Consumption of non-antibacterial drugs may have negative impact on Helicobacter pylori colonization in the stomach

Background Nineteen non-antibacterials were examined to show that their consumption for treatment of other diseases may inhibit Helicobacter pylori. Four antibiotics were used for comparison. Materials and methods Agar dilution method was used to examine the susceptibility of 20 H. pylori isolates to 4 antibiotics; metronidazole (MTZ), clarithromycin (CLR), amoxicillin (AMX), tetracycline (TET) and 19 non-antibacterials; proton pump inhibitors (PPIs), H2-blockers, bismuth subsalicylate (BSS), antifungals, statins, acetaminophen (ACE), aspirin (ASA), B-vitamins (B-Vits; Vit B1, Vit B6 and Vit Bcomplex) and vitamin C (Vit C). Blood agar plates were prepared with different concentrations of drugs and spot-inoculated with bacterial suspensions. Plates were incubated at 37 °C under microaerobic conditions and examined after 3–5 days. The isolate #20 that was mucoid and resistant to 19 drugs, including MTZ and SMV was tested against combined MTZ (8 μg/mL) and SMV (100 μg/mL). Results were analyzed statistically. Results Minimum inhibitory concentrations (MICs, μg/mL) of drugs and the frequency of susceptible H. pylori were determined as MTZ (8, 80%), CLR (2, 90%), AMX (1, 100%), TET (0.5, 70%), PPIs (8–128, 80%), H2-blockers (2000–8000, 75–80%), BSS (15, 85%), antifungals (64–256, 30–80%), statins (100–250, 35–90%), ACE (40, 75%), ASA (800, 75%), B-Vits (5000–20000, 80–100%) and Vit C (2048, 85%). Susceptibility of H. pylori isolates to 16 out of 19 non-antimicrobials (75–100%) was almost similar to those of antibiotics (70–100%) (P-value >0.05). The highest susceptibility rate (100%) belonged to Vit B1, Vit B6 and AMX. Out of 20 H. pylori isolates, 17 (85%) were susceptible to ≥13 non-antimicrobials and 3 (15%) were susceptible to < 13 (P-value <0.05). Mucoid H. pylori showed susceptibility to combination of MTZ and SMV. Conclusions Most of non-antibacterials inhibited H. pylori isolates, similar to antibiotics but their MICs exceeded those of antibiotics and their plasma concentrations. At low plasma concentration, non-antimicrobials may act as weak antibacterials, antibiotic adjuvants and immunostimulators.


