Synergistic Effect of Combined Antibiotic and Methanolic Extracts of Withania somnifera and Catharanthus roseus against MDR Salmonella enterica Serovar Typhi

Chauhan, Neha; Khan, Azhar; Farooq, Umar

doi:https://doi.org/10.1155/2023/8836886

Advanced Gut & Microbiome Research

On this page

Abstract Introduction Methods Results Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 8836886 | https://doi.org/10.1155/2023/8836886

Synergistic Effect of Combined Antibiotic and Methanolic Extracts of Withania somnifera and Catharanthus roseus against MDR Salmonella enterica Serovar Typhi

Neha Chauhan,¹Azhar Khan,¹and Umar Farooq¹

Academic Editor: Jiong Yu

Received07 Apr 2023

Revised10 Jun 2023

Accepted05 Aug 2023

Published25 Aug 2023

Abstract

Typhoid has become the most neglected and majorly affecting disease of tropical and subtropical countries, including India, and is among the most important global health problems, as the emergence of multidrug resistance has shut down the effect of antimicrobial agents and makes it difficult to control the bacteria inside the host. Hence, there is a great need to develop some natural-based drugs, which will be new promising natural therapeutic interventions with high efficacy and lesser side effects in comparison to synthetic drugs already available for typhoid treatment. The present study completely focused on the synergistic effect of bioactive constituents of medicinal plants in combination to synthetic drugs for enhancing the bacterial eradication mechanism. The methanolic extracts of 25 medicinal plants were screened for their antisalmonella effect and out of which only 2 plants were studied further on the basis of their high effectiveness against 17 MDR isolates using well diffusion assay and MIC/MBC determination. The synergistic activity was assessed with two nonantibiotics (ibuprofen and paracetamol) and 3 antibiotics (ceftazidime chloramphenicol, and trimethoprim) using methods of growth inhibitory indices (GIIs) and fractional inhibitory concentration index (FICI). The highly effective methanolic extracts were of W. somnifera and C. roseus, and the synergism was obtained in terms of GIIs and FICI values of 0.9 and 0.3 and 0.9 and 0.5, respectively, fulfilling the criteria for both extracts, respectively. The results for combinations of plant extracts and antibiotics ceftazidime, trimethoprim, and chloramphenicol and nonantibiotics (analgesic drugs) ibuprofen and paracetamol showed good synergistic activity (100%, 88%, 50%, 45%, and 35%, respectively) against the MDR isolates of S. Typhi. The present study also suggests that in the future, combined treatment with the antibiotics and the bioactive compounds can enhance the immune system to perform better action against the external pathogens as well as in the treatment of internal pathogens, and the combinations will be the successful immune modulators.

1. Introduction

Salmonella Typhi is an intracellular pathogen that causes typhoid fever in humans (the only natural hosts and reservoir of infection) [1]. This disease has become a major public health problem in developing countries [2]. The emergence of MDR strains of S. Typhi has encouraged urgency to develop more effective typhoid vaccines [3]. Despite recent advances in the area of vaccinology [4, 5], the pace of progress is not fast enough and needs to be accelerated [6]. Hence, there is a need of continuous surveillance and sharing of resistant data for Salmonella among countries worldwide [7] to ensure the effectiveness of control programs [8].

The global emergence of multidrug-resistant typhoid bacilli has become the most threatening issue and has limited the effectiveness of current drugs, causing explorative treatment failures [9]. The containment of this drug resistance requires thrust for the development of new potent antimicrobial compounds as alternatives to existing antibiotics [10]. However, the development of new antimicrobial drugs is not encouraging with only a few new drugs being licensed in the last few years [11, 12]. This mismatch between the slow development of new drugs and the fast emergence of resistant strains makes the disease management miserable. As an alternative or perhaps a sustainable option and attempts to improve the efficacy of available antibiotics, particularly the older and cheaper drugs have been suggested [13].

Medicinal plants as an alternative continue to play an important role in the healthcare systems from ancient times, particularly in the developing countries, where the herbal medicine has a longer and uninterrupted history of use [14]. The medicinal plants are the target for new therapeutic interventions due to the production of a wide variety of secondary metabolites, many of which have been reported to be of therapeutic value. This raises the prospects of obtaining novel chemotherapeutic compounds if these vastly untapped resources could be adequately explored. Indeed, the plants might be a source of biocompounds that may potentiate the activity of antibiotics against resistant pathogens. These compounds may be useful as resistance-modifying, modulating, or reversal agents. While the routine practice just screens the plant extracts for direct antimicrobial compounds, searching of resistance-modifying compounds from natural resources may also improve the efficacy of antibiotics when used in combination. These compounds must be more effective and attractive as they allow the recycling of old and relatively cheaper antibiotics that have been rendered ineffective due to resistance. Several studies have proposed that plant-derived compounds in combination with antibiotics are the novel methods for developing therapies against bacterial infections as they may enhance the effect of antibiotics in combination due to their synergistic effects [15–17]. Hence, the present study was also focused on the use of medicinal plants in combination with the synthetic drugs as alternative method of treatment against the emergence of MDR S. Typhi strains.

2. Material and Methods

2.1. Ethical Justification

The project was approved by institute’s ethical committee wide IEC no. SUIEC/15/04 (attached certificate in supplementary data).

2.2. Collection, Maintenance of Clinical Isolates, and Screening of MDR Phenotype

A total of 40 clinical isolates of S. Typhi processed from the confirmed patient’s blood and studied for their cultural characteristics and 1 positive control MTCC-733 strain obtained from IMTECH, Chandigarh, were taken for the study. All the isolates were inoculated in glycerol stocks and transported to the MIPL laboratory, Shoolini University (Solan). The isolates were further confirmed based on selective culture characterization, staining, and biochemical identifications. The blood samples from which the isolates were obtained were also studied for WIDAL (Table-2 supplementary data) and ELISA using specific serotyped antigens.

