2024 Volume 72 Issue 3 Pages 253-257
This study focused on the electrochemical properties of tetrazolium salts to develop a simple method for evaluating viable bacterial counts, which are indicators of drug susceptibility. Considering that the oxidized form of tetrazolium, which has excellent cell membrane permeability, changes to the insoluble reduced form formazan inside the cell, the number of viable cells was estimated based on the reduction current of the tetrazolium remaining in the bacterial suspension. Dissolved oxygen is an important component of bacterial activity. However, it interferes with the electrochemical response of tetrazolium. We estimated the number of viable bacteria in the suspension based on potential-selective current responses that were not affected by dissolved oxygen. Based on solubility, cell membrane permeability, and characteristic electrochemical properties of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, we developed a method for rapidly measuring viable bacteria within one-fifth of the time required by conventional colorimetric methods for drug susceptibility testing.
Bacteria that cause food poisoning and infectious diseases are detrimental to the society, making them a serious issue. Among them, Staphylococcus aureus is a common cause of serious nosocomial and community-acquired infections in medical settings.1,2) Adhesion can cause skin infections, and contaminants can accumulate in medical devices, such as artificial heart valves, artificial joints, cardiac pacemakers, and catheters inserted into blood vessels through the skin. Such accumulation can lead to infections of the heart valves, bones, and pneumonia.3–7) Because S. aureus poses a high risk of infection, its growth must be controlled. Penicillin was accidentally discovered in a blue mold in 1928 and has since been put into practical use as an antibiotic. Nearly a century later, penicillin is still considered the most common antibiotic.8–10) The cell wall of S. aureus is composed of alternating chains of N-acetylglucosamine and N-acetylmuramic acid, which are reinforced by the enzyme D-alanyl-D-alanine peptidase (DD peptidase). Normally, during bacterial growth, the DD-peptidase recognizes and incorporates the D-Ala-D-Ala structure, and the cross-linking reaction proceeds. However, bacteria mistakenly recognize penicillin G (PCG) as D-Ala-D-Ala, which inactivates DD peptidase and inhibits the cross-linking reaction (Fig. 1). In other words, PCG inhibits bacterial cell wall biosynthesis and ultimately lyses and kills bacteria. Common methods for testing the effectiveness of such antibiotics include incubating S. aureus (approximately 104–105 colony forming unit (CFU)·mL−1) at 308 K for 18 to 24 h in a medium containing a predetermined concentration of antibiotics, and the inhibition of bacterial growth is evaluated by checking the turbidity of the bacterial suspension.
We have previously developed a method to evaluate bacterial activity based on intracellular electron mediators.11) The reduced coenzymes nicotinamide adenine dinucleotide (NADH) and FADH2 exist in living cells and their function is to transfer electrons through redox reactions.12–15) The tetrazolium salt16–21) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), which is soluble and has excellent cell membrane permeability, permeates the cell membrane and forms a reduced complement in living cells.22–29) MTT receives electrons from the enzyme and is reduced to insoluble formazan, which is then deposited (Fig. 2). In our previous work, we determined the number of viable bacteria based on the reduction current of MTT remaining in the bacterial suspension and established an electrochemical method for evaluating the activity of viable bacterial species with different metabolic processes and cell wall structures.30) In the present study, we electrochemically measured the residual amount of MTT in the bacterial suspension with and without PCG and evaluated the effect of PCG based on the difference in MTT reduction. Furthermore, the effectiveness of this method for drug susceptibility testing was verified by comparison with the conventional turbidity method.
Ultrapure water (>18 MΩcm) was sterilized using an autoclave (393 K, 20 min). Reagent-grade MTT (Dojindo, Kumamoto, Japan) and animal-free penicillin G potassium salt (PCG; Nacalai Tesque, Kyoto, Japan) were used. All the reagents were purchased without further purification. After dissolving MTT in a small amount of dimethyl sulfoxide (5.0 µL), the MTT solution was adjusted to 20 mM with 0.10 M potassium chloride (KCl) aqueous solution. PCG salt (0.20 mg) was dissolved in sterilized water (0.020 mL) to prepare a 10 mg·mL−1 PCG solution. The PCG solution and 0.20% glucose-added nutrient broth medium (2.5 mL) were added to 2.5 mL of 0.10 M phosphate-buffered saline (PBS) solution (pH 7.4) to obtain 5.0 mL of 0.040 mg·mL−1 PCG solution.