Introduction
Helicobacter pylori is a spiral epsilon-proteobacterium that has been evolved to colonize the gastric epithelium of humans as its unique niche.Microscopic observation of spiral bacteria in gastric epithelium was first reported in1893 in dogs [1] and thirteen years later in humans [2].However, failure in culturing H. pylori from human stomach hampered identification of the bacterium and discovery of its correlation with gastric diseases.The initial cultivation of H. pylori from gastric biopsies of dyspeptic patients and relief after bacterial eradication with antibiotics confirmed the implication of H. pylori infection in gastritis [3] with the possible severe consequences as peptic ulcer and gastric cancer [4].On the other hand, reduction in the frequency of gastric cancer in several countries with widespread use of antibiotics for treatment of different infectious diseases led to the conclusion that cure of a primary infection may also eliminate H. pylori from the stomach of patients [5].Accordingly, antibiotic therapy for patients complaining of dyspepsia started with two antibiotics and a proton pump inhibitor.However, despite a high rate of success in initial antibiotic therapies, H. pylori started to show resistance [6] that is now a worldwide problem [7,8].
Globally reported resistance of different bacteria to a number of commonly used antibiotics has urged many investigators around the world to evaluate the antimicrobial potential of many currently used non-antimicrobial drugs.These drugs may have direct antimicrobial activity or when used in combination with antibiotics, they disrupt bacterial resistance or enhance patient's immune response [9,10].Reports published in recent years indicate that an increasing number of investigations have focused on antibacterial properties of non-antimicrobial drugs, ranging from vitamins to antineoplastics.These drugs in addition to their typical action on targets in human body have shown promising antimicrobial activity against Gram-positive and Gram-negative bacteria [11].It has been suggested that bacterial cell components have a common evolutionary origin with those of mammalian cells.These components that are often involved in vital activities of bacteria are the important targets of non-antibiotics.The antibacterial activity of these drugs may be non-specific, targeting multiple functions in bacterial cells or specific, inhibiting a single bacterial activity.Examples of these functions include; phosphorylation and dephosphorylation as a common signaling mechanism, initiation and propagation of electrical signaling through voltage-gated channels and metalloenzymes that have a metal center for catalytic activity [12].Furthermore, results of several reports indicated that non-antibacterial drugs often exert their inhibitory action through disrupting of bacterial cell plasma and outer membranes [11,13,14].
There is limited number of studies on anti-H.pylori activity of non-antibacterial drugs.These studies often used a single or one group of drugs such as bismuth subsalicylate (BSS) [15,16] aspirin (ASA) [17], vitamin C (Vit C) [18][19][20], proton pump inhibitors (PPIs) [21][22][23] and antifungals [24,25].Results of these studies showed the high potential of such drugs for successful eradication of H. pylori.However, there is no comprehensive study that describes the anti-H.pylori activity of a wider range of non-antibacterial drugs.On the other hand, investigators are trying to find more effective strategies to overcome H. pylori resistance to therapeutic regimens.In a recent study, antibiotic-free nanoparticles (NPs) were constructed by assembling metformin-linoleic acid (ML) and linoleic acid (LA) NPs that encapsulated urease inhibitor ebselen (EB) and was coated by fucoidan (FU).The FU/ML-LA/EB NPs destroyed H. pylori biofilm in cultured human gastric mucosal epithelial cells, killed dispersed bacteria by inhibiting urease and when taken up by epithelial cells, caused lysosomal acidification and activation of adenosine monophosphate-activated protein kinase (AMPK) that degraded intracellular H. pylori.Furthermore, EB and LA fractions in NPs with antioxidant properties alleviated the excessive oxidative stress in response to H. pylori infection and its severe consequences such as gastric mucosal damage [26].
In this study, antibacterial efficacy of nineteen commonly used non-antimicrobial drugs and vitamins against 20 H. pylori was investigated in order to evaluate whether consumption of such compounds for treatment of non-infectious diseases exert effective antimicrobial activity against H. pylori in the stomach and reduce the risk of development of severe gastric diseases.These drugs included 3 proton pump inhibitors; omeprazole (OMP), lansoprazole (LPZ) and pantoprazole (PAN), 3H 2 -blockers ; famotidine (FAM), cimetidine (CIM) and ranitidine (RAN), bismuth subsalicylate (BSS), 3 antifungals; ketoconazole (KTC), fluconazole (FLC) and amphotericin B (AMB), 3 statins; atorvastatin (ATV), simvastatin (SMV) and rosuvastatin (RSV), acetaminophen (ACE), aspirin (Acetylsalicylic acid, ASA), vitamin B1(Vit B1, thiamine), vitamin B6 (Vit B6, pyridoxine) and vitamin B complex (Vit B complex ) and vitamin C (Vit C, L-ascorbic acid).The 4 commonly used anti-H.pylori antibiotics; metronidazole (MTZ), clarithromycin (CLR), amoxicillin (AMX) and tetracycline (TET) were also examined for comparison.The range of concentrations used for drugs included their published minimum inhibitory concentration (MIC).Furthermore, plasma concentration of drugs in the body of normal individuals was used for comparison.Information related to mechanism of action of drugs and the susceptible bacteria are summarized in Table 1.

