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Background
The prevalence of antibiotic resistance has become a critical global health concern. In particular, resistant strains of Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) against commonly prescribed antibiotics for various infections are on the rise. These pathogens are frequently implicated in severe and complex infections, e.g., diabetic foot infections (DFI), posing a significant antimicrobial challenges during treatment. Frequently used food grade product, i.e., apple cider vinegar (ACV) carries promising antimicrobial potential. Therefore, the study designed to investigate the potential of ACV in combination with antibiotics to determine the effectiveness of the combination in overall pathogenic burden.
Results
Antimicrobial sensitivity was evaluated using disk diffusion and broth dilution techniques, revealing that at 2.5% acidity, ACV has prominent inhibitory potential against SA and PA. The fractional inhibitory concentration (FIC) index further confirmed synergistic interplay of ACV in combination with antibiotics. The results for minimum bactericidal concentration (MBC) showed when ACV is added to existing antibiotics the MBC value after checkerboard analysis method comes out to be, 128 µg/mL, 128 µg/mL, 64 µg/mL, and 64 µg/mL for amoxicillin, cefotaxime, imipenem, and vancomycin, respectively, against SA whereas concentration of 128 µg/mL, 256 µg/mL, 256 µg/mL, and 128 µg/mL MBC values for respective antibiotics against PA. Quantitative PCR analysis has demonstrated a substantial reduction in the expression of resistance-conferring genes when ACV was combined with antibiotics. Furthermore, molecular docking analysis showed ACV’s active constituents, such as acetic acid and chlorogenic acid, exhibited strong binding affinities against resistant conferring genes and subsequent proteins expression. These findings suggest that ACV may alter permeability of the outer membrane porin channels, thereby improving antibiotic penetration and augmented antimicrobial efficacy.
Conclusion
The study demonstrated that ACV not only improves antibiotic permeability within bacterial cell but also significantly augments bactericidal activity of these agents against resistant strains of SA and PA. The combination of various concentrations of ACV with antibiotics presents an innovative therapeutic strategy to combat current antimicrobial resistance, particularly in the treatment and management of complex DFI. These findings underscore the potential of integrating food grade products with conventional antibiotics to address the growing challenges of antibiotic resistance.
Background
The discovery of antibiotics in the 20th century brings revolution in medical field and had always been considered as greatest medical triumph [1]. The ready availability of antibiotics had certainly eradicated significant number of morbidities and mortalities [2]. The problem begins with the emergence of resistance to these antibiotics whether hospital acquired or through communities and linked environments. The mechanism of antibiotic resistance is not a single phenomenon rather it is a blend of several mechanisms that imparts resistance [3]. The inactivation of chief enzymes whichever way either by hydrolysis or by structural modification is the major cause of drug resistance [4]. Additionally, the alteration in Penicillin-binding protein (PBP) causes modification in the target site [5]. Moreover, spontaneous mutation of a bacterial gene on a chromosome also results in the changes in target site [6]. Furthermore, modification in the metabolic pathway as shown by sulfonamide-resistant bacteria, because they do not oblige para-amino benzoic acid (PABA) which is an essential precursor for the production of folic acid and nucleic acid in bacteria [7]. Additionally, biofilm formation by certain bacteria incur resistance against existing antibiotics [8, 9]. Despite the availability of different antibiotics, multidrug efflux pump (EP) helps bacterial population to survive and incur resistance [10, 11–12].
Diabetic foot infection (DFI) is the major complication associated with type-I and type-II diabetes mellitus. It leads to hospitalization to most of the patient due to symptoms of neuropathy which can cause tingling, burning, weakness in the foot, and ultimately loss of sensation leading to non-traumatic amputation [13]. Osteomyelitis is the major complication of DFI necessitating surgical procedure [14]. The most frequently isolated bacteria from DFI are Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). The former is a gram-positive, opportunistic bacterium that is commonly present in the epithelial mucous membrane. The latter is a gram-negative, drug-resistant, and food spoilage pathogen which is considered very notorious for causing both the in vitro and in vivo health problems [15]. These microbes pose challenge to currently available antimicrobials by conferring resistance [16]. The chief mechanism of resistance for these microbes is the change in permeability of the outer membrane which is considerably low in resistant strains compared to normal sensitive species [17]. A part of this also includes an active efflux mechanism that too largely contributes to the resistance mechanism of various β-lactam antibiotics [18].
