Through a liquid-liquid extraction procedure, pyocyanin was quantified in cultures that were incubated at 30°C, 37°C, and 40°C and in the presence of Staphylococcus aureus spent media. In addition, culturing methods for the measurement of pyocyanin under hypoxic conditions were analyzed. I hypothesized that environmental conditions such as temperature, co-infection with S. aureus, and oxygen depletion would influence pyocyanin production. It was found that overall, 30°C incubation produced statistically significant decrease in pyocyanin production compared with incubation at 37°C. These findings will help to determine how phenotypes are affected by conditions in the CF lung. In addition, these conclusions will help direct metabolic analysis and to identify volatile biomarkers of pyocyanin production for future use in breath-based diagnostics of CF lung infections.
Exchangeable Ions Are Responsible for the In Vitro Antibacterial Properties of Natural Clay Mixtures
We have identified a natural clay mixture that exhibits in vitro antibacterial activity against a broad spectrum of bacterial pathogens. We collected four samples from the same source and demonstrated through antibacterial susceptibility testing that these clay mixtures have markedly different antibacterial activity against Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Here, we used X-ray diffraction (XRD) and inductively coupled plasma - optical emission spectroscopy (ICP-OES) and - mass spectrometry (ICP-MS) to characterize the mineralogical and chemical features of the four clay mixture samples. XRD analyses of the clay mixtures revealed minor mineralogical differences between the four samples. However, ICP analyses demonstrated that the concentrations of many elements, Fe, Co, Cu, Ni, and Zn, in particular, vary greatly across the four clay mixture leachates. Supplementation of a non-antibacterial leachate containing lower concentrations of Fe, Co, Ni, Cu, and Zn to final ion concentrations and a pH equivalent to that of the antibacterial leachate generated antibacterial activity against E. coli and MRSA, confirming the role of these ions in the antibacterial clay mixture leachates. Speciation modeling revealed increased concentrations of soluble Cu2+ and Fe2+ in the antibacterial leachates, compared to the non-antibacterial leachates, suggesting these ionic species specifically are modulating the antibacterial activity of the leachates. Finally, linear regression analyses comparing the log(10) reduction in bacterial viability to the concentration of individual ion species revealed positive correlations with Zn2+ and Cu2+ and antibacterial activity, a negative correlation with Fe3+, and no correlation with pH. Together, these analyses further indicate that the ion concentration of specific species (Fe2+, Cu2+, and Zn2+) are responsible for antibacterial activity and that killing activity is not solely attributed to pH.