Unveiling the Antibacterial Properties of Statins: An In Vitro Study on Helicobacter pylori

Background Helicobacter pylori (H. pylori), a widespread bacterial pathogen, is associated with various gastrointestinal diseases, including gastric cancer. Statins, widely prescribed cholesterol-lowering agents, have demonstrated pleiotropic effects, including potential antimicrobial properties. This in vitro study investigated the direct antibacterial effects of three clinically approved statins, simvastatin, atorvastatin, and rosuvastatin, against H. pylori isolates. Methods H. pylori strains were isolated from gastric biopsies of dyspeptic patients and identified by microbiological techniques. The minimum inhibitory concentrations (MICs) of statins were determined using the agar dilution method, and their antimicrobial activity was evaluated by the disc diffusion method using different concentrations of simvastatin, atorvastatin, rosuvastatin, tetracycline, and amoxicillin. Scanning electron microscopy (SEM) was employed to examine the morphology of H. pylori cells. Results The minimum inhibitory concentration (MIC) values (μg/mL) of atorvastatin, rosuvastatin, simvastatin, tetracycline, and amoxicillin against H. pylori were 240 ± 20, 450 ± 20, 460 ± 15, 155 ± 30, and 140 ± 20, respectively. In the disc diffusion assay, atorvastatin and rosuvastatin produced significantly larger inhibition zones compared to simvastatin at all concentrations tested (p < 0.05). The inhibition zone diameters (mm) increased with higher statin concentrations, ranging from 9 ± 1.4 to 13 ± 1.4 for atorvastatin, 8 ± 0.9 to 11 ± 0.6 for rosuvastatin, and 5 ± 1.3 to 6 ± 1.4 for simvastatin at the highest tested concentration (1200 μg/ml). SEM analysis revealed the characteristic spiral morphology of H. pylori cells. Conclusion Statins demonstrated varying degrees of antibacterial activity against H. pylori isolates, with atorvastatin exhibiting the highest potency. While the observed effects were lower than those of conventional antibiotics, these findings suggest the potential of statins as adjunctive agents or alternative therapeutic options, warranting further investigation through in vivo studies and clinical trials.


Introduction
Helicobacter pylori (H.pylori) is a spiral Gram-negative bacterium usually acquired in childhood through the orofecal route.Without therapeutic eradication, H. pylori can persist in the human stomach for an entire lifetime [1,2].H. pylori infection is known to infuence more than 50% of the worldwide population.Te pathogen is assumed to be associated with an increased risk of gastrointestinal illnesses, including peptic ulcers, gastritis, and mucosa-associated lymphoid tissue (MALT) lymphoma.H. pylori infection also results in chronic oxidative stress, which eventually may lead to the development of gastric adenocarcinoma [3,4].Te most important cause of stomach cancer, which is known as the third cause of cancer-related death, can be considered H. pylori infection, whose eradication alone can reduce the risk of stomach cancer by 47% [5].Besides, several studies have demonstrated a link between H. pylori infection and extragastric diseases such as vitamin B12 defciency anemia, iron defciency anemia, primary immune thrombocytopenia (PIT), Parkinson's disease, psoriasis, and Guillain-Barrè syndrome [6,7].H. pylori eradication therapies include triple or even quadruple antibiotic-based regimens.Despite the efort, eradication failure has increased due to escalating antimicrobial resistance of H. pylori, which has reached alarming rates worldwide [8,9].Also, some factors, such as more acidic gastric juice and being overweight, can contribute to a lower eradication rate [10,11].In addition to antimicrobial resistance, antibiotic overuse may cause unintended consequences such as shortterm impairment of the gut normal fora and increased risk of asthma and allergy [12].
Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and were discovered in the 1970s [13].Tey are among the most commonly prescribed medications due to their cholesterol-lowering efects.In the guidelines, they have been suggested as a frontline treatment for primary and secondary prevention of cardiovascular diseases [14,15].In addition, statins have been demonstrated to have some cholesterol-independent or pleiotropic impacts, including anti-infammatory, anticancer, and immunomodulatory functions [16].Studies have demonstrated that statins have a positive efect on reducing the risk of severe bacterial infections, such as Chlamydia pneumoniae, Clostridium difcile, Staphylococcus aureus, and Streptococcus pneumoniae infections [17][18][19].In addition, various studies have reported that statins can improve the outcome of bacterial infections such as wound infections, pneumonia, and sepsis [20][21][22][23][24]. Clinical trials have investigated adding statins in H. pylori eradication regimen, and the results have shown the efectiveness of diferent statins in H. pylori eradication [25][26][27].Furthermore, there is growing evidence suggesting that statins have a direct inhibitory efect on the in vitro growth and virulence of diferent bacterial pathogens [28,29].
Te emergence of multidrug-resistant bacteria, such as new H. pylori strains, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa compromises our ability to treat common infections [30,31].As a result, there is a need to develop new strategies to address the issue of antimicrobial resistance.One strategy is repurposing existing commercial drugs, such as statins, as a therapeutic alternative to antimicrobial resistance [32].Although statins have been shown to cause myopathy and type 2 diabetes mellitus, these are the only reliably proven adverse efects.However, some studies suggest that statin use may also increase the risk of hemorrhagic stroke, cataracts, cognitive impairment, and liver injury [33][34][35].
In this study, we investigated the direct antibacterial efect of three clinically approved statins, including simvastatin (SMV), atorvastatin (ATV), and rosuvastatin (RSV) against H. pylori, one of the most common pathogens in the world.A total of 40 patients with dyspeptic symptoms were recruited from the endoscopy units of Shahid Beheshti and Shahid Yahyanejad hospitals in Babol, Iran.Gastric antral and corporal biopsies were obtained from these patients during upper gastrointestinal endoscopy, and clinical diagnoses were made based on endoscopic fndings by experienced gastroenterologists.Te inclusion criteria were patients with dyspeptic symptoms undergoing upper gastrointestinal endoscopy.Exclusion criteria were as follows:

