Available online on 15.04.2021 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

© 2011-21, publisher and licensee JDDT, This is an Open Access article which permits unrestricted non-commercial use(CC By-NC), provided the original work is properly cited

Open Access  Full Text Article                                                                      Research Paper

The Ethanolic Leaf Extracts of Dissotis multiflora (Sm) Triana and Paullinia pinnata Linn Exert Inhibitory Effect on Escherichia coli Through Membrane Permeabilization, Loss of Intracellular Material, and DNA Fragmentation

Afagnigni Alian Désiré^1,2*, Nyegue Maximilienne Ascension¹, Pracheta Janmeda², Vinay Sharma³, Etoa François-Xavier ¹

¹ Department of Microbiology, Faculty of Science, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon

² Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali, Rajasthan-304022, India

³ Amity Institute of Biotechnology, Amity University, Jaipur, Rajasthan-303002, India

INTRODUCTION

Infectious diseases remain one of the major public health problems that lead to human morbidity and mortality ¹. Escherichia coli is one of the microorganisms that cause significant health threat and responsible for a number of human illness conditions especially in developing countries ^2-4. Currently, a wide range of antimicrobials have shown efficacy against bacterial infections. However, most of them have shown several undesirable side effects ⁵. Furthermore, pathogen resistance due to over prescription and overuse of drugs add to the existing causes ^6,7. It is therefore urgent to find novel and efficient agents to control emerging pathogens ⁸.

The mechanisms by which antimicrobials act are mediated through their ability to inhibit the synthesis of the cell wall and proteins, chromosome replication, and modify cell membrane permeability ⁹. The cell wall provides shape and cell integrity. Therefore, the disturbance of cell wall and membrane integrity, and its disruption can lead to metabolic dysfunction and cell death ^10,11. The mode of action of plant extracts is similar to that of antibiotics for it has been shown that they disturb cell structures, increase their permeability, and lead to release of intracellular material ^12-14. The intensive search and employment of innovative research strategies to explore newer, safe, and effective antimicrobial agents which are now being sourced from bioactive components of medicinal plants are increasing ^6,15-18. Plant extracts are used as alternative to antibiotics to prevent and control various diseases, especially in the developing countries because it is relatively cheaper and easily accessible ^16,19,20.

The leaves of Dissotis multiflora (Sm) Triana (Melastomataceae) are widely used in the Cameroon traditional medicine to treat infectious diseases. In our previous findings, we reported the presence of phytochemicals, antibacterial, antioxidant, cytotoxicity, genotoxicity, and effect of the ethanolic leaf extract of D. multiflora in infectious diarrhea ^21-23. Paullinia pinnata Linn (Sapindaceae) family is an important medicinal plant used in many indigenous medical systems for the treatment of infectious diseases ²⁴. Several studies revealed antibacterial and antidiarrheal activities of extracts and isolated compounds from P. pinnata ^25-30. D. multiflora and P. pinnata have shown promising antibacterial activities. However, the information available regarding their mode of action is not yet reported. Therefore, it is necessary to explore the target sites of these two extracts towards E. coli. The present study is a follow up on the previously reported antibacterial activity of the two extracts. It was designed to determine the mechanisms of action of D. multiflora and P. pinnata in antimicrobial potential against E. coli through kill-time analysis, interferences in the synthesis of cell wall, the permeability of cell membrane, the release of cell constituents, and the DNA degradation assay, in order to better understand their antibacterial activity.

MATERIALS AND METHODS

Reagents and chemicals

2,3,5-triphenyltetrazolium Chloride (TTC) (Himedia, India), Dimethylsulfoxide (DMSO), sorbitol (0.8 M) (Srichem, India), agarose (Himedia, India), Ethidium bromide (Himedia, India), Bromophenol blue (Himedia, India), xylene cyanol FF (Himedia, India), glycerol (Himedia, India), Tris, base (Himedia, India), Tris-Hcl (Himedia, India), Ethylenediamine teraacetic acid (EDTA) dihydrate (Fischer Scientific), Mueller-Hinton agar (Himedia, India), Mueller-Hinton broth (Himedia, India), Genomic DNA extraction kit (GeneiTM, India), DNA ladder (1 Kb, Sigma, India),  Whatman No. 1 filter paper (Whatman GE Healthcare Company). All the other chemicals of analytical grade were purchased from Sigma-Aldrich India Limited, Hi Media, India and Merck, India.

Microorganism and Culture

A clinical strain of E. coli was used in this study and provided by the Department of Bioscience and Biotechnology, Banasthali Vidyapith, Banasthali, India and was stored at -80°C in 15% glycerol stocks to be used in the study. Colonies grown on a Mueller-Hinton agar plate incubated overnight at 37°C were inoculated in 10 mL of Mueller-Hinton broth, and incubated at 37°C under shaking and used.