Patients and H. pylori isolates
Twenty H. pylori strains used in this study were isolated from gastric biopsies of 20 dyspeptic patients that exhibited positive rapid urease test.Patients included 9 women (45%) and 11 men (55%) with the age range of 7-86 yr.They were referrals to the endoscopy unit of digestive disease research institute, Shariati hospital, Tehran, Iran.All patients signed informed consent and the study was approved by the research ethics committee of Tehran University of Medical Sciences (code number: IR.TUMS.DDRI.REC.1400.023).To obtain reliable results from H. pylori culture, patients were recommended to stop taking proton pump inhibitor and antibiotics 2 weeks before endoscopy [54].Gastric biopsies were cultured on selective brucella agar (Pronadisa, Spain), supplemented with 7% defibrinated sheep blood, vancomycin (10 mg/L), trimethoprim (5 mg/L), polymyxin B (50 μg/L) and amphotericin B (4 mg/L).All the used antibiotics were purchased from Sigma Company (Sigma, Germany).Cultures were examined for H. pylori growth after 3-5 days incubation under microaerobic atmosphere at 37 • C. Out of 20 bacterial isolates, 19 produced glistening pin-pointed colonies on blood agar and examination of their Gram-stained smears with the light microscope showed Gram-negative spirals.The # 20 bacterial isolate showed mucoid growth on blood agar and appeared as Gram-negative bacteria with spiral morphology when examined with the light microscope.All the 20 bacterial isolates exhibited positive catalase, oxidase, and urease activities.

Table 1
Summarized properties of 19 non-antibacterial and 4 antibiotics; diseases used for, mechanism of action in humans, mechanism of anti-microbial action and susceptible bacteria.

Drug name
Diseases used for Mechanism of action in humans Mechanism of anti-microbial action Susceptible bacteria MTZ Oral infections and parasite related diseases [27].
-Breakage in DNA strands and inhibition of nucleic acid synthesis [27].
-Inhibition of protein synthesis by interfering with aminoacyl translocation in ribosomes [28].
-Inhibition of protein synthesis by preventing the attachment of charged aminoacyl-tRNA to the A-site on the ribosomes [30].
Inhibition of bacterial growth and replication with unknown mechanism [31].
Reducing gastric acid secretion by blocking H 2 -receptors in parietal cells and inhibiting human carbonic anhydrase [33].
Increasing mucosal protective factors, including prostaglandin with no toxicity due to low absorption in mammalian cells [35].
Depletion of ergosterol from fungal plasma cell membrane that alters its structure and function.Bacterial lysis due to inhibition of cholesterol incorporation into cell membrane [25].

AMB
Fungal infections [39].Interacts with cholesterol in human cell membrane, leading to loss of minerals, anaphylaxia, fever and renal failure [39].
Interacts with sterols in fungal cell membrane causing cytoplasmic leakage and death [39]. -
Reduction of cholesterol availability to H. pylori with negative effect on bacterial colonization, innate immune evasion and antibiotic resistance [42].
Inhibition of bacterial growth and replication with unknown mechanism [45].
By inhibition cyclooxygenase activity, causes inhibition of prostaglandin formation and thus reduction of inflammation, swelling, pain and fever [46].
Inhibition of bacterial growth and replication with unknown mechanism.

B-Vit
Food supplement for improving human health [48].
Involved in vital activities of all living cells [48], with immunomodulatory property that helps to fight the pathogens [49].
Vit C Food supplement for improving human health [52].
Serves as an antioxidant and a cofactor of several enzymes.Also enhances immune response and iron absorption [52].
Inhibition of bacterial growth and replication with unknown mechanism.
products were electrophoresed on 1% agarose gel and amplicons of isolates # 19 and 20 were sequenced and matched with reference sequences in the GenBank by BLAST program.

Susceptibility tests for 20 H. pylori isolates
Fresh sub-cultures of all 20 H. pylori isolates on blood agar were used for preparation of bacterial suspensions with the turbidity of MacFarland standard No.2 (6 × 10 8 cell/mL).A 10-μL volume of each bacterial suspension was spot-inoculated on the blood agar containing different concentrations of drugs.Plates were incubated as mentioned earlier and examined after 3-5 days.All the susceptibility tests were performed in triplicates and the results were recorded as susceptible (no growth) or resistant (growth).

