Dihydrotanshinone I Is Effective against Drug-Resistant Helicobacter pylori In Vitro and In Vivo

Helicobacter pylori is a major global pathogen and has been implicated in gastritis, peptic ulcer, and gastric carcinoma. The efficacy of the extensive therapy of H. pylori infection with antibiotics is compromised by the development of drug resistance and toxicity toward human gut microbiota, which urgently demands novel and selective antibacterial strategies.

ABSTRACT

Helicobacter pylori is a major global pathogen and has been implicated in gastritis, peptic ulcer, and gastric carcinoma. The efficacy of the extensive therapy of H. pylori infection with antibiotics is compromised by the development of drug resistance and toxicity toward human gut microbiota, which urgently demands novel and selective antibacterial strategies. The present study was mainly performed to assess the in vitro and in vivo effects of a natural herbal compound, dihydrotanshinone I (DHT), against standard and clinical H. pylori strains. DHT demonstrated effective antibacterial activity against H. pylori in vitro (MIC_50/90, 0.25/0.5 μg/ml), with no development of resistance during continuous serial passaging. Time-kill curves showed strong time-dependent bactericidal activity for DHT. Also, DHT eliminated preformed biofilms and killed biofilm-encased H. pylori cells more efficiently than the conventional antibiotic metronidazole. In mouse models of multidrug-resistant H. pylori infection, dual therapy with DHT and omeprazole showed in vivo killing efficacy superior to that of the standard triple-therapy approach. Moreover, DHT treatment induces negligible toxicity against normal tissues and exhibits a relatively good safety index. These results suggest that DHT could be suitable for use as an anti-H. pylori agent in combination with a proton pump inhibitor to eradicate multidrug-resistant H. pylori.

INTRODUCTION

Helicobacter pylori is a common human pathogen colonizing approximately half of the world’s population (1). Infection with H. pylori causes chronic gastritis and peptic ulcer disease and significantly increases the risk of developing gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer (2, 3). Many studies have demonstrated that eradication of H. pylori could prevent relapse and accelerate the healing of peptic ulcer disease (4) and, more importantly, reduce the incidence of gastric cancer (5, 6). In general, the standard triple therapy combining two broad-spectrum antibiotics with a proton pump inhibitor (PPI) is still recommended as a first-line regimen for H. pylori eradication (7). However, H. pylori eradication rates have been increasingly falling due to the rapid emergence of antibiotic-resistant H. pylori strains and poor patient compliance (8, 9). Besides genetic mutation, the ability of H. pylori to form biofilm has been shown to contribute to drug resistance in conventional therapy (10, 11). Hence, it is of great significance to develop novel and efficient anti-H. pylori agents with potentially diminished resistance and antibiofilm activity for improving the treatment success rates in clinical practice.

Danshen, the dried root and rhizome of Salvia miltiorrhiza Burge, is a well-known traditional Chinese medicine herbal remedy for treating cardiovascular- and cerebrovascular-related disorders (12, 13) as well as preventing inflammation (14). With a relatively good safety profile, it has been officially listed in the Chinese Pharmacopeia (2015 edition), U.S. Pharmacopeia (USP39-NF34), and European Pharmacopeia (EP6.1) (52–54). More recently, a phase III clinical trial has been completed with T89 (Dantonic; also known as compound danshen dripping pills) to evaluate its efficacy and safety in patients with chronic stable angina pectoris (Clinicaltrials.gov identifier NCT01659580). To date, the main bioactive constituents of danshen have been well identified, including lipid-soluble diterpene quinone pigments known as tanshinones (15) and water-soluble compounds that mainly have a phenolic acid structure (16) (danshensu and salvianolic acids A to K, etc.). Phenolic acids possess antioxidant and anticoagulant activities (17), whereas tanshinones show antibacterial, antioxidant, antiatherosclerosis, and antitumor activities (18, 19).

