Antimicrobial Effects of Medicinal Plant Species on Salmonella typhimurium Strains Isolated from Poultry Feces Samples

authors:

avatar Mehdi Dehghani 1 , * , avatar Saeide Saeidi ORCID 2

Department of Biology, Faculty of Science, University of Zabol, Zabol, Iran
Biotechnology Research Institute, University of Zabol, Zabol, Iran
Jentashapir Journal of Cellular and Molecular Biology: Vol.14, issue 4; e135947
published online: November 19, 2023
article type: Research Article
received: February 25, 2023
revised: October 15, 2023
accepted: October 22, 2023
DOI: https://doi.org/10.5812/jjcmb-135947

how to cite: Dehghani M, Saeidi S. Antimicrobial Effects of Medicinal Plant Species on Salmonella typhimurium Strains Isolated from Poultry Feces Samples. Jentashapir J Cell Mol Biol. 2023;14(4):e135947. https://doi.org/10.5812/jjcmb-135947.

Abstract

Background:

It has been proven that plant extracts show great promise in fighting pathogenic microorganisms. This study aimed to evaluate the resistance of 20 strains of Salmonella typhimurium extracted from poultry feces against conventional antibiotics and the antibacterial activity of 10 medicinal plant extracts, including Hibiscus sabdariffa L., Capparis spinosa L., Azadirachta indica A. Juss., Eryngium planum L., Rumex acetosa L., Calotropis procera (Aiton) Dryand, Psidium guajava L., Malva sylvestris L., Urtica dioica L., and Alcea setosa Alef., against the extracted strains.

Methods:

The susceptibility of S. typhimurium strains against tested antibiotics was determined using disk diffusion, and the antibacterial activity of medicinal plant extracts was evaluated using well diffusion and broth microdilution assays.

Results:

The extracted S. typhimurium strains showed high resistance to cephalosporin (100%) and gentamicin (40%); however, all plant extracts examined in this study were influential in inhibiting the growth of the tested strains. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of tested plant extracts ranged from 6.25 to 25 mg/mL and 12.5 to 50 mg/mL, respectively. The most effective plant extracts in inhibiting bacterial growth in the agar well diffusion method were P. guajava, H. sabdariffa, and A. setosa; nevertheless, the most potent bactericidal activity was recorded for M. sylvestris and A. setosa in the broth microdilution method. The examined strains showed 80% and 85% sensitivity to the MBC of alcoholic extracts of M. sylvestris and A. setosa (50 mg/mL), respectively, which is worthy of further exploration by scientists.

Conclusions:

The results of this study represent the high potency of M. sylvestris and A. setosa extracts as appropriate medicinal and/or food supplements to replace ineffective antibiotics in bird breeding.

1. Background

Antibiotics are artificially or naturally synthesized organic substances that have been used for over 70 years in a wide variety of fields, including industrial production, agriculture, and medicine. Antibiotic use is the primary cause of the emergence of antibiotic-resistant microorganisms. Resistance to antibiotics is increasingly happening both in benign and pathogenic bacteria, giving rise to a growing global concern for humans, animals, and environmental health (1, 2). Antibiotic resistance is becoming more prevalent across various antibiotic classes, and some scholars argue that the threat and cost of antibiotic resistance are comparable to that of climate change (3). Global warming and antimicrobial resistance are closely intertwined; accordingly, rising temperatures raise the growth rate of bacteria and infections and horizontal gene transfer, which is a significant factor in the occurrence of antibiotic resistance (4-6).

Salmonella, named after the veterinarian Daniel Elmer Salmon, is a non-sporing Gram-negative bacillus genus in the family Enterobacteriaceae. Salmonella contains two species of Salmonella enterica and Salmonella bongori, with S. enterica being further divided into six additional subspecies and more than 2,600 serotypes (7). Salmonella species are abundantly represented in the environment and can cause a wide range of illnesses in both humans and animals. Generally, infection occurs via the ingestion of foods or water contaminated with the feces of infected humans or animals (7-9). The emergence of Salmonella serotypes resistant to multiple antibiotics is a major public health concern due to the remarkable food safety hazard that can happen (10). Salmonella is also able to form biofilms on a variety of biotic and abiotic surfaces, which might explain its survival in food production processes and clinical settings (11, 12).

Plant extracts and phytochemicals are considered promising new antimicrobial agents due to several reasons. They are cost-effective and readily available with no or negligible side effects in proper dosage. They also show great structural diversity and are less prone to produce antibiotic resistance than synthetic antibiotics. On the other hand, plant-based antimicrobial agents, unlike synthesized chemicals that pollute soil and water and move through ecological food chains, are environmentally friendly. Plants’ secondary metabolites generally kill the bacteria by disruption of the cell envelope, metabolism, or intracellular communication (12, 13).

2. Objectives

Considering the above-mentioned issues, this study aimed primarily to determine the antibiotic resistance of Salmonella typhimurium isolates extracted from poultry feces in the Zabol region, Sistan and Baluchestan province, Iran, and evaluate the in vitro antimicrobial properties of 10 medicinal plant species on the S. typhimurium isolates.

