Garlic (Allium sativum), an extensively cultivated aromatic plant belonging to the Amaryllidaceae family, is highly regarded for its diverse bioactive constituents, including allicin, sulfur, zinc, and calcium. These bioactive molecules exhibit potent antioxidant, antimicrobial, and antifungal properties, underpinning its broad utility in pharmacological and dermatological applications. Owing to its remarkable adaptability and extensive health-promoting effects, garlic remains an integral component of both traditional and contemporary therapeutic paradigms. Garlic (Allium sativum) is widely acknowledged for its diverse therapeutic properties, including antiviral, antifungal, and antibacterial effects (Kshirsagar et al., 2018). Due to its minimal side effects and cost-effectiveness, medicinal plants, including garlic, have been extensively utilized in various traditional medicinal systems. Among herbal remedies, garlic is one of the oldest and most highly regarded for its numerous health benefits. Scientific and clinical investigations have demonstrated garlic's potential to enhance immune function, combat infections and inflammation, and reduce cancer risk (Karwan, 2013; Chekki et al., 2014). Furthermore, garlic has shown efficacy against drug-resistant bacterial strains. Studies indicate that during the metabolic breakdown of allicin in the body, sulfonic acid is produced, which neutralizes free radicals at a rate unparalleled by any other known compound. To activate its health-promoting effects, garlic cloves must be crushed or finely chopped, which triggers the release of the enzyme alliinase, subsequently catalyzing allicin synthesis (Al Bayati et al., 2018). The emergence of antimicrobial resistance, driven by bacteria with novel resistance mechanisms, continues to challenge the effective treatment of common infections (Yasin et al., 2022; Uddin et al., 2023).

The therapeutic properties of garlic encompass a wide range of benefits, including positive effects on the cardiovascular and central nervous systems, anticancer activity, anti-inflammatory properties, antimicrobial efficacy, and immune system enhancement (Abidullah et al., 2021). Methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) are among the most commonly identified skin pathogens. Despite advancements in modern medicine, a significant portion of the global population continues to rely on traditional medicinal practices to meet their healthcare needs. Substantial efforts have been directed toward identifying novel antimicrobial agents derived from diverse medicinal plant species (Abdulrahman et al., 2017). Based on above research discussion here are several specific research gaps in Bangladesh related to the antimicrobial activity of garlic (Allium sativum) extract against clinical isolates from diabetic foot infections (DFIs) was noted including clinical validation of garlic extract efficacy on diabetic foot infections in human or animal models, limited focus on multidrug resistance strains, unexp-lored synergistic effects with antibiotics, geographical and ethnobotanical variations, wound care formula-tions (ointments, gels, dressings) for diabetic patients, which could aid in real-world application, and poor integration with traditional and modern medicine. Meanwhile, garlic is widely used in traditional Bangladeshi medicine, yet there is minimal interdisciplinary research linking ethnomedicine knowledge with microbiological and pharmacological evaluation.

Therefore, the objective of this study is to assess the antibacterial properties of garlic (Allium sativum) extract against common pathogenic bacteria, including Methicillin resistant Staphylococcus aureus and Escherichia coli. This research also aims to evaluate the effectiveness of garlic extract as a potential natural substitute for conventional antibiotics in inhibiting the growth of these bacterial pathogens.

Study design and sampling 

A Cross-sectional research was carried out during January 2023 to December 2023 for antimicrobial activity analysis of garlic on clinical isolates such as MRSA and E. coli. A total of 20 isolates including 10 Methicillin-Resistant Staphylococcus aureus (MRSA) and 10 E. coli were isolated from wound samples from Diabetic Foot Infections patients from Gonoshasthaya Samajvittik Medical College Hospital. All Isolates were microbiologically identified. We selected Bangladeshi small size garlic with strong pungency for antimicrobial efficacy determination of MRSA and E. coli. The current research was ethically approved by the Department of Microbiology, Gono Bishwabidyalay, Savar, Dhaka and assigned Hospital authority's ethical committee. 

