Exploring the Selective Antibacterial Effects of Natural Products Against Pathogenic and Beneficial Bacteria

Abstract

Objectives: To determine the selective antibacterial effects of medicinal plants alone and in combination with prescribed antibiotics. Methods: Using in vitro well diffusion assays and turbidity measurements, we assessed the antibacterial activity of herbal extracts against gram-positive and gram-negative bacteria, including beneficial bacteria like Lactobacilli. Measuring the inhibition zone diameters provided initial screening for the natural products with antibacterial properties. Results: Among 31 tested herbal extracts, only six demonstrated notable antibacterial activity when used alone or in combination with prescribed antibiotics. These were effective against both gram-positive and gram-negative bacteria, except for radish seed, which was active only against E. coli. Most drug-herb combinations did not enhance the efficacy of antibiotics. Among the tested substances, only capparis spinosa root, vinegar, and bicarbonate inhibited the growth of Lactobacilli, whereas other effective herbal extracts had no harmful effects on beneficial bacteria. Conclusion: This study highlights specific herbal compounds with selective antibacterial effects that may offer therapeutic value, particularly in addressing antibiotic resistance. Notably, five active compounds did not inhibit the growth of Lactobacilli, suggesting their potential to target pathogens without disrupting the beneficial microbiota. Our findings support the use of natural products as antibacterial agents, although the absence of minimum inhibitory concentration (MIC) testing and phytochemical profiling of specific bioactive compounds limits the study. Further studies are needed to explore the clinical relevance and underlying mechanisms of these natural products.

Graphical Abstract.

Selective Antimicrobial Activity of Herbal Extracts Against Pathogens

Impact Statement

The study holds both socio-economic and global importance. It supports the development of affordable, plant-based antibacterial treatments that are accessible in low-resource settings. The identification of herbal compounds with selective antibacterial activity contributes to the continuous fight against antimicrobial resistance. By preserving beneficial bacteria such as Lactobacilli, these microbiota-friendly treatments address the need for safer and more effective antimicrobial therapies. Additionally, the findings of the study offer valuable insights into herb-drug interactions, providing a basis for the potential integration of natural products into modern antimicrobial strategies.

Introduction

Current antibiotics are effective against a wide range of pathogenic and beneficial bacteria, thereby affecting microbiome homeostasis. ¹ The burden of antibiotic resistance continues to grow over time as pathogenic bacteria develop resistance against common synthetic antibiotics. Many natural products have been investigated for their antibacterial activity against multidrug-resistant bacteria as an alternative approach to conventional antibiotics. ² Bioactive compounds, such as allicin in garlic and flavonoids in lemon, primarily mediate the antibacterial effects of natural products. ³ Several common bioactive compounds interfere with bacterial growth, including flavonoids, phenolic acids, allicin, organic acids, and antimicrobial peptides like defensins.^4-6 Many studies on the antibacterial activity of natural products focus on their effects against resistant pathogenic bacteria, such as Escherichia coli (E. coli) or methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial effect of natural products against beneficial bacteria, such as Lactobacillus species, has not been thoroughly investigated. Beneficial bacteria, such as Lactobacilli and Bifidobacterium species, perform various functions in the gut, including the prevention of pathogenic bacterial colonization, facilitating nutrient absorption, and stimulating immune responses.^7,8 In addition, beneficial bacteria like Bifidobacterium synthesize essential vitamins, including vitamin K and B-group vitamins, such as riboflavin, niacin, and cobalamin.^9,10 Beneficial bacteria also play a role in mental health through microbiota-gut-brain crosstalk. ¹¹ Psychobiotics like Lactobacillus plantarum, Lactobacillus brevis, and Bifidobacterium dentium produce serotonin and gamma-aminobutyric acid (GABA), which play a significant role in modulating mental health.^12-14 Beneficial bacteria also reduce inflammation by suppressing pro-inflammatory cytokines like Interleukin-1 (IL-1) and IL-6.^15,16 There are many other functions besides immune regulation that beneficial bacteria perform to maintain body homeostasis.

Many herbs and natural products have antioxidants, anti-aging, and anti-inflammatory properties.^17,18 In addition, several herbs and natural products such as garlic (Allium sativum), Quercus coccifera, Ocimum gratissimum, and Curcuma longa have been found to effectively inhibit bacterial growth.^19-21 These plant extracts have been shown to inhibit important pathogenic bacteria, such as Pseudomonas aeruginosa, E. coli, and Staphylococcus aureus (S. aureus).^20,21 Although herbal extracts are an effective alternative approach for suppressing pathogenic bacteria, the effect of herbs on microbiota is only beginning to be explored.^22,23 Here, we focused on natural products that inhibit pathogenic bacteria while allowing beneficial ones to thrive.