Bacterial CultureStaphylococcus aureus was purchased from the National Institute of Technology and Evaluation Biological Resource Center (NBRC, Tokyo, Japan). Bacterial cultures were prepared, and all experiments were performed in a Biosafety Level 2 laboratory, and managed in accordance with the appropriate safety regulations (WHO Laboratory Biosafety Manual). The bacterial strain was cultured in agar growth medium (E-MC35, Eiken Chemical, Tokyo, Japan) at 310 K for 18 h. A single colony was selected, placed in liquid growth medium (5.0 mL), and incubated at 308 K for 5 h. The precipitate was dispersed in sterile ultrapure water, and the concentration of the S. aureus suspension was adjusted to 1.0 × 104 CFU·mL−1.
Electrochemical MeasurementsAll electrochemical experiments were conducted using a single-channel potentiostat electrochemical analyzer (CHI 840D, CH Instruments, Austin, TX, U.S.A.). A glassy carbon disk electrode (surface area: 0.28 cm2) was used as the working electrode, a platinum coil as the counter electrode, and Ag|AgCl as the reference electrode (EC Frontier Co., Ltd., Kyoto, Japan). The bacterial suspension (50 µL) was added to PBS (5.0 mL) with and without PCG and incubated at 308 K for a predetermined time. Electrochemical measurements were carried out in a bacterial sample (5.0 mL) containing 0.10 mM MTT solution (0.025 mL) before and after incubating the suspension at 308 K for 1 h. Cyclic voltammograms (CVs) were obtained by sweeping the potential from +0.20 V (vs. Ag|AgCl) toward the cathode at a scan rate of 100 mV·s−1.
Drug susceptibility testing typically evaluates bacterial growth inhibition in designated media. However, whether antibiotic-induced bacterial growth inhibition can be assessed in media containing MTT is generally unknown. Therefore, we evaluated turbidity to determine whether MTT had an effect on the inhibition of bacterial growth by antibiotics. Bacteria in liquid growth medium (1 × 104 CFU·mL−1) with and without PCG (40 µg·mL−1) were incubated for 6 h (308 K), and then MTT (0.10 mM) was added and cultured. Changes in turbidity of the suspension without PCG were visually observed using a turbidity meter (at 650 nm). A visual observation of turbidity revealed that the PCG-free suspension started to appear turbid after incubation for 5 h. The bacterial suspension immediately changed color from yellow to purple following the addition of MTT. However, no change in turbidity or color based on the MTT of the suspension was observed after treatment with PCG. Therefore, we conclude that S. aureus is a PCG-susceptible bacterium (at 40 µg·mL−1 PCG), and bacterial growth is suppressed only when PCG is added, regardless of the presence or absence of MTT. The evaluation of the changes in turbidity using a turbidity meter revealed that the turbidity of the PCG-free suspension increased after incubation for 4 h, which could not be observed with the naked eye (Fig. 3). The increase in turbidity was similar in the solutions with and without MTT. These results suggest that bacterial growth inhibition can be evaluated by PCG even in the presence of MTT.
Time represents the time elapsed after the addition of MTT.
Absorbance measurements were performed to examine whether the inhibition of bacterial growth by PCG could be evaluated based on residual MTT without bacterial uptake. Bacterial suspensions (1 × 104 CFU·mL−1) with and without PCG treatment were cultured for a predetermined time at 308 K. The suspensions were centrifuged (10000 × g, 278 K, 10 min) to obtain the supernatant and the absorbance was measured (Fig. 4). According to the absorbance measurement results, the absorbance of the bacterial suspensions decreased slightly and no difference was observed between the bacterial suspensions with and without PCG treatment after 5 h of incubation. A significant decrease in absorbance was observed in the suspension without PCG after incubation for 10 h. This indicates that the bacteria in the growth phase absorbed MTT in the bacterial suspension without PCG treatment, thereby making it possible to measure the reduction in residual MTT spectroscopically. In contrast, no decrease in MTT absorbance was observed in PCG-treated bacterial suspensions, indicating that the bacteria did not grow. These results indicate that growth inhibition by PCG can be evaluated using the residual MTT assay.
The supernatant was obtained by centrifuging the suspension (1 × 104 CFU·mL−1).
Dissolved oxygen often interferes with electrode reactions; however, it is an important factor in the survival of bacteria.31–35) In a previous study, a distinct current peak (Ic) obtained at −0.1 V (vs. Ag|AgCl) in the sweep range of −0.3 to 0.7 V, which is based on the reaction in which MTT was reduced to form the intermediate radical (MTTH+.),23) is not affected by dissolved oxygen and obtains constant current responses regardless of the number of sweeps.30) Quantitative assessment of cellular activity based on the current response of Ic within 1 h (103–107 CFU·mL−1) was achieved by measuring the amount of residual MTT before and after the addition of bacteria. In addition, the results revealed that the cell membrane permeability and intracellular reduction rate of MTT were the same regardless of the bacterial species, including enterohemorrhagic Escherichia coli K12, E. coli O26, and Salmonella typhimurium (facultatively anaerobic bacteria) as representative examples of Enterobacteriaceae; Pseudomonas fluorescens (obligately aerobic bacterium) as a glucose-non-fermentative Gram-negative bacillus; and S. aureus as a Gram-positive bacterium. This suggests that the current response to MTT is more dependent on bacterial activity than on attributes, such as respiration or cell membrane structure. Therefore, this method can be used to assess cell activity and viability regardless of the bacterial type, group, or biological function. In other words, the differences between the activated and PCG-inactivated bacteria can be evaluated.