Bacterial pathogens identification 

All clinical isolates were identified by cultural test, microscopy and biochemical tests under aseptic conditions in microbiology laboratory, Gono Bishwabidyalay. After collecting wound samples were primarily identified on Nutrient agar (NA), Nutrient broth, MacConkey agar, Mannitol Salt Agar (MSA), Eosin Methylene Blue agar (EMB) and Blood Agar media by cultural tests followed by previously published studies Mohammed et al. (2021). After inoculation of wound swab samples were aerobically incubated at 37 °C for overnight. A group of standard biochemical tests such as Methyl red, Voges- Proskauer (MR-VP), Indole, Oxidase, Catalase, Triple Sugar Iron (TSI), Motility Indole Urease (MIU) and Citrate utilization tests were used for identification of isolates. According to (Mohammed et al., 2021) Muller-Hinton agar containing Oxacillin discs (1 µg) using for sensitivity test and 12 mm zone of inhibition indicates sensitive and 11-12 mm define as inter-mediate. All microbiological media were purchased from Oxoid, UK, and Hi Media Private Ltd., India.

Evaluation of MRSA 

According to (Mohammed et al., 2021) protocol identification of MRSA using oxacillin was done by following guidelines of NCCLS. A suspension equivalent to 0.5 McFarland standard, prepared from each bacterial strain, was uniformly inoculated across the entire surface of Mueller-Hinton agar plates (Oxoid, UK) supplemented with 4% NaCl and 6 μg/mL oxacillin, using sterile swabs. The plates were incubated at 35°C for 24 hours. The presence of growth (>1 colony) indicated oxacillin/methicillin resistance, as described by Genç et al. (2008). A zone of inhibition greater than 12 mm was classified as sensitive, while zones measuring 11–12 mm were considered intermediate.

Biofilm Formation Analysis (Congo Red Agar)

Biofilm formation was evaluated using Congo Red Agar, modified with Brain Heart Infusion (BHI) Agar and Blood Agar Base (BAB), following the protocol outlined by Lee et al. (2016). The BHI agar medium was formulated by incorporating 0.8 g of Congo red dye and 36 g of sucrose (both sourced from Baker, UK) into 52 g of BHI agar (Oxoid, UK) dissolved in 1000 mL of distilled water. Similarly, the Blood Agar Base medium was prepared by combining 0.4 g of Congo red dye, 10 g of glucose, and 42 g of BAB (Oxoid, UK) in 1000 mL of distilled water. Bacterial isolates were inoculated onto these media, and the plates were incubated at 37°C for 48 hours, followed by an additional maturation period at room temperature for 2–4 days. Slime-producing isolates were identified by the appearance of black colonies, while non-slime-producing isolates exhibited red colonies on the agar plates.

Garlic extract preparation

For preparation of garlic extracts first we purchased fresh garlic from local market in Savar region of Bangladesh and garlic bulbs were peeled, weighted and cleaned (100gm). Then clean cloves were mashed by using a sterile mortar and pestle and then the mixture was filtered through a cheese cloth. After filtered garlic juice we prepared different concen-trations including 100% (v/v), 75% (v/v), 50% (v/v) and 25% (v/v) respectively. Then 100% concentrated garlic extract was dilatated and prepared further concentration by adding sterile distilled water. Each 100-gram raw garlic finally converted 50% yield of garlic juice (2 g/ml) (Yadav et al., 2015). Finally, the garlic extract was stored at -20°C for further processes (Yadav et al., 2015).

Standard inoculum preparation

For standard inoculum preparation first pure colonies were taken from selective culture media of test isolates with sterile loop and inoculated on sterile normal saline solution and overnight incubated at 37°C for incubator (Biobase, China). After overnight incubation match the turbidity of 0.5 McFarland standard containing 1 × 107 CFU/ml approximately.

Zone detection by well diffusion method for garlic extract 

For detection of garlic extract zone of inhibition prepared Muller-Hinton agar plates with 3-4 mm depth. Then inoculum of test isolates was taken from normal saline and spread on Muller-Hinton agar carefully. The agar plates were dry for 20 minutes under laminar airflow and then prepared 5 mm well by using cutter on Muller-Hinton agar surface. Furthermore, 30 microliter of garlic extract containing 25% (v/v), 50% (v/v), 75% (v/v) and 100% (v/v) solutions were added on prepared well on Muller-Hinton agar. For control in one well we loaded normal saline. After loaded specific concentration of garlic extract all plates were incubated at 37°C for 24 hours. According to (Yadav et al., 2015; Wayne, 1999) the zone of inhibition was measured by a millimeter scale.

