Novel MAXPOWER biological antibacterial liquid for eradicating oral Helicobacter pylori – BMC Infectious Diseases

Materials

Trimethoprim lactate (T9170), vancomycin (V8050), amphotericin B (A8251), and polymyxin B sulfate (P8350) were purchased from Sigma‒Aldrich Corp. (Shanghai, China). The Cell Counting Kit-8 (CK04), Urease Activity Assay Kit (No. BC4110), and Live/Dead Cell Staining Kit (No. 40747ES76) were purchased from Yeasen Biotechnology Co. Ltd. (Shanghai, China). The Urea Test Kit was purchased from Shandong Boan Biotech Co. Ltd. (Nanjing, China). The SYTO 9/PI Live/Dead Bacterial Double Stain Kit (No. MX4234-40T) was purchased from Shanghai Moukang Biotechnology Co. (Shanghai, China). The bicinchoninic acid (BCA) protein assay kit (No. 23,227) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The H. pylori strain was provided by the Department of Gastroenterology of the First Affiliated Hospital of Nanchang University (Jiangxi, China).

Cell culture and H. pylori strains

Mouse fibroblasts (L929) were purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin/streptomycin in an incubator under a 5% CO₂ atmosphere at 37 °C. The H. pylori strain CagA + ATCC43504 used in the present study was provided by Prof. Chuan Xie of the First Affiliated Hospital of Nanchang University. All H. pylori strains were stored at -80 °C. Before use, they were revived and cultured for two generations on blood agar plates containing 5% (v/v) sheep blood and 1% (w/v) mixed antibiotics under a 10% CO₂ and 5% O₂ atmosphere at 37 °C. The H. pylori liquid culture system comprised Brucella broth, 10% (v/v) FBS, and 0.5% (w/v) mixed antibiotics, and the cultures were incubated on microshakers.

Mice

All animal experiments were conducted per the protocols approved by the Laboratory Animal Center of Changhai Hospital of Naval Medical University (Shanghai, China) (SYXK2020-0033) and are reported in accordance with ARRIVE guidelines. Kunming mice were purchased from the Shanghai Laboratory Animal Center (Shanghai, China) and maintained under specific pathogen-free (SPF) conditions at the Institute of Pancreatic Diseases of Shanghai Changhai Hospital (Shanghai, China). They were housed at ~ 55% relative humidity and 18–22 °C under a 12-h light/12-h dark cycle.

In vitro cytocompatibility

In vitro cytocompatibility was assessed by Cell Counting Kit-8 (CCK-8) and live/dead cell staining assays. L929 cells were seeded in 96-well plates at a density of 10⁴/well. The cells were allowed to adhere overnight, and different concentrations of MAXPOWER biological antibacterial solution (original solution, 1:1, 1:2, 1:4, 1:8, 1:16) were added to the culture medium to explore the optimal concentration with cell safety. The cells were then incubated for 24 h, and each culture medium was replaced with 100 µL of 10% (w/v) CCK-8 dye solution per well. Incubation was then resumed for another 2 h. Cell viability was calculated by measuring the absorbance of each well at 450 nm (OD₄₅₀) in a microreader (SpectraMax® i3; Molecular Devices LLC, San Jose, CA, USA). There were three replicate wells per group, and cell viability was calculated as shown in Eq. (1) below:

The live/dead cell staining assay was performed by coculturing the MAXPOWER Biological Antibacterial Liquid with the L929 cells and then replacing the medium with calcein AM and propidium iodide (PI) dyes at 37 °C for 30 min. The cells were then observed under an inverted-phase contrast microscope (DMIL LED, Leica Microsystems, Wetzlar, Germany).

