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

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Department of Chemistry, Oregon State University, Corvallis, Oregon 97331, United States
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, United States
§ Department of Restorative Dentistry, Oregon Health and Science University, Portland, Oregon 97239, United States
*E-mail: [email protected]. Phone: +1-541-737-0791.
Cite this: Anal. Chem. 2017, 89, 20, 11044–11052
Publication Date (Web):September 18, 2017
Copyright © 2017 American Chemical Society

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    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.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.7b03050.

    • Schematics of the SECM setup, probe approach curve recorded with the pH microprobe on the bacteria hydrogel biofilm in 1 mM ferrocenemethanol in artificial saliva solution (pH 7.2), calibration of the pH microprobe in Britton–Robinson buffer, calibration of the pH microprobe in artificial saliva before and after the SECM experiment, calibration curve of the pH microprobe in the presence of 80 μM H2O2 and 1 mM RuHex, pH-dependent metabolic activity of S. gordonii and H2O2 sensor calibration, fixed-distance hydrogen peroxide and pH measurement 50 μm above the dual-bacteria (Sm–Sg–Sm) biofilm, calibration curve obtained in artificial saliva with the 2 μM LysoSensor yellow/blue dextran pH probe, viability of S. gordonii and S. mutans in alginate gel, RNA isolation, cDNA synthesis, and real-time PCR, and H+ concentration vs z-distance profile estimated from the z-direction pH profile obtained on the S. mutans (single biofilm) and S. gordonii in dual biofilm (PDF)

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