2.3. Antibiotic Susceptibility Assay

All the isolates were screened to know the MDR phenotype by disc diffusion assay [18] which was performed with 12 different groups of antibiotics. The complete growth inhibition around each of the discs was measured by using a transparent plastic ruler. Zone diameters of inhibition were compared with the standards as given by HiMedia (Zone Scale). The percent inhibition of diameter growth was calculated in the following manners:

The minimum inhibitory concentration against S. Typhi was determined by broth microdilution reference method (CLSI M7-A7) after the determination of the antimicrobial activity by agar well diffusion method. The turbidity of the wells in the microtiter plate was interpreted as visible growth of microorganisms, and for quantitative analysis, OD was taken at 595 nm.

2.4. Collection of Plant Material

A total of 25 medicinal plants were collected from Distt. Solan (HP) area (medicinal plants used in the study enlisted in Table-1 of supplementary data). The plants were authenticated from the Faculty of Basic and Environmental Sciences, Shoolini University wide Herbarium nos. SUBMS/BOT-S203 and SUBMS/BOT-S272. As per their antityphoid efficacy and phytochemical-based analysis, only two plants, i.e., Withania somnifera and Catharanthus roseus, were further studied for synergistic activity against the MDR strains.

2.5. Study of Combined Effect of Extracts and Antibiotics [19]

2.5.1. GII (Growth Inhibitory Index) Method

Antibacterial activity was measured using well diffusion method according to National Committee for Clinical Laboratory Standard 2000. Presence of turbidity was adjusted according to 0.5 McFarland standards, and the Mueller-Hinton agar plates were prepared.

The growth inhibitory indices (GIIs) were calculated for the well diffusion method as [20], to corroborate the synergistic activity (in the forms of ZDI) of the extraction combination with the antibiotics and nonantibiotics (analgesic drugs) as control. The effect was said to be synergistic if the value of , additive if , or antagonistic effect was measured in terms of [21].

2.5.2. FIC (Fractional Inhibitory Concentration) Method: Efficacy of Plant Extracts in Lowering the MIC of Antibiotics

Firstly, MIC of antibiotics and plant extract was determined separately. To determine the combined effect of antibiotic and plant extract, combinations of different concentrations ranging from to of each were used. This assay was performed in 96-well ELISA plate. By this assay, a fixed concentration of active compound was determined which decreased the MIC of the antibiotic. The following formula was used for the determination of FICI:

Combinations were classified as follows: synergistic as , additive as , indifferent as , and antagonistic as ([21–23] and Salaria and [23]).

2.5.3. Bactericidal Kinetic Assay

Bactericidal kinetic assays were performed by the method of Gadhi et al. [24] against S. Typhi with minor modifications. A series of tubes containing nutrient broth and sterile extracts at varying concentrations (5-50 mg/ml) and both positive (antibiotic) and negative (broth culture of S. Typhi) controls was inoculated with 10⁵ CFU of S. Typhi and incubated at 37°C. After 0, 1, 2, 4, 6, 8, 10, and 12 h of incubation, bacterial inoculums from each tube were plated on triple sugar iron agar. Plates were incubated overnight at 37°C, and numbers of viable bacteria were counted by colony counter.

2.5.4. Fractionation of Crude Plant Extracts

The methanolic leaf extracts (ME) were fractionated by solvent–solvent partitioning to obtain five water (WtF), ethyl acetate (EaF), chloroform (CfF), n-butanol (BtE), and n-hexane (HxF) fractions [25] (further isolated by column chromatography).

2.5.5. Synergistic Assay of Active Fractions with Antibiotic and Nonantibiotics

The combined effect of active fractions was studied in combination to antibiotics and nonantibiotics as described in GII and FICI analysis methods.

2.5.6. FTIR Analysis of Active Fraction and Bioassay-Guided Fractions

The Agilent Cary 630 Series FTIR spectrometer (Agilent Technologies) was used to study functional group present in chloroform fraction and their subtractions. The main purpose of this study was to identify the presence of certain functional groups in a molecule and the unique collections of the absorption bands to confirm the identity of a pure compound. The frequencies and intensities of the infrared bonds provide information about the nature of the molecular IR spectroscopy [26].

2.5.7. Statistical Analysis

The obtained results were analyzed statistically, and values were represented as . Statistical analysis of collected data was also conducted using CRD three factorial analysis carried out on three factors. The least significant difference at 5 percent level was used for the analysis of significant data among treatments [27].

3. Results

3.1. Isolation, Characterization, and Identification of Salmonella Typhi Isolates

A total no. of 40 suspected typhoid bacillus samples were cultured on the blood agar, incubated at 37°C for 24 h, and then examined macroscopically and microscopically. The isolates were grown on general purpose media (nutrient agar), differential media (MacConkey agar), and selective media (bismuth sulphite agar and XLD). Primarily, S. Typhi was identified based on colony characteristics and further subjected to microscopic and biochemical identifications. The growth was observed based on colony characteristics produced by S. Typhi on various culture media. It produces dome or disc-shaped colonies on nutrient agar, nonlactose fermenting colorless colonies on MacConkey agar, black-colored colonies on bismuth sulphite agar, and pink- or black-centered colonies on XLD agar. The Gram-stained smear was examined under the microscopic oil immersion lens that revealed Gram-negative rod-shaped bacilli. The isolates were further characterized by biochemical tests. The isolates were found to be catalase positive, indole nonfermenting, methyl red positive, and negative for the VP test. Furthermore, the bacilli were found to be noncitrate utilizing and nonurease degrading but nitrate reducing. The bacilli ferment glucose, mannitol, maltose, and sucrose by producing acid but were nonlactose fermenting. The bacilli were found to be highly motile as they had spread-type growth on the semisolid medium. The typhoid bacilli had high agglutination titer in WIDAL and ELISA (profile available as supplementary data Table-2).