Among the tanshinone derivatives, tanshinone IIA (TIIA) (20), tanshinone I (TI) (21), 15,16-dihydrotanshinone I (DHT) (22), and cryptotanshinone (CT) (23) are the most extensively studied. They have been shown to exhibit moderate antibacterial activity against some Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus (24, 25). Thus, the objective of the present study was to investigate whether tanshinone derivatives have anti-H. pylori activities. We report that DHT possesses strong in vitro antibacterial and antibiofilm activities against various standard and drug-resistant strains of H. pylori. Furthermore, in vivo efficacy studies were performed to demonstrate significant clearance of multidrug-resistant H. pylori with inhibited toxicity against normal tissues after dual therapeutic dosing with DHT and a PPI. These results suggest that DHT may represent a promising, effective, and safe therapeutic agent to eradicate multidrug-resistant H. pylori.

RESULTS

In vitro antimicrobial activity of DHT.

The anti-H. pylori potential of 13 tanshinone derivatives was assessed using a broth microdilution assay against all 25 strains. The MIC_50/90 values of these compounds against all H. pylori isolates ranged from 0.25 to 64 μg/ml (Table 1; see also Table S1 in the supplemental material). Six tanshinone derivatives appeared to have efficient anti-H. pylori activities with MIC₉₀ values of <4 μg/ml, including dihydrotanshinone I, dihydroisotanshinone I, tanshinone I, isotanshinone I, tanshinone IIA, and cryptotanshinone (Fig. 1). In particular, the MIC_50/90 values of DHT were 0.25/0.5 μg/ml (range, 0.125 to 0.5 μg/ml), indicating that DHT showed the most potent activity. Hence, we focused on DHT after screening the tanshinone derivatives against H. pylori.

TABLE 1.

Antibacterial activities of tanshinone derivatives against H. pylori standard strains and drug-resistant clinical isolates^a

Compoundb	MIC (μg/ml)
	Range	50%	90%
DHT	0.125–0.5	0.25	0.5
DHIT	0.125–1	0.25	0.5
TI	0.25–4	1	2
ITI	0.25–4	2	4
TIIA	0.5–4	1	2
ITIIA	2–16	4	8
CT	0.5–2	1	2
ICT	8–32	32	32
NCT	32–64	64	64
MET	16–64	32	32
THT	4–16	8	8
TIIB	4–16	16	16
MIT	4–16	8	8
MTZ	0.5–64	4	64

^aA total of 25 strains (4 standard and 21 clinical strains) were tested.

^bDHT, dihydrotanshinone I; DHIT, dihydroisotanshinone I; TI, tanshinone I; ITI, isotanshinone I; TIIA, tanshinone IIA; ITIIA, isotanshinone IIA; CT, cryptotanshinone; ICT, isocryptotanshinone; NCT, neocryptotanshinone; MET, methyl tanshinonate; THT, tetrahydro tanshinone I; TIIB, tanshinone IIB; MIT, miltirone; MTZ, metronidazole.

FIG 1.

Chemical structures of six tanshinone derivatives.

The rate and extent of killing of H. pylori by DHT were then assessed against the reference strain G27 to determine its effectiveness as a bactericidal agent (Fig. 2). DHT showed rapid bactericidal activity, with a 99.9% reduction in CFU observed within 4 h at 8× MIC (2 μg/ml). The same amount of killing was also achieved at 4 to 5 h upon incubation with 1×, 2×, and 4× the MIC of DHT. Complete killing was observed at 8 h, and no recoverable colonies were present at 36 h of exposure (Fig. 2). These results suggested that the anti-H. pylori activity of DHT is concentration independent but time dependent. Note that as a control experiment, metronidazole (MTZ) exhibited much slower killing against strain G27 than did DHT and showed concentration- and time-dependent bactericidal activity.

FIG 2.

Kill kinetics of DHT against H. pylori G27. Bactericidal curves are shown for the control culture (DMSO treated) and cultures treated with DHT or metronidazole (MTZ) at the indicated concentrations. The MICs of DHT and MTZ were considered 0.5 and 1 μg/ml, respectively. Data represent medians ± standard deviations (SD) of the results from three independent experiments.