Many conventional antibiotics, such as amoxicillin and penicillin, are effective against a wide range of bacteria, including beneficial bacteria like Lactobacillus species. ²⁴ Eliminating beneficial bacteria or losing microbiota diversity in the gut results in dysbiosis, which is linked with many diseases. ²⁵ In our study, we investigated the antibacterial effects of several natural products, including garlic, dried lemon, pomegranate peel powder, capparis spinosa root, galangal root, radish seed, and myrtle leaf. All of these products induce antimicrobial effects. Here, we aimed to explore the antibacterial activity of various natural products against both beneficial and pathogenic bacteria. We focused on natural products that may enhance the effects of conventional antibiotics and inhibit pathogenic bacteria without affecting the beneficial ones. Our study could help identify novel approaches to eliminate microbes while preserving a healthy microbiome during bacterial infections.

Materials and Methods

Plant Material

All products used for antibiotic evaluation in the experiments were obtained commercially, including onion, garlic, cumin, dried lemon, costus root, frankincense, artemisia monosperma, black pepper, cymbopogon schoenanthus, paronychia argentea, myrrh, pomegranate peel powder, teucrium polium, capparis spinosa root, wild thyme, mahaleb prunus, galangal root, lyakinfirst, basil seeds, nettle powder, radish seed, yellow sulfer, black alum stone, shamatri powder, ground indian costus root, bicarbonate, rosemary powder, camphor, myrtle leaf, mastic powder, khella seeds and vinegar. All plant materials were soaked in distilled water according to the indicated concentrations to prepare the water extract solutions. The concentration used in the experiments was the weight of extract per mL. The extract solutions were incubated overnight at room temperature. Then, using a 10 mL tube, they were centrifuged at 3000 rpm for 10 min. For all experiments, 130 μL of the supernatant was dispensed into each agar well.

Antibacterial Activity and Media

The inhibition zone diameter was measured in mm using the well diffusion method as previously described ²⁶ to determine the antibacterial activity of each extract. The antibiotic (Amoxicillin) was used as a positive control, while distilled water was used as a negative control. The agar well diffusion method was performed using Mueller-Hinton agar (MHA) for pathogenic bacteria and De Man, Rogosa, and Sharpe (MRS) agar for beneficial bacteria (Lactobacillus). A concentration of 100 μg/μL (130 μL/well = 13 mg) was used for amoxicillin. A concentration of 500 μg/ μL (130 μL /well = 65 mg) was used for plant extracts. Only a concentration of 5 μg/μL (130 μL = 650 μg) of amoxicillin was used in the antibiotic + plant extract mixture.

Well Diffusion Method

Agar wells with an 8-millimeter diameter were created using a cork borer. A sterile cotton swab was used to spread bacterial suspensions (adjusted to a 0.5 McFarland standard) evenly onto Mueller-Hinton agar plates. The plates were allowed to air dry for 15 min before adding 130 μL of plant extraction or antibiotics. Amoxicillin served as a positive control, while distilled water was used as a negative control. Following an incubation period of 18–24 h, the diameter of the inhibition zone surrounding each well was measured, and photographic documentation of the plates was performed. ²⁶

Bacterial Strains Used

Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), and clinical isolates of S. aureus and E. coli were used to test the antibacterial activity of natural products. As beneficial bacteria, Jamieson Probiotic 30 Billion, which contains nine strains of Lactobacillus species, including Lactobacillus paracasei, Lactobacillus gasseri, Lactobacillus rhamnosus (UALr-06), Lactobacillus rhamnosus (UALr-18), Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus reuteri, and Lactococcus lactis, was used. For pathogenic bacteria, incubation was performed at 37 °C for 24 h under aerobic conditions. For Lactobacillus spp., cultures were incubated at 37 °C for 24–48 h in a CO₂-enriched environment using a sealed jar system to support optimal growth of anaerobic or microaerophilic conditions. Bacterial suspensions were prepared by selecting single colonies from agar plates and adjusting the turbidity to 0.5 McFarland standard, corresponding to approximately 1.5 × 10⁸ CFU/mL.