Plant Material and extraction

30. multiflora and P. pinnata leaves were collected and treated as previously described ^23,30. The leaves of D. multiflora and P. pinnata were air-dried for two weeks and ground separately. Then, fine dried material powder was soaked for 48 h in 95% ethanol with frequent stirring. The mixture was filtered with Whatman No. 1 filter paper and concentrated using a Rotavapor (Buchi) at 55°C. Then, each extract was collected in Eppendorf tube and preserved in a refrigerator at 4°C for further use. Sample was diluted ten-fold in 1% (v/v) dimethylsulfoxide (DMSO) and used in the assays.

Determination of the Minimum Inhibitory Concentrations (MICs) and the Minimum Bactericidal Concentrations (MBCs)

The MICs and the MBCs of the crude extracts to E. coli were determined by microbroth dilution method in 96 plates as previously described ³¹. Briefly, each well was filled with 100 µL of Mueller Hinton broth. A series of two-fold dilution of each crude extract was made with final concentrations ranged from 25 x 10³ to 24 μg/mL and from 25 to 0.048 μg/mL for ciprofloxacin used as reference. A 100 µL standardized suspension of E. coli (1.5 x 10⁶ CFU/mL) was mixed with the above samples. After 24 h incubation at 37°C, MICs were determined after the addition of 40 µL of 2,3,5-triphenyltetrazolium chloride (TTC) at 0.01% in sterile distilled water and incubation at 37°C for 30 min. Wells that contained only extract and nutrient broth were used as negative control. The MIC was defined as the lowest concentrations those inhibited visible bacterial growth. The MBCs were determined by picking 50 μL aliquots from each well having no growth in MICs assay plate to new plate with each 100 μL of Mueller-Hinton broth, and the lowest concentration of the well which did not show colour change by addition of TTC.

Bacterial Growth Curve

The effects of the crude extracts on E. coli growth were evaluated following a previous report by Babii et al. ³². Briefly, a volume of 150 µL from an overnight preculture of E. coli containing 1.5 x 10⁶ CFU/mL was added in 15 mL of Mueller Hinton Broth with 1/2 MIC, MIC, and 2 MIC values as final concentrations of D. multiflora and P. pinnata. Treated samples were incubated on an orbital shaker (190 rev/min, 37°C) for 10 h. Growth rates were determined by measuring OD600 at regular intervals using UV-vis spectrophotometer. 1% DMSO was supplemented to control.

Sorbitol Protection Assay

The effects of sorbitol on antibacterial activity of the crude extracts were evaluated by microbroth dilution method according to the previous report by Frost et al. ³³ with slight modifications. Briefly, duplicate plates (96 wells) containing test samples (100 µL) were prepared; one containing two-fold dilutions of test extracts and the other containing test extracts plus 0.8 M sorbitol (Srichem, India) as an osmoprotectant. 100 μL of cell suspension (0.5 McFarland) was added in all the wells, plates were then incubated at 37°C (Cooling Incubator, REMI). The plates were read at 2 and 7 days.

Release of 260 nm-Cellular Absorbing Materials

The measurement of the release of 260 nm-absorbing-materials from E. coli cell was performed as previously described by Bennis et al.³⁴ with slight modifications. Briefly, a suspension of E. coli (2.5 x 10⁷ CFU/mL) grown in Mueller Hinton Broth (Himedia, India) at 37°C for 48 h was treated with D. multiflora and P. pinnata extracts at 1/2 MIC, 1 MIC, 2 MIC for 1 h or a fixed concentration (1 MIC and 2 MIC) for different time intervals (30, 60, 90, and 120 min). Suspension without extract was run as negative control. After treatment, samples were centrifuged at 10,000 x g for 10 min (Cooling centrifuge, REMI), and the absorbance of supernatant was read at 260 nm using UV-vis spectrophotometer.

Outer Membrane Permeability

The outer membrane permeability was determined according to Hao et al. ³⁵ with slight modifications as described by Da Silva et al. ³⁶. Briefly, a 18 h E. coli culture (5 x 10⁷ cells/mL) was inoculated into Mueller-Hinton broth containing extract at 0 MIC, 1 MIC, and 2 MICs on the sterilized test tubes. The mixture was incubated at 37°C for 24 h. E. coli growth was measured at 450 nm using UV-vis spectrophotometer.

Fluorescent Microscopic Analysis

The bacterial membrane integrity was appreciated by capturing confocal photomicrographs of the uptake of the fluorescent nuclear dye ethidium bromide for E. coli as previously described by Lambert et al. ³⁷ with slight modifications. Exponential phase cells were centrifuged at 4000 x g for 5 min (Cooling centrifuge, REMI, India), washed several times and suspended in phosphate buffer saline (PBS, pH-7.4). 10 µg/mL of ethidium bromide was added to 200 µL aliquots of these cells, then extracts were added for final concentrations equal to 1 MIC and 2 MICs values of DMSO. During the 37°C incubation, samples of treated and untreated cells were taken at 2 h and the fluorescent cells were observed using a Leica Confocal Laser Scanning Microscope (Leica DM 1000). At least three random, independent images were captured per sample. The percentage of fluorescent cells was determined by calculating the ratio between fluorescent cells and total cells visualized with differential interference contrast (DIC).

