Synergistic potentiation of antibiotics by chamomile phytochemicals against multidrug-resistant Helicobacter pylori

Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. Briefly, 200 µL of ATL buffer and 20 µL of proteinase K were added to 200 µL of the sample to lyse it, and then the mixture was incubated for 10 min at 56 °C. To assist the binding of DNA to the QIAamp Mini Spin Column, 200 µL of ethanol (96%) was added. After vortexing for 15 s, the mixture was transferred to the QIAamp Mini spin column (in a 2 mL collection tube) and centrifuged at 6000 x g (8000 rpm) for 1 min to allow the DNA to adhere to the silica membrane within the column. Subsequently, three consecutive washing steps were performed using different buffers provided in the kit to purify the genomic DNA, according to the manufacturer’s instructions. Finally, after the purification, the eluted DNA was used as the template for the PCR reaction.

For the molecular detection of H. pylori, two specific genes were targeted: the glmM gene and the 16 S rRNA gene. The glmM gene is a housekeeping gene present in all H. pylori strains. It encodes phosphoglucosamine mutase, an enzyme that converts glucosamine-6-phosphate into glucosamine-1-phosphate, a precursor in the biosynthesis of N-acetylglucosamine. This pathway is essential for peptidoglycan synthesis, which maintains bacterial cell wall integrity and supports growth [19]. Conventional PCR assays were performed at the Animal Health Research Institute, Dokki, Giza, Egypt. Both genes were amplified in identical reaction mixtures with a final volume of 25 µL, including 5 µL of extracted DNA, 12.5 µL of EmeraldAmp GT PCR Master Mix (Takara), and 20 pmol of each primer. Primers for the glmM gene were adopted from Arfaee et al. [20], and primers for the 16 S rRNA gene from Ho et al. [21]. The sequences of the forward (F) and reverse (R) primers are listed in Table 2, and the PCR cycling conditions are summarized in Table 3. The expected amplicon sizes were 296 bp for glmM and 109 bp for 16 S rRNA. PCR products were analyzed on 2% agarose gels stained with ethidium bromide, using a 100 bp ladder as the molecular size marker. Each assay included both positive and negative controls. Electrophoresis was run for 30 min, after which the gels were visualized under UV light and photographed with a gel documentation system.

Dried chamomile flowers (Matricaria chamomilla) were purchased from local markets in Cairo, Egypt, for testing against H. pylori. Ethanolic extract was prepared using the soaking method with a 1:10 (w/v) plant-to-solvent ratio. Specifically, 10 g of dried flowers were soaked in 100 mL of 99.8% ethanol for 48 h in a sterilized flask sealed with a cork [22]. After soaking, the mixture was filtered through Whatman No. 1 filter paper, and the filtrate was sterilized by passing through a 0.22 μm Millipore filter. The final extract concentration was adjusted to 100 mg/mL.

The tolerance level of chamomile extract was determined using the ratio of MBC to MIC (MBC/MIC). This ratio was used to classify the antimicrobial activity of the extract as either bacteriostatic or bactericidal. Extracts with an MBC/MIC ratio ≥ 4 were considered bacteriostatic, while those with an MBC/MIC ratio < 4 were considered bactericidal. This method provides a straightforward way to evaluate whether the extract inhibits growth or kills bacterial cells [25].

Fourier Transform Infrared Spectroscopy (FTIR) was used to identify the functional groups in the chamomile extract. This technique detects infrared light absorption at specific wavelengths that correspond to characteristic vibrations of chemical bonds, producing a spectrum with peaks indicating particular functional groups. The analysis was performed at the Central Laboratory, Faculty of Science, Ain Shams University, Cairo, Egypt.

Before gas chromatography–mass spectrometry (GC–MS) analysis, the chamomile extract was dried and derivatized by suspending it in 50 µL of bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and 50 µL of pyridine to convert functional groups into trimethylsilyl (TMS) derivatives. GC–MS analysis was performed using an Agilent Technologies system, consisting of a 7890B gas chromatograph coupled with a 5977 A mass selective detector, at the Central Laboratories Network of the National Research Centre, Cairo, Egypt. A DB-5MS column (30 m × 0.25 mm internal diameter, 0.25 μm film thickness) was used. The temperature program included an initial 60 °C for 1 min, followed by a ramp to 320 °C at 10 °C/min, with a 10-minute hold. A splitless injection of 1 µL was used, with hydrogen as the carrier gas at a flow rate of 1.0 mL/min. The injector and detector temperatures were maintained at 300 °C and 320 °C, respectively. Mass spectra were recorded by electron ionization (EI) at 70 eV over the m/z range of 50–800, with a solvent delay of 6 min, a quadrupole temperature of 150 °C, and a mass transfer line temperature of 230 °C. Compounds were identified by comparing fragmentation patterns with those in the Wiley and NIST Mass Spectral Libraries.