Hemolysis evaluation

Fresh blood was collected from the rats, centrifuged at 5,000 rpm for 3 min, and washed thrice with phosphate-buffered saline (PBS). Then, 2 mL of blood was diluted to 50 mL with saline, and 300 µL of the diluted blood was combined with 1.2 mL of saline solution containing various MAXPOWER Biological Antibacterial Liquid concentrations (original solution, 1:1, 1:2, 1:4, 1:8, 1:16). The negative and positive controls were prepared by mixing 300 µL erythrocytes with 1.2 mL saline and 1.2 mL deionized water, respectively. The mixtures were incubated at 37 °C for 2 h, and the supernatant absorbance was measured in a microplate reader at 540 nm (OD₅₄₀). The rate of hemolysis was calculated as shown in Eq. (2) below:

Antibacterial assay against H. pylori

H. pylori were harvested in Brucella broth, and their density was adjusted to 10⁶ CFU/mL. The H. pylori suspension and 2 mL MAXPOWER Biological Antibacterial Liquid were added to a liquid culture system consisting of 5 mL Brucella broth, 500 µL FBS, and 25 µL mixed antibiotics (40 mg of doxycycline, 60 mg of metronidazole, 40 mg of amphotericin B, and 50 mg of vancomycin dissolved in 200 ml of sterile distilled water). The mixtures were then incubated under a 10% CO₂ and 5% O₂ atmosphere at 37 °C with shaking at 150 rpm for 0.5 min, 1 min, and 1.5 min. After diluting the suspension 200-fold, spread it on Columbia agar plates and then incubate for 3–4 days before counting. The absorbances of the suspensions were also measured at 600 nm (OD₆₀₀).

Live/dead fluorescence staining

H. pylori were harvested in Brucella broth, and their density was adjusted to 10⁶ CFU/mL. Then, 1 mL H. pylori was cocultured with 2 mL MAXPOWER Biological Antibacterial Liquid (a concentration of 25% of the original solution) for different lengths of time (0.5 min, 1 min, 1.5 min). Then, SYTO 9/PI Live/Dead Bacterial Double Staining Reagent was added to the suspensions, and the mixtures were incubated under low light for 15 min. Finally, the suspensions were photographed under an orthofluorescence microscope (No. MF43-N; Micro-Shot Technology (MSHOT), Guangzhou, China).

Antibacterial mechanism study

Helicobacter pylori was dispersed in Brucella broth and incubated with PBS (control group) or MAXPOWER Biological Antibacterial Liquid (a concentration of 25% of the original solution) for 0.5 min, 1 min, and 1.5 min. Each sample was then centrifuged at 5,000 rpm for 10 min, and the supernatant was removed. (i) H. pylori morphological examination by scanning electron microscopy (SEM). The supernatant-free bacteria were fixed with 2.5% (v/v) glutaraldehyde and incubated at 4 °C overnight. The bacterial suspensions were then centrifuged at 3,000 rpm for 10 min and dehydrated in a gradient of 50% (v/v), 70% (v/v), 90% (v/v), and 100% (v/v) ethanol for 10 min/step. The samples were then freeze-dried and sputter-plated with gold. The morphology of H. pylori was observed under SEM, and 6 visual fields from each group were randomly selected to count the percentage of fragmentation bacteria to the total number of bacteria in that field of view. ii) H. pylori protein leakage. After aspiration of the above suspension, the leaked protein concentration was measured using the BCA Kit according to the instructions provided in the manual and detected using a spectrophotometer at 562 nm. iii) Urease activity of H. pylori. The urease activity of H. pylori subjected to MAXPOWER Biological Antibacterial Liquid (a concentration of 25% of the original solution) for different times (0.5 min, 1 min, 1.5 min) was measured with a Urease Kit and Urease Detection Solution (0.9% (w/v) NaCl, 20 mM urea, and 14 µg/mL phenol red). The OD₅₅₀ was measured in a spectrophotometer.