DNA degradation assay

The effect of D. multiflora and P. pinnata on DNA integrity was evaluated as previously reported by Babii et al. ³². The genomic DNA was isolated after 3 h treatment of E. coli with extracts or DMSO using a ZR Bacterial DNA MiniPrep kit (Zymo Research, Reiburg, Germany). The DNA samples were electrophoresed in 1% agarose gel at 120 V until sufficient resolution was obtained. A 1 kb ladder (New England Biolabs, Ipswich, MA) was used as standard size marker. The bands were visualized by ethidium bromide (EB) in gel staining under UV light (GeNei^TM, UVITEC, Cambridge).

Statistical Analysis

Data are expressed as mean ± standard deviation of triplicate determinations. Microsoft Excel was used to enter and capture data from which graphs and tables were extracted. Differences between groups were assessed by One-way analysis of variance (ANOVA) with post hoc comparisons. All analyses were carried out using GraphPad Prism 7.  Significant differences were considered at P ˂ 0.05.

RESULTS

MIC and MBC

The antibacterial activity of D. multiflora and P. pinnata leaves extracts was performed using clinical isolate of E. coli. Both plant exhibited potent inhibitory effect as shown by their various MIC and MBC values. In this assay, the MIC values of D. multiflora and P. pinnata against the tested pathogen were found to be 390.62 and 781.25 µg/mL respectively, while the MBC values were found to be 781.25 and 1562.5 µg/mL respectively. Ciprofloxacin exhibited a MIC and MBC value of 1.62 µg/mL. A bactericidal activity of all the extracts was recorded.

Bacterial Growth Curve

1. multiflora and P. pinnata leaves extracts inhibited the growth of E. coli at different concentrations used with immediate decrease of bacterial load 1 h after incubation. Treatment with D. multiflora extract showed a decrease in bacterial load at different times in concentration dependent manner (Figure 1A). As from 6 h, an increase of bacterial load was noted with 1/2 MIC. P. pinnata extract showed a decrease in bacterial load from 0 to 2 h of incubation (Figure 1B). However, an increase in bacterial load was noted from 2 to 6 h with 1 MIC and 2 MICs.

Sorbitol Protection Assay

The effect of the ethanolic extracts of D. multiflora and P. pinnata on bacterial cell wall was determined in the presence and absence of sorbitol (Table 1). The antibacterial activity of D. multiflora decreased in the presence of sorbitol, an osmoprotectant with increasing MIC observed. The MIC of D. multiflora increased 4x as from day-7 with MIC varying from 390.62 µg/mL after 48 h of incubation to 1562.5 µg/mL after 7 days. However, the MIC of P. pinnata remained the same in the presence and absence of sorbitol. These results showed that D. multiflora extract was implicated in the inhibition of the synthesis of cell wall constituents of E. coli while P. pinnata had no effect on the cell wall.

Table 1: Effect of the ethanolic extracts of D. multiflora and P. pinnata on the cell wall of E. coli in the presence and absence of sorbitol

Values are mean ± standard deviation of independent triplicate measurements. Values in the same column with different superscripts are significantly different according to Post hoc multiple comparison test (P ˂ 0.05).

Release of 260nm-Cellular Absorbing Material

The OD260 values of the filtrates from E. coli control suspension remained approximately the same after 30, 60, 90, and 120 min. Upon treatment with the 1/2 MIC, 1 MIC, and 2 MICs of D. multiflora and P. pinnata, the OD values of the filtrates from E. coli suspensions increased after 1 h in concentration-dependent manner. The OD values from suspensions treated by the extracts were greater than values from control (Figure 2).

The release of 260 nm-cellular absorbing materials was assessed with extracts at concentrations corresponding to 1 MIC and 2 MICs. No increase of OD was observed from 30 to 90 min with 1 MIC and 2 MICs after exposure to D. multiflora (Figure 3A). However, an increase of OD was noted with increasing concentration of P. pinnata (Figure 3B).

Outer Membrane Permeability

The permeability of external membrane was determined at different concentrations (1/2 MIC, 1 MIC, and 2 MICs). A decrease of OD of external membrane constituents of E. coli exposed to D. multiflora and P. pinnata compared with control which showed constant absorbance (Figure 4). D. multiflora and P. pinnata increased membrane permeability of E. coli that led to the leakage and release of cellular material with activity most pronounced at 1/2 MIC and 1 MIC than 2 MICs.

Fluorescent Microscopy

As D. multiflora and P. pinnata were able to reduce cultivable cell number of E. coli, it was of interest to examine whether they had an effect on biofilm viability. The ability of both the plant extracts to damage cellular membrane and make cellular nucleic acids accessible to fluorescent dye was determined after ethidium bromide staining. Intense fluorescent cells were observed after 2 h treatment with D. multiflora (Figure 5). The percentage of fluorescent cells increased with increasing concentration (1 MIC and 2 MICs) for D. multiflora (Figure 6). Similar results were observed after exposure with P. pinnata extract especially at 2 MICs (Figure 6). However, treatment with P. pinnata extract did not cause an intense increase in mean fluorescence percentage.