The three-dimensional (3D) structures of H. pylori protein targets were obtained in PDB format from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) [47]. The proteins listed in Table 4 were prepared by removing water molecules, adding hydrogen atoms and Kollman charges, and checking for and correcting any missing residues. Subsequently, the protein structures were converted to PDBQT format using AutoDock Vina 1.5.7 for further molecular docking studies.

After GC–MS analysis of chamomile extract, compounds present at higher concentrations and known for their antibacterial activity were selected as ligands for docking studies against H. pylori targets. Ligand preparation involved retrieving the 3D structures of these compounds from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) [48] in Structure Data File (SDF) format. Geometry optimization and energy minimization were then performed using the Auto Optimization tool, followed by the Merck Molecular Force Field 94 (MMFF94) in Avogadro 1.2.0, to generate stable, low-energy conformations. The optimized ligand structures were subsequently converted to Protein Data Bank (PDB) format for use in molecular docking studies.

Molecular docking was performed using AutoDock Vina 1.5.7, which requires both the receptors and ligands to be in PDBQT format. Protein binding sites were identified by visualizing interactions with control ligands and based on information from previous studies involving these proteins. For each target, a grid box was created to cover the active site and facilitate ligand binding. Grid center coordinates and box dimensions for each target are listed in Table 5. The binding energy (kcal/mol) between ligand and protein served as the main parameter for docking. Ligand–protein interactions were visualized with Discovery Studio (DS) Visualizer software 2021 to examine how compounds bind to specific amino acid residues within the target proteins.

The pharmacokinetic profiles of the selected compounds were assessed by first applying Lipinski’s rule of five to evaluate drug-likeness, followed by in-silico ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) predictions. Compounds with favorable docking scores and interactions were then prioritized for further pharmacokinetic analysis.

Although all thirty samples tested positive for RUT, only twenty produced positive cultures. The colonies obtained were characteristic of H. pylori, as they were small, transparent, and tested positive for urease and catalase, as shown in Figs. 1 and 2. Under microscopic examination, the cells appeared in the typical form of H. pylori, which is a gram-negative spiral or curved rod.

Since neither EUCAST nor CLSI guidelines specify disc diffusion breakpoints for H. pylori, we relied on previous studies to interpret our results. Resistance was defined as an inhibition zone diameter of less than 21 mm for clarithromycin, less than 17 mm for metronidazole, rifampicin, and levofloxacin, and less than 14 mm for amoxicillin and tetracycline [53]– [54]. Based on these criteria, all isolates were resistant to clarithromycin, metronidazole, amoxicillin, tetracycline, and rifampicin, while 60% remained susceptible to levofloxacin, as shown in (Figs. 3 and 4; Tables 6 and 7). The isolates were grouped into four groups based on antibiotic sensitivity results: Group 1 (four isolates) showed inhibition zones of 10.33 ± 0.33 mm (TE, 30 µg), 12.33 ± 0.33 mm (RA, 30 µg), and 10 ± 0.00 mm (LEV, 5 µg), with no inhibition for other antibiotics; Group 2 (four isolates) exhibited 13 ± 0.00 mm (TE, 30 µg) and 13 ± 0.00 mm (RA, 30 µg), with no inhibition for LEV (5 µg), CLR (15 µg), MET (5 µg), or AX (25 µg); Group 3 (four isolates) demonstrated inhibition zones of 13 ± 0.00 mm (TE, 30 µg), 14 ± 0.00 mm (RA, 30 µg), and 17.33 ± 0.33 mm (LEV, 5 µg); and Group 4 (eight isolates) showed inhibition zones of 12.33 ± 0.33 mm (TE, 30 µg), 14 ± 0.00 mm (RA, 30 µg), and 22.66 ± 0.33 mm (LEV, 5 µg), as shown in Fig. 4.