3.2. Screening of MDR Strains of Salmonella Typhi

All the 40 S. Typhi isolates were processed for ASA (antimicrobial susceptibility assay) using disc diffusion method. It was found that out of 40 strains, 42% (17/40) were MDR (antibiogram pattern available as supplementary data Table-3 and Table-4). The results were interpreted as resistant, intermediate, and sensitive as per the CLSI guidelines, 2012.

Antibiogram profile of the resistant isolates against the antimicrobials was studied (description given but the data not shown here). Antibiotic sensitivity assay was carried out for S. Typhi isolates () using more than 20 antibiotic discs (HiMedia, Mumbai) belonging to different classes; out of the screened isolates, only 42% (17/40) were found MDR (supplementary data Table-3 and Table-4). The isolates were found to be highly resistant to penicillin and vancomycin (62.5%) followed by ofloxacin and tetracycline (47.5%); kanamycin and ampicillin (45%); trimethoprim, sulfanilamide, and cotrimoxazole (42.5%); and amikacin (40%), while amoxicillin showed 39.5% and clindamycin and chloramphenicol 35%; low resistance was found against ciprofloxacin (10%). In addition, the isolates were found to be 99% resistant to azithromycin and gentamicin, while 100% sensitive for ceftazidime, levofloxacin, and cefotaxime.

The minimum inhibitory concentration (MIC) of MDR S. Typhi isolates () was determined. The MIC μl/ml were considered as borderline (BL) resistant, whereas the increase in MIC values was designated as highly resistant and all the isolates were found with MIC values above this considered MDR. It was found that none of the isolates showed inhibition at the MIC conc. of 0.5-8.0 μl/ml. The isolates found beyond the MIC conc. of 256 μl/ml were highly resistant towards the antibiotics studied. The predominant MIC concentration was found to be ≥256 μl/ml for S. Typhi isolates against the resistant antibiotics followed by 128 μl/ml (Table-4 in supplementary data).

3.3. Antityphoid/Antisalmonella Assay of Plant Extracts

The antibacterial activity of methanolic extracts (yield enlisted in Table-5 supplementary data) was performed on 17 MDR S. Typhi isolates. A total of 25 methanolic extracts obtained from different parts of traditionally used medicinal plants were evaluated for their antisalmonella activity. Out of these 25 methanolic extracts (MEs), the MDR isolates were found to be highly sensitive towards 10/25 plants: D. purpurea (), C. roseus (), R. serpentine (), W. somnifera (), G. glabra (), C. sinensis (), T. chebula (), J. regia (), C. sativa (), and P. granatum (), although the MDR isolates showed intermediate sensitivity towards 6/25 medicinal plants: C. citratus (), N. jatamansi (), M. officinalis (), F. vulgare (), A. paniculata (), and S. cumini (). In addition to this, we have observed that the MDR isolates were found resistant to 9/25 medicinal plants: C. longa (), C. pseudolimon (), B. suaveolens (), C. annum (), A. nilotica (), O. vulgare (), F. religiosa (), A. racemosus (), and O. tenuiflorum (). Among the methanolic extracts of medicinal plants which showed sensitivity towards the MDR S. Typhi isolates, the predominant plant extract was found to be of W. somnifera, J. regia, and C. sativa followed by C. sinensis, T. chebula, C. roseus, D. purpurea, R. serpentine, P. granatum, and G. glabra.

3.4. Minimum Inhibitory Concentration and Minimum Bactericidal Count (MIC/MBC in mg/ml) of the Most Effective Methanolic Extracts

The most effective plant extracts found in the zone diameter of inhibition (ZDI) pattern were processed for their MIC and MBC determination. The results of MIC and MBC revealed that the methanolic extract of W. somnifera found to have the lowest MIC and MBC values (conc. 0.156 mg/ml-0.625 mg/ml) followed by C. roseus (conc. 0.156 mg/ml-1.25 mg/ml), C. sativa (conc. 0.156 mg/ml-2.5 mg/ml), J. regia (conc. 0.156 mg/ml-2.50 mg/ml), R. serpentine (conc. 0.312 mg/ml-2.50 mg/ml), T. chebula (conc. 0.312 mg/ml-2.50 mg/ml), and D. purpurea (conc. 0.312 mg/ml-5.0 mg/ml), and the less effective MIC and MBC were found for P. granatum (0.625 mg/ml-5.0 mg/ml) against the MDR isolates studied. The results conclude that the highly active plants were W. somnifera, C. roseus, C. sativa, J. regia, R. serpentine, T. chebula, and D. purpurea, and the least effective was P. granatum.

3.5. Synergistic Assay of Medicinal Plants with or without Antibiotics and Nonantibiotics

Methanolic extracts of medicinal plants () were evaluated for synergistic activity with 3 antibiotics (trimethoprim, chloramphenicol, and ceftazidime) and two nonantibiotics (paracetamol and ibuprofen) against MDR S. Typhi isolates (). The methanolic extracts of W. somnifera and C. roseus were found most synergistic based on GII (growth inhibitory index) and FICI (fractional inhibitory concentration index) values (0.9 and 0.3 and 0.9 and 0.5, respectively) (Table-6 supplementary data).