DNA degradation assay

This test was performed to detect damages of genomic DNA of E. coli by D. multiflora and P. pinnata extracts by observing bands on agarose gel after 3 h of exposure. The agarose gel electrophoresis of extraction products from treated samples showed no bands while that of DNA sample from E. coli not exposed to extracts showed bands similar to that of DNA ladder (Figure 7). These observations evidence the fact that both extracts maybe implicated in the hydrolysis of DNA.

Figure 1: The growth curves of E. coli cells exposed to different concentrations (1/2 MIC, 1 MIC, and 2 MICs) of extracts. ♦ Control;  ■ 1/2 MIC; ▲ 1 MIC; ■ 2 MICs. (A): D. multiflora; (B): P. pinnata. Values are mean ± standard deviation of independent triplicate measurements. Values in the same column with different superscripts are significantly different according to Post hoc multiple comparison test (P ˂ 0.05).

Figure 2: Release of cellular material of E. coli absorbing at 260 nm following treatment with D. multiflora and P. pinnata extracts for 60 minutes. ■ D. multiflora; ■ P. pinnata; ■ Control. Values are mean ± standard deviation of independent triplicate measurements. Values in the same column with different superscripts are significantly different according to Post hoc multiple comparison test (P ˂ 0.05).

Figure 3: Release of 260 nm-absorbing material of E. coli following treatment with D. multiflora and P. pinnata extracts at different times. ■ D. multiflora; ■ P. pinnata; ■ Control. (A): 1 MIC; (B): 2 MICs. Values are mean ± standard deviation of independent triplicate measurements. Values in the same column with different superscripts are significantly different according to Post hoc multiple comparison test (P ˂ 0.05).

Figure 4: Outer membrane permeability of E. coli following treatment with D. multiflora and P. pinnata extracts at 1/2 MIC, 1 MIC, and 2 MICs. ♦ D. multiflora; ■ P. pinnata. Values are mean ± standard deviation of independent triplicate measurements. 

Figure 5: Inner membrane permeabilization induced by plant extracts. E. coli cells membrane disruption effect due to 2 h exposure to D. multiflora. Visualised by efflux of ethidium bromide (10 µg/mL), a nuclear fluorescent dye. Images are representative of typical results. (A): image of contrast of differential interference 1 min after exposure; (B): fluorescent image 1 min after exposure; (C):   fluorescent image 2 h without exposure (Control); (D):  fluorescent image 2 h after D. multiflora exposure; excitation line: 488 nm.

Figure 6: Mean fluorescence intensity in E. coli treated with different plant extracts. ■ Control; ■ D. multiflora; ■ P. pinnata. At least three random, independent images were captured per sample and the ratio between fluorescent cells and total cells visualized with differential interference contrast (DIC) was calculated as percentage. Values in the same column with different superscripts are significantly different according to Post hoc multiple comparison test (P ˂ 0.05). 

Figure 7: Agarose gel electrophoresis 1% of genomic DNA from E. coli after exposure with ethanolic extracts of D. multiflora and P. pinnata. M: DNA ladder (1 Kb); Lanes 1: DNA sample from E. coli treated with D. multiflora for 3 h; Lanes 2: DNA sample from E. coli treated with P. pinnata for 3 h; Lanes 3 : DNA from E. coli not exposed to extracts.

DISCUSSION

Despite diversity and aggressiveness of current antimicrobial treatment, bacterial infections still remain a public health concern in developing countries that face a lot of difficulties to solve the problem to which can be added resistance to current antibacterial agents used ^38-39.  Natural products are reservoirs of novel efficient compounds with diverse biological properties that contribute significantly to the discovery of antibacterial substances ^40-41. D. multiflora and P. pinnata are used to treat diarrhea and other infectious diseases. To date, few studies revealed pharmacological properties of D. multiflora while many of them focussed on biological properties of P. pinnata ^42-44. However, no previous study was carried out to understand their mechanism of action in order to clarify their biological activity.

MIC and MBC

The antibacterial assay shows that D. multiflora and P. pinnata exhibited antibacterial activity with bactericidal effects. These results are in accordance with the similar reports that have confirmed their antibacterial efficacy ^21,29.

Bacterial Growth Curve

In the present study, the bacterial growth curve of E. coli decreased with increasing concentrations and time of exposure to D. multiflora and P. pinnata leaves extracts. The results obtained revealed that both plant extracts exhibited bactericidal effects as shown by the immediate decrease of bacterial load 1 h after incubation. Hence, the ability of the plant extracts to inhibit the growth or cause death of bacteria is an indicator of their efficacy ³². These observations are consistent with the previous report by Zhang et al. ⁴ who evidenced the shortening of kill-time following treatment of meat-borne E. coli with black pepper essential oil.