In this study, conducted at a single hospital in Egypt, H. pylori isolates were grouped based on their antibiotic sensitivity profiles. One isolate from each group was randomly chosen for PCR detection of the H. pylori-specific glmM and 16 S rRNA genes. Four isolates with typical colony morphology and positive results in both RUT and catalase assay were analyzed. Only the isolate from group 1 produced the expected 296 bp glmM band, as shown in Fig. 5. The other three isolates were negative for glmM, and all four tested negative for the H. pylori-specific 16 S rRNA gene.

The tested plant extract showed anti-H. pylori activity, with an average inhibition zone diameter of 16.73 ± 0.87 mm. The inhibition zones for groups 1–4 were 24.33 ± 0.33, 14.00 ± 0.00, 15.33 ± 0.33, and 15.00 ± 0.00 mm, respectively, as shown in Fig. 6 and Table 7. When combined with antibiotics, chamomile extract exhibited varying effects depending on the H. pylori isolate, revealing two types of synergism. In groups 2 and 4, chamomile extract alone produced zones of 14.00 ± 0.00 mm and 15.00 ± 0.00 mm, while (CLR, 15 µg) and (MET, 5 µg) alone have no inhibition; however, when combined with chamomile extract, inhibition zones increased to 16.33 ± 0.33 mm and 16.00 ± 0.00 mm in group 2 respectively, and 15.33 ± 0.33 mm for both antibiotics in group 4, indicating antibiotic synergistic to chamomile effect. Mutual synergism was also observed: (TE, 30 µg) and (RA, 30 µg) in group 2 increased from 13.00 ± 0.00 mm alone to 28.66 ± 0.88 mm (2.2-fold) and 27.33 ± 0.33 mm (2.1-fold), respectively, when combined with chamomile extract. In group 4, (TE, 30 µg), (RA, 30 µg), and (LEV, 5 µg) alone produced zones of 12.33 ± 0.33 mm, 14.00 ± 0.00 mm, and 22.66 mm, which increased to 28.66 ± 0.33 mm (2.3-fold), 29.33 ± 0.33 mm (2.1-fold), and 38.33 ± 0.88 mm (1.7-fold), respectively, when combined with chamomile extract. For isolates in group 3, LEV (5 µg) alone produced a zone of 17.33 ± 0.33 mm, which increased to 34.33 ± 0.33 mm (1.9-fold) with the addition of chamomile extract, highlighting the enhanced activity of both agents.

Additionally, two distinct types of antagonism were observed between chamomile extract and antibiotics. In the first type, antibiotics antagonistic to chamomile (CLR, 15 µg; MET, 5 µg; AX, 25 µg) showed no inhibitory effect when tested alone, whereas the chamomile extract demonstrated strong inhibition. When combined, the inhibition zones were smaller than those of chamomile alone, indicating reduced sensitivity of H. pylori. For example, in group 1, chamomile alone produced a zone of 24.33 ± 0.33 mm, while combinations with clarithromycin (CLR, 15 µg), amoxicillin (AX, 25 µg), and metronidazole (MET, 5 µg) resulted in zones measuring 13.33 ± 0.33 mm, 11.33 ± 0.33 mm, and 11 ± 0.00 mm, respectively. In group 3, chamomile alone produced a zone of 15.33 ± 0.33 mm, while the combination treatments yielded zones of 14.33 ± 0.33 mm (CLR, 15 µg) and 14 ± 0.00 mm (AX, 25 µg; MET, 5 µg), which were again smaller than the zones for chamomile alone.

The second type, mutual antagonism, was observed with tetracycline (TE, 30 µg) and rifampicin (RA, 30 µg), where both the antibiotic and chamomile extract individually inhibited H. pylori. However, the combined zones were smaller than the sum of their individual effects. For example, in Group 1, TE, RA, and levofloxacin (LEV, 5 µg) alone produced diameters of 10.33 ± 0.33 mm, 12.33 ± 0.33 mm, and 10.00 ± 0.00 mm, respectively. When combined with chamomile, the inhibition zones measured 14.66 ± 0.33 mm, 15 ± 0.00 mm, and 16.66 ± 0.33 mm for TE, RA, and LEV, respectively. Similarly, in Group 3, TE and RA alone produced 13 ± 0.00 mm and 14 ± 0.00 mm, respectively, resulting in zones of 24.33 ± 0.66 mm (TE, 30 µg) and 22 ± 0.00 mm (RA, 30 µg) with chamomile, still less than the sum of their individual effects, confirming mutual antagonism. Synergistic and antagonistic interactions of chamomile with antibiotics are represented in Table 8; Figs. 7 and 8. The effect of chamomile and its combination with antibiotics was statistically significant (P < 0.05).