3.6. Synergistic Effect Using Growth Inhibitory Indices (GIIs) and Fractional Inhibitory Concentration Indices (FICI)

The methanolic extracts from the plants showed synergistic effect (W. somnifera and C. roseus) and were further studied to determine the synergism, antagonism, and indifferent activity between the extracts and antibiotics/nonantibiotics. The zone of inhibition (for GIIs) and MIC (for FICI) were determined separately and then in combination (Figure 1 and Tables 1–4). The results revealed that the methanolic plant extracts in combination with antibiotics chloramphenicol (50%), ceftazidime (100%), and trimethoprim (88%) and nonantibiotics paracetamol (35%) and ibuprofen (45%) showed potent synergistic activity against the MDR isolates of S. Typhi. The synergistic effect was found to be effective on more strains by the GII method (56.9%) than the FICI method (36.4%) (Tables 1–4). Furthermore, the results also revealed that the synergistic effect was found higher in combination to antibiotics (100%) than the nonantibiotics (61%) (Tables 1–4). Hence, the findings suggest that the methanolic extracts of W. somnifera and C. roseus contain bioactive compounds which have properties to enhance the effect of antimicrobials on the MDR S. Typhi isolates or are potent enhancers of the effectivity to decrease the resistance among the S. Typhi isolates.

(a)

(b)

3.7. Bactericidal Kinetic Assay of Methanolic Extracts of W. somnifera and C. roseus against MDR S. Typhi Isolates

The bactericidal kinetic assay was performed for highly effective/synergistic plants: W. somnifera and C. roseus. Four different concentrations (12.5 mg/ml, 25 mg/ml, 50 mg/ml, and 100 mg/ml) of methanolic extracts were studied for the time-dependent inhibition of the MDR strains ( CFU/ml). The results of time-kill kinetic study of the methanolic extract against S. Typhi showed 99.9% reduction at 50 mg/ml concentration. The maximum reduction of 2 log₁₀ at 50 mg/ml was achieved after 12 h by the methanolic extracts of W. somnifera and C. roseus for controlling the microbes. Approximately 98% reduction in bacterial growth was observed after 24 hours of incubation (Figures 2(a) and 2(b)). It was found from the present study that plants showed bacteriostatic activity up to 6-8 h and after 8 h. The plant extracts completely inhibit the growth of MDR strains of S. Typhi (Figures 2(a) and 2(b)).

(a)

(b)

3.8. Antisalmonella Assay of Fractions against MDR S. Typhi Isolates

The fractions were obtained using the separating funnel method and further studied for antisalmonella activity on 17 MDR S. Typhi isolates. Only 57% (4/7) plant fractions were found active, of which the chloroform fractions from both plants, W. somnifera and C. roseus, were found to be highly sensitive (ZDIs and , respectively) against MDR isolates followed by ethyl acetate, butanol, and aqueous extracts, respectively (Figure 1 and Table-7, 8 supplementary data).

3.9. Synergistic Effect of Fractions of W. somnifera and C. roseus against MDR S. Typhi Isolates

The ethyl acetate, chloroform, butanol, and aqueous fractions of W. somnifera and C. roseus were evaluated for synergistic activity with three antibiotics, i.e., trimethoprim, chloramphenicol, and ceftazidime, and two nonantibiotics, i.e., paracetamol and ibuprofen, against 17 MDR S. Typhi isolates. The chloroform fractions of W. somnifera and C. roseus were found most synergistic based on growth inhibitory index (GII, 0.7) and fractional inhibitory concentration (FIC, 0.5) values. The GII values of chloroform fraction were observed as 0.7 and 0.9, higher than the normal value and FIC value of 0.5, respectively, indicating the synergistic effect followed by the ethyl acetate fraction where the GII value was 0.6 and FIC was 0.9, whereas the effect of aqueous fraction was found antagonistic as it gives GII values 0.45 and FIC 2.0. However, the effect of butanol fraction was found to be additive since here the GIIs and FIC were 0.5 and 1.0, respectively (Table 5).

3.10. FTIR Analysis of Chloroform Fractions of W. somnifera and C. roseus for the Detection of Functional Groups

The chloroform fractions were found to be most effective and synergistic towards the MDR isolates of S. Typhi. Hence, the chloroform fractions of both plants, W. somnifera and C. roseus, were studied for FTIR analysis. The FTIR study showed the differences between the organization and positioning of functional groups of various bioactive compounds present in chloroform fractions of both plants (Figures 3(a) and 3(b) and Table 6).

(a)

(b)

4. Discussion

Typhoid fever is known to be a major public health problem in tropical and subtropical countries including India [28]. The emergence of multidrug resistance strains of Salmonella against existing antibiotics is going to cause serious problems, and spread of drug-resistant strains throughout the endemic regions is an alarming feature. Hence, there is a need to discover and develop reliable and cost-effective drugs to overcome the problem of multidrug resistance. To overcome the problem of antibiotic resistance, medicinal plants have been extensively studied as an alternative treatment for infectious diseases.

Synergism is said to be a phenomenon in which two different compounds combine in such manners so that they can enhance their individual activity or effectiveness. The medicinal plants were screened in a systematic manner to obtain the most synergistic plants in combination to antibiotics and nonantibiotics. A total of 25 medicinal plants were obtained for the study based on their ancient uses in the treatment of fever and as a booster of the immune system. The different parts of the plants selected for the study were dried, and the extract yield was obtained in the range of 100 mg to 2.0 g from the 10 g powder.