Among all Staphylococci species, approximately ~ 23.7% are found to be methicillin-resistant Staphylococcus aureus. Methicillin, the initial semi-synthetic penicillinase-resistant penicillin, has been discontinued in the USA due to a notable prevalence of interstitial nephritis linked to its usage. This antibiotic is administered either intramuscularly or intravenously to address infections caused by gram-positive aerobes. Therefore, newer drugs, e.g., vancomycin have shown to be effective therapy for the treatment DFI. However, it is a narrow spectrum antibiotic and can only effectually cover gram-positive spectrum [19].
Extensive work in recent past has demonstrated antimicrobial efficacy of various naturally occurring food grade products. The limited data regarding mechanistic insights of these substances urge scientists to employ these agents simultaneously with synthetically produced antimicrobial agents to curb existing microbial resistance [20]. Currently, owing to the increased antibiotic resistance, these naturally occurring substances are being frequently employed/evaluated as an adjunct to augment the sensitivity of existing antimicrobial agents. The crucial components from these natural sources used as antimicrobial components include various phytochemicals and some essential oils which are complex mixture of volatile secondary metabolites [21, 22]. Apple cider vinegar (ACV) is produced by fermentation of ripped apples. It is an excellent source of potassium which is necessary for soft tissue repair and worn-out tissues [23]. The previous studies have endorsed that ACV has shown standalone antimicrobial activity and was occasionally employed in the cleaning of wounds [24]. However, the mechanism of synergy of this agent with antibiotics is yet to be identified. Therefore, combining these food grade products with existing antibiotics in order to mitigate or combat microbial resistance could provide astonishing approach to overcome currently prevailing antibiotic resistance.
Material and methods
Chemicals, reagents, and equipment
Nutrient agar media, staph 110 media, cetrimide agar media, and antibiotic disks for amoxicillin (25 µg), cefotaxime (30 µg), vancomycin (30 µg), and imipenem (10 µg) were acquired from (Bio-Analyze Limited). ELISA microplate reader (Bio-Tek), compound microscope (Nikon Japan), SimpliAMP PCR machine (Singapore), Double-beam spectrophotometer (PerkinElmer), 96-well microplate (corning), 6-well plates (corning), L-shaped glass spreader, wire loop, Whatman no. 4 filter paper aluminum foil, glass stirrers, NaOH, pipettes, and sucker were taken from the Department of Pharmacology and Toxicology, UVAS, Lahore.
Experimental design
The study was designed to isolate and identify Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) from clinical samples obtained from patients with diabetic foot infections. The minimum selection parameters were, confirmed diabetic (type-I and type-II) patients of either sex, age between 18 and 60 years, have no other major complication except diabetes, have not taken any antibiotic during the past 30 days, willingness to comply with study protocols in the form of informed documented consent, ulcer duration of minimum 2 weeks. The excluded patients were those having not given consent to participate, patients requiring surgical procedure to combat current situation, already using topical antibacterial, pregnant or lactating females, patients taking any immunosuppressants, non-diabetic foot ulcer patients, and already taking any antibiotic either self-administering or prescribed. The antimicrobial susceptibility of the isolates was then evaluated against existing antibiotics, including amoxicillin, cefotaxime, imipenem, and vancomycin, both individually and in combination with apple cider vinegar (ACV). In vitro assays, including disk diffusion and broth dilution methods, were employed to assess the sensitivity patterns and potential synergistic effects between the antibiotics and ACV. The experimental work was carried out at the Postgraduate Laboratory of the Department of Pharmacology and Toxicology, in collaboration with the Quality Operations Laboratory (QOL) at the University of Veterinary and Animal Sciences, Lahore, Pakistan.
Preparation of apple cider vinegar
The apple cider vinegar (ACV) was prepared through an artisanal process, utilizing 5 distinct varieties of apples, e.g., Gacha, kala kulu, red delicious, pink lady, and gajra; abundantly grown in northern part of the country [25]. The acidity of selected varieties of samples was determined by titration with 0.1 N NaOH. The results were expressed as a percentage of acetic acid equivalent.