Isolation and Identifcation of H. pylori.
Gastric biopsy samples were transported to the microbiology laboratory at the School of Allied Sciences, Babol University of Medical Sciences, in 9% saline solution at 4 °C within 2 hours of collection.Biopsies were homogenized, and a portion was used for the urease test, while the remaining tissue was cultured for H. pylori isolation.For culture, homogenized samples were inoculated onto selective Brucella agar plates (Merck, Germany) supplemented with 7% (v/v) defbrinated sheep blood, 10 mg/L vancomycin, 5 mg/L trimethoprim, and 2.5 mg/L amphotericin B. Plates were incubated at 37 °C for 3-7 days under microaerobic conditions (80-90% N 2 , 5-10% CO 2 , and 5-10% O 2 ) using Gas Pack C (Anaerocult C, Merck) in an anaerobic jar.After 3 days of incubation, plates were examined for the presence of circular, smooth, and translucent H. pylori colonies (0.5-1 mm diameter).Presumptive identifcation of H. pylori was based on colony morphology, gram staining (curved or straight Gram-negative rods), and positive results for catalase, oxidase, and urease tests.Plates with no visible growth were incubated for up to 7 days before being discarded as negative.

Scanning Electron Microscopy (SEM).
For SEM analysis, H. pylori bacterial suspension was prepared in Brucella blood broth medium at a turbidity equivalent to 0.5 McFarland standard and cultured at 37 °C.Te bacterial cells were washed with phosphate-bufered saline (PBS) and fxed with 3% glutaraldehyde solution for 2 hours at room temperature.After fxation, the cells were washed again with PBS and dehydrated using a graded ethanol series (50%, 70%, 90%, and 100%).Te dehydrated specimens were airdried for 24 hours.Prior to SEM examination, the dried bacterial samples were sputter-coated with gold-palladium to enhance conductivity.Te surface morphology of H. pylori cells was then investigated using a scanning electron microscope (SNE-4500M, SEC CO., LTD, Suwon, Korea) [36,37].3 shows the average MICs attained for ATV, SMV, and RSV against H. pylori.Te sterile medium without any drug did not interfere with bacterial growth.Te disc difusion method was employed to evaluate the antimicrobial activity of the statins by measuring the diameter of the inhibition zones produced against H. pylori.As shown in Table 2, increasing concentrations of ATV, RSV, and SMV (400, 800, and 1200 μg/ml) resulted in larger inhibition zones, ranging from 5 ± 1.3 mm to 13 ± 1.4 mm.ATV and RSV exhibited signifcantly larger inhibition zones compared to SMV at all tested concentrations (p < 0.05).Larger inhibition zones of AMOX at concentrations less than 320 μg/ml were not signifcantly diferent from ATV 800 and 400 μg/ml and RSV 400 μg/ml.TET showed signifcantly larger inhibition zones compared to SMV at all tested concentrations (p < 0.05).Te bacterial growth inhibition zone is raised with increasing statin concentrations.