3. Methods

3.1. Plant Sample Preparation

Plant organs of Hibiscus sabdariffa L., Capparis spinosa L., Azadirachta indica A. Juss., Eryngium planum L., Rumex acetosa L., Calotropis procera (Aiton) Dryand, Psidium guajava L., Malva sylvestris L., Urtica dioica L., and Alcea setosa Alef. were collected in 2021 from the Baqiyatallah Al-Azam Educational and Recreational Complex, belonging to the University of Zabol, Zabol, Iran (Table 1). The plant species were identified by the first author, a botanist at the Department of Biology of the University of Zabol, using regional and online floras. Additionally, the voucher specimens of each species were deposited at the University of Zabol Herbarium, Zabol, Iran. The plant materials were dried in a shade away from direct light before converting to a fine powder using an electric mill. The scientific, English, and local names, distribution ranges, and some characteristics of the studied plant species are presented in Table 1.

Table 1.

Characteristics of Investigated Medicinal Plants, Including Names, Habitats, Distribution, and Parts Used in This Study

No.TaxaFamilyHabitatLocal NameEnglish NameDistributionPart Used
1Hibiscus sabdariffa L.MalvaceaeAnnual, up to 2 m tallChaye TorshRoselleNative to tropical AfricaSepals
2Capparis spinosa L.CapparaceaeShrubs, prostrate or hanging, up to 100 cmKabarCaperS. Europe eastward to AustraliaFruit
3Azadirachta indica A. Juss.MeliaceaeTree, up to 15 m tallDerakhte AzadNeemNative to the Indian subcontinentFlower, Fruit, Leaf
4Eryngium planum L.ApiaceaePerennial to 1 mShishaghBlue eryngoEurope and Central AsiaLeaf
5Rumex acetosa L.PolygonaceaePerennial up to 120 cm highTorshakSorrelEurasiaLeaf
6Calotropis procera (Aiton) Dryand.ApocynaceaeSubshrubs to 2 m or more highEstabraghSodom appleNative to dry tropical Asia and AfricaFlower
7Psidium guajava L.MyrtaceaeTreeGovavaCommon guavaNative to the Caribbean, Central, and South AmericaFruit
8Malva sylvestris L.MalvaceaeBiannual up to 1.5 mPanirakCommon mallowEurasia and AfricaLeaf
9Urtica dioica L.UrticaceaePerennial herb 50-150 cmGazanehCommon nettleEurope, Asia, and North AfricaLeaf
10Alcea setosa Alef.MalvaceaePerennial up to 2 mKhatmiBristly hollyhockWestern AsiaFlower

3.2. Preparation of Plant Extracts and Salmonella typhimurium Strains

In this study, 10 grams of each plant powder was soaked in 100 mL of 96% ethanol and mixed with a shaker machine (Azma Pars, Iran) at a speed of 130 rpm for 24 hours at room temperature. The obtained solution was filtered through Whatman No. 2 filter paper, and the filtrate was dried using a rotary device (Heidolph, Germany) and a vacuum pump (distillation in a vacuum) and stored in the dark at 4°C until needed for experiments. The dried extract was then dissolved in dimethylsulfoxide (DMSO) (10%) to prepare a stock solution at a concentration of 200 mg/mL from each plant extract, from which desired concentrations were made (14). In the Agar well diffusion method, the extract concentration of 50 mg/mL was used. For calculating minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), the extract concentration in the first row of the 96 well-plate was 200 mg/mL, which was subsequently diluted serially into 100, 50, 25, 12.5, 6.25, and finally 3.125 mg/mL in the last row. Additionally, 20 pure Salmonella typhimurium strains, isolated from fresh poultry feces from Zabol, were obtained from the microbiology section in the Central Laboratory of the University of Zabol.

3.3. Determining the Sensitivity of Bacterial Strains to Conventional Antibiotics

The sensitivity of bacterial strains to tetracycline, gentamicin, cephalosporin, and ciprofloxacin antibiotics was evaluated using the disk diffusion assay following the instructions of Bauer (15). Bacterial suspensions equivalent to a 0.5 McFarland turbidity were made from all bacterial strains in Mueller Hinton broth liquid medium and cultured on Muller Hinton’s agar medium. Antibiotic disks were precisely placed at proper distances from one another on the surface of the previously inoculated cultures, followed by incubation for 24 hours at 37ºC. The inhibition zones around the discs were measured to determine the resistance and sensitivity of 20 strains of S. typhimurium to the applied antibiotics. The absence of inhibition zones around the discs indicated complete resistance of the bacterial strain to the corresponding antibiotic.