Patients sample collection
After taking verbal and written informed consent from the patients of different age groups, the pus samples were directly collected with the help of a sterile cotton swab from the infected foot of diabetic patients and labeled accordingly. The minimum selection parameters opted were, confirmed diabetic (type-I and type-II) patients of either sex, age between 18 and 60 years, have no other major complication except diabetes, have not taken any antibiotic during the past 30 days, willingness to comply with study protocols in the form of informed and documented consent, DFI, and ulcer duration of minimum 2 weeks. The excluded patients were those having not given consent to participate, patients requiring surgical procedure to combat current situation, already using topical antibacterial, pregnant or lactating females, patients taking any immunosuppressants, non-diabetic foot ulcer patients, and already taking any antibiotic either self-administering or prescribed. The ethical approval for the said purpose was taken from institutional review board, vide approval letter number (ORIC/IRB/UVAS-JA235773/23) University of Veterinary and Animal Sciences, Lahore. The procedure was done before washing and applying any kind of antiseptic solution or sterile dressing to the patient’s affected area on skin (n = 63). The samples were collected from outpatient department of orthopedic, medical, and surgical wards of Sir Ganga Ram Hospital, Lahore, Pakistan, in accordance with previously described protocols [26, 27].
Streak plate method: isolation and identification of bacterial strain
The collected samples were inoculated separately by streak plate method on nutrient agar media primary culture. The bacterial colonies formed were then taken and separately inoculated on a selective cetrimide agar medium and Staph 110 medium (subculture) and incubated for 24 h at 37 °C. In this way, selective samples of both bacterial species were isolated. In order for the confirmation of isolated samples, gram staining and other biochemical identification tests, e.g., oxidase, catalase, and urease test for PA and catalase, coagulase, and mannitol fermentation test for SA were performed in accordance with the methods described earlier [28, 29].
Antimicrobial assay
Bacterial culture preparation
After isolation and identification of specific bacterial strains, fresh cultures of pure PA and SA were grown on nutrient agar media containing culture plates by streaking each bacterium separately and subsequently incubated overnight at 37 °C. Single colony from overnight culture were transferred into a 5 mL test tube containing sterile saline solution adjusted to a turbidity equivalent to 0.5 McFarland standard (1.5 × 108 CFU/mL) using spectrophotometer at 600 nm.
Spread plate method
Mueller–Hinton agar (MHA) plates were prepared according to manufacturer instructions. The plates were allowed to solidify in a laminar flow hood prior use. Approximately 100 µl of the standardized bacterial suspension of each strain were separately inoculated and spread evenly on to the surface of each MHA plates. A sterile L-shaped glass spreader was used to evenly distribute the bacterial suspension across the agar surface. During the process, care was taken to ensure uniform coverage of bacterial suspension without damaging the agar surface.
Disk diffusion assay
In order to evaluate extent of microbial sensitivity/resistance against under consideration antibiotics, e.g., amoxicillin, imipenem, cefotaxime, and vancomycin, the antibiotic disk was introduced on to the culture plates and incubated for 24 h at 37 °C. Later, the plates were examined to evaluate antibiotic efficacy by measuring diameter of zones of inhibition using calibrated ruler. The zone of inhibition established against each disk was then compared according to “The Clinical and Laboratory Standards Institute” (CLSI) guidelines [30].
Agar well diffusion assay
The agar gel surface in a culture dish is inoculated by spreading a specified volume of the microbial suspension over the entire surface. Then, a hole of about 6–8 mm in diameter is aseptically punched with a sterile cork borer or a sterile pipette tip. A 100 µl volume of the antimicrobial agent alone or prepared sample of food grade product and antimicrobial agent at (1:1) at desired concentrations was introduced into the corresponding well accordingly. Then, agar plates were incubated for 24 h at 37 °C. Later on, zone of inhibition was measured for each sample with the help of calibrated scale.
Initially, apple cider vinegar at concentration of 5%, 2.5%, 1.25%, and 0.625% was added into the wells already cultured for PA and SA bacterial strains. The zone of inhibition was measured with the help of a scale after 12, 24, and 48 h. Later on, selected concentration of ACV along with antibiotic 512 µg (amoxicillin, vancomycin, imipenem, and cefotaxime) was added into separate wells and incubated for a period of 24 h and 48 h at 37 °C. The zone of inhibition was measured accordingly, marked, and analyzed.