Sorbitol Protection Assay

The rigid cell wall of Gram-negative bacteria does not allow penetration of antibacterial agents contributing to resistance ⁴⁵. In order to determine the effect of the plant extracts on bacterial cell wall, an osmotic stabilizer, namely sorbitol was added in the medium. It has been shown that several antibacterial agents’ action can be reduced in the medium containing an osmotic stabilizer ³³. The present study revealed a marginal increase of the MIC observed in the presence of D. multiflora extract and sorbitol signifying that the macromolecules and enzymes which ensure the synthesis of E. coli cell wall constituents are D. multiflora extract’s targets ⁴⁶.  The findings of this study are similar to the observation of Frost et al. ³³ who reported the inhibitions of fungal cell wall synthesis and assembly. It has been demonstrated that flavonoids can complex soluble extracellular proteins and bacterial cell wall ^20,47. No evidence of inhibition of synthesis and cell wall assemblage was noted in the presence of P. pinnata extract and sorbitol. However, this extract causes membrane permeability suggesting that it should be its primary target site.

Release of 260nm-Cellular Absorbing Material

Exposure of E. coli to crude extracts of D. multiflora and P. pinnata led to rapid loss of 260 nm absorbing molecules in time and concentration-dependent manner. These results indicate eventual rupture of cellular membrane permeability control ⁴⁸.  Consistent with these findings, the release of intracellular materials was reported by previous study ³. In fact, the nucleic acids and proteins are essential constituents of bacteria cells. The loss of nucleic acids leads to dysfunction of DNA and proteins synthesis and inhibition of cellular growth. The disruption of cellular membrane releases DNA and RNA with high absorbing potential at 260 nm ³. Furthermore, saponins found in these extracts can cause lysis of certain membrane and plasmatic proteins while tannins can inactivate microbial adhesion, enzymes, and proteins of cellular envelope ^49,50. The plants’ extracts may have cause damage of membrane that lead to efflux of nucleotides and then a complete lysis of cell resulting to death ^36,51,52. It has been demonstrated that phenols and flavonoids possess detergent properties which exert action on the destruction of cellular membrane ²⁰. Moreover, alkaloids were shown to intercalate between cellular membranes ⁵³.

Outer Membrane Permeability

The outer membrane permeability of E. coli was evaluated in the presence of D. multiflora and P. pinnata extracts. The differential sensitivity of E. coli to antibiotics should be due to the fact that the peptidoglycan of Gram-negative bacteria is surrounded by external membrane that limits the diffusion across its covering lipopolysaccharides (LPS) that prevent penetration of numerous toxic compounds ^54,55. Phenolic compounds can extract LPS from bacterial membranes and intervene in their destruction ⁵⁶. The increase of membrane permeability of   E. coli in the presence of D. multiflora and P. pinnata extracts was noted with 1/2 MIC and 1 MIC. Therefore, at low concentrations, these extracts effectively permeate the intact outer membrane of Gram-negative bacteria. This may be due to the presence of these bioactive secondary metabolites that facilitates the entry of antibacterial substances in the cell and affect the permeability and integrity of membrane followed by the loss of intracellular substances ^57,58. These results are similar with the previous report by Da Silva et al. ³⁶ who showed that the hydroethanolic extract of Piper umbellatum increases permeabilization of outer membrane of Shigella flexneri, facilitating the entry of the antibiotics.

Fluorescent Microscopy

The effect of D. multiflora and P. pinnata extracts on membrane integrity was further confirmed by fluorescence microscopy using a fluorescent dye, ethidium bromide. Increased fluorescent was noted following treatment with the plant extracts. These results are consistent with the membrane disruption and bactericidal effects reported on E. coli in the present study. The fluorescent cells observed without exposure to the plant extracts may be due to natural death of E. coli. Moreover, the mean fluorescent intensity in E. coli treated with different plant extracts increased with increasing concentration and duration of exposure, and was most pronounced in the presence of D. multiflora. These results are similar with the previous results. ⁵⁹ The fluorescent cells observed owed to plant extracts exposure indicated damage of cellular membrane which makes cellular nucleic acids accessible to the ethidium bromide ³².  

DNA degradation assay

In our study, genomic DNA was extracted in order to determine the effect of D. multiflora and P. pinnata extracts on DNA degradation. It is evident from the results that both extracts were implicated in bacterial DNA degradation. This may be attributed to the phenolic components which can alter the structure and morphology of DNA ⁶⁰.

The findings of this study suggests that the antimicrobial action of D. multiflora and P. pinnata could be due to their efficacy to alter cell membrane permeability parameters of the bacteria, and provoke leakage and release of cellular materials. These modifications of membrane permeability and the release of the cellular material lead to disorder, decomposition, and event death corresponding to a similar reduction in the time kill of viable bacteria. Due to the heterogeneous compositions of both extracts it is suggested that compounds from these plants products should be screened to obtain novel and effective antibacterial agents.

CONCLUSION

In summary, we report that the ethanolic leaf extracts of D. multiflora and P. pinnata exhibited antibacterial activity on E. coli with action in time and concentration dependent. This inhibitory activity of D. multiflora is through inhibition of cell wall constituents, while both the plant extracts act by permeabilization of cell membrane, release of intracellular materials, and DNA fragmentation.

LIMITATIONS OF THE STUDY

Only one strain (clinical isolate) of E. coli was used as test microorganism. Positive and negative control drugs were not included in the experiments as well as a membrane disturbing agent. In fact, this is a preliminary study which determined the susceptibility of the above strain in the presence and absence of the plant extracts. The upcoming conclusive study will include both the isolates and characterized strains, positive and negative control drugs, and bioactive isolated compounds.