Zone determination by disk diffusion method of antibiotics 

According to CLSI guidelines antibiotic sensitivity tests was Performed in Muller-Hinton agar plates followed by Kirby Bauer disk diffusion method. Antibiotic sensitivity test for MRSA and E. coli commercially available antibiotics including Amoxicillin, Ampicillin, Streptomycin, Ciprofloxacin and Azithromycin were applied on Muller-Hinton agar plates. Pure colonies of MRSA and E. coli from selective culture media were spread on Muller-Hinton agar plates by using spreader. After aseptically spreading of culture on Muller-Hinton agar plates, antibiotics disk was placed on agar plates and incubated at 37°C for 24 hours. After overnight incubation according to CLSI guidelines the zone of inhibition was measured. All commercially available antibiotic discs were purchased from Hi Media Private ltd. India.

Statistical analysis 

All data were inputted in excel spread sheet and SPSS version 25 was applied for data analysis. A one-way ANOVA followed by post hoc tests was used to compare two mean values as a measure of statistical significance. The zone of inhibition values is presented as mean ± standard deviation and standard errors of the mean, with a significance threshold set at P < 0.05. 

Bacterial isolates screening and prevalence 

According to cultural, morphology and biochemical tests MRSA and E. coli isolates identified with different characteristics in Fig.  1. MRSA showed yellow colonies on Mannitol Salt agar (MSA) Fig. 1a, strong biofilm formation with black colonies on Congo red agar Fig. 1b, moderate biofilm formation with light radish colonies on Congo red agar Fig. 1c and E. coli produce metallic shine colonies on Eosin Methylene Blue Agar (EMB) Fig.1d respectively. In this study, a total of 20 isolates including 10 MRSA and 10 E. coli was isolated from Gonoshasthaya Samajvittik Medical College Hospital Savar, Dhaka. 

Fig. 1: 1a: MRSA showed yellow colonies on Mannitol Salt agar, 1b: strong biofilm formation with black colonies on Congo red agar, 1c: moderate biofilm formation with light radish colonies on Congo red agar, 1d: E. coli produce metallic shine colonies on Eosin Methylene Blue Agar.

Detection of Biofilm formation MRSA 

In this study, a total of 10 biofilm producing MRSA isolated from DFIs patients that categories as strong biofilm and moderate biofilm producing capabilities. Out of 10 MRSA 8(80%) was detected as strong biofilm formation and remaining 2(20%) recorded as moderate biofilm producing. 

Sociodemographic Study

For garlic extract experiment we isolated MRSA and E. coli from Gonoshasthaya Samajvittik Medical College Hospital and sociodemographic parameters observed in Table 1. Table 1 explained that 7(70%) MRSA isolated from male patients whereas only 3(30%) from female patients and 6(60%) E. coli was found in male and 4(40%) was found in female patients respectively. Out of 20 isolates the highest number of E. coli 7(70%) was recorded in 61-70 years of age patients followed by 6(60%) MRSA whereas in age group 51-60 years 4(40%) MRSA and 3(30%) E. coli was recorded. In addition, wound size is most important variables and in this study we revealed that the most prevalent MRSA and E. coli was observed in more than 8mm wound size where 4(40%) of each isolates was found in ≤7 mm wound size. 

Table 1: Sociodemographic study of DFIs patients based on different categories. 

Zone of inhibition determination of garlic extract (MRSA)

According to well diffusion method antimicrobial efficacy determination of garlic extract against pathogens from diabetic foot infections patients and the zone of inhibition with different concentrations are demonstrated in Table 2. 

Table 2: Effect of different garlic extract concentration and commercial antibiotics on MRSA.

Legends: Values are expresssed as mean±standard deviation; NS: Statistically not significant (p>0.05)

abcd Means having different superscript in the same row differed significantly (p<0.05)

p<0.05 indicates significant; p<0.01 indicates highly significant; p<0.001 indicates extremely significant; SD: Standard deviation; SEM: Standard errors of mean

MRSA showed statistically significant results on different concentrations of garlic extract. In addition, 100% concentration of garlic extract showed 23.66 mm zone of inhibition which mostly significant compare to commercial antibiotics amoxicillin and ampicillin followed by 75% concentration of garlic extract 21.66 mm zone and in 50% concentration 20.33 mm zone of inhibition was observed. Similarly, 100% and 75% concentrated garlic extract showed highly significant value that follow p<0.05 and p<0.01 level of significant. On the other hand, commercial antibiotics are statistically nonsignificant. The highest mean±SD value of zone inhibition of MRSA on 100% garlic extract 23.66± 0.57 mm followed by 20.00±0.33 mm Table 2. Table 3 represented the mean±SEM values for different samples with statistical significant of MRSA.