In the present study, the antibacterial activity of methanolic extracts was studied on 17 MDR S. Typhi isolates. A total of 25 methanolic extracts of medicinal plants were evaluated for their antisalmonella activity. Out of these 25 methanolic extracts (MEs), 10 plants, i.e., D. purpurea, C. roseus, R. serpentine, W. somnifera, G. glabra, C. sinensis, T. chebula, J. regia, C. sativa, and P. granatum, were found to be highly sensitive against MDR isolates of S. Typhi. In support, Arora et al. [29] have suggested that the methanolic and hexane extracts of medicinal plants are much more effective against the bacterial strains by studying the antimicrobial efficacy of methanolic and hexane extracts against S. Typhimurium and E. coli.

The phytochemical analysis also revealed the presence of all secondary metabolites studied in W. somnifera. Similar findings were revealed by Akinpelu et al. [30] that alkaloid and flavonoids present in the plant parts contribute to their biological effectiveness. They further stated that the most common biological properties of alkaloids and flavonoids are antimicrobial efficacy and toxicity against cells of foreign organisms.

The methanolic extracts analyzed for synergistic effect (W. somnifera and C. roseus) were further studied to determine the synergism, antagonism, and indifferent activity between the extracts and antibiotics/nonantibiotics against the MDR strains on individual basis. The zone of inhibition (for GIIs) and MIC (for FIC) were observed and calculated separately as well as in combination. The results revealed that the methanolic plant extracts in combination with antibiotics chloramphenicol (50%), ceftazidime (100%), and trimethoprim (88%) and nonantibiotics paracetamol (35%) and ibuprofen (45%) showed potent synergistic activity against the MDR isolates of S. Typhi. The synergistic effect was found to be effective on more strains by GII method (56.9%) than the FIC method (36.4%). Furthermore, the results also revealed that the synergistic effect was found high in combination to antibiotics (100%) than the nonantibiotics (61%). Hence, the findings suggested that the methanolic extracts of W. somnifera and C. roseus contain bioactive compounds which have properties to enhance the effect of antimicrobials on the MDR S. Typhi isolates or are potent enhancers of the efficacy to decrease the resistance among the S. Typhi isolates. The C. roseus has been observed with potent bioactivity, including anticancer, anti-inflammatory, antimicrobial, and antidiabetic activity in several studies [31–33], thus justifying its use as ethnomedical plant. C. roseus have majority of alkaloids, while besides alkaloids, it also produces other compounds, including anthocyanins, flavonoids, iridoids, and steroids [33, 34].

Accordingly, Muhammad et al. [35] reported the antibacterial potential of crude extracts of different parts (viz., leaves, stem, root, and flower) of C. roseus against clinically important bacteria. Similarly, Zhang et al. [36] reported the antibacterial potential of the root extract of W. somnifera against S. Typhi. The leaves of the W. somnifera (Indian chemotype) contain 5 unidentified alkaloids, 12 withanolides, several free amino acids glycosides, chlorogenic acid, condensed tannins, flavonoids, and glucose [37]. Several studies have been reported that the alkaloid withaferin A is able to inhibit the growth of various Gram-positive bacteria, acid-fast bacteria, aerobic bacilli, and the pathogenic fungi. The studies also suggested the active effect of alkaloids on Micrococcus pyogenes varaureus and partial inhibition of Bacillus subtilis glucose-6-phosphatedehydrogenase. Withaferin A also reported in the similar studies to inhibit the growth of Ranikhet virus, and the shrub extract was found to inhibit Vaccinia virus and Entamoeba histolytica [37–39]. In the present study, the inhibition was observed to be significantly very high against S. Typhi and other strains used in this study which concordance to the previous studies.

The bactericidal kinetic assay was performed for highly effective/synergistic plants: W. somnifera and C. roseus. The present study revealed that the time-kill kinetic assay of methanolic extract against MDR S. Typhi showed significantly very high (99.9%) reduction with 50 mg/ml concentration. The maximum reduction of 2 log₁₀ at 50 mg/ml was analyzed after 12 h by the methanolic extracts of W. somnifera and C. roseus for controlling the microbes. Approximately 98% reduction in bacterial growth was observed after 24 h of incubation. It was found from the present study that plants showed bacteriostatic activity up to 6 h-8 h, and after 8 h, the plant extracts completely inhibit the growth of MDR strains of S. Typhi. Furthermore, the fractions of methanolic extract have also been studied to know the sensitivity of a particular fraction for the isolation and extraction of bioactive compound. The present study revealed that the chloroform fraction of methanolic extract of W. somnifera and C. roseus was observed highly sensitive against the MDR isolates of S. Typhi followed by ethyl acetate, butanol, and aqueous extracts, respectively, while the other fractions were not found active against the MDR isolates. The chloroform fractions were observed to be the most effective and synergistic towards the MDR isolates of S. Typhi. Hence, the chloroform fractions of both plants, W. somnifera and C. roseus, were further analyzed by physical properties and FTIR. The color of the fractions was found to be black and mist green along with 265°C (C. roseus) and 276-278°C (W. somnifera) melting temperature, respectively. The fractions have solubility with methanol, chloroform, and DMSO. The FTIR analysis revealed the differences between the positioning and organization of functional groups of various bioactive compounds present in chloroform fractions of both plants.

Similarly, a study conducted on similar group of microbes with medicinal plant extracts showed stimulated effect on the activity of antibiotics to inhibit the cytoplasmic targets [40, 41], while a similar study conducted by Baddley and Poppas on the fungal strains revealed the synergy of broader spectrum activity between plant extracts and antibiotics and a decreased risk of emergence of resistant strains [42]. They also stated that the synergistic effect shortens the total duration of therapy and decreases drug-related toxicities by allowing the use of lower doses. Hence, in the current system, isolating, identifying, and evaluating the promising bioactive phytoconstituents from the plant extracts become essential [43].

