Helicobacter pylori Eradication Using a Light-Emitting Diode and Methylene Blue

Abstract

Background and aims

Eradication failures are increasing with the increasing antibiotic resistance of Helicobacter pylori.

We examined the basic effect of methylene blue (MB) with sodium bicarbonate (NaHCO₃) on H. pylori eradication using antimicrobial chemotherapy activated by light.

Material and methods

When NaHCO₃ was added to MB, the pH became basic. We smeared H. pylori on a medium with basic MB and irradiated it using a red light-emitting diode with a wavelength of 660 nm. The applied energy fluencies were 10 J/cm² and 15 J/cm². After 4 days of culture, the effect of this intervention was determined according to the bacterial growth area.

Results

The basic effect of MB appeared between a pH of 8.6 and 9.0. The NaHCO₃ concentration was between 4% and 6%. The basic effect at 15 J/cm² was greater than that at 10 J/cm².

Conclusions

We concluded that antimicrobial chemotherapy activated by light with basic MB was effective in H. pylori eradication.

Introduction

Helicobacter pylori was first reported by Barry J Marshall and J. Robin Warren in 1984 ¹⁾. H. pylori are spiral or curved bacilli that are gram-negative, flagellate, and microaerophilic ¹⁾. These bacteria are present in almost all patients with active chronic gastritis, duodenal ulcers, or gastric ulcers ¹⁾. H. pylori is considered as an important etiology of these diseases.

The combination of eradication therapy with proton pump inhibitors and antibiotics is used worldwide. However, because of an increase in antibiotic-resistant bacteria, it has become difficult to achieve complete eradication.

Therefore, a new eradication method that is independent of antibiotic resistance is required to destroy the resistant bacteria.

MB generates active oxygen in the presence of light energy ²⁾. We developed basic pH conditions by adding NaHCO₃ to MB and consequently devised a new photochemical eradication method using basic MB. We then performed antimicrobial chemotherapy activated by light and verified its bactericidal effect on H. pylori.

Material and methods

We used H. pylori strain JCM No.12093, which was obtained from National Research and Development Agency, Institute of Physical and Chemical Research ( Japan). H. pylori was identified using the API Campy kit (SYSMEX bioMerieux Co., Ltd.).

We used 500 mL of stock solution containing 25 g of MB (Wako Pure Chemical Industries, Ltd.) and NaHCO₃, which was purchased from Nichi-Iko Pharmaceutical Co., Ltd.

We regulated the MB concentrations (0.01%, 0.05%, 0.1%, 0.2%, 0.5%, and 1%) in a sterile test tube with sterile water for injection. We also regulated the NaHCO₃ concentrations (2%, 3%, 4%, 5%, 6%, and 6.5%) similarly.

We adjusted the H. pylori count to 2.0 × 10⁸ cells/ml in sterile physiological saline in a sterile test tube.

Using a sterile pipette tip, 100 µl of MB, 20 µl of H. pylori , and 10 µl of NaHCO₃ were taken out of the test tubes and smeared on a helicobacter agar medium (Nissui Pharmaceutical Co., Ltd.) using a sterile bacteria spreader.

The added NaHCO₃ was diluted 13-fold in the medium, and the final concentrations were 0.15%, 0.23%, 0.30%, 0.38%, 0.46%, and 0.50%.

After placing it in the dark for 5 min, the bacterial culture was irradiated from a distance of 5 cm using a red light-emitting diode (LED; CCS Inc.) with a wavelength of 660 nm. The energy fluencies were 10 J/cm² (at 2 min 40 s) and 15 J/cm² (at 4 min).

After irradiation, we placed the culture in an incubator at 37°C for 4 days. The bactericidal effect was evaluated according to the growth area of H. pylori, which was categorized into six grades.

The criteria for effectiveness were as follows:

The bactericidal effect in the absence of irradiation and MB was ineffective.

The bactericidal effect of irradiation in the absence of MB was ineffective.

The blank test included the bacteria and each MB concentration with no irradiation.

(−) indicated ineffectiveness.

(1+), (2+), (3+), and (4+) indicated a decrease in growth areas by 20%, 40%, 60%, and 80%, respectively, compared with the blank test using an ineffective concentration of 0.01% MB.

(5+) indicated no growth area.

We added 2000 µl of MB, 400 µl of sterile physiological saline, and 200 µl of NaHCO₃ to a test tube. We measured the pH using Seven Compact instruments ( METTLER TOLEDO International Inc.).

