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

Abstract

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.

Figure 1.. Potentiometric characterization of the pH sensor:

(A) Schematics of H⁺ ion selective membrane electrode (H⁺ ISME or pH microprobe). Here, DOS represents dioctyl sebacate. (B) Calibration of pH microprobe in universal buffer from pH 10 to pH 4 and artificial saliva (AS) solution from pH 7.2 to 4.0. (C) pH microprobe calibration in artificial saliva in the presence of the redox mediator RuHex, ferrocenemethanol and with and without O₂ in AS solution. (D) Response time of the pH sensor.

Figure 2.. Amperometric characterization of pH sensor:

(A) Cyclic voltammogram recorded at the pH sensor in N₂-purged 1 mM RuHex in 0.1 M KCl. (B) Square wave voltammetric (SWV) curves recorded in various concentrations of RuHex in a 0.1 M KCl solution at the pH sensor. (C) Linear relation of current obtained from the SWV and i-t curves with various concentration of RuHex. (D) PAC recorded at the glass surface with a pH probe.

Figure 3.. 3D pH mapping above S. mutans biofilm:

(A) Schematic of S. mutans bacteria gel biofilm substrate and pH microprobe used in SECM experiments. (B) Z-directional pH profile mapping from 50 μm above the S. mutans biofilm to 1000 μm in the bulk solution after addition of 30 mM sucrose in artificial saliva (pH 6.0) at 37 °C. Scan speed was 6 μm/s. (C) 3D morphological image of the S. mutans bacteria gel biofilm substrate recorded 60 μm above the biofilm with pH microprobe (scan speed, 30 μm/s) in 1 mM ferrocenemethanol in AS (pH 7.2) at 23 °C. (D) 3D pH image recorded 60 μm above the biofilm with pH microprobe (scan speed, 30 μm/s) after addition of 30 mM sucrose in artificial saliva (pH 6.0) at 37 °C.

Figure 4.. Metabolic activity of S. gordonii and S. mutans in a dual biofilm in artificial saliva.

(A) Schematic of dual-bacteria gel biofilm substrate: S. gordonii surrounded by S. mutans (Sm) used in SECM experiments. White dashed line represents the x-direction probe scan curve recorded with H₂O₂ and pH ultramicrosensors to measure the H₂O₂ and pH profile on the dual biofilm. (B) X-direction pH profile 150 μm above the dual-bacteria biofilm in the presence of glucose (G), sucrose (S), and glucose+sucrose (G+S) at pH 6.0 and 7.2. Glucose and sucrose concentrations are 1 and 30 mM, respectively. (C) X-direction H₂O₂ profile 150 μm above the dual-bacteria biofilm in the presence of G and G+S at pH 6.0 and 7.2. (D) Z-direction H₂O₂ and pH profile from 50 μm above S. gordonii in dual-bacteria biofilm to 1000 μm above in the bulk solution in the presence of G+S at pH 6.0 (solid lines) and 7.2 (dashed lines).

Figure 5.. Confocal fluorescence pH mapping and spxB gene expression study.

(A) Confocal fluorescence pH mapping at the top and bottom of dual (S. mutans-S. gordonii-S. mutans) biofilm (average of two sets of experiments) after exposure to glucose + sucrose in pH 7.0 artificial saliva for 3 h at 37 °C (n = 3 for each set). (B) Representative image (n = 3) of H₂O₂ production of S. gordonii during aerobic growth at pH 7 and pH 6. (C) spxB gene expression in S. gordonii at pH 7 and after a shift from pH 7 to pH 6 determined at the indicated time intervals; mean and standard deviation are shown (n = 3). One-way analysis of variance; * p < 0.046.