Synergistic antibacterial effect of MTZ and SMV on mucoid H. pylori
The mucoid H. pylori (#20) that showed resistance to 19 out of 23 drugs, including SMV and MTZ was examined for the synergistic antibacterial effect of MTZ and SMV, using combined disc diffusion and agar dilution methods [61].First, H. pylori susceptibility to MTZ and SMV was tested by disc diffusion and agar dilution method, respectively.Bacterial suspension with the turbidity of Mac-Farland standard No.2 was surface-inoculated on blood agar plates and sterile blank paper discs were superimposed on their surface and impregnated with different concentrations of MTZ (8, 4, 2 and 1 μg/mL).Second, different concentrations of SMV were added to cooled blood agar to reach the final concentrations of 100, 50, 25 and 12.5 μg/mL.SMV-containing plates were inoculated with 6 H. pylori isolates, including 5 non-mucoids (No:1-5) and the mucoid one (No:6).Third, combined susceptibility test was performed by first surface-inoculating the bacterial suspension on the blood agar plates containing 100, 50, 25 and 12.5 μg/mL of SMV and then superimposing the blank paper discs impregnated with different concentrations of MTZ (8 and 4 μg/mL).Drugs' combinations included: (8 μg/mL MTZ +100 μg/mL SMV), (8 μg/mL MTZ +50 μg/mL SMV), (4 μg/mL MTZ +25 μg/mL SMV) and (4 μg/mL MTZ +12.5 μg/mL SMV).Blood agar with no added drug was used as a control.Plates were incubated as mentioned earlier and examined after 3-5 days.

Statistical analysis
Non-parametric tests, including the Chi-Square and Mann-Whitney U were employed to compare various groups within our research.For our statistical analysis, we relied on IBM SPSS Statistics version 26.P-value <0.05 was considered as significant.

Molecular identification of H. pylori isolates
Electrophoresis of PCR products amplified from all H. pylori isolates showed amplicons with the size of 521 bp.Amplicons of isolates #19 and #20 (Fig. 1) were sequenced and matched with sequences in GenBank by the BLAST program.Results of BLAST analysis showed 99%-100% sequence homology between amplified fragments from isolates #19 and #20 and the corresponding sequences of the reference H pylori in GenBank, confirming the identity of isolates as H. pylori.