Results

Table 1 shows the pH of MB (C₁₆H₁₈ClN₃S) ³⁾, sodium bicarbonate (NaHCO₃) ⁴⁾, and MB with the addition of NaHCO₃.

As the MB concentration increased from 0.01% to 1%, the pH decreased from 7.435 to 5.890.

As the NaHCO₃ concentration increased from 2% to 6.5%, the pH decreased from 8.141 to 8.045.

When NaHCO₃ was added to MB, the pH increased to in the range of 8.550–9.0.

Table 2 shows that, at an energy fluence of 10 J/cm², the MB concentration of > 0.2% was graded as 5+. With the addition of 2% and 3% NaHCO₃, the MB concentration of > 0.2% was graded as 5+. With the addition of 4% and 6% NaHCO₃, the MB concentration of > 0.1% was graded as 5+.

With the addition of 5% NaHCO₃, the MB concentration of > 0.05% was graded as 5+.

With the addition of 6.5% NaHCO₃, the MB concentration of > 0.2% was graded as 5+. The effects on these media are shown in Figures 1, 2, and 3.

Table 3 shows that, at an energy fluence of 15 J/cm², the MB concentration of > 0.05% was graded as 5+. With the addition of 2% and 3% NaHCO₃, the MB concentration of > 0.05% was also graded as 5+.

Table 1: The pH of MB with the addition of NaHCO₃.

NaHCO3 concentration (%)	MB concentration%
	0	0.01	0.05	0.1	0.2	0.5	1
0		7.435	6.997	6.856	6.962	6.505	5.89
2	8.141	8.55	8.574	8.589	8.62	8.708	8.869
3	8.109	8.598	8.612	8.617	8.658	8.748	8.939
4	8.082	8.605	8.619	8.672	8.653	8.782	9
5	8.037	8.6	8.617	8.632	8.674	8.785	8.929
6	8.03	8.617	8.624	8.628	8.671	8.777	8.972
6.5	8.045	8.668	8.667	8.701	8.739	8.849	9

Table 2: The basic effect of MB with the addition of NaHCO₃ at 10 J/cm².

NaHCO3 concentration (%)	MB concentration%
	0	0.01	0.05	0.1	0.2	0.5	1
0	—	(4+)	(4+)	(4+)	(5+)	(5+)	(5+)
2	—	(4+)	(4+)	(4+)	(5+)	(5+)	(5+)
3	—	(3+)	(4+)	(4+)	(5+)	(5+)	(5+)
4	—	(3+)	(4+)	(5+)	(5+)	(5+)	(5+)
5	—	(4+)	(5+)	(5+)	(5+)	(5+)	(5+)
6	—	(3+)	(4+)	(5+)	(5+)	(5+)	(5+)
6.5	—	(3+)	(4+)	(4+)	(5+)	(5+)	(5+)
blank test (no irradiation)	—	(—)	(2+)	(3+)	(4+)	(5+)	(5+)

Table 3: The basic effect of MB with the addition of NaHCO₃ at 15 J/cm².

NaHCO3 concentration (%)	MB concentration%
	0	0.01	0.05	0.1	0.2	0.5	1
0	—	(4+)	(5+)	(5+)	(4+)	(5+)	(5+)
2	—	(4+)	(5+)	(4+)	(5+)	(5+)	(5+)
3	—	(3+)	(5+)	(5+)	(5+)	(5+)	(5+)
4	—	(5+)	(4+)	(5+)	(5+)	(5+)	(5+)
5	—	(5+)	(5+)	(5+)	(5+)	(5+)	(5+)
6	—	(5+)	(5+)	(5+)	(5+)	(5+)	(5+)
6.5	—	(4+)	(4+)	(5+)	(5+)	(5+)	(5+)
blank test (no irradiation)	—	(—)	(2+)	(3+)	(4+)	(5+)	(5+)

Figure 1:

No irradiation (blank test)

Figure 2:

The effect of MB irradiated at the fluence of 10 J/cm²

Figure 3:

The basic effect of MB with the addition of 5%NaHCO₃ irradiated at the fluence of 10 J/cm²

With the addition of 4%–6% NaHCO₃, the MB concentration of > 0.01% was graded as 5+.

With the addition of 6.5% NaHCO₃, the MB concentration of > 0.1% was graded as 5+.