DHT was then examined for the possible development of insensitivity toward H. pylori upon repeated subculture (Fig. 3). No change in the MIC value of DHT was observed with strain G27 during continuous serial passaging over 48 days. In contrast, the MIC of metronidazole was observed to increase 64-fold after 24 passages (Fig. 3). In addition, DHT was tested for activity against 21 bacterial species to determine its antimicrobial spectrum. DHT had antibacterial activity against a broad range of Gram-positive bacteria and some Gram-negative bacteria, including Moraxella catarrhalis, Haemophilus influenzae, and Campylobacter jejuni (Table S2).

FIG 3.

Development of resistance to DHT in H. pylori G27. The fold change is the normalized ratio of the MIC obtained for a given subculture to the MIC that was obtained for first-time exposure. Serial passaging of H. pylori with subinhibitory concentrations of MTZ (metronidazole) leads to high-level resistance against metronidazole. Representative results from three independent experiments are shown.

In vitro antibiofilm activity of DHT.

Targeting biofilm formation by H. pylori has become an effective strategy to improve the eradication rate (10, 26, 27). Crystal violet assays were first used to evaluate the ability of DHT to disrupt mature biofilms of H. pylori. Figure 4A shows that DHT removed H. pylori mature biofilms more effectively than MTZ. DHT and MTZ eradicated 85% and 71% of H. pylori G27 biofilms at 1× MIC, respectively. In addition, we used a clinical isolate, the BYES604D strain, to confirm that DHT was able to disperse biofilms formed by drug-resistant clinical isolates of H. pylori (Fig. S1).

FIG 4.

Antibiofilm activity of DHT determined by crystal violet staining (A), SYTO9-PI double staining (B), and viable colony count methods (C). DMSO and MTZ (metronidazole) served as negative and positive controls, respectively. Data are presented as averages ± SD from three independent experiments analyzed by Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

SYTO9-propidium iodide (PI) double staining was then performed to examine the effect of DHT on the cell viability of preformed biofilms. Under a fluorescence microscope, H. pylori G27 in the control group showed viable cells with green fluorescence (SYTO9 stained) and almost no red fluorescence. DHT at 1 μg/ml killed almost all H. pylori (PI-stained) (red) cells within the biofilms (Fig. 4B). The antibiofilm activity of DHT was further confirmed by performing viable cell counts. H. pylori biofilms treated with ∼0.5 to 2 μg/ml DHT showed a significant ∼2.1- to 3.0-log₁₀ decrease in CFU per milliliter compared with dimethyl sulfoxide (DMSO)-treated biofilms (Fig. 4C), in accordance with the results of the SYTO9-PI staining assays. As a positive control, MTZ also showed moderate killing activity against H. pylori but was less effective than DHT. The above-described results suggest the potential application of DHT in disrupting structural components of the biofilm and killing biofilm-encased H. pylori.

Combination effect of antibiotics with DHT.

To assess whether DHT can manifest a synergistic effect with antibiotics used to treat H. pylori infection, a checkerboard titration analysis was carried out against a panel of 10 strains. Of the 10 strains used for this investigation, one standard strain, G27, is antibiotic sensitive, whereas all nine clinical isolates are single-, dual-, or triple-drug resistant. As evident from Table 2, a combination of DHT and each antibiotic demonstrated additive effects against most H. pylori strains. Interestingly, DHT showed synergism with MTZ in the case of 10% of strains (fractional inhibitory concentration [FIC] index, 0.5). It was also found that the MTZ-DHT combination exhibited an FIC index of 0.625 to 0.75 in 50% (5/10) of the strains, all of them being MTZ resistant. Taken together, DHT appears to lack the potential to exhibit synergism with the five antibiotics tested.

TABLE 2.

In vitro combinatory anti-H. pylori effect of DHT with each antibiotic

FIC index (interpretation)	No. of isolates (%)a
	DHT+AMX	DHT+CLR	DHT+MTZ	DHT+LVX	DHT+TET
≤0.5 (synergistic)	0	0	1 (10)	0	0
0.5 to ≤1 (additive)	7 (10)	9 (10)	8 (10)	6 (10)	8 (10)
1 to ≤4 (indifferent)	3 (10)	1 (10)	1 (10)	4 (10)	2 (10)
>4 (antagonistic)	0	0	0	0	0

^aAMX, amoxicillin; CLR, clarithromycin; MTZ, metronidazole; LVX, levofloxacin; TET, tetracycline.