Percentage inhibition of microbial growth
In a 96-well plate, 50 µl of broth was added till 10th column. Thereafter, 50 µl of standard bacterial suspension of turbidity 0.5 McFarland and 100 µl of drug solution having concentrations of 512 µg/mL, 256 µg/mL, and 128 µg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL, 8 µg/mL, and 4 µg/mL were added into the rows, respectively, till 10th column. In the 11th column, 150 µl of broth and 50 µl of bacterial suspension were added, and in 12th column, only 200 µl of broth was added as a control. The plates were incubated for a period of 48 h. The absorbance was measured at 590 nm wavelength with the help of Bio-Tek microplate reader in accordance with the established protocols. The experiment was repeated with all groups by adding ACV 2.5% such that drug and ACV (1:1).
Fractional inhibitory concentration (FIC) index
The fractional inhibitory concentration index (FIC Index) was measured using checkerboard method. Serially diluted antibiotics were added along abscissa (X-axis) while the second agent, i.e., ACV was diluted and added along the ordinate (Y-axis). The fractional inhibitory concentration (FIC) index for all of the combinations was then determined using the following formula:
1
where FICA and FICB are the fractional inhibitory concentration of drugs A and drug B, MICA and MICB are the minimum inhibitory concentration of drugs A and B, whereas symbols [A] and [B] are the concentration of drugs A and B, respectively. A FIC index of < 0.5 was interpreted as synergistic, between 0.5 and 4 was interpreted as additive or indifferent, and a value of > 4 was interpreted as antagonistic.Minimum bactericidal concentration (MBC)
After determining the percentage inhibition of combined treatment with antibiotic and ACV at required concentrations, the aliquots of 50 μl from dilutions tubes showed that no visible bacterial growth was seeded on nutrient agar plates and incubated for 24 h at 37 °C. The plates showing no bacterial growth were regarded as MBC end point for said combination. The microbial examination was done under an inverted microscope by observing pre-incubated and post-incubated agar plates for the presence or absence of bacterial growth.
Quantitative polymerase chain reaction (PCR) analysis
Bacterial strains were isolated from clinical settings and cultured on appropriate growth media. Genomic DNA was extracted from overnight cultures using the AccuPrep genomic DNA extraction kit; Bioneer, as per the manufacturer’s instructions. DNA concentration and purity were determined using a Nanodrop spectrophotometer (Thermo Scientific) at 260/280 nm. The primers for antibiotic-resistant genes, such as blaZ, β-lactamase gene (S. aureus), mecA Methicillin resistance gene (S. aureus), blaIMP-1, Carbapenemase gene (P. aeruginosa), and OprD, Outer membrane porin gene associated with imipenem resistance (P. aeruginosa), were designed from the National Center for Biotechnology Information (NCBI) database. The internal control used were 16 s rRNA gene because of its universality and presence across large number of bacterial strains. The conditions were initial denaturation temperature 95 °C for 5 min, cycling (denaturation 95 °C, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s) and, finally, curve analysis were performed at 70 °C for 35 cycles. The forward primer sequence designed for evaluation in S. aureus is 5´-AGAGTTTGATCCTGGCTCAGA-3´, and the reverse primer sequence for the internal control gene is 5´-GGTTACCTTGTTACGACTTCG-3´. Whereas primer sequence for P. aeruginosa for forward primer sequence is 5´-TGGCACCCAGCACAATGACGA-3´ and reverse primer sequence arrangement comprises of 5´-CTAGTATAGTCGCTAGAAGCA-3´. The primers were synthesized by Gene Pharma Pvt. Ltd., P.R China.
Primer sequences used are listed in below table.
Microorganism | Target gene | Forward primer | Reverse primer |
|---|---|---|---|
Staphylococcus aureus and Pseudomonas aeruginosa | blaZ | 5´-ATGAAGAAGCTGATTTTTCTA-3´ | 5´-CATTTCACTTCGCAAAAGCTT-3´ |
mecA | 5´-ATGAAGAAGATCAAAATTGTT-3´ | 5´-CAAGATCAAATGGTCCTAGA-3´ | |
OprD | 5´-ATGAAGTGATGAAGTGGATGC-3´ | 5´ GTCGGCGTTGGCACGGTGCTC-3´ | |
blaIMP | 5´ TGAGCAAGTTATCTGTATTCG 3´ | 5´ TAGTTGCTTGGTTTTGATGCA 3´ | |
Internal control S. aureus | 16s rRNA | 5´-AGAGTTTGATCCTGGCTCAGA-3´ | 5´-GGTTACCTTGTTACGACTTCG-3´ |
Internal control P. aeruginosa | 16s rRNA | 5´-TGGCACCCAGCACAATGACGA-3´ | 5´-CTAGTATAGTCGCTAGAAGCA-3´ |
Molecular docking analysis
The AutoDock Vina 1.1.2 and AutoDock Tool 1.5.6 (ADT) were utilized to carry out molecular docking analysis. The antibiotic-resistant genes in under consideration bacterial species, e.g., mecA, blaZ, blaIMP-1, and OprD receptor crystal structure (PDB: 1DJA, 5M18, 2ODJ, and 1WUO), respectively, were obtained from the protein data bank. Subsequently, ADT was applied to remove water molecules and remove co-crystallized ligands from the crystal structure. Consequently, Gasteiger charges and polar hydrogens were added, and the resulted receptors were saved in PDBQT format. Energy minimized and 3D of amoxicillin, cefotaxime, imipenem, and vancomycin, acetic acid, chlorogenic acid, and quercetin, were built and documented in Protein Data formatted by applying Chem-Office software.