The ethanolic extract of chamomile inhibited H. pylori, with MICs varying among isolate groups, indicating differences in susceptibility (range: 1.56–6.25 mg/mL). The lowest MIC (1.56 mg/mL) was found in the second group of isolates (four isolates), while the highest MIC (6.25 mg/mL) was observed in the third group (four isolates). Corresponding MBCs ranged from 3.12 to 12.5 mg/mL, with groups 1, 2, and 4 showing MBCs of 3.12 mg/mL, and group 3 having an MBC of 12.5 mg/mL (Supplementary Table S1). The MBC/MIC ratios, shown in Table 9, indicate that chamomile extract displayed bactericidal activity against all H. pylori groups at concentrations close to their MICs.

The FT-IR spectrum of chamomile ethanolic extract, as shown in Fig. 9, reveals various functional groups. A peak at 3272 cm⁻¹ is typically linked to the stretching vibration of hydrogen-bonded hydroxyl groups (-OH), while the peak at 2924 cm⁻¹ corresponds to the C–H stretching vibrations of alkanes. Another peak at 2854 cm⁻¹ is also related to C–H stretching, specifically the symmetric stretching of CH₂ groups in alkanes. The peak at 1712 cm⁻¹ is commonly attributed to the carbonyl (C = O) stretching vibration of ketones, aldehydes, or esters, whereas the peak at 1606 cm⁻¹ indicates the C = C stretching vibration of aromatic rings. Additionally, the peak at 1030 cm⁻¹ is often associated with C–O stretching vibrations in functional groups such as phenols and ethers. Notably, hazardous groups like cyano (C ≡ N: 2220–2260 cm⁻¹) and acetylenic (C ≡ C: 2100–2200 cm⁻¹) were absent, suggesting that chamomile ethanolic extract is safe, consistent with previous studies using FT-IR for safety assessment [55].

GC-MS analysis identified thirty-eight compounds in the chamomile extract, as shown in Fig. 10, including saturated and unsaturated fatty acids, carboxylic acids, sugar alcohols, glycosides, amino acids, and sugars. Among these, fourteen compounds such as palmitic acid, oleic acid, 9,12-octadecadienoic acid, stearic acid, benzoic acid, xylonic acid, myo-inositol, myristic acid, erythritol, butanedioic acid, heptanoic acid, octanoic acid, and nonanoic acid have been previously reported to possess antibacterial activity, enhance the activity of other compounds, or exhibit synergistic effects with antibiotics, as shown in Table 10.

An in silico study showed that chamomile extract can significantly impact H. pylori by targeting various essential cellular components, especially enzymes. Of the fourteen antibacterial compounds found in the extract, eight demonstrated high binding affinities with specific H. pylori targets compared to control ligands. Lower binding energy values represent more stable ligand–protein complexes, as summarized in Table 11, while detailed interactions between ligands and target proteins are illustrated in Table 12; Fig. 11. These promising compounds include myo-inositol, xylonic acid, stearic acid, palmitic acid, cis-11,14-eicosadienoic acid, oleic acid, octadecadienoic acid, and benzoic acid. Interactions between control ligands and target proteins are shown in Supplementary Table S2 and Fig. S1.

The eight compounds from chamomile extract that showed strong binding affinities with various H. pylori targets were further evaluated for their ADMET properties, as shown in Table 13. Lipinski’s rule of five was also used to assess the drug-likeness of each compound, ensuring their potential safety and effectiveness. All eight compounds exhibited either no violations or only one violation of Lipinski’s rules, as demonstrated in Table 14.

GIA (gastrointestinal absorption). BBB (Blood Brain Barrier). PgP (P-glycoprotein transporter). CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4 are the five types of cytochromes P450 (CYP). PAINS (Pan Assay Interference).