Discussion
Te escalating rates of antibiotic resistance and the drugs' adverse efects indicate a rising need to fnd new ways to face infections with fewer adverse events and higher or similar benefcial properties.A part of statins' favorable role in infectious diseases may arise from their antimicrobial properties.In our study, all three statins showed some in vitro antibacterial activity against H. pylori.ATV was the most potent, followed by RSV and SMV, respectively.RSV achieved better MIC values and inhibition zone diameter than SMV.Results of the current study demonstrate that the antibacterial efect increases dose-dependently.
To the best of our knowledge, no previous studies have investigated the in vitro antibacterial efect of statins on H. pylori.However, some reports have demonstrated that statins have a direct antibacterial efect against other bacterial strains.Te MIC values reported against diferent pathogens were inconsistent and ranged from 15 to 500 μg/ ml [24,28,29,[39][40][41][42][43].According to a study conducted by Masadeh et al., it was found that ATV and SMV were more  4 Advances in Pharmacological and Pharmaceutical Sciences efective in inhibiting the growth of bacterial strains in lower MICs compared to RSV.Tese results were consistent with our fndings.In contrast, the same values for ATV (108.33 and 216.67 μg/ml) and SMV (116.67 and 291.67 μg/ml) were signifcantly lower.We utilized the same agar medium supplemented with drugs as the study mentioned earlier; however, diferent bacterial isolates exhibited varying degrees of susceptibility to the same antimicrobial drugs [29].
Te emergence of multidrug-resistant strains of H. pylori, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa restricts our ability to treat common infections.None of the tested statins was found to have any practical antibacterial efects against Escherichia coli, Pseudomonas aeruginosa, or Serratia marcescens, at least in tested concentrations [42].In a study, RSV and ATV were observed to inhibit the growth of methicillin-susceptible and methicillin-resistant S. aureus, P. aeruginosa, E. coli, vancomycin-sensitive E. faecalis, and vancomycin-resistant E. faecalis strains at 100 and 250 μg/ml concentrations, respectively.In this study, RSV was more potent in inhibiting bacterial growth than ATV, which was in contrast with our results [43].Statins inhibit HMG-CoA reductase, the enzyme necessary for the biosynthesis of isoprenes in bacterial cells.However, in prokaryotes, this enzyme is of a diferent structural class and has a 10,000 times lower afnity for statins; thus, the antibacterial efect of statins is unlikely to be related to the suppression of HMG-CoA reductase [44].
Statins are also known for pleiotropic efects such as cytotoxicity and apoptosis-promoting efects and suppress cell growth.Te antimicrobial action of statins might be related to these properties [45][46][47].Clinical trials have been conducted to assess the impact of adding ATV and SMV to H. pylori eradication therapy based on their antimicrobial and pleiotropic efect, and they observed improved eradication rates in experiment groups [25][26][27]48].Some researchers demonstrated the MIC value of around 30 μg/ml for SMV against S. aureus strains [24,40].At the same time, other studies noted 64 and 75 μg/ml as MIC values for SMV against the same strains [28,42].Using diferent solvents and culture media might be the reason for this confict.Te mechanism of the antibacterial properties of statins still needs elucidating.SMV, pravastatin (PRV), and lovastatin (LVS) are type 1 statins that are considered to have fungal origins.According to prior studies, unlike PRV and LVS, SMV exhibited antibacterial efects [24].ATV and RSV are classifed as type 2 statins, which are synthetic compounds, and both have shown antibacterial activity [29].Hence, the fungal origin might not be related to statins' antibacterial properties.Te presence of two methyl groups with a tetrahedral or similar trigonal pyramidal in molecular geometry, evident in statins with antibacterial activity (ATV, RSV, SMV, and fuvastatin), has been hypothesized as  Advances in Pharmacological and Pharmaceutical Sciences a critical mechanism of the statin's antibacterial properties [49].Te mentioned structure can infuence the bacteria in diferent ways, such as hydrogen bonds with lipopolysaccharide structures leading to Gram-negative bacterial surface breakdown, disruption in cellular controlling functions through nonpolar forces between statins and teichoic acids, and lipoteichoic acids in the surface of the Gram-positive bacteria.Lastly, the potential van der Waals forces and hydrogen bonds with surface proteins in other Gramnegative and Gram-positive bacteria interfere with surface consistency [49,50].In a study, 100 μM of statins (SMV, LVS, and mevastatin) could decrease the swarming motility of P. aeruginosa in diferent strains [41].It has been elucidated that bacteria motility is intrinsically related to other essential traits of bacterial virulence phenotypes, such as quorum sensing [51] and the ability to form a bioflm [52].
In the present study, the inhibition zones produced by statins were not more extensive than those of current antibiotics, and our reported MICs were higher than those in some previous studies.Tis discrepancy may be attributed to the emergence of new H. pylori-resistant strains, failed H. pylori eradication therapy, uncontrolled use of empirical antibiotic therapy for treating respiratory, genital, and urinary infections, as well as potential variations due to race and ethnicity [9,53].In addition, the H. pylori bacteria investigated in this study were retrieved from gastric biopsies, and clinical isolates have been observed to be less susceptible to antimicrobial agents compared to standard isolates [29].
We anticipated difculties in receiving H. pylori eradication therapy due to the drug's side efects, such as diarrhea, taste disturbance, nausea, and abdominal pain [54].Statins are a potential option for H. pylori eradication clinical trials to decrease the current drug dosage and treatment duration.However, our study has some limitations.Te polymerase chain reaction method could have aided in identifying specifc resistant strains and clarifying the efects of statins on the most frequent local strains.Furthermore, using standard-type isolates of various pathogens and clinical isolates could have improved our understanding of the antibacterial properties of statins.