The present study showed that the combination of chloroform fractions of methanolic extract of W. somnifera and C. roseus with the antibiotics was more synergistic than being indifferent or antagonistic. The antibacterial combinations resulted in synergy that strongly inhibited the growth of the bacterial isolates. In accordance with our findings, Gaur et al. [44] have observed the synergistic effect of C. roseus against Xanthomonas, P. aeruginosa, and S. Typhi. In addition to this, Muddukrishnaiah and Singh [45] have reported the synergistic effect of W. somnifera against MDR strains of E. coli and S. aureus. The FTIR spectroscopy of the chloroform fractions of C. roseus and W. somnifera revealed the major independent peaks of probable functional groups of bioactive compounds present in the leaves of the plants.

In the present study, the major FTIR peaks were obtained at 3357 cm^-1, 2933 cm^-1, 2929 cm^-1, 2877 cm^-1, 1734 cm^-1, 1693 cm^-1, etc. which belong to the predominant functional groups, alcohols, carboxylic acids, alkanes, aldehydes, and alkyls. In accordance with our findings, Rajeev [46] obtained the FT-IR spectrum of the Withania somnifera (leaves) samples between 3320.29 cm^-1, 1652.83 cm^-1, 2945.67 cm^-1, 2834.64 cm^-1, 1449.39 cm^-1, 1113.62 cm^-1, 575.61 cm^-1, 1417.14 cm^-1, 1016.45 cm^-1, 755.15 cm^-1, 546.14 cm^-1, 534.78 cm^-1, and 510.12 cm^-1, respectively, which indicate the presence of flavonoids in the leaf extracts.

The present study gives a clue to develop new antimicrobials based on the combination of bioactive compounds and synthetic drugs, which can be used for the treatment of MDR strains and also to boost the immunity of the host. The study also highlighted the role of secondary metabolites and bioactive compound present in the plant leaves as the most effective antimicrobial constituents. The next necessary step in the study is to isolate these bioactive compounds from the fractions of medicinal plants.

5. Conclusion

The study suggested that to overcome the problems regarding the emergence of MDR strains, the plant derivatives as alternatives in combination to synthetic drugs can help in the development of much effective therapeutics to treat the MDR strains of S. Typhi and other bacteria. The present study also suggests that the combined treatment with these can enhance the property of the immune system to perform better action against the external pathogens as well as in the treatment of internal pathogens. The study also suggested that synergistic drugs may be the golden standard in the inhibition of protective biofilms of resistant bacteria. Further studies are needed with these herbs to isolate, characterize, and elucidate the structure of the bioactive compounds of the herbs which are responsible for the antimicrobial activity and other therapeutic value.

Data Availability

The data (figures and tables) used to support the findings of this study are included within the article as well as in the supplementary data.

Conflicts of Interest

All authors have none to declare.

Acknowledgments

We are thankful to Shoolini University for the support and facilities. We are highly grateful to the Department of Science and Technology (DST) for the funding under DST-INSPIRE fellowship (INSPIRE regd.140568).

Supplementary Materials

Table 1: antityphoid/antisalmonella assay of 25 plant extracts under study. Table 2: reactivity of different antigens O, H, and AH with the serum samples by slide agglutination test in both genders. Table 3: antibiogram profile of MDR S. Typhi isolates. Table 4: determination of MIC of resistant antimicrobials against S. Typhi isolates (). Table 5: yield of methanolic extracts of medicinal plants obtained from dried extracts. Table 6: synergistic assay of methanolic extracts against MDR S. Typhi isolates. Table 7: antisalmonella activity of the various fractions of Withania somnifera. Table 8: antisalmonella activity of the various fractions of Catharanthus roseus. Figure 1: antisalmonella effect of methanolic extract fractions (Ch = chloroform; ChB = basified chloroform; Bt = butanol; EA = ethyl acetate; Aq = aqueous) against MDR S. Typhi isolates. (Supplementary Materials)