Bacteria grow and proliferate in four stages: lag, logarithmic, stationary, and death phases.36) During the lag phase, the first phase of the bacterial growth cycle, bacteria take in medium components, activate their metabolism, and produce energy. The length of the lag phase is called lag time. During the logarithmic phase, the second phase of the bacterial growth cycle, bacterial cells use energy to divide into two daughter cells, which then undergo repeat division. The time required for bacteria to divide during the logarithmic phase is called the generation time. During the stationary phase, the third phase of the bacterial growth cycle, excessive accumulation of metabolites derived from bacterial metabolism causes environmental degradation, leading to a decrease in the cell division rate and an increase in the number of dead bacteria. Consequently, the total number of bacteria appears to be constant because both live and dead bacteria coexist. Finally, during the death phase, the fourth phase of bacterial growth, cell decay and death predominate. To evaluate the effectiveness of antibiotics, we used bacteria in the logarithmic phase, which contains more active cells than those in the stationary phase that has less active and dead cells.30) The electrochemical response of MTT in S. aureus suspension (1.6 × 108 CFU·mL−1) is shown in Fig. 5. The voltammogram obtained without incubation shows one pair of redox peaks for MTT. During incubation, the current response gradually decreased, indicating that MTT had moved into the cells, in turn, leading to a decrease in the net concentration of MTT in the suspension.11,30) The suspension, which was yellow before incubation, gradually started to change color and finally became a dark-purple suspension after 60 min of incubation. The MTT-based color of the supernatant obtained by centrifugation disappeared, suggesting that most MTT molecules were converted to formazan in the living cells. After 60 min of incubation, the electrochemical response almost disappeared.
The potential sweep ranges from −0.3 to 0.7 V. The potential sweep direction is indicated by arrows. The scan rate was 100 mV·s−1.
The electrochemical responses of the viable and inactivated bacteria were examined. Voltammograms obtained before and after incubation for 1 h with the addition of MTT to the bacterial fluids that had been incubated with and without PCG for 5 h, are shown in Fig. 6. The current response of MTT with PCG treatment decreased by 0.045 µA, whereas that of MTT without PCG treatment decreased by 0.96 µA. This was because PCG treatment inhibited cell proliferation and reduced MTT uptake by the bacteria. The current response of MTT to the viable and inactivated bacterial suspensions at each incubation time is shown in Fig. 7. The difference in current responses between the viable and inactivated bacteria increased with an increase in the culture time. The observation suggests that the cells entered the logarithmic phase from the lag phase after being cultured for a few hours and cell activity increased with an increase in the culture time. Estimation of the growth rate of S. aureus in the 0.10 mM MTT solution by colony counting revealed that the lag time was 1.2 h and generation time was 22 min. Therefore, the number of bacteria was estimated to be more than 4000 times the initial value after 5 h of incubation. However, no change was observed in the current response of the suspension containing PCG. Significant differences in the current responses of the viable and inactivated bacteria were observed after incubation for 2 h, which was much faster than spectroscopic detection that requires a 10-h incubation period. Therefore, evaluation of bacterial activity based on the electrochemical properties of MTT is a new effective evaluation method that can replace traditional turbidity and MTT colorimetric methods.
The bacterial suspensions with and without PCG were cultured for 5 h at 308 K.
Error bars indicate the differences between electrodes (n = 4).
We developed a new electrochemical method using an MTT assay to assess bacterial viability during drug susceptibility testing with the antibiotic PCG. Based on the characteristic electrochemical properties of MTT, the differences between live and PCG-inactivated bacteria can be assessed within 2 h. Unlike existing technologies, which require long inspection times (18–24 h), the method developed in this study is expected to be used for the rapid evaluation of antibiotics. The development of effective and accurate testing technology not only facilitates drug susceptibility testing, but also ensures safety at food and drug manufacturing sites, thereby contributing to a safe, secure, and comfortable life.
This study was financially supported by the JST START Grant (Grant No. JPMJST1916). We gratefully acknowledge the financial support provided by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (A) (KAKENHI Grant No. 21H04963) and Grants-in-Aid for Challenging Exploratory Research (Grant No. 22K18442).
The authors declare no conflict of interest.