Statistical Analysis

Statistical analyses were performed using a 2-tailed unpaired t-test (2 groups). p < 0.05 was considered statistically significant. Data are expressed as the mean ± SD in the manuscript.

Results

Natural products are widely used to treat various diseases due to their bioactive compounds, such as allicin in garlic. In some cases, natural products are used in combination with prescribed antibiotics. People usually consume natural products without further purification and sterilization techniques. Therefore, unpurified aqueous extracts of natural products are used in this study. Here, we evaluated the efficacy of certain natural products, both alone and in combination with antibiotics, to determine their impact on antibiotic resistance. After combining amoxicillin with natural products, only garlic, dried lemon, pomegranate peel powder, capparis spinosa root, galangal root, radish seed, and myrtle leaf, in addition to bicarbonate and vinegar, exhibited antibacterial activity against a clinical isolate of S. aureus (Figures 1 & 2). The zone of inhibition diameter for garlic, dried lemon, capparis spinosa root, galangal root, radish seed, and myrtle leaf were 31 mm, 25 mm, 25.5 mm, 18.5 mm, 20 mm, and 25 mm, respectively, compared to the control sample, which has a diameter of 13–15 mm. These results indicate that only eight natural products have antibacterial activity when combined with antibiotics (amoxicillin).

Figure 1.

Evaluation of Antibacterial Effects of Common Natural Products in Combination With Antibiotics. (A) Several Products (1-onion, 2-garlic, 3-cumin, 4-dried lemon, 5-costus root, 6-frankincense, 7-artemisia monosperma, 8-black pepper, 9-cymbopogon schoenanthus, 10-paronychia argentea, 11-myrrh, 12-pomegranate peel powder, 13-teucrium polium, 14-capparis spinosa root, 15-wild thyme, 16-mahaleb prunus, 17-vinegar) were Tested for Antibacterial Effects using the Well Diffusion Method with a Concentration of 65 μg/well for Plant Extract and 13 mg/well for Amoxicillin. (B) Bar Graph Showing the Zone of Inhibition (in mm) for the Tested Samples from (A).

Figure 2.

Evaluation of Antibacterial Effects of Common Natural Products in Combination With Antibiotics. (A) Several Products (17-galangal root, 18-lyakinfirst, 19-basil seeds, 20-nettle powder, 21-radish seed, 22-yellow sulfer, 23-black alum stone, 24-shamatri powder, 25-ground indian costus root, 26-bicarbonate, 27-rosemary powder, 28-camphor, 29-myrtle leaf, 30-mastic powder, 31-khella seeds) were Tested for Antibacterial Effects Using the Well Diffusion Method With a Concentration of 65 μg/Well for Plant Extract and 13 mg/Well for Amoxicillin. (B) Bar Graph Showing the Zone of Inhibition (in mm) for the Tested Samples from (A).

To test whether the antibacterial effect induced by natural products is due to combining them with antibiotics, we evaluated the antibacterial effects of natural products alone (Figure 3). The results show that only six products have antibacterial effects against gram-positive bacteria, while seven products were effective against gram-negative bacteria. Dried lemon, capparis spinosa root, pomegranate peel powder, myrtle leaf, bicarbonate, and vinegar increased the antibacterial effect of amoxicillin by ∼4–10 mm. Surprisingly, radish seed inhibited bacterial growth of only gram-negative bacteria and was not affected by antibiotics. Interestingly, when used alone, galangal root did not inhibit bacterial growth. These results suggest that combining certain natural products with prescribed antibiotics can significantly enhance their antibacterial efficacy. Among the tested natural products, dried lemon showed an 11 mm increase in the inhibition zone when combined with amoxicillin, whereas garlic's antibacterial efficacy remained unaffected by the antibiotics.

Figure 3.

Antibacterial Effects of Certain Natural Products Against Gram-Positive and Gram-Negative Bacteria. (A) Certain Products (1-garlic, 2-dried lemon, 3-capparis spinosa root, 4-pomegranate peel powder, 5-radish seed, 6-galangal root, 7-myrtle leaf, 8-bicarbonate, and 9-vinegar) were Tested for their Antibacterial Effects Alone Using The Well Diffusion Method With A Concentration of 26 μg/well for Plant Extract and 13 mg/well for Amoxicillin. (B) Bar Graph Showing the Zone of Inhibition (in mm) for the Tested Samples from (A).

Next, to test whether combining natural products induces robust antibacterial effects, we combined certain products to test their synergistic effects (Supplementary Figure 1). Combining natural products did not enhance antibacterial efficacy; in fact, the antibacterial effect was lower than that of garlic alone (Figure 1).