Statistical analysis
Statistical analysis of the results was performed using one-way ANOVA followed by LSD and Tukey post hoc analysis using SPSS 22 software (SPSS Inc., Chicago, IL, USA) and GraphPad prism version 9.0. The p-values of < 0.05 were considered to be statistically significant.
Results
Patient sample analysis
A total of 63 patient’s samples were collected and distributed accordingly against each strain and from each ward. Only 10 samples were decisively identified as PA, and 25 samples were identified as SA. The remaining samples showed insignificant growth on selective growth media (Staph 110 and cetrimide) in screening analysis and were excluded from further analysis as shown in Fig. 1 (A–C), respectively.
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Fig. 1
Patient sample analysis A Total number of patient samples taken from surgical, medical, and orthopedic wards, B bacterial strain isolation, i.e., S. aureus and P. aeruginosa from the total number of sample, and C distribution frequency of isolated bacterial strain from each ward
Food grade sample characterization
The acidity analysis results showed apple variety of sample S4 (red delicious) possess the highest percentage of acidity, i.e., (4.9 ± 0.07%). The characterization and acidity analysis of different vinegar samples were performed according to the previously described protocols [31, 32–33] and shown in Table 1.
Table 1. Acidity profile of different apple varieties
Sample | Origin | Variety | Source purchased | Acidity (%) |
|---|---|---|---|---|
S1 | Swat valley | Gacha | Hyper mart | 2.9 ± 0.04 |
S2 | Swat valley | Kala Kulu | Hyper mart | 2.5 ± 0.06 |
S3 | Swat valley | Gajra | Hyper mart | 1.7 ± 0.03 |
S4 | Hunza valley | Red delicious | Hyper mart | 4.9 ± 0.07 |
S5 | Hunza valley | Pink lady | Hyper mart | 1.2 ± 0.06 |
The bold identifies the highest acidity content out of all samples
Patient sample characterization and biochemical analysis
Isolation of bacterial strains showed PA appears in blueish–green color colonies whereas SA appears in golden–yellow in respective culture dishes. The gram staining results showed golden–red to pinkish rods for PA and round purple colonies of SA, respectively, as shown in Supplementary Fig. 1 (A&E), respectively. Further biochemical analysis results, e.g., oxidase and urease test confirmed presence of SA whereas mannitol fermentation, coagulase, and catalase test confirmed presence of PA strain in sample as shown in Supplementary Fig. 1 (B, C, D, F, G, H), respectively.
Disk diffusion assay results
In total of 25 samples identified as SA, all were resistant against imipenem and cefotaxime, 23 were resistant against amoxicillin, and only 1 was resistant against vancomycin. Likewise for PA from a total of 10 samples, all were resistant against vancomycin and amoxicillin, 9 were resistant against cefotaxime; conversely, all samples have shown sensitivity against imipenem when compared in accordance with CLSI guidelines as shown in Fig. 2 (A, B) and supplementary Fig. 2(A–D).
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Fig. 2
Disk diffusion assay result A Antibiotic sensitivity pattern of SA against different antibiotics at specified concentration. B Antibiotic sensitivity pattern of PA against different antibiotics
Antimicrobial potential of apple cider vinegar
In an effort to evaluate antimicrobial potential of under investigation food grade product ACV, we ran a screening analysis assay for calculation of optimum concentration of these agents showing growth inhibitory potential against selected bacterial strains. The results showed ACV at concentration 2.5% and 5% showed significant bacterial growth inhibition at 24h and 48h as shown in Fig. 3.