In vivo efficacy of DHT.

Due to the strong anti-H. pylori activity in vitro, we turned to a mouse model of H. pylori infection to determine whether DHT was effective in an in vivo model. This model used BHKS159, a mouse-adapted multidrug-resistant strain (28), and mimics drug-resistant H. pylori infection in humans. Three weeks after bacterial inoculation, a rapid urease test (RUT) and colony counts after enumerating H. pylori bacteria in the mouse stomach confirmed an abundance of H. pylori in the stomach tissue of infected mice. The infected mice were then randomly divided into three groups and treated with phosphate-buffered saline (PBS) (control), triple therapy (omeprazole plus amoxicillin [AMX] and clarithromycin [OPZ+AC]), or DHT plus omeprazole (OPZ+DHT). In terms of clearance as measured 48 h after the last treatment, the OPZ+DHT-treated groups exhibited significant reductions in colony counts by 3.2 orders of magnitude, which was superior to the triple therapy, which reduced them by 2.4 orders of magnitude (Fig. 5B). Thus, the high therapeutic efficacy of DHT in vivo is in accordance with the in vitro MIC results, confirming the anti-H. pylori potential of DHT.

FIG 5.

In vivo efficacy of DHT against H. pylori infection in a mouse model. (A) Schematic diagram for examining the efficacy of DHT in mouse models infected by a drug-resistant strain, BHKS159. (B) Quantification of the bacterial burdens in the stomachs of H. pylori-infected mice treated with PBS, triple therapy (OPZ+AC), and omeprazole plus DHT (OPZ+DHT). If no colonies were present, calculations were made using the limit of detection (10² CFU/g stomach). Error bars represent the SD derived from 8 mice per group. *, P < 0.05; **, P < 0.01.

In vivo safety evaluation of DHT.

Finally, the potential adverse effects of DHT were assessed. There were no notable changes in animal body weight following treatment with PBS, triple therapy, or omeprazole plus DHT (Fig. S2), indicating the low toxicity of DHT in vivo. The safety of DHT toward the stomach was evaluated by hematoxylin and eosin (H&E) staining of the gastric tissues obtained from the mice. Histological examination revealed that H. pylori infection (treated with PBS) caused inflammation in gastric pit cells along with glandular atrophy and infiltration of inflammatory cells, compared with uninfected controls (Fig. 6A). However, the gastric tissues treated with DHT plus omeprazole maintained undisturbed structural integrity with well-organized gastric pits, similar to the gastric samples from the uninfected controls. In addition, the mice treated with DHT plus omeprazole exhibited levels of gastric epithelial apoptosis comparable to those in mice treated with PBS (Fig. 6B), as measured by a terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay. The low toxicity of DHT was further evaluated by overdose (285 mg/kg of body weight [10 times the effective dosage]) DHT treatment of uninfected mice. There were no notable changes in behavior, body weight (Fig. S3), and gastric histopathological examination (Fig. S4) with 5-day overdose treatment compared with the control groups. These results collectively suggest that orally administered DHT is safe.

FIG 6.

Representative images of H&E-stained (A) or TUNEL-stained (B) stomachs from uninfected mice (control) and infected mice treated with PBS, triple therapy (OPZ+AC), or omeprazole plus DHT (OPZ+DHT). OPZ+DHT treatment showed the same level of safety as the control (n ≥ 5). All images were taken at a ×100 magnification.