To determine whether tested natural products could inhibit the growth of bacterial clinical isolates, we tested garlic alone and a mixture of pomegranate peel powder, radish seed, and myrtle leaf against drug-resistant S. aureus and E. coli (Figure 4). Garlic alone had a similar or higher antibacterial effect to amoxicillin; however, the mix showed a lower or similar antibacterial effect against standard and clinical S. aureus. The antibacterial effect of garlic was lower compared to amoxicillin when tested against standard E. coli. Surprisingly, garlic was significantly better in inducing antibacterial effects compared to amoxicillin against a clinical isolate of E. coli. Collectively, these results indicate that the tested plant extracts do not exhibit synergistic effects in inhibiting bacterial growth. Moreover, amoxicillin works better against standard but not clinical isolates of E. coli, which is more clinically relevant, as E. coli is more prone to developing resistance than S. aureus. Some plant extracts, such as garlic, are highly effective in inhibiting the growth of bacterial clinical isolates.

Figure 4.

Antibacterial Effects of Garlic and a Mix of Natural Products Against Gram-Positive and Gram-Negative Bacteria Clinical Isolates. (A) Certain Products, Including Pomegranate Peel Powder (18%), Radish Seed (11%), And Myrtle Leaf (14%) (Mix), Were Tested for their Antibacterial Effects Using the Well Diffusion Method. Garlic Alone and the Mix were Tested at A Concentration of 26 μg/well for Plant Extracts and 26 mg/well for Amoxicillin. (B) Bar Graph Showing the Zone of Inhibition (in mm) for the Tested Samples From (A).

Certain natural products are highly effective in inhibiting the growth of pathogenic bacteria; however, their impact on beneficial bacteria remains unclear. Here, we examined the effects of natural products on common commensal bacteria species, specifically Lactobacilli. We tested the antibacterial effects of natural products using the well diffusion method on MRS agar, a medium selective for Lactobacilli species. Only capparis spinosa root, bicarbonate, and vinegar inhibited the growth of Lactobacilli (Figure 5A & B). Other natural products, including garlic, dried lemon, pomegranate peel powder, radish seed, and myrtle leaf, did not affect Lactobacilli growth, suggesting that these products only inhibit pathogenic but not beneficial bacteria. To determine whether the observed effects are due to changes in acidity, we measured the pH of natural products (Figure 5C). Acidity and alkalinity did not contribute to the antibacterial effects. These results suggest that the tested natural products exhibit selective antibacterial activity by inhibiting pathogenic bacteria while sparing beneficial ones, and that this effect occurs independently of pH level (Figures 3 & 5).

Figure 5.

Antibacterial Effects of Natural Products Against Lactic Acid Bacteria (Lactobacilli). (A) Certain Products (1-garlic, 2-dried lemon, 3-capparis spinosa root, 4-pomegranate peel powder, 5-radish seed, 6-myrtle leaf, 7-bicarbonate, and 8-vinegar) were Tested for their Impact on Beneficial Bacteria (Lactobacilli) using a Concentration of 26 μg/well. (B) Bar Graph Showing the Zone of Inhibition (in mm) for the Tested Samples from (A). (C) pH Measurement for the Tested Samples in (A) and (B).

Discussion

Our study highlights the role of natural products in enhancing antibiotic activity against pathogenic bacteria while sparing beneficial ones. Numerous studies have demonstrated the antibacterial effects of natural products (Supplementary Table 1).^27-54 Our findings show that certain natural products work synergistically with antibiotics, improving their efficacy against pathogenic bacteria. This finding aligns with a previous study suggesting that plant-derived compounds can enhance antibiotic action. ⁵⁵ Nevertheless, the effectiveness of this synergy is selective since not all plant mixtures resulted in stronger antibacterial effects (Figure 4). In our study, only garlic, dried lemon, pomegranate peel powder, capparis spinosa root, radish seed, and myrtle leaf enhance the antibacterial effects of amoxicillin. Garlic was the most effective antibacterial compound against pathogenic bacteria due to the presence of the bioactive compound allicin, which is consistent with prior studies conducted by T. Wolde and others.^56-58 Based on the results in Figure 4, garlic exhibited antibacterial activity against standard S. aureus similar to amoxicillin; however, its effect was stronger against the clinically isolated, more resistant strain. As expected, amoxicillin induced strong bacterial inhibition against laboratory E. coli compared to garlic. Interestingly, garlic strongly inhibited the growth of a clinically isolated strain of E. coli, suggesting that E. coli acquires more resistance to antibiotics.