3.4. Antibacterial Activity of Plant Extracts Against Salmonella typhimurium Assay

The sensitivity of bacterial isolates to the plant extracts was determined using the broth microdilution method with the help of 96-well microplates. An amount of 10 microliters of 0.5 McFarland microbial suspension was added to Mueller Hinton broth nutrient liquid medium (MHB) and incubated at 37°C for 24 hours. Bacterial growth or inhibition was determined by the visual evaluation of turbidity, and the lowest concentration of the plant extract that inhibited the bacterial growth was considered the MIC. For the determination of MBC, 10 μL of the content of each clear well was transferred to Mueller Hinton agar medium and incubated for 24 hours at 37°C. The plant extract concentration (corresponding well) at which 99.9% of the bacteria were eliminated was regarded as the MBC.

3.5. Agar Well Diffusion Method

The entire surface of the Mueller Hinton agar culture medium was inoculated by 50 microliters of 0.5 McFarland concentration of bacterial suspension. Wells of 5 × 4 mm were created on the medium, and 50 μL of each extract solution (the concentration of 50 mg/mL) was added to the wells (16). Then, the plates were kept at 37°C for 24 hours, and the inhibition zone of the extracts was measured.

3.6. Statistical Analyses

All tests were performed in three repetitions. The data obtained from the agar well diffusion method were analyzed statistically using two-way analysis of variance (ANOVA) and Tukey post hoc tests at a significance level of 0.05 via SPSS statistical software (version 16).

4. Results

The in vitro evaluation of the efficacy of tested antibiotics in S. typhimurium strains showed the highest antibiotic resistance to cephalosporin (100%), followed by gentamicin (40%), tetracycline (20%), and ciprofloxacin (5%) respectively; however, the most sensitivity was observed to ciprofloxacin (90%) and tetracycline (70%) (Table 2).

Table 2.

Percentage of Sensitivity and Resistance of Salmonella typhimurium Strains to Tested Antibiotics

Resistance Level TetracyclineGentamicinCephalosporinCiprofloxacin
Sensitive7035090
Intermediate102505
Resistant20401005

The two-way ANOVA analysis indicated that different plant species showed a significant impact on the inhibition zone diameter (f = 5.760, P = 0.000); nevertheless, the bacterial strains and species bacterial interaction revealed no significant differences (P = 0.378 and P = 0.078, respectively). To look for differences between the groups, a Tukey post hoc test was run with a total alpha of 0.05. The pairwise comparisons revealed significant differences between P. guajava and all other species except for A. setosa. Additionally, A. setosa was significantly effective in inhibiting bacterial growth in comparison to C. spinose (P = 0.003). There were no significant differences among other species based on the pairwise comparisons using the Tukey post hoc test.

The results of the antibacterial assay of ethanolic extracts of tested plant species on 20 isolated strains of S. typhimurium based on the agar well diffusion method are summarized in Table 3. All alcoholic plant extracts were more or less influential in inhibiting the growth of the tested strains except for C. procera, which was ineffective on strains number 1 and 3. However, the plant extracts affected different strains inconsistently. The best result was obtained with A. setosa on strain number 5 (13 mm) and H. sabdariffa on strain number 10 (10 mm). The most effective plant extract in inhibiting bacterial growth was P. guajava (Table 3).

Table 3.

Average Diameter of Inhibition Zone (mm) and Corresponding Standard Deviation of Ethanolic Extracts of Investigated Medicinal Plants Against 20 Strains of Salmonella typhimurium Based on the Agar Well Diffusion Method