References

E. Mweu and M. English, “Typhoid fever in children in Africa,” Tropical Medicine & International Health, vol. 13, no. 4, pp. 532–540, 2008.
View at: Publisher Site | Google Scholar
F. N. Qamar, A. Azmatullah, A. M. Kazi, E. Khan, and A. K. Zaidi, “A three-year review of antimicrobial resistance of Salmonella enterica serovars Typhi and Paratyphi A in Pakistan,” The Journal of Infection in Developing Countries, vol. 8, no. 8, pp. 981–986, 2014.
View at: Publisher Site | Google Scholar
M. M. Levine, J. E. Galen, C. O. Tacket, E. M. Barry, M. F. Pasetti, and M. B. Sztein, “Attenuated strains of Salmonella enterica serovar Typhi as live oral vaccines against typhoid fever,” in New Generation Vaccines, M. M. Levine, J. B. Kaper, R. Rappuoli, M. Liu, and M. Good, Eds., pp. 479–486, Marcel Dekker, New York, 3rd edition, 2004.
View at: Google Scholar
R. Wahid, R. Salerno-Gonçalves, C. O. Tacket, M. M. Levine, and M. B. Sztein, “Cell-mediated immune responses in humans after immunization with one or two doses of oral live attenuated typhoid vaccine CVD 909,” Vaccine, vol. 25, no. 8, pp. 1416–1425, 2007.
View at: Publisher Site | Google Scholar
C. O. Tacket, M. B. Sztein, S. S. Wasserman et al., “Phase 2 clinical trial of attenuated Salmonella enterica serovar typhi oral live vector vaccine CVD 908-htrA in U.S. volunteers,” Infection and Immunity, vol. 68, no. 3, pp. 1196–1201, 2000.
View at: Publisher Site | Google Scholar
M. Sztein, “Cell-mediated immunity and antibody responses elicited by attenuated Salmonella enterica serovar typhi strains used as live oral vaccines in humans,” Clinical Infectious Diseases, vol. 45, Supplement 1, pp. S15–S19, 2007.
View at: Publisher Site | Google Scholar
F. A. De Oliveira, A. P. Pasqualotto, W. P. da Silva, and E. C. Tondo, “Characterization of Salmonella Enteritidis isolated from human samples,” Food Research International, In press.
View at: Publisher Site | Google Scholar
C. F. Pui, W. C. Wong, L. C. Chai et al., “Salmonella: a food borne pathogen,” The International Food Research Journal, vol. 18, no. 2, pp. 465–473, 2011.
View at: Google Scholar
E. W. Hancock, “Mechanisms of action of newer antibiotics for Gram-positive pathogens,” Lancet Infectious Diseases, vol. 5, no. 4, pp. 209–218, 2005.
View at: Publisher Site | Google Scholar
K. M. Overbye and J. F. Barrett, “Antibiotics: Where did we go wrong?” Drug Discovery Today, vol. 10, no. 1, pp. 45–52, 2005.
View at: Publisher Site | Google Scholar
S. B. Levy and B. Marshall, “Antibacterial resistance worldwide: causes, challenges and responses,” Nature Medicine, vol. 10, no. S12, pp. S122–S129, 2004.
View at: Publisher Site | Google Scholar
S. R. Norrby, C. E. Nord, R. Finch, and European Society of Clinical Microbiology and Infectious Diseases, “Lack of development of new antimicrobial drugs: a potential serious threat to public health,” The Lancet Infectious Diseases, vol. 5, no. 2, pp. 115–119, 2005.
View at: Publisher Site | Google Scholar
O. Lomovskaya and K. A. Bostian, “Practical applications and feasibility of efflux pump inhibitors in the clinic—A vision for applied use,” Biochemical Pharmacology, vol. 71, no. 7, pp. 910–918, 2006.
View at: Publisher Site | Google Scholar
S. Koduru, D. S. Grierson, and A. J. Afolayan, “Ethnobotanical information of medicinal plants used for treatment of cancer in the Eastern Cape Province, South Africa,” Current Science, vol. 92, no. 7, pp. 906–908, 2007.
View at: Google Scholar
E. Garo, G. R. Eldridge, M. G. Goering et al., “Asiatic acid and corosolic acid enhance the susceptibility of Pseudomonas aeruginosa biofilms to tobramycin,” Antimicrobial Agents and Chemotherapy, vol. 51, no. 5, pp. 1813–1817, 2007.
View at: Publisher Site | Google Scholar
H. D. M. Coutinho, J. G. M. Costa, E. O. Lima, V. S. Falcão-Silva, and J. P. Siqueira-Júnior, “Enhancement of the Antibiotic Activity against a Multiresistant Escherichia coli by Mentha arvensis L. and Chlorpromazine,” Chemotherapy, vol. 54, no. 4, pp. 328–330, 2008.
View at: Publisher Site | Google Scholar
H. D. M. Coutinho, J. G. M. Costa, and E. O. Lima, “Herbal therapy associated with antibiotic therapy: potentiation of the antibiotic activity against methicillin-resistant Staphylococcus aureus by Turneraulmi folia L,” Journal of Alternative and Complementary Medicine, vol. 9, p. 35, 2009.
View at: Publisher Site | Google Scholar
Clinical and Laboratory Standards Institute, CLSI document M100-S25, Clinical and Laboratory Standards Institute, Wayne, PA, USA, 2015.
CLSI (formally NCCLS), “Clinical and Laboratory Standards Institute Standards Development Policies and Process,” CLSI, vol. 32, no. 3, 2012.
View at: Google Scholar
S. Mandal, M. DebMandal, N. K. Pal, and K. Saha, “Synergistic anti–Staphylococcus aureus activity of amoxicillin in combination with Emblica officinalis and Nymphae odorata extracts,” Asian Pacific Journal of Tropical Medicine, vol. 3, no. 9, pp. 711–714, 2010.
View at: Publisher Site | Google Scholar
O. O. Olajuyigbe and A. J. Afolayan, “Synergistic interactions of methanolic extract of Acacia mearnsii De Wild. with antibiotics against bacteria of clinical relevance,” International Journal of Molecular Sciences, vol. 13, no. 7, pp. 8915–8932, 2012.
View at: Publisher Site | Google Scholar
R. Rolta, A. Sharma, A. Sourirajan, P. K. Mallikarjunan, and K. Dev, “Combination between antibacterial and antifungal antibiotics with phytocompounds of Artemisia annua L: A strategy to control drug resistance pathogens,” Journal of Ethnopharmacology, vol. 266, article 113420, 2021.