DISCUSSION

The growing resistance of H. pylori to the antibiotics commonly used in standard combination therapies is a major reason for the failure to eradicate H. pylori. Clarithromycin-resistant H. pylori has been categorized by the World Health Organization (WHO) as a priority pathogen for which new antibiotics are urgently needed (29). In the present study, we first investigated the in vitro anti-H. pylori activity of a subset of tanshinones, natural herbal compounds in traditional Chinese medicine widely used for the treatment of cardiovascular and cerebrovascular diseases. Of the 13 molecules, DHT exhibited the highest antimicrobial activity against clinical isolates of H. pylori, including multidrug-resistant strains; no signs of cross-resistance between DHT and commercially available anti-H. pylori antibiotics were observed. Looking at the structural features of the tanshinones, it seems that ortho-quinones confer higher activity than para-quinones. Our results also demonstrated that dual therapy (DHT plus a PPI) afforded in vivo killing efficacy superior to that of the current triple therapy in a mouse model of multidrug-resistant H. pylori infection. Thus, DHT may be suitable as a therapeutic agent for the treatment of drug-resistant H. pylori infection.

The frequency and level of resistance of H. pylori to antibiotics are important considerations when selecting drugs in the design of therapeutic regimens. DHT did not show any tendency to develop resistance against H. pylori (Fig. 2). Actually, after 48 days of continuous serial passaging, we failed to isolate spontaneous DHT-resistant mutants in three independent experiments performed with DHT added to the screening Columbia blood agar medium. Therefore, DHT could provide an advantage in the prevention or treatment of drug resistance of H. pylori. The rate of development of drug resistance is highly influenced by the magnitude of the fitness cost (30). Thus, a high fitness cost may explain the scarcity of DHT-resistant H. pylori strains in the population. Another possible reason for the ultralow prevalence of DHT resistance could be that DHT impacts multiple targets within H. pylori and that alterations in several functions may be essential for resistance to emerge. Actually, the selectivity and specificity for DHT targets in human cells are relatively low. Hypoxia-inducible factor 1α (HIF-1α) (31), human antigen R (HuR) (32, 33), and acetylcholinesterase (AchE) (34, 35) have been identified as its potential targets. Thus, the mechanism of the anti-H. pylori action of DHT remains to be clarified.

Biofilm formation has been suggested to play an important role in H. pylori colonization and resistance to antibacterial therapy (11, 36–38). Thus, eradication of H. pylori infection could benefit from novel antibiofilm approaches dispersing the biofilm and/or killing biofilm-encased bacteria. In the present study, DHT exhibited such potent antibiofilm activity and effectively acts in a dose-dependent manner. Moreover, the eradication efficacy of DHT was superior to that of the conventional antibiotic metronidazole. Thus, the current study significantly establishes the usage of DHT as potentially a safe and effective alternative for the treatment of H. pylori infection through its antibiofilm property.

DHT is one of the main lipophilic components of danshen with high medicinal value. Accumulated studies have shown that DHT exhibits a wide range of pharmacological activities such as cardiovascular protection (39, 40) and antibacterial (24), anticarcinogenic (41), and anti-inflammatory (42) properties. For instance, DHT demonstrated significant inhibition of the production of nitric oxide and the release of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-12, and IL-1β in lipopolysaccharide (LPS)-induced immune cells (43, 44). Actually, all these effectors produced in response to H. pylori infection are generally believed to contribute to disease pathogenesis (45, 46). As such, it would be interesting to determine whether DHT has protective effects against the H. pylori-induced proinflammatory response.

In summary, we report that DHT, a natural product, showed potent antimicrobial and antibiofilm activities against H. pylori. No obvious safety or toxicity signals for DHT were observed from early toxicity and safety evaluations. DHT might be a promising lead candidate for the development of anti-H. pylori therapy. Moreover, the mechanism of action of DHT, different from those of conventional antibiotics used to treat H. pylori infections, would avoid cross-resistance to established targets.

MATERIALS AND METHODS

Chemicals and antimicrobial agents.