 AUTHOR CONTRIBUTIONS

AA, MN and FE designed the study. JP and SV supervised experiments. AA carried out the experiments, analyse data, and wrote the manuscript. JP critically revised the paper and intellectual content.

FUNDING

This study was financially supported by The Department of Biotechnology, Government of India (DBT), India, and The World Academy of Science (TWAS) for the advancement of science in developing countries (Grant number: 3240293146).

ACKNOWLEDGMENTS

The authors are very gratefull to the University of Yaounde I, Cameroon and Banasthali Vidyapith, Rajasthan, India for providing facilities.

CONFLICT OF INTEREST

The authors declare that there is no potential conflict of interest regarding the publication of this paper.

REFERENCES

1. Chitemerere TA, Mukanganyama S. Evaluation of cell membrane integrity as a potential antimicrobial target for plant products. BMC Complement Altern Med 2014; 14:278.
2. Mueller M, Tainter CR. Escherichia coli. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021.
3. Bajpai VK, Shukla S, Paek WK, Lim J, Kumar P, Kumar P, et al. Efficacy of (C)-Lariciresinol to control bacterial growth of Staphylococcus aureus and Escherichia coli O157:H7. Front Microbiol 2017; 8:804.
4. Zhang J, Ye KP, Zhang X, Pan DD, Sun YY, Cao JX. Antibacterial Activity and mechanism of action of Black pepper essential oil on meat-borne Escherichia coli. Front Microbiol 2017; 7:2094.
5. Reduan FH, Shaari RM, Sayuti NS Acute and subacute dermal toxicity of ethanolic extract of Melastoma malabrathricum leaves in Sprague-Dawley rats. Toxicol Res 2020; 36:203-210.
6. Hana MA, Insha S, Vijay K, Irfan AR, Hashem AS, Arif TJ et al. Plant-based phytochemicals as possible alternative to antibiotics in combating bacterial drug resistance. Antibiotics 2020; 9:480.
7. Mohammed KO, Abdulrahman AA, Saud SA, Walid HS, Altaf A, Litaker MS et al. Antibacterial and antifungal activity of the extracts of different parts of Avicennia marina (Forssk.) Vierh. Plants 2021; 10:252.
8. Ting SU, Ye Q, Xuesi H, Bi Y, Haoming L, Da L et al. Novel opportunity to reverse antibiotic resistance: to explore traditional chineses medicine with potential activity against antibiotics-resistance bacteria. Front Microbiol 2020; 11:610070.
9. Bahman K, Milad I, Vahid S, Bibi SF. A review on antimicrobials: a mechanistic viewpoint. Antimicrob Resist Infect Control 2019; 8:118.
10. Matijaševi´ D, Panti´ M, Raškovi´ B, Pavlovi´ V, Duvnjak D, Sknepnek A, et al. The antibacterial activity of coriolus versicolor methanol extract and its effect on ultrastructural changes of Staphylococcus aureus and Salmonella Enteritidis. Front Microbiol 2016; 7:1226.
11. Collet JF, Cho SH, Iorga BI, Goemans CV. How the assembly and protection of the cell envelope depend on cysteine residues. J Biol Chem 2020; 21:11984-11994.
12. Mani-Lopez E, Cortes-Zavaleta O,Lopez-Malo A. A review of the methods used to determine the target site or the mechanism of action of essential oils and their components against fungi. SN Appl Sci 2021; 3:44.
13. Lv F, Liang H, Yuan Q, Li C. In vitro antimicrobial effects and mechanism of action of selected plant essential oil combinations against four food-related microorganisms. FoodRes Int 2011; 44:3057-3064.
14. Bajpai VK, Sharma A, Baek KH. Antibacterial mode of action of Cudrania tricuspidata fruit essential oil, affecting membrane permeability and surface characteristics of food-borne pathogens. Food Control 2013; 32:582-590.
15. Militello M, Settanni L, Aleo A, Mammina C, Moschetti G, Giammanco GM, et al. Chemical composition and antibacterial potential of Artemisia arborescens L. essential oil. Curr Microbiol 2011; 62:1274-1281.
16. Akinpelu DA, Alayande KA, Aiyegoro OA, Akinpelu OF, Okoh AI. Probable mechanisms of biocidal action of Cocos nucifera Husk extract and fractions on bacteria isolates. BMC Complement Altern Med 2015; 15:116.
17. Robert EM, Ngono BD, Auguste AA, Mbabi NN, Moni NE, Betote DP et al. Rauvolfianine, a new antimycobacterial glyceroglycolipid and other constituents from Rauvolfia caffra. Sond (Apocynaceae). Nat Prod Res 2017; 1-6.