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Fig. 3
Agar well diffusion assay. ACV concentration 0.625, 1.25, 2.5, and 5% was evaluated against AS. aureus strain and BP. aeruginosa and zone of inhibition were measured
Agar well diffusion assay results
The addition of ACV with existing antibiotics, e.g., amoxicillin, imipenem, cefotaxime, and vancomycin against selected bacterial strains has found to significantly enhance sensitivity of SA and PA strains. The zone of inhibition measured at 24 h and 48 h showed significantly greater inhibition against SA compared to inhibition against PA when combination was evaluated via agar well diffusion assay as shown in Fig. 4. The agents were evaluated for its inhibitory potential alone and in combination with antibiotics.
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Fig. 4
Agar well diffusion assay. A, C Different antibiotic combinations with ACV different concentrations showed augmented zone of inhibition against S. aureus as compared to individual inhibitory potential of each agent. B, D Combination of antibiotics with different ACV concentrations showed enhanced zone of inhibition as compared to their individual inhibitory potential
Percentage inhibition of microbial growth:
The bacterial sensitivity against combination therapy via agar well assay was calculated in terms of bacterial growth. The results showed that combination of antibiotics with ACV has shown significant inhibition in microbial growth. The assay results have shown that the growth of SA, Fig. 5 (A, B, C, and D) and PA, Fig. 5 (E, F, G and H) were significantly reduced against combination of ACV 2.5% and various antibiotics combination.
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Fig. 5
Percentage inhibition of microbial growth S. aureusA–D and P. aeruginosaE–H when evaluated against amoxicillin, cefotaxime, imipenem, and vancomycin, respectively, alone or in combination with 2.5% ACV
Checkerboard method for synergy interpretation
The checkerboard method was performed for four combinations of ACV with amoxicillin, cefotaxime, vancomycin, and imipenem separately, and FIC index was calculated. The interpretation of the test is such that; FIC Index ≤ 0.5 synergism, FIC Index ˃ 0.5– ≤ 1 partial synergism, FIC Index ˃ 1– < 2 indifference, and FIC Index ≥ 2 Antagonism. In our results, the combination of ACV with antibiotics gives FIC index nearly ≤ 1 stating that the combination has shown partial synergism as shown in Fig. 6.
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Fig. 6
FIC index calculation. A–D FIC index of various antibiotics with ACV against S. aureus.E–H FIC index of various antibiotics with ACV against P. aeruginosa
Minimum bactericidal concentration
The results for minimum bactericidal concentration (MBC) showed when ACV is added to existing antibiotics the MBC value after checkerboard analysis method comes out to be, 128 µg/mL, 128 µg/mL, 64 µg/mL, and 64 µg/mL for amoxicillin, cefotaxime, imipenem, and vancomycin against SA whereas concentration of 128 µg/mL, 256 µg/mL, 256 µg/mL, and 128 µg/mL MBC values for respective antibiotics against PA.
Quantitative PCR analysis
The results of qPCR showed significant decrease in the expression of resistance conferring genes in both bacterial strains after the combination therapy has been utilized. We have evaluated four different resistant gene expressions against using combination of ACV 2.5% and stated concentrations of under consideration antibiotics. The results of resistant gene expression are shown in Fig. 7.
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Fig. 7
Quantitative PCR analysis for antibiotics resistant conferring genes, e.g., mecA, blaZ, blaIMP-1, and OprD that aid pathogen to escape against bacteriostatic or bactericidal effect against existing antibiotics. A mecA, B blaZ, C bla-IMP-1, and D OprD expression of resistant genes against combination therapy
Molecular docking
In order to find out the precise target for enhanced antimicrobial sensitivity mechanism, we carried out molecular docking analysis. The target proteins and ligand for docking studies were selected and prepared using online database, i.e., Protein data base (PDB) and PubChem online platform. The docking score for selected proteins with various ligands is summarized in Table 2.
