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.

Scanning electrochemical microscopy and its potential for studying biofilms and antimicrobial coatings

“…The same group mapped with the carbon-based pH microsensor topography and pH profiles of pathogenic Streptococcus mutans biofilms, which belong to acid-producing bacteria as shown in Fig. 5a–c [ 88 ]. The authors also studied the metabolic interplay between the H 2 O 2 -producing S. gordonii and S. mutans organized in an alginate gel (Fig.…”

“…( d ) z -direction H 2 O 2 and pH profile from 50 μm above S. gordonii in the dual bacterial 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). ( b ), ( c ), ( e ), ( f ) Reprinted from Joshi et al [ 88 ], with permission from the American Chemical Society (copyright 2017) …”

“…2d ). Although pH changes are frequently studied with fluorescence probes as spatiotemporal resolution can be achieved [ 83 , 84 ], potentiometric probes for pH measurements such as Sb/SbO 2 [ 85 ], Ir/IrO x [ 86 ], polyaniline [ 87 ] and ultramicroelectrodes (UME) and carbon-based pH microsensors [ 88 ] are attractive as no labelling is required. Also, dual ultramicroelectrodes (Fig.…”

“…Joshi et al [88] and Ummadi et al [106] recently presented potentiometric microsensors that could be also used for amperometric measurements (e.g. for positioning the microsensor in SECM experiments) by mixing ionophores with conductive carbon materials.…”

Biological imaging with scanning electrochemical microscopy

“…SECM study entries into the literature databases that underline well the wide applicability of scanning electrochemical probe-based cellular analysis are examinations of the differentiation status of embryonic stem cells [72], evaluation of the respiration activity of blastocysts [73], monitoring intracellular enzyme activity of HeLa cells [74], analysis of beat fluctuations and oxygen consumption of cardiomyocytes [75], characterization of Ag + toxicity on fibroblast cells [76], inspection of the multi-drug resistance of adenocarcinoma cervical cancer cells [77], the detection of reactive oxygen species in prostate cancer cells [78], the creation of insulin release profiles of single pancreatic islets [79] and the disclosure of Cd 2+ -induced variability in the permeation properties of human bladder cancer cells [80,81]. Recent activities also addressed topics such as SECM-based inspections of heavy metal (chromium) cell toxicity [82,83], quantitative detection of protein markers for lung cancer [84], real-time monitoring of metabolic crosstalk between neighbouring bacterial cells [85], spatial mapping of molecular uptake of chemical species at living cells [59] and the pH dependence of yeast cell viability and metabolism [66].…”