The bactericidal effect was attenuated compared with that of MB alone. The effects on these media are shown in Figures 4, 5, and 6.

Figure 4:

No irradiation (blank test)

Figure 5:

The effect of MB irradiated at the fluence of 15 J/cm²

Figure 6:

The basic effect of MB with the addition of 5%NaHCO₃ irradiated at the fluence of 15 J/cm²

Discussion

The chemical structure of MB ³⁾ is shown in Figure 7. In general, dyes are divided into acidic, basic, and amphoteric types ⁵⁾. Acidic dyes are negatively charged in an aqueous solution ⁵⁾, whereas basic dyes are positively charged ⁵⁾. Amphoteric dyes have both properties and maintain an equilibrium state ⁵⁾.

Figure 7:

The chemical structure of MB (C₁₆H₁₈ClN₃S)

MB is a water-soluble and basic dye ⁵⁾. The positive charge of MB binds to a bacterium containing a negative charge ⁵⁾. Stainability is enhanced in the basic state ^{5, 6)}.

We demonstrated the relationships between the minimal MB concentration for achieving a grade 5+ bactericidal effect, NaHCO₃ concentration, energy fluence, and pH (Figures 8 and 9). The pastel part of the figure is the region where a basic effect appeared and increased. We estimated that the basic effect of MB was present at 4%–6% NaHCO₃ concentration and a pH of 8.6–9.0 at 10 J/cm² and 15 J/cm². The actual NaHCO₃ concentration was in the range of 0.30%–0.46%.

Figure 8:

The basic effect at 10 J/cm²

•The minimal MB concentration for achieving a grade 5+ bactericidal effect

Figure 9:

The basic effect at 15 J/cm²

•The minimal MB concentration for achieving a grade 5+ bactericidal effect

Figure 10 depicts the comparison of the basic effects of the two energy fluencies. The basic effect appears most strongly at pH 8.6. At the 4%–6% NaHCO₃ concentration, the increasing energy decreases the MB's effective minimal concentration to 0.01%. The basic effect is enhanced by the increasing the energy.

Figure 10:

Comparison of the basic effects between Figures 8 and 9

•Comparison of the minimal MB concentration for achieving the grade 5+ bactericidal effect observed in Figures 8 and 9.

The basic effect of MB is related to the following features:

1. NaHCO₃ concentration

2. basic pH

3. adjustment and balance

It is estimated that the basic effect appears and increases when the adjusted NaHCO₃ concentration and the basic pH are balanced.

Even when MB is sprayed in the stomach, the normal gastric mucosa is not stained ⁷⁾. In cases of intestinal metaplasia, it is absorbed from the intestinal epithelium ⁷⁾ and is thus, useful for the diagnosis of intestinal metaplasia.

NaHCO₃, the proteolytic enzyme pronase, and stomach mucus antifoaming agents are orally administered in the form of aqueous solutions as a pretreatment during pigment endoscopy ⁸⁾. In such cases, NaHCO₃ is used to increase the enzyme activity and decrease the mucus viscosity ⁹⁾.

This is because the optimal pH of the proteolytic enzyme is between 7 and 10, and the enzyme activity is unstable under acidic conditions ⁹⁾.

After sufficiently suctioning the dissolved mucus using endoscopic forceps, MB with NaHCO₃ is sprayed. Light is irradiated from the endoscopic light source after these procedures. NaHCO₃ is administered twice to achieve a basic pH because of the following reasons:

1. the optimal pH of pronase and

2. the enhancement of MB stainability.

The generation and maintenance of basic pH is particularly important for this new photochemical therapy.

Conclusions

Our eradication method is effective against antibiotic-resistant H. pylori and does not result in the production of resistant bacteria. This new photochemical method is a viable option for the eradication of H. pylori.

Endoscopic H. pylori eradication using basic MB can be applied. We propose that this method is termed as endoscopic antimicrobial chemotherapy activated by light.

Authors

Kouji Ogasawara: Sarufutsu Village National Health Insurance Hospital, Japan

Asahikawa Medical University, Respiratory Center, Japan

Yoshinobu Osaki: Asahikawa Medical University, Respiratory Center, Japan

Mizuki Sasaki: Asahikawa Medical University, Parasitology course, Japan

Susumu Nakajima: Moriyama Memorial Hospital, Japan

Hiroshi Sato: Sarufutsu Village National Health Insurance Hospital, Japan