Discussion
Several studies have focused on the problem of antimicrobial resistance of bacterial pathogens, examining the antimicrobial and immunological properties of different non-antibacterial drugs alone or in combination with antibiotics.Although a considerable number of investigators have succeeded to show the antibacterial effect of non-antibacterial drugs on a wide range of pathogenic bacteria, the mechanism of action of these drugs has remained largely unknown.It has been suggested that non-antibiotics interact with common prokaryotic and eukaryotic targets because of their common evolutionary origin [12].However, there is no report to show that results of in vitro studies correspond with those of in vivo studies.In this study the antibacterial effect of 19 non-antibiotic drugs and 4 currently used antibiotics against H. pylori isolates was examined to propose that when these drugs are used for treatment of non-infectious diseases, they may exert effective antibacterial activity on the existing bacterial infections such as that of H. pylori.
FMT and CIM at MIC 2000 μg/mL inhibited 75% and 80% of H. pylori isolates, respectively.However, RAN inhibited 80% of the isolates with 4-times higher concentration (8000 μg/mL).FMT with the MIC of 8000-64000 μg/mL exhibited a significant antibacterial activity against E. coli, S. aureus and Pseudomonas aeruginosa while RAN and CIM showed no inhibitory effect [34].MICs of H 2blockers obtained in this study (2000-8000 μg/mL) were much higher than their related plasma concentration (μg/mL): FMT 0.02 [62], CIM 1.5 [63] and RAN 0.896 [64].Results of a recent study have indicated that antibacterial effect of FAM combined with rifampicin on Acinetobacter baumannii and Salmonella typhimurium was more effective than when they were used alone [74].It was suggested that FMT by disrupting bacterial cell membrane in A. baumannii, exerted a negative impact on bacterial virulence, drug resistance (efflux pump) [75], and release of damaging inflammatory factors [76].Furthermore, inhibition of efflux pump in A. baumannii by usnic acid, turned the tigecycline-resistant bacteria to susceptible [77].
BSS with the concentration of 15 μg/mL inhibited 85% of H. pylori isolates.This inhibitory concentration of BSS was among the lowest effective antibacterial concentrations found in this study, after antibiotics (0.5-8 μg/mL) and LPZ (8 μg/mL).Results of a study demonstrated the inhibitory effect of BSS on H. pylori growth with the concentration of 4-8 μg/mL [15].Since H. pylori does not exhibit primary and secondary resistance to BSS, it is often included in different therapeutic regimens for effective eradication of antibiotic-resistant H. pylori [16].The MIC obtained for BSS in this study (15 μg/mL) was much higher than its related plasma concentration (0.05 μg/mL) [16].Synergistically-enhanced antibacterial activity of BSS-norfloxacin [78] and BSS-ciprofloxacin has been reported against different bacteria, including E. coli and S. aureus.[36].
Results of a similar study showed that KTC inhibited H. pylori with the MIC range of 8-32 μg/mL [24].In a study by our group, KTC inhibited the growth of MTZ-resistant H. pylori with the MIC of 8 μg/mL [25].The MICs found for KTC (64 μg/mL) and FLC (256 μg/mL) in this study exceeded their respective plasma concentrations, KTC 3-4.5 μg/mL [65] and FLC 0.5-9 μg/mL [66].Reports showed that azole antifungal could enhance the antibacterial effectiveness of TET against methicillin-resistant S. aureus [79].Furthermore, KTC could enhance the antibacterial activity of fluoroquinolones against multi-drug resistant S. aureus by inhibiting efflux pump and biofilm formation [38].
Vit C inhibited 85% of H. pylori isolates at concentration of 2048 μg/mL.This antibacterial effect of Vit C could be due to the activity of Vit C oxidation products, L-dehydroascorbic acid or L-diketogluconic acid as described in an in vitro study on Campylobacter jejuni [84].In a study on 64 H. pylori isolates, 90% were inhibited at Vit C concentration of 2048 μg/mL [18].In another study, Vit C at 10, 000-20,000 μg/mL effectively inhibited H. pylori growth [19].Similarly, antibacterial activity of Vit C was determined as 5000-20000 μg/mL against K. pneumoniae and E. coli [85].Vilchèze et al. reported that L-ascorbic acid killed the strains of Mycobacterium tuberculosis that were resistant to a wide range of antibiotics [53].Furthermore, the synergistic effect of L-ascorbic acid combined with deferoxamine has been found against Proteus mirabilis and S. aureus [86].The anti-H.pylori MIC of Vit C determined in this study (2048 μg/mL) exceeded its reported plasma concentration (>4 μg/mL) [72].In this regard, secretion of Vit C from plasma to gastric juice that reaches 0-28.8 μg/mL [18] and is 3-7 times higher than plasma levels [20] might play a protective role against H. pylori.On the other hand, in an in vitro study, it has been demonstrated that treatment with Vit C could interfere with development of dormant multidrug resistant H. pylori that emerged either as viable but nonculturable (VBNC) due to nutrient starvation or as persister when treated with 10× AMX.This interference led to bacterial resuscitation and increased susceptibility to antibiotics.Accordingly, pretreatment with Vit C was proposed for selection of vegetative forms of H. pylori that show higher susceptibility to antibacterial regimens [87].
The mucoid H. pylori that showed susceptibility to AMX, CLR, Vit B1 and Vit B6 was effectively inhibited when treated with 8 μg/ mL of MTZ combined with 100 μg/mL of SMV.In our previous studies we have reported isolation of 3 mucoid H. pylori strains from gastric biopsies of dyspeptic patients, two with spiral [88] and one with coccoid [89] morphology, showing resistance to currently used antibiotics.It was revealed that accumulation of cholesterol and production of exopolysaccharides in mucoid H. pylori could lead to decrease in antibiotic uptake and emergence of resistance [90].Further results revealed that compared with H. pylori isolates, mucoid H. pylori besides accumulating high amounts of cholesterol, showed high content of unsaturated fatty acids (82%).Unsaturated fatty acids in bacteria have been implicated in antioxidant activity [91] and antibiotic efflux [92].In the present study, inhibition of mucoid H. pylori with a combination of high concentrations of MTZ (8 μg/mL) and SMV (100 μg/mL) might indicate the disruption of permeability barrier of bacterial cell that led to the synergistic effect of both drugs.It has been demonstrated that H. pylori uses cholesteryl-αglucoside transferase (CGT) to glycosylate the exogenous cholesterol and then incorporate the glycosylated cholesterol into its cell membrane to maintain its spiral morphology and cell wall integrity.However, any alteration in cholesteryl glucoside content affects H. pylori morphology and renders the bacterium susceptible to various antibiotics.It has also been demonstrated that depletion of cholesteryl glucoside in H. pylori due to deletion of CGT-encoding gene, hp0421 or bacterial cultivation in the absence of cholesterol led to a significant increase in cell membrane permeability and susceptibility to antibiotics [93].Statins are renowned for their ability to lower cholesterol by binding to the active site of HMG-CoA reductase, a rate-limiting enzyme involved in cholesterol biosynthesis [94].Statins are also known for their anti-inflammatory, immunomodulatory, anticancer and antimicrobial effects.Although the mechanisms of their action on humans are well studied, their target (s) in bacterial cell has remained unknown [41].