18. Afagnigni AD, Nkonpa KR, Teugwa MC. Phytochemical analysis, antimicrobial, and radical scavenging properties of methanol extracts of Dracaena deisteliana (Dracaenaceae) and Sporobulus indicus (Poaceae). Global J Med Res 2020; 20(6): 41-50.
19. Yamani HA, Pang EC, Martin N, Deighton MA. Antimicrobacterial activity of Tulsi (Ocimum tenuiflorum) essential oil and their major constituents against three species of bacteria. Front Microbiol 2016; 7:681.
20. Saritha K, Rajesh A, Manjulatha K, Setty OH, Yenugu S. Mechanism of antibacterial action of the alcoholic extracts of Hemidesmus indicus (L.) R. Br. ex Schult, Leucas aspera (Wild.), Plumbago zeylanica, and Tridax procumbens (L.) R. Br. ex Schult. Front Microbiol 2015; 6:577.
21. Afagnigni AD, Nyegue MA, Ndoye FC, Voundi SH, Fonkoua MC, Etoa F-X. Antibacterial and antioxidant activities of ethanolic leaves extracts of Dissotis multiflora Triana (Melastomataceae). Int J Pharm Sci Drug Res 2016; 8:50-56.
22. Afagnigni AD, Nyegue, MA, Ndoye, FC, Njankouo, NY, Njayou, FN, Fonkoua, MC et al. Antidiarrheal activity of Dissotis multiflora (Sm) Triana (Melastomataceae) leaf extract in Wistar rats and sub-acute toxicity evaluation. Evid Based Complement Alternat Med 2017; 1-9.
23. Afagnigni AD, Nyegue MA, Djova SV, Etoa FX. LC-MS analysis, 15-lipoxygenase inhibition, cytotoxicity, and genotoxicity of Dissotis multiflora (Sm) Triana (Melastomataceae) and Paullinia pinnata Linn (Sapindaceae). J Trop Med 2020; 1-8.
24. Lasisi A.A., Ayinde B.W., Adeleye A.O., Onocha P.A., Oladosu I.A., Idowu P.A. New triterpene isovanniloyl and antibacterial activity of constituents from the roots of Paullinia pinnata Linn (Sapindaceae). J Saudi Chem Soc 2011;19:117-122.
25. Lunga PK, Tamoukou JD, Fodouop SP, Kuiate JR, Gatsing TD. Antityphoid and radical scavenging properties of the methanol extracts and compounds from the aerial part of Paullinia pinnata Linn (Sapindaceae). Springerplus 2014; 3: 302.
26. Yusuf AZ, Zakir A, Shemau Z, Abdullahi M, Halima SA. Phytochemical analysis of the methanol leaves extracts of Paullinia pinnata Linn. J Pharmacognosy Phytother 2014; 6:10-16.
27. Jackson N, Annan K, Mensah AY, Ekuadzi E, Mensah ML, Habtemariam S. A novel triterpene from the roots of Paullina pinnata: 6α-(3’-Methoxy-4’-hydroxybenzoyl)-lup-20(29)-ene-3-one. Nat Prod Commun 2015; 10:563564.
28. Lunga PK, Nkodo JM, Tamokou JD, Kuiate JR, Gatsing G, Tchoumboue J. Post-treatment evaluation of the side effects of methanol leaf extract from Paullinia pinnata Linn, an antityphoid plant. Pharmacologia 2015; 6:264-272.
29. Afagnigni AD, Nyegue MA, Ndoye FC, Voundi OS, Fonkoua MC, Etoa F-X. In vitro assessment of antibacterial and antioxidant activities of ethanolic leaves extracts of Paullinia pinnata Linn (Sapindaceae). World J Pharm Sci 2016; 4:173-182.
30. Nyegue MA, Afagnigni AD, Ndam YN, Djova SV, Fonkoua MC, Etoa F-X. Toxicity and activity of ethanolic leaf extract of Paullinia pinnata (Sapindaceae) in Shigella flexneri-induced diarrhea in Wistar rats. J Evid-Based Integr Med 2020; 25:1-9.
31. Eloff JN. A sensitive and quick microplate method to determine the minimum inhibitory concentration of plant extracts for bacteria. Plant Med 1998; 64:711-713.
32. Babii C, Bahrin LG, Neagu AN, Gostin I, Mihasan M, Birsa LM et al. Antibacterial activity and proposed action mechanism of a new class of synthetic tricyclic flavonoids. J Appl Microbiol 2016; 120:630-637.
33. Frost DJ, Brandt KD, Cugier D, Goldman R A whole-cell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis and assembly. J Antibiot 1995; 48:306-310.
34. Bennis S, Chami F, Chami N, Bouchikhi T, Remmal A. Surface alteration of Saccharomyces cerevisiae induced by thymol and eugenol. Lett Appl Microbiol 2004; 38:454-458.
35. Hao G, Shi YH, Tang YI, Le GW. The membrane action mechanism of analogs of the antimicrobial peptide Buforin 2. Peptides 2009; 30:1421-1427.
36. Da Silva IF, De Oliveira RG, Soares IM, Alvim TC, Ascêncio SD, Martins DT. Evaluation of acute toxicity, antibacterial activity and mode of action of the hydroethanolic extract of Piper umbellatum L. J Ethnopharmacol. 2014 ; 151:137-143.
37. Lambert RJ, Skandamis PN, Coote PJ, Nychas GJ. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. J Appl Microbiol 2001; 91:453-462.