Table 2. Summary of ligand vs protein binding energy
Ligand vs Protein Binding energy (kj/mol) | ||||
|---|---|---|---|---|
Ligands | mecA | blaZ | bla-IMP-1 | OprD |
Amoxicillin | − 8.8 | − 7.9 | − 7.6 | − 7.5 |
Cefotaxime | − 7.9 | − 7.2 | − 7.4 | − 7.2 |
Imipenem | − 7.0 | − 6.3 | − 8.6 | − 8.7 |
Vancomycin | − 9.5 | − 9.9 | − 12.7 | − 10.7 |
Acetic acid | − 3.3 | − 3.4 | − 3.6 | − 3.2 |
Chlorogenic acid | − 8.2 | − 7.9 | − 8.4 | − 7.8 |
Quercetin | − 8.7 | − 8.2 | − 8.3 | − 8.4 |
The maximum binding affinity of selected ligands results for antimicrobial sensitivity mechanism of action are shown in Fig. 8A and B. For preparing protein for mecA (PDB: 1IAS), amoxicillin showed highest binding affinity of (− 11.5 kJ/mol). For blaZ (PDB: 1KS6), amoxicillin showed binding affinity of (− 7.2 kJ/mol) as shown in Fig. 8A.
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Fig. 8 A
Molecular docking analysis. Antibiotic resistant conferring gene binding energy relationship with existing antibiotics and active constituents of ACV B Molecular docking analysis. Antibiotic resistant conferring gene binding energy relationship with existing antibiotics and active constituents of ACV
In a similar way for preparing blaIMP-1 (PDB: 1IVO), amoxicillin showed significant binding affinity of (− 9.8 kJ/mol). For OprD protein (PDB: 1HHN), amoxicillin showed binding affinity of (− 7.2 kJ/mol) as shown in Fig. 8B.
Therefore, the docking results showed that antibiotics have great affinity toward resistant gene. The increased sensitivity of antibiotics in combination with ACV implies that, latter has significantly contributed toward outer membrane penetration of antibiotics to produce their bactericidal effects.
Discussion
Antimicrobial resistance (AMR) poses a significant challenge in the management of infections caused by SA and PA. These pathogens are particularly notorious for their ability to resist the effects of commonly used antibiotics, including penicillin, cephalosporin, imipenem, and vancomycin, due to mechanisms such as enzymatic degradation, efflux pumps, and biofilm formation [34, 35]. This resistance is especially problematic in patients suffering from DFIs, where these pathogens are frequently isolated and found to be associated with poor clinical outcomes [36, 37–38].
The findings of this study suggested that combining ACV along with existing antibiotics enhances bactericidal activity of these antibiotics against SA and PA pathogens. ACV’s antimicrobial effects are attributed to its acetic acid content, which disrupts bacterial cell membranes, alters intracellular pH, and inhibits essential metabolic pathways [39]. These mechanisms likely complement the action of traditional antibiotics, thereby reducing the MIC required to achieve bactericidal effects [40, 41, 42, 43, 44–45].
The present study evaluated the food grade product ACV synergistic interplay with existing antibiotics, e.g., amoxicillin, cefotaxime, imipenem, and vancomycin against resistant strains of SA and PA. The incidence of these microbes in DFI imparts serious threats toward wound healing [36]. Therefore, evaluating effectiveness of this combination of ACV with antibiotics is novel approach to mitigate overall resistant microbial burden.
The pus samples were initially grown on nutrient agar media where they showed multiple bacterial growth which were further purified by resuspending them on selective Staph 110 and cetrimide agar growth media. From a total number of samples, i.e., (n = 63) around 25 samples (~ 40%) showed measurable growth on selective Staph 110 agar media, and only 10 samples (~ 16%) showed measurable growth on selective cetrimide agar media. However, in around 28 samples (~ 44%), insignificant to no growth were observed on selective culture medium and, therefore, were excluded from the study.
These samples showing confirmed growth were further accessed by gram staining technique. The gram staining procedure confirmed that the 25 samples showed growth on selective Staph 110 agar were all gram positive. Whereas 10 samples collected from selective cetrimide agar media were confirmed gram negative. These samples were further analyzed through specific biochemical testing to identify specific specie. From 25 samples identified as gram-positive biochemical tests, e.g., catalase test, coagulase test, and mannitol test were performed. The catalase, coagulase, and mannitol red test all gave positive result in all the samples confirming the presence of SA. In order to differentiate which gram-negative microbe was involved, oxidase test, and urease and catalase test were performed. The samples (n = 10) showed positive results for oxidase and catalase test but negative for urease test confirming the presence of gram-negative PA.