Interestingly, radish seed extract was a highly selective compound by inducing antibacterial effects against gram-negative but not gram-positive bacteria (Figure 3). This suggests that radish seed may selectively interfere with or disrupt the structural composition of gram-negative bacterial cell walls, such as outer membranes or lipopolysaccharides.

Natural products are widely used to treat various diseases. The antibacterial activity is due to the presence of bioactive compounds, such as gingerol in ginger and allicin in garlic. Many natural products tested for antibacterial activity require further purification methods and protocols to display their antibacterial effects, unlike the aqueous extracts used in this study. In this study, the antibacterial effects of natural products were observed in their natural state without the need for purification or isolation, as each product was simply dissolved in water. This is of importance since people typically consume these products in aqueous forms. Although some natural products enhanced antibiotic efficacy when used alone, their combinations did not result in a stronger antibacterial effect, suggesting that possible antagonistic interactions may occur when natural products are used simultaneously. The highest antibacterial effect enhancement was observed with dried lemon, where the inhibition zone diameter increased by 11 mm when combined with antibiotics. Other extracts also showed an increase in antibacterial activity, with zone diameters ranging from 4 to 11 mm. These effects could be attributed to bioactive phytochemicals in the plant extracts, such as phenolic compounds, flavonoids, and organic acids that may disrupt bacterial cell walls or alter membrane permeability, thereby inducing a stronger antibacterial effect. However, radish seed and galangal root did not enhance antibacterial activity when combined with antibiotics, probably due to the lack of bioactive compounds that synergize efficiently with antibiotics. Further studies are necessary to determine the possible specific interactions between natural compounds and antibiotics.

The impact of natural products on beneficial bacteria was also evaluated in this study. The results in Figure 5 show that garlic, dried lemon, pomegranate peel powder, radish seed, and myrtle leaf do not inhibit the growth of beneficial bacteria (Lactobacilli), although they induce antibacterial effects against S. aureus and E. coli. Acidity is closely correlated with higher bacterial inhibition; therefore, we measured the pH of the products to determine if it has any role in bacterial inhibition.^59,60 As shown in Figure 5, pH did not contribute to the antibacterial activity of natural products.

It is essential to acknowledge the limitations of this study. The use of the inhibition zone diameter method is qualitative and only shows the presence or absence of bacterial growth without fully reflecting the true antibacterial potency of the examined natural product extracts. It does not show the minimum concentration required to inhibit bacterial growth. In addition, this method can be affected by some factors, such as the diffusion rate of the compound through the agar. It is recommended to use complementary methods such as MIC testing. Future studies should include MIC testing to determine the antibacterial potency and optimal therapeutic concentrations for clinical use. The incorporation of both qualitative and quantitative assays will provide a more comprehensive assessment of the antibacterial activity and therapeutic potential of natural products. In addition, the study is limited by the absence of phytochemical profiling of the tested compounds. It is not clear which specific bioactive component is responsible for the observed antibacterial effects. Future studies should incorporate chromatographic techniques to identify and quantify major specific bioactive components. Additionally, herbs were collected commercially at a single time point, which may not reflect the seasonal changes in their properties. Also, since the materials were processed (dried or powdered), preparation of herbarium vouchers for taxonomic verification was not practical. Overcoming these limitations is crucial for investigating the underlying mechanisms and achieving consistent and reproducible outcomes in clinical use.

Conclusion

In conclusion, our findings support the use of natural products as an alternative approach to conventional antibiotics. The selective activity of radish seed extract, the robust efficacy of garlic, and the ability of natural compounds to maintain their effects in water highlight their relevance in both dietary and therapeutic contexts. In the future, it is important to include MIC testing and phytochemical profiling to study the mechanisms of antibacterial action. Future studies should also focus on exploring the potential role of herbs and natural products in combating antibiotic-resistant bacteria while preserving beneficial bacterial strains.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251387863 - Supplemental material for Exploring the Selective Antibacterial Effects of Natural Products Against Pathogenic and Beneficial Bacteria

Supplemental material, sj-docx-1-npx-10.1177_1934578X251387863 for Exploring the Selective Antibacterial Effects of Natural Products Against Pathogenic and Beneficial Bacteria by Hamad H. Alanazi in Natural Product Communications