Dihydrotanshinone I (CAS no. 87205-99-0), dihydroisotanshinone I (CAS no. 20958-18-3), tanshinone I (CAS no. 568-73-0), tanshinone IIA (CAS no. 568-72-9), cryptotanshinone (CAS no. 35825-57-1), and five antibiotics (amoxicillin, metronidazole, clarithromycin, levofloxacin [LVX], and tetracycline [TET]) were purchased from MedChem Express (Monmouth Junction, NJ, USA). Isotanshinone I (CAS no. 20958-17-2), isocryptotanshinone (CAS no. 22550-15-8), isotanshinone IIA (CAS no. 20958-15-0), neocryptotanshinone (CAS no. 109664-02-0), methyl tanshinonate (CAS no. 18887-19-9), tetrahydro tanshinone I (CAS no. 126979-84-8), tanshinone IIB (CAS no. 17397-93-2), and miltirone (CAS no. 27210-57-7) were purchased from ChemFaces (Wuhan, Hubei, China). Other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). The stock solutions of tanshinones were reconstituted in dimethyl sulfoxide (DMSO).

Bacterial strains and culture conditions.

H. pylori strains ATCC 43504 (47), 26695 (48), G27, and NSH57 (49) and 21 clinical isolates were used in this study and were routinely cultured in either brain heart infusion (BHI) broth (Becton, Dickinson, Sparks, MD, USA) medium containing 10% fetal calf serum (FCS) or Columbia blood agar (Oxoid, Basingstoke, UK) plates containing 5% FCS, supplemented with Dent selective supplement (Oxoid). All of the plates or media were incubated at 37°C for 48 to 72 h under microaerophilic conditions (10% CO₂, 85% N₂, and 5% O₂ at 90% relative humidity) using a double-gas CO₂ incubator (model CB160; Binder, Germany). Standard strains G27 and NSH57 were kindly provided by Nina R. Salama, Fred Hutchinson Cancer Research Center, Seattle, WA. A total of 21 local strains of H. pylori were isolated from biopsy samples from 21 patients with gastritis or gastric cancer using standard protocols. The specimens were obtained between 2018 and 2019 from Sir Run Run Hospital and the First Affiliated Hospital of Nanjing Medical University, China. The strains were identified based on colony appearance, Gram staining, and positive reactions with the rapid urease test. The following 21 reference strains of different species were also used: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 35657, Pseudomonas aeruginosa PAO1, Salmonella enterica serovar Typhimurium ATCC 14028, Shigella dysenteriae Sd197, Acinetobacter baumannii ATCC 19606, Proteus mirabilis ATCC 29906, Enterobacter cloacae ATCC 13047, Stenotrophomonas maltophilia ATCC 51331, Moraxella catarrhalis ATCC 25238, Bacillus subtilis 168, Enterococcus faecium ATCC 19434, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Staphylococcus aureus ATCC 25923, Staphylococcus haemolyticus ATCC 29970, Listeria monocytogenes EGDe, Morganella morganii ATCC 25830, Haemophilus influenzae ATCC 49766, Campylobacter jejuni NCTC11168, and Mycobacterium smegmatis MC²155.

Drug susceptibility test.

The MICs against H. pylori were determined by the broth microdilution assay in 96-well microtiter plates as described previously (28). Twofold serial dilutions of the test compounds were prepared in a 96-well microtiter plate containing 100 μl of BHI broth supplemented with 10% FCS. An H. pylori liquid culture grown overnight was diluted 10 times in BHI broth and inoculated into each well to yield an initial cell density of 5 × 10⁵ to 1 × 10⁶ CFU/ml. The plates were incubated for 3 days in a microaerophilic atmosphere at 37°C. After incubation, the plates were examined visually, and the MIC was determined to be the lowest concentration at which the compound inhibited visible bacterial growth. For quality control and comparative analyses, the antibiotic metronidazole was also tested with each batch of tanshinones. MICs for aerobic bacteria were determined by the agar dilution streak method recommended in Clinical and Laboratory Standards Institute document M07-A7 (55). The broth was diluted with saline and applied to plates, delivering a final concentration of approximately 10⁵ CFU/spot. All MIC assays were performed in at least triplicate.

Drug resistance study.