The pure sample of isolated pathogens was analyzed for sensitivity against amoxicillin, cefotaxime, imipenem, and vancomycin via disk diffusion assay. The sample showed resistance against these antibiotics, and it was found that gram-positive samples of SA were mainly resistant against cefotaxime, imipenem, and amoxicillin. Whereas gram-negative samples were found to be resistant against amoxicillin and vancomycin.
After carefully evaluating the bacterial resistance against antibiotics, initially, the antimicrobial activity of ACV at various concentrations was evaluated using the agar well plate method, with observations recorded over 48 h. Results indicated that ACV at a concentration of 2.5% exhibited commendable microbial suppression both at 24 and 48 h, demonstrating its inherent antimicrobial properties.
Subsequently, ACV was applied to bacterial cultures of SA and PA in combination with antibiotics. This combination displayed significant bactericidal potential, as evidenced by the suppression of bacterial growth pattern. The fractional inhibitory concentration (FIC) index further supported these findings, confirming partial synergism between ACV and antibiotics. The probable mechanism underlying this synergistic bactericidal activity was proposed to involve enhanced permeability of antibiotics into bacterial cells, facilitated by the presence of ACV.
To further investigate the mechanism of this partial synergism, PCR analysis was conducted. The results revealed a marked suppression of key resistance-conferring genes, such as mecA, BlaIMP, OprD, and BlaZ, when ACV was used in combination with antibiotics. This suppression suggests that ACV not only augments antibiotic penetration but also mitigates bacterial resistance at the genetic level, thereby improving the overall efficacy of the antibiotics.
To validate whether this enhanced bactericidal activity effectively reduced the pathogenic burden, molecular docking analysis was performed. Docking results indicated that while antibiotics demonstrated higher binding affinities to bacterial targets compared to the active constituents of ACV, the simultaneous use of ACV significantly improved antibiotic permeability into bacterial cells. This enhanced permeability likely contributed to the observed increase in intracellular antibiotic concentration, which, in turn, accelerated bacterial cell death. In the future, however, further analysis for the precise measurement of ligand targeting intracellular protein is required. Techniques such as immunofluorescence, protein expression analysis, and gene knockdown studies could further strengthen the concept of this synergistic approach.
Conclusion
The combination of apple cider vinegar with existing antibiotics presents a novel and promising approach to combat antimicrobial resistance in S. aureus and P. aeruginosa, particularly in the context of diabetic foot infections. This strategy highlights the potential of integrating natural products with conventional therapeutics to address the growing challenge of AMR. Future clinical trials and intracellular mechanistic studies will be essential to establish the feasibility and safety of this synergistic treatment approach.
Acknowledgements
The authors with affiliation1 are thankful to the CCL Pharmaceuticals, Department of Microbiology to provide resources to conduct experiments and data curation.
Author contributions
S.M and M.A.R helped in conceptualization; S.M and M.A.G helped in methodology, M.A.G, S.A, and R.M worked in software; R.M, M.A.G, and S.A helped in validation; R.M and S.A helped in formal analysis; S.M and S.A helped in investigation; M.O.O and M. A.R helped in resources, S.M and M.A.G helped in data curation, M.A.G, S.A, and S.M. contributed to writing—original draft; R.M and M.A.G contributed to writing—review and editing, M.A.G, S.A, S.M, and R.M helped in visualization; M.A.R worked in supervision; and M.A.R worked in project administration. All authors have read and agreed to publish latest version of the manuscript.
Funding
None.
Data availability
Data and material are available upon request.
Declarations
Ethics approval and consent to participate
The ethical approval for conducting this study was taken from university institutional review board, (University of Veterinary and animal sciences) with the approval letter number ORIC/IRB/UVAS-JA235-773/23 for this study. The research study was performed in accordance with the Declaration of Helsinki. An informed consent was taken from all the patients and/or their legal guardians prior initiating sample collection.
Consent for publication
Not applicable.
Competing interests
All authors declare no competing interest.
Abbreviations
Apple cider vinegar
Antimicrobial resistance
Analysis of variance
Colony-forming units
Clinical and laboratory standards institute
Diabetic foot infections
Enzyme-linked immunosorbent assay
Efflux pump
Fractional inhibitory concentration
High-pressure liquid chromatography
Minimum bactericidal concentration
Minimum inhibitory concentration
National center for biotechnology information
Pseudomonas aeruginosa
Para-amino benzoic acid
Polymerase chain reaction
Protein data base
Quality operations laboratory
Staphylococcus aureus
Publisher's Note
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