StrainH. sabdariffaC. spinoseA. indica flowerA. indica leafA. indica fruitE. planumR. acetosaC. procera.P. guajavaM. sylvestrisU. dioicaA. setosa
15 ± 0.71 ± 0.21 ± 0.12 ± 0.41 ± 0.13 ± 0.54 ± 0.50 ± 0.04 ± 0.61 ± 0.21 ± 0.15 ± 0.7
24 ± 0.52 ± 0.33 ± 0.42 ± 0.21 ± 0.32 ± 0.31 ± 0.17 ± 0.65 ± 0.51 ± 0.13 ± 0.43 ± 0.4
38 ± 0.53 ± 0.33 ± 0.41 ± 0.31 ± 0.32 ± 0.35 ± 0.70 ± 0.06 ± 0.53 ± 0.43 ± 0.44 ± 0.6
43 ± 0.61 ± 0.13 ± 0.41 ± 0.31 ± 0.11 ± 0.36 ± 0.55 ± 0.78 ± 0.62 ± 0.23 ± 0.55 ± 0.7
52 ± 0.21 ± 0.23 ± 0.41 ± 0.31 ± 0.13 ± 0.46 ± 0.64 ± 0.47 ± 0.61 ± 0.12 ± 0.313 ± 1.1
62 ± 0.21 ± 0.21 ± 0.33 ± 0.61 ± 0.24 ± 0.62 ± 0.51 ± 0.15 ± 0.51 ± 0.21 ± 0.27 ± 0.8
73 ± 0.61 ± 0.31 ± 0.31 ± 0.11 ± 0.21 ± 0.33 ± 0.61 ± 0.26 ± 0.41 ± 0.22 ± 0.41 ± 0.2
86 ± 0.61 ± 0.23 ± 0.41 ± 0.31 ± 0.12 ± 0.32 ± 0.31 ± 0.15 ± 0.723 ± 0.54 ± 0.6
97 ± 0.61 ± 0.13 ± 0.42 ± 0.31 ± 0.34 ± 0.65 ± 0.71 ± 0.26 ± 0.63 ± 0.62 ± 0.31 ± 0.1
1010 ± 1.11 ± 0.35 ± 0.71 ± 0.21 ± 0.12 ± 0.32 ± 0.31 ± 0.18 ± 0.65 ± 0.62 ± 0.34 ± 0.5
115 ± 0.71 ± 0.13 ± 0.51 ± 0.21 ± 0.21 ± 0.22 ± 0.23 ± 0.55 ± 0.71 ± 0.11 ± 0.31 ± 0.1
123 ± 0.61 ± 0.21 ± 0.32 ± 0.41 ± 0.32 ± 0.51 ± 0.31 ± 0.37 ± 0.72 ± 0.52 ± 0.24 ± 0.5
135 ± 0.62 ± 0.22 ± 0.32 ± 0.22 ± 0.42 ± 0.22 ± 0.21 ± 0.37 ± 0.63 ± 0.61 ± 0.36 ± 0.5
146 ± 0.51 ± 0.31 ± 0.31 ± 0.32 ± 0.42 ± 0.21 ± 0.32 ± 0.45 ± 0.52 ± 0.35 ± 0.51 ± 0.3
152 ± 0.53 ± 0.63 ± 0.61 ± 0.35 ± 0.71 ± 0.23 ± 0.51 ± 0.28 ± 0.62 ± 0.24 ± 0.43 ± 0.6
161 ± 0.31 ± 0.34 ± 0.65 ± 0.51 ± 0.31 ± 0.22 ± 0.41 ± 0.27 ± 0.52 ± 0.31 ± 0.14 ± 0.4
173 ± 0.54 ± 0.41 ± 0.24 ± 0.52 ± 0.23 ± 0.61 ± 0.31 ± 0.25 ± 0.61 ± 0.11 ± 0.11 ± 0.1
185 ± 0.62 ± 0.21 ± 0.23 ± 0.61 ± 0.34 ± 0.42 ± 0.41 ± 0.23 ± 0.63 ± 0.34 ± 0.64 ± 0.4
195 ± 0.61 ± 0.25 ± 0.65 ± 0.61 ± 0.31 ± 0.31 ± 0.32 ± 0.44 ± 0.54 ± 0.62 ± 0.33 ± 0.6
202 ± 0.21 ± 0.22 ± 0.25 ± 0.51 ± 0.32 ± 0.21 ± 0.31 ± 0.12 ± 0.42 ± 0.33 ± 0.61 ± 0.1

The MIC and MBC of the ethanolic extracts of 10 investigated medicinal plants in 20 isolated strains of S. typhimurium from poultry feces are shown in Table 4. The results showed that the lowest and highest inhibitory concentrations of H. sabdariffa L. were 12.5 and 100 mg/mL, respectively, where one strain of S. typhimurium (strains number 9 and 19) was inhibited by the ethanolic extract of the flowers.

Table 4.

Minimum Inhibitory Concentration (MIC, mg/mL) and Minimum Bactericidal Concentration (MBC, mg/mL) of Ethanolic Extracts of 10 Medicinal Plants Against 20 Salmonella typhimurium Strains Showing Quantitative Antimicrobial Activity

H. sabdariffaC. spinoseA. indica FlowerA. indica LeafA. indica FruitE. planumR. acetosaC. proceraP. guajavaM. sylvestrisU. dioicaA. setosa
MICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBCMICMBC
150100255025-5050255025502550100200501002550255025502550
250100255025-505050100501002550100200255012.52525506.2512.52550
350100501005010050100255025505010025502550255025502550
4501005010025-5050501005010050100501005010050100501005010050100
5501005010025-50502550501002550100200501005010025501002002550
650100255025-505025505010012.5255010012.5252550255025502550
7501005010050-10010025502550255010020025502550255025502550
82550255050100501002550255050100255050100255025502550
9100200255050100255025502550100200255050100255025502550
10501005010050100255050100255050100501002550501005010050100
112550501005010025505010010020010020050100255050100501002550
12255050100501005010025502550501002550255025505010050100
13255025505010012.52550100100200255012.52512.5252550501002550
14255025506.2512.525502550255050100255012.5252550501002550
1525505010012.52525502550255050100255012.525255025502550
162550501002550255012.5252550255012.52512.525255025502550
1725502550501006.2512.5255012.525255025502550255025502550
182550100200501002550255012.52512.525255012.525255025502550
1912.525501005010025505010025502550255012.5252550501002550
202550255050100255050100255025502550ngNg5010025502550

Abbreviations: MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration.