The development of drug resistance was investigated using a previously reported method (28), with minor modifications. DHT or metronidazole was prepared at concentrations of 0.25×, 0.5×, 1×, 2×, and 4× the MIC in BHI broth containing 10% FCS in 96-well microtiter plates. H. pylori strain G27 was inoculated into each dilution series to give a final concentration of approximately 2 × 10⁷ CFU/ml. After a 2-day incubation at 37°C with continuous shaking, growth was determined with a microplate reader as the optical density at 600 nm (OD₆₀₀), and cells from the second-highest concentration showing visible growth were subcultured in a fresh series of the same sample. This procedure was repeated for up to 24 cycles (48 days), and the MIC values after every 2 cycles during the course of continued exposure were determined with the broth microdilution method described above. All measurements were performed with three biological replicates.

Bactericidal kinetics.

The rate and extent of killing of H. pylori by DHT were assessed in BHI broth medium containing 10% FCS at 37°C with constant shaking. Liquid medium in the presence of DHT (at 1×, 2×, 4×, or 8× MIC) or an equivalent amount of DMSO (which served as a vehicle control) was inoculated with H. pylori strain G27 obtained from a culture grown overnight to yield an initial cell density of ∼10⁶ CFU/ml. After incubation at different time points (0, 1, 2, 4, 6, 8, 10, 12, 14, 18, 24, 30, and 36 h), aliquots (100 μl) of the culture were taken out to monitor growth by direct microscopic examination, and the viable cells were counted by serial dilutions onto Columbia blood agar plates. The numbers of CFU were determined after 4 days of incubation, and the bactericidal activities were assessed in terms of the decrease in the cell count (CFU per milliliter).

Antibiofilm assay.

To assess the ability of DHT to eradicate preformed biofilms, H. pylori G27 or BYES604D cells were grown overnight in brucella broth supplemented with 7% FCS, diluted to an OD₆₀₀ of 0.15 with the fresh medium described above, and then used to fill triplicate wells of a sterile 96-well polystyrene microtiter plate. After culture in microtiter plates for 3 days to allow biofilm formation, the medium was aspirated, and the plates were then washed with PBS twice. Fresh brucella broth supplemented with 7% FCS and various doses of DHT or MTZ (which served as a positive control) or an equivalent amount of DMSO (which served as a vehicle control) was added to the wells for another 24 h. The dispersal of mature biofilms was measured using the crystal violet staining method (50). All data points are expressed as means ± standard deviations (SD) from three separate experiments performed in triplicate.

Quantifying the effect of DHT on bacterial viability in biofilms.

Bacterial viability within the biofilm was assessed by using a Live/Dead BacLight bacterial viability kit (Invitrogen, Molecular Probes, USA), which consists of two fluorescent dyes, SYTO9 and propidium iodide (PI). Biofilms of H. pylori G27 cells were prepared and treated with various doses of DHT or MTZ (which served as a positive control) or an equivalent amount of DMSO (which served as a vehicle control). Following incubation for 24 h under microaerobic conditions as described above, nonadherent cells were washed three times with PBS and then stained with the two fluorescent dyes for 30 min at room temperature in the dark. After rinsing, the images were observed using a confocal laser scanning microscope (LSM710; Carl Zeiss, Oberkochen, Germany), and more fields of view were examined randomly. SYTO9 and PI differentiate live (green) and dead (red) cells because the former is membrane permeable in both live and dead cells, while the latter is membrane impermeable and stains cells with damaged membranes.

The effect of DHT or MTZ on the cell viability of preformed biofilms was also investigated using colony count methods. Biofilms of H. pylori G27 cells were prepared and treated, followed by 24 h under microaerobic conditions, as described above. After incubation, additives were removed, and the wells were rewashed with PBS, followed by the addition of 250 μl BHI broth to every well. The biofilms were detached by scraping wells with a 200-μl pipette tip 15 times across the well. The scraped biofilms were then homogenized in solution by repeatedly pipetting the solution several times. Finally, the homogenized biofilm suspensions were serially diluted, spread out onto Columbia blood agar plates containing Dent supplement, and then incubated at 37°C under microaerobic conditions for 4 days. Viable bacterial colonies on plates were enumerated and expressed as CFU per milliliter.

Combination assay.