Conclusion
Results of this study showed that susceptibility of 20 H. pylori isolates to 16 out of 19 non-antimicrobials (75-100%) was almost similar to their susceptibility to 4 antibiotics (70-100%) (P-value >0.05).The highest susceptibility rate (100%) belonged to Vit B1, Vit B6 and AMX followed by CLR (90%) and SMV (90%) and the highest resistance rate (100%) belonged to AMB, followed by FLC (70%), RSV (65%) and TET (30%).Furthermore, out of 20 H. pylori isolates, 17 (85%) were susceptible to >13 non-antimicrobials and 3 (15%) were susceptible to < 13 (P-value <0.05).Furthermore, the effective antibacterial concentration of non-antibiotics was much higher than those of antibiotics and also exceeded their plasma concentration.It has been proposed that non-antimicrobial drugs in their low plasma concentration, may act as weak antibacterials, antibiotic adjuvants and immunostimulators.The synergy between antibiotic and adjuvant could effectively reduce the dosing and also negatively affect the emergence of resistance [95].Overall, it was revealed that the majority of non-antimicrobial drugs used in this study exerted effective antibacterial activity against H. pylori isolates.Although H. pylori susceptibility rates to non-antimicrobials were similar to those of currently used antibiotics, their mechanism of action remains to be elucidated.Considering the limitation of our study, we can mention the lack of direct in vivo studies to confirm the potential antimicrobial effects of drugs' metabolites or indirect effects via stimulation of the immune system.Furthermore, there is a need for in vitro investigating the anti-H.pylori effect of all the tested non-antibacterials when combined with antibiotics.Because consumption of high concentrations of non-antimicrobials is associated with cytotoxicity and risk of debilitating side effects, combining their sub-inhibitory concentrations with antibiotics would be an important subject for intensive research on these drugs with such a wide range of diversity.

Table 2
Susceptibility of 20 H. pylori isolates to 19 non-antibacterial and 4 antibiotics, their minimum inhibitory concentration (MIC) and plasma concentration.

Table 3
Classification of 20 H. pylori isolates into 9 groups according to the number of drugs they showed susceptibility (+) or resistance (− ) to.