The lowest and highest inhibitory concentrations of C. spinosa L. were at 25 and 100 mg/mL, inhibiting 9 and 1 strains in these concentrations, respectively. The lowest inhibitory concentration of A. indica flower extract was 6.25 mg/mL, which inhibited one strain growth; nevertheless, its highest inhibitory concentration was 50 mg/mL, in which 12 strains were inhibited. The lowest and highest inhibitory concentrations of A. indica leaf extract were recorded at 6.25 and 50 mg/mL, where 1 and 5 strains were inhibited, respectively. On the other hand, the lowest and highest inhibitory concentrations of fruit extract of A. indica were observed to be 12.5 and 50 mg/mL, and 1 and 9 strains were inhibited consecutively (Table 4). The E. planum leaf extract showed the lowest and highest inhibitory concentrations of 12.5 and 100 mg/mL, in which 3 strains were inhibited. The lowest and highest inhibitory concentrations of R. acetosa were also shown to be 12.5 and 100 mg/mL, and the growth of 3 and 6 strains was inhibited accordingly (Table 4).

The lowest and the highest inhibitory concentrations of the alcoholic extracts of both C. procera and P. guajava were 12.5 and 50 mg/mL; nonetheless, the first species inhibited the growth of 3 and 5, and the second species inhibited 4 and 7 strains serially (Table 4). Table 4 also shows that the lowest and highest inhibitory concentrations were 25 and 50 mg/mL for M. sylvestris and A. setosa, respectively, where the first species inhibited the growth of 16 and 4 strains, and the latter inhibited the growth of 18 and 2 strains successionally. U. dioica in concentrations of 100 and 6.25 mg/mL exhibited the highest and lowest inhibitory concentrations and inhibited the growth of 1 strain each (Table 4).

The lowest MBC of the alcoholic extracts of all examined species was 12.5 mg/mL for A. indica flower and leaf and U. dioica, and only one strain was inactivated in each case. The lowest MBC of 25 mg/mL was observed in H. sabdariffa, A. indica fruit, E. planum, R. acetosa, C. procera, and P. guajava, in which 1, 1, 2, 1, and 3 strains were destroyed serially. The highest MBC among all tested species was observed to be 50 mg/mL in C. spinose, M. sylvestris, and A. setosa, where 9, 16, and 17 strains were eliminated, respectively.

5. Discussion

The results of this study showed that the 20 isolated strains of S. typhimurium from the poultry feces of Zabol chickens are 100% and 40% resistant to cephalosporin and gentamicin, respectively. Cephalosporin and gentamicin are widely used to treat human and animal bacterial infections worldwide. In line with the current study’s results, there are several reports on Salmonella resistance to these vital antibiotics (17-20). However, high sensitivity to antibiotics was expected due to the traditional way of breeding poultry and the limited use of drugs and antibiotics in this region. It is recommended to use ciprofloxacin as the best option against S. typhimurium infection among the other tested antibiotics. The susceptibility of S. typhimurium isolates to ciprofloxacin has already been reported (21).

Despite the absolute resistance of S. typhimurium strains against cephalosporin and relatively high resistance against tetracycline, almost all tested strains were inhibited by the ethanolic extracts of examined medicinal plants. The most effective plant extracts in inhibiting Salmonella growth in the disk diffusion method were those of P. guajava and A. setosa. The lowest MIC of the alcoholic extracts of tested medicinal plants varies from 6.25 (U. dioica and A. indica flower and leaf extracts) to 25 mg/mL (C. spinosa, M. sylvestris, and A. setosa); nevertheless, the lowest MBC ranged from 12.5 (U. dioica and A. indica flower and leaf extracts) to 50 mg/mL (C. spinose, M. sylvestris, and A. setosa). Although A. setosa and M. sylvestris showed higher MIC and MBC than some other examined plants in this study (A. indica, P. guajava, H. sabdariffa, E. planum, R. acetosa, U. dioica, and C. procera), they manifested the best efficacy against various strains with different levels of drug resistance (Table 4). A. setosa and M. sylvestris were capable of eliminating 16 and 17 out of 20 S. typhimurium strains, respectively, at concentration of 50 mg/mL. Then, the tested S. typhimurium strains were 80% and 85% sensitive to alcoholic extracts of M. sylvestris and A. setosa, respectively, which candidate them as appropriate medicinal and/or food supplements in bird breeding in the Zabol region.

Numerous studies have been conducted to discover effective medicinal plants on Salmonella species and strains and their mechanism of action. The effectiveness of P. guajava leaf extract was shown against the clinical isolates of S. Typhi with a much higher zone of inhibition (15 mm) than the results of this study. The reported MIC and MBC were also much lower than the present study’s results (3.13 and 6.25, respectively) (22). These results validate the traditional use of P. guajava as anti-diarrheal and anti-typhoid fever in tropical countries (23, 24). A. indica also showed a broader inhibition zone (11 mm), lower MIC (1.56), and higher MBC (25) in comparison to the results of this study (22).

