Real-Time Metabolic Interactions between Two Bacterial Species Using a Carbon-Based pH Microsensor as a Scanning Electrochemical Microscopy Probe.

We have developed a carbon-based, fast-response potentiometric pH microsensor for use as a scanning electrochemical microscopy (SECM) chemical probe to quantitatively map the microbial metabolic exchange between two bacterial species, commensal Streptococcus gordonii and pathogenic Streptococcus mutans. The 25 μm diameter H+ ion-selective microelectrode or pH microprobe showed a Nernstian slope of 59 mV/pH and high selectivity against major ions such Na+, K+, Ca2+, and Mg2+. In addition, the unique conductive membrane composition aided us in performing an amperometric approach curve to position the probe and obtain a high-resolution pH map of the microenvironment produced by the lactate-producing S. mutans biofilm. The x-directional pH scan over S. mutans also showed the influence of the pH profile on the metabolic activity of another species, H2O2-producing S. gordonii. When these bacterial species were placed in close spatial proximity, we observed an initial increase in the local H2O2 concentration of approximately 12 ± 5 μM above S. gordonii, followed by a gradual decrease in H2O2 concentration (>30 min) to almost zero as lactate was produced, and a subsequent decrease in pH with a more pronounced metabolic output of S. mutans. These results were supported by gene expression and confocal fluorescence microscopic studies. Our findings illustrate that H2O2-producing S. gordonii is dominant while the buffering capacity of saliva is valid (∼pH 6.0) but is gradually taken over by S. mutans as the latter species slowly starts decreasing the local pH to 5.0 or less by producing lactic acid. Our observations demonstrate the unique capability of our SECM chemical probes for studying real-time metabolic interactions between two bacterial species, which would not otherwise be achievable in traditional assays.