Antimicrobial interactions between DHT and each antibiotic (AMX, CLR, LVX, TET, or MTZ) against H. pylori strains were evaluated by the standard checkerboard titration method (51). The test compounds represented by each antibiotic were serially diluted on the x axis, ranging from 1 to 1/64× MIC, with decreasing concentrations of DHT ranging from 1 to 1/32× MIC on the y axis. For evaluation of the synergistic effect of DHT (A) with the antibiotic tested (B), the fractional inhibitory concentrations (FICs) were calculated as ∑FIC = FIC_A + FIC_B, where FIC_A = MIC_A (in the presence of B)/MIC_A (alone) and FIC_B = MIC_B (in the presence of A)/MIC_B (alone). The FIC indices were interpreted as follows: ≤0.5 for synergy, >0.5 to 1 for an additive effect, >1 to 4.0 for indifference, and >4.0 for antagonism. All MIC determinations were performed in triplicate for each strain.

Anti-H. pylori efficacy of DHT in vivo.

Six-week-old specific-pathogen-free female C57BL/6 mice were used as the hosts, and the H. pylori BHKS159 strain (28) (with resistance to metronidazole, clarithromycin, and levofloxacin) was used as the infecting strain. Mice were obtained from the Animal Core Facility, Nanjing Medical University, China, and housed at the same place. The process of infection and treatment is illustrated in Fig. 5A. Each mouse was challenged with 0.3 ml of 1 × 10⁹ CFU/ml H. pylori BHKS159 in BHI broth intragastrically by oral gavage every 48 h, repeated four times (on days 1, 3, 5, and 7). Three weeks after inoculation, the mice were randomly divided into three groups and treated as follows: DHT plus omeprazole, triple therapy, or PBS (n = 8). Omeprazole (400 μmol/kg/day) was fed by oral gavage 30 min before the administration of the assigned treatments. DHT (28.5 mg/kg) and the triple-therapy formulation (amoxicillin at 28.5 mg/kg and clarithromycin at 14.3 mg/kg) were administered once daily for 3 consecutive days by oral gavage. The control group received an equivalent volume of PBS. Forty-eight hours after the last treatment, mice were killed, and the stomach, liver, spleen, kidney, and ileum were harvested from the abdominal cavity. The stomach was cut into two longitudinal sections along the greater curvature, and each section was weighed after the gastric contents were removed and rinsed with PBS. The sections were used for assessments of bacterial colonization and histology/epithelial apoptosis. For bacterial colonization, the gastric tissue section was suspended in 1 ml BHI broth and homogenized gently for H. pylori recovery. The suspensions were serially diluted, streaked onto a Columbia blood agar plate containing Dent supplement, and then incubated at 37°C under microaerobic conditions for 5 days. Viable bacterial colonies were counted and expressed as CFU per gram of stomach.

In vivo toxicity study.

DHT overdose (285 mg/kg [10-fold effective dosage]) treatment was performed in C57BL/6 female mice (n = 8) at 6 to 8 weeks of age to evaluate DHT toxicity in vivo. Mice were given a daily gavage of DHT or PBS for 5 consecutive days. Mice were sacrificed on day 7, and the stomach, liver, spleen, and kidney were harvested from the abdominal cavity for histological analysis. The longitudinal sections of each tissue were fixed in buffered paraffin, embedded in paraffin wax, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E) to analyze tissue inflammation. Epithelial cell apoptosis was examined by a terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay (R&D Systems, Minneapolis, MN, USA). Sections were visualized with a Hamamatsu NanoZoomer 2.0HT instrument, and the images were processed using NDP viewing software. Mouse body weight was monitored during the experimental period by weighing the mice daily.

Statistical analysis.

Statistical analyses were performed and graphs were generated by using GraphPad Prism version 7.0 (GraphPad, San Diego, CA, USA). For all experiments, statistical analyses were performed using Student’s t test. A P value of <0.05 was considered a statistically significant difference.

Ethics.

Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (IACUC approval no. 1707025) and were conducted in accordance with international standards for animal welfare and institutional guidelines.