The H. sabdariffa calyx extract efficacy against Salmonella strains in this study coincides with previous studies in which H. sabdariffa calyx extracts exhibited antimicrobial activity against 13 multidrug-resistant Salmonella strains extracted from raw carrots (25). In addition, acetone extract and hibiscus acid extracted from H. sabdariffa calyces exhibited potent antimicrobial activity against multidrug-resistant Salmonella strains (26). In another study, H. sabdariffa ethanolic extract was employed successfully as a natural preservative to extend the shelf-life of beef by removing foodborne bacteria (27). However, the ethanolic leaf extract of H. sabdariffa was reported to be ineffective against the clinical isolates of S. typhi (28).

The potent antibacterial potential of M. sylvestris, as witnessed in the present study, agrees with the reports on it against various bacteria, including Salmonella (29, 30). Moreover, the MIC/MBC of M. sylvestris extract against the standard and clinically isolated Salmonella enterica from diarrheic lambs in Urmia, Iran, were reported to be 50/100 and 42/80 mg/mL, respectively (31). Additionally, M. sylvestris contains various chemical ingredients, such as carbohydrates, tannins, flavonoids, phenolic compounds, and ascorbic acid, denoting its multiple pharmaceutical properties. Additionally, Malvone (a phytoalexin) is found in M. sylvestris with a potent antimicrobial effect and might be a candidate for its prominent action against Salmonella (32, 33).

To the best of our knowledge, there is no scientific report on the effects of A. setosa on Salmonella in the literature. However, contrary to the present study’s results, weak to moderate antioxidant potential and no significant antimicrobial for A. setosa have been reported (34, 35). On the other hand, the chemical composition of the methanolic extract of Alcea setosa from Jordan showed 290 compounds, among which flavonoids (flavones) were diversified (34). Phenolic compounds, including flavonoids, exhibit various biological activities and might explain the potent anti-Salmonella effect of this species.

5.1. Conclusions

The current study showed that bacterial resistance to conventional antibiotics is expanding even in regions with low antibiotic consumption. Moreover, the tested medicinal plant extracts revealed effective antimicrobial properties against resistant Salmonella strains, with M. sylvestris and A. setosa as the most active bactericide extracts at a concentration of 50 mg/mL. The alcoholic extracts of these two Malvaceae species are remarkably more effective than tetracycline, gentamicin, and cephalosporin and almost as potent as ciprofloxacin against Salmonella strains extracted from poultry feces. Due to the growing ineffectiveness of antibiotics against infectious diseases, the introduction of new antibiotics or complementary agents with fewer risks (e.g., drug resistance, allergies, and cancers) is of high necessity. It is recommended that M. sylvestris and A. setosa extracts containing useful antimicrobial agents be used not only as treatment or preventive supplements in poultry food but also to combat the present health challenge due to the antimicrobial resistance of foodborne pathogens. However, the results obtained in laboratory conditions should be redone and confirmed in vivo to evaluate the possible toxicity, side effects, or adverse reactions with foods or animals.

Acknowledgements

References

  • 1.

    Knapp CW, Dolfing J, Ehlert PA, Graham DW. Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Environ Sci Technol. 2010;44(2):580-7. [PubMed ID: 20025282]. https://doi.org/10.1021/es901221x.

  • 2.

    Kuppusamy S, Kakarla D, Venkateswarlu K, Megharaj M, Yoon Y, Lee YB. Veterinary antibiotics (VAs) contamination as a global agro-ecological issue: A critical view. Agric Ecosyst Environ. 2018;257:47-59. https://doi.org/10.1016/j.agee.2018.01.026.

  • 3.

    Rodriguez-Verdugo A, Lozano-Huntelman N, Cruz-Loya M, Savage V, Yeh P. Compounding Effects of Climate Warming and Antibiotic Resistance. iScience. 2020;23(4):101024. [PubMed ID: 32299057]. [PubMed Central ID: PMC7160571]. https://doi.org/10.1016/j.isci.2020.101024.

  • 4.

    Burnham JP. Climate change and antibiotic resistance: a deadly combination. Ther Adv Infect Dis. 2021;8:2049936121991370. [PubMed ID: 33643652]. [PubMed Central ID: PMC7890742]. https://doi.org/10.1177/2049936121991374.

  • 5.

    Pietikainen J, Pettersson M, Baath E. Comparison of temperature effects on soil respiration and bacterial and fungal growth rates. FEMS Microbiol Ecol. 2005;52(1):49-58. [PubMed ID: 16329892]. https://doi.org/10.1016/j.femsec.2004.10.002.

  • 6.

    Philipsborn R, Ahmed SM, Brosi BJ, Levy K. Climatic Drivers of Diarrheagenic Escherichia coli Incidence: A Systematic Review and Meta-analysis. J Infect Dis. 2016;214(1):6-15. [PubMed ID: 26931446]. [PubMed Central ID: PMC4907410]. https://doi.org/10.1093/infdis/jiw081.

  • 7.