View at: Publisher Site | Google Scholar
R. Rolta, M. Goyal, S. Sharma et al., “Bioassay guided fractionation of phytocompounds from Bergenia ligulata: a synergistic approach to treat drug resistant bacterial and fungal pathogens,” Pharmacological Research-Modern Chinese Medicine, vol. 3, article 100076, 2022.
View at: Publisher Site | Google Scholar
C. Gadhi, R. Hatier, F. Mory et al., “Bactericidal properties of the chloroform fraction from rhizomes of Aristolochia paucinervis Pomel,” Journal of Ethnopharmacology, vol. 75, no. 2-3, pp. 207–212, 2001.
View at: Publisher Site | Google Scholar
Z. P. Ruan, L. L. Zhang, and Y. M. Lin, “Evaluation of the antioxidant activity of Syzygium cumini leaves,” Molecules, vol. 13, no. 10, pp. 2545–2556, 2008.
View at: Publisher Site | Google Scholar
L. Nyquist, D. Bogard, H. Takeda, B. Bansal, H. Wiesmann, and C. Y. Shih, “Crystallization, recrystallization, and impact-metamorphic ages of eucrites Y792510 and Y791186,” Geochimica et Cosmochimica Acta, vol. 61, no. 10, pp. 2119–2138, 1997.
View at: Publisher Site | Google Scholar
A. K. Nigam and U. K. Gupta, Handbook on Analysis of Agriculture Experiments, IASRI Publication, 1979.
J. A. Crump, S. P. Luby, and E. D. Mintz, “The global burden of typhoid fever,” Bulletin of the World Health Organ, vol. 82, no. 5, pp. 346–353, 2004.
View at: Google Scholar
S. Arora, S. Dhillon, G. Rani, and A. Nagpal, “The in vitro antibacterial/synergistic activities of Withania somnifera extracts,” Fitoterapia, vol. 75, no. 3, pp. 385–388, 2004.
View at: Publisher Site | Google Scholar
D. A. Akinpelu, O. A. Aiyegoro, and A. I. Okoh, “In vitro antimicrobial and phytochemical properties of crude extract of stem bark of Afzelia africana (Smith),” African Journal of Biotechnology, vol. 7, no. 20, 2008.
View at: Google Scholar
Z. Guo, R. Xing, S. Liu et al., “The influence of molecular weight of quaternized chitosan on antifungal activity,” Carbohydrate Polymers, vol. 71, no. 4, pp. 694–697, 2008.
View at: Publisher Site | Google Scholar
M. I. Van der Kraan, J. Groenink, K. Nazmi, E. C. Veerman, J. G. Bolscher, and A. V. Amerongen, “Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin,” Peptides, vol. 25, no. 2, pp. 177–183, 2004.
View at: Publisher Site | Google Scholar
N. Nejat, A. Valdiani, D. Cahill, Y. H. Tan, M. Maziah, and R. Abiri, “Ornamental exterior versus therapeutic interior of madagascar periwinkle (Catharanthus roseus): the two faces of a versatile herb,” The Scientific World Journal, vol. 2015, Article ID 982412, 19 pages, 2015.
View at: Publisher Site | Google Scholar
N. R. Mustafa and R. Verpoorte, “Phenolic compounds in Catharanthus roseus,” Phytochemistry Reviews, vol. 6, no. 2-3, pp. 243–258, 2007.
View at: Publisher Site | Google Scholar
H. S. Muhammad and S. Muhammad, “The use of Lawsonia inermis Linn.(henna) in the management of burn wound infections,” African Journal of Biotechnology, vol. 4, no. 9, 2005.
View at: Google Scholar
H. Zhang, X. Xie, M. S. Kim, D. A. Kornyeyev, S. Holaday, and P. W. Paré, “Soil bacteria augment Arabidopsis photosynthesis by decreasing glucose sensing and abscisic acid levels in planta,” The Plant Journal, vol. 56, no. 2, pp. 264–273, 2008.
View at: Publisher Site | Google Scholar
C. Khare, Indian medicinal plants-an illustrated dictionary, Springer, New Delhi, India, 1st edition, 2007.
Anonymous, The Wealth of India. Vol. X (Sp-W), Publications and Information Directorate, Council of Scientific and Industrial Research (CSIR), New Delhi, 1982.
R. P. Rastogi and B. N. Mehrotra, Compendium of Indian Medicinal plants, vol. 5, Central Drug Research Institute Lucknow & NISC, New Delhi, 1998.
J. Reichling, P. Schnitzler, U. Suschke, and R. Saller, “Essential oils of aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties – an overview,” Complementary Medicine Research, vol. 16, no. 2, pp. 79–90, 2009.
View at: Publisher Site | Google Scholar
N. C. Silva and J. A. Fernandes, “Biological properties of medicinal plants: a review of their antimicrobial activity,” Journal of Venomous Animals and Toxins including Tropical Diseases, vol. 16, no. 3, pp. 402–413, 2010.
View at: Publisher Site | Google Scholar
J. W. Baddley and P. G. Poppas, “Antifungal combination therapy,” Drugs, vol. 65, no. 11, pp. 1461–1480, 2005.
View at: Publisher Site | Google Scholar
W. N. Setzer, J. M. Schmidt, J. A. Noletto, and B. Vogler, “Leaf oil compositions and bioactivities of abaco bush medicines,” Pharmacology Online, vol. 3, pp. 794–802, 2006.
View at: Google Scholar
A. Gaur, A. D. Bholay, and M. Ganeshan, “Antimicrobial activity of Catharatnhus roseus against human microbial pathogens,” Journal for Advanced Research in Applied Sciences, vol. 3, pp. 120–129, 2016.
View at: Google Scholar
K. Muddukrishnaiah and S. Singh, “Antimicrobial, synergistic activity and antioxidant studies on multidrug resistance human pathogen using crude extract of Azadirachta indica leaf and Withania somnifera rhizome,” Journal of Plant Pathology and Microbiology, vol. 6, pp. 1–3, 2015.
View at: Google Scholar
R. Nema, P. Jain, S. Khare, A. Pradhan, A. Gupta, and D. Singh, “Study of Withania somnifera with the spatial reference of phytochemical, FTIR and flavonoids quantification,” International Journal of Pharmacy and Life Sciences, vol. 3, no. 3, pp. 1530–1532, 2012.
View at: Google Scholar

Copyright

Copyright © 2023 Neha Chauhan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

701

Downloads

298

Citations