gas on antibiotic activities when combined. Furthermore, conjunction of CORM-2 or CORM-3 with doxycycline, minocycline, gentamicin or chloramphenicol revealed that minimal inhibitory concentration (MIC) values of the antibiotics decreased between 16 - 640 fold with CORM-2 and 13 - 60 fold with CORM-3; the minimal bactericidal concentration (MBC) values were also reduced 2 - 6 fold with CORM-2 and 1 - 150 fold with CORM-3. Moreover, the drug interactions between CORMs and antibiotics were assessed using checkerboard microdilution methods, time-killing curves and Etests; the results revealed that CORMs have synergistic interactions with several antibiotics such as trimethoprim, novobiocin and doxycycline with fractional inhibitory concentration indexes (FICi) between 0.35 and 0.50. Real-time PCR data showed that CORM-2 significantly upregulates the transcription of recA and spy, which encode proteins for DNA recombination and repair and an envelope-stress-induced periplasmic protein respectively, However, there were no significant changes in the transcriptional level of sodA and katG, which encode for oxidative stress enzymes. Furthermore, ruthenium-based CORMs caused DNA degradation and also membrane leakage of nucleic acids in a concentration-dependent manner. Finally, CORM-2 produces a significant amount of ROS when assayed using a DCF-DC probe. However, using the HFP probe that selectively measures the generation of hydroxyl radical (OH^), we showed that CORM-2 does not produce OH^. Furthermore, the Amplex Red assay revealed that CORM-2 at lower concentrations was not able to produce H2O2 while 4 μM H2O2 was produced at 150 μM CORM-2. In conclusion, these findings reveal that the CO released from CORMs, especially CORM-2 and CORM-3 has bactericidal activity and, more importantly, these CORMs showed synergistic interactions with various antibiotics against both strains of E. coli.