    Gal-Mor O, Boyle EC, Grassl GA. Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front Microbiol. 2014;5:391. [PubMed ID: 25136336]. [PubMed Central ID: PMC4120697]. https://doi.org/10.3389/fmicb.2014.00391.

  • 8.

    Ryan MP, O'Dwyer J, Adley CC. Evaluation of the Complex Nomenclature of the Clinically and Veterinary Significant Pathogen Salmonella. Biomed Res Int. 2017;2017:3782182. [PubMed ID: 28540296]. [PubMed Central ID: PMC5429938]. https://doi.org/10.1155/2017/3782182.

  • 9.

    Silva C, Calva E, Maloy S. One Health and Food-Borne Disease: Salmonella Transmission between Humans, Animals, and Plants. Microbiol Spectr. 2014;2(1):OH-20-2013. [PubMed ID: 26082128]. https://doi.org/10.1128/microbiolspec.OH-0020-2013.

  • 10.

    V. T. Nair D, Venkitanarayanan K, Kollanoor Johny A. Antibiotic-Resistant Salmonella in the Food Supply and the Potential Role of Antibiotic Alternatives for Control. Foods. 2018;7(10). [PubMed ID: 30314348]. [PubMed Central ID: PMC6210005]. https://doi.org/10.3390/foods7100167.

  • 11.

    Merino L, Procura F, Trejo FM, Bueno DJ, Golowczyc MA. Biofilm formation by Salmonella sp. in the poultry industry: Detection, control and eradication strategies. Food Res Int. 2019;119:530-40. [PubMed ID: 30884686]. https://doi.org/10.1016/j.foodres.2017.11.024.

  • 12.

    Sakarikou C, Kostoglou D, Simoes M, Giaouris E. Exploitation of plant extracts and phytochemicals against resistant Salmonella spp. in biofilms. Food Res Int. 2020;128:108806. [PubMed ID: 31955766]. https://doi.org/10.1016/j.foodres.2019.108806.

  • 13.

    Tian M, He X, Feng Y, Wang W, Chen H, Gong M, et al. Pollution by Antibiotics and Antimicrobial Resistance in LiveStock and Poultry Manure in China, and Countermeasures. Antibiotics (Basel). 2021;10(5). [PubMed ID: 34066587]. [PubMed Central ID: PMC8148549]. https://doi.org/10.3390/antibiotics10050539.

  • 14.

    Ebrahimnezhad Z, Dehghani M, Beyzaei H. Assessment of Phenolic and Flavonoid Contents, Antioxidant Properties, and Antimicrobial Activities of Stocksia Brahuica Benth. Int J Basic Sci Med. 2022;7(1):34-40. https://doi.org/10.34172/ijbsm.2022.07.

  • 15.

    Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1966;45(4):493-6. [PubMed ID: 5325707].

  • 16.

    Manandhar S, Luitel S, Dahal RK. In Vitro Antimicrobial Activity of Some Medicinal Plants against Human Pathogenic Bacteria. J Trop Med. 2019;2019:1895340. [PubMed ID: 31065287]. [PubMed Central ID: PMC6466868]. https://doi.org/10.1155/2019/1895340.

  • 17.

    Wong MH, Kan B, Chan EW, Yan M, Chen S. IncI1 Plasmids Carrying Various blaCTX-M Genes Contribute to Ceftriaxone Resistance in Salmonella enterica Serovar Enteritidis in China. Antimicrob Agents Chemother. 2016;60(2):982-9. [PubMed ID: 26643327]. [PubMed Central ID: PMC4750657]. https://doi.org/10.1128/AAC.02746-15.

  • 18.

    Goncuoglu M, Ormanci FB, Uludag M, Cil GI. Prevalence and Antibiotic Resistance of Salmonella SPP. and Salmonella Typhimurium in Broiler Carcasses Wings and Liver. J Food Safety. 2016;36(4):524-31. https://doi.org/10.1111/jfs.12272.

  • 19.

    Ferrari RG, Rosario DKA, Cunha-Neto A, Mano SB, Figueiredo EES, Conte-Junior CA. Worldwide Epidemiology of Salmonella Serovars in Animal-Based Foods: a Meta-analysis. Appl Environ Microbiol. 2019;85(14). [PubMed ID: 31053586]. [PubMed Central ID: PMC6606869]. https://doi.org/10.1128/AEM.00591-19.

  • 20.

    Lee LA, Puhr ND, Maloney EK, Bean NH, Tauxe RV. Increase in antimicrobial-resistant Salmonella infections in the United States, 1989-1990. J Infect Dis. 1994;170(1):128-34. [PubMed ID: 8014487]. https://doi.org/10.1093/infdis/170.1.128.

  • 21.

    Wang X, Biswas S, Paudyal N, Pan H, Li X, Fang W, et al. Antibiotic Resistance in Salmonella Typhimurium Isolates Recovered From the Food Chain Through National Antimicrobial Resistance Monitoring System Between 1996 and 2016. Front Microbiol. 2019;10:985. [PubMed ID: 31134024]. [PubMed Central ID: PMC6514237]. https://doi.org/10.3389/fmicb.2019.00985.