Gut microbiota analysis and in vivo animal tissue safety assessment

Specific pathogen-free (SPF) male Kunming (KM) mice (6–7 weeks old, body weight 20–25 g) were assigned to the control and experimental groups (n = 6/group). The experimental group was administered MAXPOWER Biological Antibacterial Liquid (a concentration of 25% of the original solution) by gavage at a rate of 200 µL/mouse/d every morning. Gavage was administered on alternate days, and there were four gavages. The control was administered 200 µL of 0.9% saline solution. The mice had ad libitum food access, and their weight was recorded regularly. On the morning following the final treatment, place the mice in a clean, sterile container to collect fresh fecal samples, and the number and diversity of the bacteria in them were determined by 16 S rRNA sequencing performed at Shanghai Daixuan Biotechnology Co. (Shanghai, China). The mice were euthanized on day 28 after gavage, and their sera were collected for biochemical and routine blood tests. Hearts, livers, spleens, lungs, and kidneys were excised, dissected, and stained with hematoxylin-eosin (HE) to assess the safety of MAXPOWER Biological Antibacterial Liquid.

Transcriptomic analysis

Helicobacter pylori (OD₆₀₀ = 1) was coincubated with PBS for 2 min and MAXPOWER Biological Antibacterial Liquid (a concentration of 25% of the original solution) for 2 min, collected by centrifugation and subjected to high-throughput sequencing. The experiment utilized the TruSeq™ Stranded Total RNA Library Prep Kit (Illumina, 20,020,594) for library construction. Briefly, after removing rRNA, mRNA fragments were obtained by adding a fragmentation buffer. Subsequently, cDNA was synthesized by reverse transcription, and adaptors were ligated using End Repair Mix. Before PCR amplification, the second cDNA strand was digested using the UNG enzyme to ensure that the library only contained the first cDNA strand. Finally, sequencing was performed on the NovaSeqXPlus platform and the DNBSEQ-T7 platform. The bioinformatics data were analyzed using the Majorbio Cloud Platform (Major Biomedical Technology Co., Shanghai, China).

Clinical study design

A prospective, randomized clinical pilot study was designed and performed per the Declaration of Helsinki, and informed consent was obtained from all patients. This study was retrospectively registered in the ClinicalTrials.gov Trial Registry on 21/09/2023 (NCT06045832). Mouthwash is a medical device and does not require ethics committee approval. The patients enrolled in the trial were oral H. pylori-positive and aged 18–70 y and had visited the outpatient clinic of Changhai Hospital between March 1, 2023, and August 1, 2023. The exclusion criteria were as follows: [1] Zollinger-Ellison syndrome, gastric cancer, upper gastrointestinal bleeding, or active peptic ulcer; [2] coexistence of significant concomitant illnesses including heart diseases, renal failure, hepatic diseases as well as previous abdominal surgery, lactation, or pregnancy; and [3] refusal to participate in the trial. Written informed consent was obtained from all patients before their participation.

The sample size of this study was calculated using PASS (Version11.0.7) according to Z-Test for two proportions. At least 34 patients would be required in randomized clinical pilot study based on power (1 − β), alpha (significance level), and effect size were 0.80, 0.05, and 0.45, respectively. Assuming a follow-up loss rate was 20%, the final sample size of this study was calculated as 42 patients.

At the start of the study, the demographic characteristics of all participants were recorded in detail and included sex, age, history of tobacco smoking, history of alcohol consumption, presence of halitosis, and GI symptoms. SPSS v. 25.0 (IBM Corp., Armonk, NY, USA) was used to randomize the patients to be administered either Novel MAXPOWER Biological Antibacterial Liquid (a concentration of 25% of the original solution) or water. The randomization ratio was 1:1. All patients were instructed to brush their teeth only once in the morning and before bedtime as usual for 7 days. Additionally, they were asked to rinse their mouth with Novel MAXPOWER Biological Antibacterial Liquid 3×/d for 2 min each time each time. Upon waking up on the morning of the 8th day, oral H. pylori saliva antigen (HPS) testing was immediately performed to detect oral H. pylori [17].

The primary outcome of the study was the oral H. pylori eradication rate based on intention-to-treat (ITT) and per-protocol (PP) analyses.

Statistical analysis

All data are presented as the means ± standard deviations (SD) and were analyzed with SPSS v. 25.0 and GraphPad Prism v. 9.5 (GraphPad Software, La Jolla, CA, USA). p