38. Tambe AB, Nzefa LD, Nchang AN. Childhood diarrhea determinants in Sub-Saharan Africa: A cross sectional study of Tiko-Cameroon. Challenges 2015; 6:229-243.
39. Mekonnen B, Asrie AB, Wubneh ZB. Antidiarrheal activity of 80% methanolic leaf extract of Justicia schimperiana. -BasedComplement. Alternat Med 2018; 3037120:1-10.
40. Ali I, Sharma P, Suri KA, Satti NK, Dutt P, Afrin A et al. In vitro antifungal activities of amphotericin B in combination with acteoside, a phenylethanoid glycoside from Colebrookea oppositifolia. J Med Microbiol 2011; 60: 1326-1336.
41. Kuete V, Fokou W F, Karaosmanoğlu O, Beng VP, Sivas H. Cytotoxicity of the methanol extracts of Elephantopus mollis, Kalanchoe crenata and 4 other Cameroonian medicinal plants towards human carcinoma cells. BMC Complement Altern Med 2017; 17:280.
42. Adeyemo SO, Makinde J. M. Acute and sub-acute toxicity studies of the methanol extract of the leaves of Paullinia pinnata Linn in Wistar mice and rats. Afr J Med Med Sci 2013; 42:81-90
43. Lunga PK, Tamoukou JD, Fodouop SP, Kuiate JR, Gatsing TD. Antityphoid and radical scavenging properties of the methanol extracts and compounds from the aerial part of Paullinia pinnata Linn (Sapindaceae). SpringerPlus. 2014; 3:302.
44. Mariame E. Aboudoulatif D, Povi LE, Kodjo A, Kossi M, Essohana P et al. Repeated toxicological study and cardiotoxicity of hydroalcoholic root extract of Paullinia pinnata Linn (Sapindaceae). J Appl Pharm Sci 2016; 6:24-28.
45. Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M, Daniele C et al. Mechanisms of antibacterial action of three monoterpenes. Antimicrob AgentsChemother 2005; 49:2474-2478.
46. Khan MS, Ahmad I, Cameotra SS. Phenyl aldehyde and propanoids exert multiple sites of action towards cell membrane and cell wall targeting ergosterol in Candida albicans. AMB Express 2013; 3:54.
47. Cowan MM. Plant’s products as antimicrobial agents. Clin Microbiol Rev. 1999; 14:564-582.
48. Carson CF, Mee BJ, Riley TV. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Agents Chemother 2002; 46:1914-1920.
49. Zablotowicz RM, Hoagland RE, Wagner SC. Effect of Saponins on the Growth and Activity of Rhizosphere Bacteria," In: Waller G.R., Yamasaki K. (eds) Saponins used in Food and Agriculture. Adv Exp Med Biol Springer, Boston MA
50. Shimada T. Salivary proteins as a defence against dietary tannins. J Chem Ecol 2006; 32:1149-1163.
51. Denyer SP. Mechanisms of action of biocides. Int Biodeterior Biodegr 1990; 26:89-100.
52. Patra JK, Baek KH. Antibacterial activity and action mechanism of the essential oil from Enteromorpha linza against foodborne pathogenic bacteria. Molecules 2016; 21:388.
53. Ghestem A, Seguin E, Paris M, Orecchioni AM. Le préparateur en Dossier 2, Botanique-Pharmacognosie-Phytothérapie-Homéopathie. Tec et Doc (ed), 2001; 272.
54. Bezic N, Skocibusic M, Dinkic V, Radonic A. Composition and antimicrobial activity of Achillea clavennae essential oil. Phytother Res 2003; 17:1037-1040.
55. Sharma A, Bajpai VK, Baek KH. Determination of antibacterial mode of action of Allium sativum essential oil against foodborne Pathogens using membrane permeability and surface characteristic parameters. J Food Saf 2013; 33:197-208.
56. Jiangwei M, Luxi J, Yu C, Jian K. Activities and mechanisms of eugenol and cinnamaldehyde against Legionella pneumophila. Int J Clin Exp Med 2017; 10:16460-16467.
57. Tassou C, Koutsoumanis K, Nychas GJ. Inhibition of Salmonella enteritidis and Staphylococcus aureus in nutrient broth by mint essential oil. FoodRes Int 2000; 33 :273-280.
58. Bajpai VK, Al-Reza SM, Choi UK, Lee JH, Kang SC. Chemical composition antibacterial and antioxidant activities of leaf essential oil and extracts of Metasequoia glyptostroboides Miki ex Hu. Food Toxicol 2009; 47:1876-1883.
59. Famuyinde IM, Fasina FO, Eloff JN, McGaw LJ. The ultrastructural damage caused by Eugenia zeyheri and Syzygium legatii acetone leaf extracts on pathogenic Escherichia coli. BMC Vet Res 2020; 16:326.
60. Wang LH, Zhang ZH, Zeng XA, Gong DM, Wang MS. Combination of microbiological, spectroscopic and molecular docking techniques to study the antibacterial mechanism of thymol against Staphylococcus aureus: membrane damage and genomic DNA binding. Anal Bioanal Chem 2017, 409:3055-3055.