Conclusion
Tis study demonstrated that statins exhibit antibacterial activity against Helicobacter pylori isolates, with atorvastatin showing the highest potency among the tested agents.Tese fndings suggest that adding statins (especially atorvastatin) as a combination therapy for H. pylori is benefcial, particularly in the context of antibiotic resistance.Further research is warranted to elucidate the mechanisms underlying the antibacterial efects of statins and explore their synergistic potential with other antimicrobial agents.

Figure 1 :
Figure 1: Isolation and identifcation of Helicobacter pylori.(a) Typical appearance of H. pylori colonies on selective Brucella agar plates.Te colonies exhibit a circular, smooth, and translucent morphology with diameters of 0.5-1 mm.(b) Gram staining of the isolated H. pylori bacteria, showing the characteristic curved or spiral-shaped, Gram-negative rods.

Figure 2 :
Figure 2: Scanning electron micrograph depicting the characteristic spiral morphology of H. pylori bacterial cells isolated from gastric biopsy samples.Te bacteria in groups A (AMOX), B (SMV), and C (ATV) were cultured, fxed, dehydrated, and sputter-coated with goldpalladium before imaging by scanning electron microscopy.

Figure 3 :
Figure 3: Te minimum inhibitory concentration (μg/ml) against H. pylori: the MICs of tetracycline and amoxicillin are included for comparison.Data are presented as mean ± standard deviation.* * * p < 0.001 compared to atorvastatin (n � 10 sample).
Of the 65 biopsy samples analyzed, 35 (53.84%) were positive for the urease test, indicating the presence of active H. pylori infection.However, only 10 samples (15.38%) yielded positive cultures for H. pylori.3.2.Isolation and Identifcation of H. pylori.Among the 65 biopsy samples analyzed, 35 (53.84%) tested positive for the urease test, indicating the presence of H. pylori infection.
(40,2.Disc Difusion Method.Te antibacterial activity of SMV, ATV, and RSV was evaluated with the Kirby-Bauer disc difusion method.Sterile blank discs (6 mm) were inoculated with 10 μl of 400, 800, and 1200 μg/ml of SMV, ATV, and RSV (400, 800, and 1200 μg/ml).AMOX and TET(40, 80, 160, and 320 μg/ml) and the no drug/solvent discs served as controls.For each case, 10 μl of 0.5 McFarland bacterial suspension was spread on Brucella blood agar's Petri dish.Discs were placed with sterilized forceps at a distance of 24 mm on a dry agar surface.After incubation for three days under microaerobic conditions, the diameter of the inhibition zones around each disc was measured using a metric ruler.Te study comprised 23 males (57.5%) and 17 females (42.5%).Endoscopic evaluation revealed gastritis as the most prevalent fnding, observed in 25 patients (62.5%), followed by peptic ulcers in 15 patients (37.5%).A total of 65 tissue samples were obtained from these patients.Te demographic characteristics and endoscopic fndings of the study participants are summarized in Table1.Subsequent culture isolation eforts yielded 10 (15.38%) positive cultures for H. pylori.SEM analysis of the cultured isolates revealed the characteristic morphology of H. pylori bacteria, which is a spiral-shaped rod (Figures1 and 2).3.3.Antibacterial Assay.Te MICs of the tested antimicrobial agents against H. pylori isolates were determined by the agar dilution method.ATV exhibited the lowest MIC (240 ± 20 μg/mL) among the statins tested, followed by RSV (450 ± 20 μg/mL) and SMV (460 ± 15 μg/mL).Te MIC values for the control antibiotics, TET and AMOX, were 155 ± 30 μg/mL and 140 ± 20 μg/mL, respectively.ATV demonstrated signifcantly lower MIC values compared to SMV and RSV (p < 0.001).In addition, TET and AMOX exhibited signifcantly lower MIC values than all three statins (p < 0.001).Figure

Table 1 :
Demographic characteristics of the patients and endoscopic fndings.