Table 3: Effect of different garlic extract concentration and commercial antibiotics on MRSA.

Legends: Values are expresssed as mean±standard errors of mean; NS: Statistically not significant (p>0.05)

abcd Means having different superscript in the same row differed significantly (p<0.05)

p<0.05 indicates significant; p<0.01 indicates highly significant; p<0.001 indicates extremely significant; SEM: Standard errors of mean

Zone of inhibition determination of garlic extract (E. coli)

According to well diffusion method antimicrobial efficacy determination of garlic extract against pathogens from diabetic foot infections patients and the zone of inhibition with different concentrations are demonstrated in Table 4. E. coli showed statistically significant results on different concentrations of garlic extract. In addition, the highest mean±SD of 100% concentration of garlic extract showed 18.66±0.57 mm zone of inhibition in sample 5 which mostly signifi-cant compare to commercial antibiotics amoxicillin and ampicillin followed by 75% concentration of garlic extract shown 13.33±1.57=2 mm zone. Similarly, 100% and 75% concentrated garlic extract showed highly significant value that follow p<0.05 and p<0.01 level of significant. On the other hand, commercial antibiotics are statistically nonsignificant. The highest mean±SD value of zone inhibition of E. coli on 100% garlic extract 18.66±0.57 mm and lowest 8.33±0.57 mm (Table 4). Table 5 represented the mean±SEM values for different samples with statistically significant of E. coli.

Table 4: Effect of different garlic extract concentration and commercial antibiotics on E. coli.

Legends: Values are expresssed as mean±standard errors of mean; NS: Statistically not significant (p>0.05)

abc Means having different superscript in the same row differed significantly (p<0.05)

p<0.05 indicates significant; p<0.01 indicates highly significant; p<0.001 indicates extremely significant; SD: Standard deviation

 

Table 5: Effect of different garlic extract concentration and commercial antibiotics on E. coli.

Legends: Values are expresssed as mean±standard errors of mean; NS: Statistically not significant (p>0.05)

abc Means having different superscript in the same row differed significantly (p<0.05)

p<0.05 indicates significant; p<0.01 indicates highly significant; p<0.001 indicates extremely significant; SEM: Standard errors of mean

Fig. 2: Left: Zone of inhibition of garlic extract 100% and 50% concentration and commercial Ampicillin antibiotic against MRSA on Muller-Hinton agar; Right: Zone of inhibition of garlic extract 100% and 50% concentration and commercial Azythromycin antibiotic against E. coli.

 Finally, our research suggested that 100% and 75℅ garlic extract concentration showed highly significant zone against pathogenic isolates including MRSA and E. coli. Nowadays DFIs causes leading threat and economic losses for diabetic patients. The most common pathogens (MRSA and E. coli) are highly resistance to 2nd and 3rd generation antibiotics and effect on diabetic foot infections patients. Our research focused on antimicrobial efficacy of garlic extract to compare commercial antibiotics and researchers have benefits and further research on combine treatment with garlic extract and antibiotics for better treatment option for DFIs in Bangladesh. 

The antimicrobial efficacy of garlic extract as a natural treatment for diabetic foot infections demonstrates significant potential against multidrug-resistant pathogens such as Methicillin-resistant Staphylococcus aureus (MRSA) and E. coli. Clinical samples indicate that garlic extract exhibits strong inhibitory effects due to its bioactive compounds, particularly allicin, which disrupts bacterial cell walls and metabolic processes. This natural remedy offers a promising, cost-effective, and less toxic alternative to conventional antibiotics, addressing the challenges posed by antibiotic resistance and improving outcomes for diabetic foot infection patients. Multi-drug resistance E. coli and Staphylococcus aureus poses significant public health impact and global threat for human being. In this study, all 20 bacterial isolates showed highly resistance to several antibiotics where E. coli showed highly sensitive to Ciprofloxacin and Azithromycin. MRSA showed highly sensitive to Ampicillin Azithromycin and Amoxicillin Respectively.  Additionally, in this research garlic extract concentration 100% and 75% showed highly sensitive against E. coli and MRSA on Muller-Hinton agar whereas the similar findings were observed in previously published studies Yadav et al., 2015. In previously published studies Yadav demon-strated that E. coli produce 26.44±0.72 mm zone of inhibition on 100% concentration of garlic juice which is closely supported our findings. Yadav et al. (2015) also observed in their research Staphylococcus aureus produce 26.44±0.57 mm zone on 100% concentrated garlic extracts where our study revealed that 23.66±0.57 mm zone of inhibition, 50% concentrated garlic extract produce 21.16±0.53 mm zone whereas in our study produce 20.33±0.57 mm zone and completely support our findings.  Previous research indicates that allicin primarily exerts its antimicrobial effects through the rapid and complete suppression of RNA synthesis, although it also partially inhibits DNA and protein synthesis, highlighting RNA as the main target of its action. Variations in the structural composition of bacterial strains may further influence their susceptibility to garlic-derived compounds (Yadav et al., 2015). In addition to its antimicrobial properties, garlic has also demonstrated significant antifungal activity (Davis et al., 2003; Islam et al., 2020)).

In this study we observed that MRSA and E. coli were highly resistance to commercial antibiotics that agreed with several previously published studies where they also found E. coli and Staphylococcus aureus mostly resistance to commercial antibiotics and garlic extract showed highly sensitive to same organisms (Li et al., 2015). Gaekwad et al. (2013) mentioned in their research the combine treatment of garlic extracts and conventional antibiotics against Staphylococcus aureus and E. coli for complete synergism. In another research Mohammed et al. (2021) observed that 100% concentration garlic extract create 17.96±1.09 mm zone against MRSA and the p-value was recorded 0.038 where our study revealed that 23.66±0.57 mm zone and p-value found in 0.015. Our research highly recommended 100% and 75% concentrated garlic extract for alternative treatment of commercial antibiotics against MRSA and E. coli. Garlic may enhance the effectiveness of standard antimicrobial agents against E. coli and MRSA, but limited research has explored this interaction and the combine treatment of garlic extract and commercial antibiotics will help to prevent DFIs in diabetic patients in Bangladesh and also reduce economic losses. 

Limitations and future research of the study

The study has several limitations that need to be addressed in future research. Firstly, the investigation did not incorporate comparisons with commercial or clinical antibiotics to comprehensively evaluate the efficacy of garlic extracts in antimicrobial applications. Additionally, the extraction methods employed may not fully preserve the bioactivity of allicin and other active compounds, raising the need for alternative extraction techniques, such as mechanical pressing, which could minimize the degradation of bioactive components. Moreover, the scope of the study was limited to specific extraction processes, underscoring the importance of broader investigations into various garlic-derived compounds to identify optimal active ingredients using standardized methodologies. These constraints highlight the necessity for further research to validate the findings and explore synergistic effects when combining garlic extracts with established antibiotics.

Garlic extract have antimicrobial activity that act as natural treatment against pathogenic bacteria like Staphylococcus aureus and E. coli. Biofilm producing Staphylococcus aureus has poses public health significant and causes several infectious diseases in human being mainly hospital acquired disease. Nowadays in Bangladesh, rapidly increased the diabetic foot infections patients and the treatment is very expensive due to antibiotic resistance pattern of bacterial pathogens. This is the right time to choose the treatment option of garlic extract as natural medicine to prevent diabetes foot infections. Compara-tively garlic extract is less expensive and easily available as well as save economic losses. To validate our research further study is needed where more data will be collected and combine treatment with garlic extract and commercial antibiotics will be applied together for best treatment against pathogenic isolates. 

The Institutional Research Ethics Committee of Gono Bishwabidyalay, Savar, Dhaka, Bangladesh approved the study. Before taken the samples all patients given their permission to collect samples and Hospital Authority also approved our research work.

M.M.S.; M.D.; S.M.: Conceptualization, original draft writing, reviewing, and editing. S.M.; M.F.A.; A.A.; M.J.M.; T.A.: Formal analysis, investigations, funding acquisition, reviewing, and editing. T.S.; M.A.; M.J.A.: Table and Figure formatting. M.D.: Resources, data validation, data curation, and supervision. 

The authors want to give special thanks to the Department of Microbiology, Gono Bishwabidyalay, Savar, Dhaka, Bangladesh, for providing their laboratory facilities, and also to the Gonoshastha Somaj Vittik Medical College Hospital for supporting us during sampling. 

The authors declare that they have no competing interests.