  • 22.

    Olubunmi OS, B OLW, Olusoji OA, Tamilore A, Temitope OD, Adewale A. In vitro and in vivo Activities of Psidium guajava and Azadirachta indica Leaf Extracts and solvent Fractions against Salmonella Typhi. European J Med Plants. 2021:56-71. https://doi.org/10.9734/ejmp/2021/v32i730405.

  • 23.

    Adetutu A, Olaniyi TD, Owoade OA. GC-MS analysis and in silico assessment of constituents of Psidium guajava leaf extract against DNA gyrase of Salmonella enterica serovar Typhi. Inform Med Unlocked. 2021;26:100722. https://doi.org/10.1016/j.imu.2021.100722.

  • 24.

    Lutterodt GD, Ismail A, Basheer RH, Baharudin HM. Antimicrobial effects of psidium guajava extract as one mechanism of its antidiarrhoeal action. Malays J Med Sci. 1999;6(2):17-20. [PubMed ID: 22589684]. [PubMed Central ID: PMC3329747].

  • 25.

    Gutiérrez‐Alcántara EJ, Gómez‐Aldapa CA, Román‐Gutiérrez AD, Rangel‐Vargas E, González‐Olivares LG, Castro‐Rosas J. Antimicrobial Activity of Roselle Hibiscus Sabdariffa Calyx Extracts on Culture Media and Carrots Against Multidrug‐Resistant Salmonella Strains Isolated from Raw Carrots. J Food Safety. 2016;36(4):450-8. https://doi.org/10.1111/jfs.12259.

  • 26.

    Portillo-Torres LA, Bernardino-Nicanor A, Gomez-Aldapa CA, Gonzalez-Montiel S, Rangel-Vargas E, Villagomez-Ibarra JR, et al. Hibiscus Acid and Chromatographic Fractions from Hibiscus Sabdariffa Calyces: Antimicrobial Activity against Multidrug-Resistant Pathogenic Bacteria. Antibiotics (Basel). 2019;8(4). [PubMed ID: 31718033]. [PubMed Central ID: PMC6963829]. https://doi.org/10.3390/antibiotics8040218.

  • 27.

    Marquez-Rodriguez AS, Nevarez-Baca S, Lerma-Hernandez JC, Hernandez-Ochoa LR, Nevarez-Moorillon GV, Gutierrez-Mendez N, et al. In Vitro Antibacterial Activity of Hibiscus sabdariffa L. Phenolic Extract and Its In Situ Application on Shelf-Life of Beef Meat. Foods. 2020;9(8). [PubMed ID: 32784385]. [PubMed Central ID: PMC7464790]. https://doi.org/10.3390/foods9081080.

  • 28.

    Arogbodo JO, Faluyi OB, Igbe FO. In vitro Antimicrobial Activity of Ethanolic Leaf Extracts of Hibiscus Asper Hook. F. and Hibiscus Sabdariffa L. on some Pathogenic Bacteria. J Sci Res Med Biol Sci. 2021;2(3):1-12. https://doi.org/10.47631/jsrmbs.v2i3.304.

  • 29.

    Mohammed HJ. Screening of Antibacterial Properties for Some Iraqi Plants Against Salmonella typhimurium. Iraqi J Vet Med. 2011;35(2):28-35. https://doi.org/10.30539/iraqijvm.v35i2.572.

  • 30.

    Shadid KA, Shakya AK, Naik RR, Jaradat N, Farah HS, Shalan N, et al. Phenolic Content and Antioxidant and Antimicrobial Activities of Malva sylvestris L., Malva oxyloba Boiss., Malva parviflora L., and Malva aegyptia L. Leaves Extract. J Chem. 2021;2021:1-10. https://doi.org/10.1155/2021/8867400.

  • 31.

    Nozohour Y, Jalilzadeh G. Antibacterial Activities of Ethanolic Extract of Malva sylvestris L. Against Salmonella enterica and Escherichia coli Isolated from Diarrheic Lambs. Iran J Med Microbiol. 2021;15(1):121-9. https://doi.org/10.30699/ijmm.15.1.121.

  • 32.

    Gasparetto JC, Martins CA, Hayashi SS, Otuky MF, Pontarolo R. Ethnobotanical and scientific aspects of Malva sylvestris L.: a millennial herbal medicine. J Pharm Pharmacol. 2012;64(2):172-89. [PubMed ID: 22221093]. https://doi.org/10.1111/j.2042-7158.2011.01383.x.

  • 33.

    Mousavi SM, Hashemi SA, Behbudi G, Mazraedoost S, Omidifar N, Gholami A, et al. A Review on Health Benefits of Malva sylvestris L. Nutritional Compounds for Metabolites, Antioxidants, and Anti-Inflammatory, Anticancer, and Antimicrobial Applications. Evid Based Complement Alternat Med. 2021;2021:5548404. [PubMed ID: 34434245]. [PubMed Central ID: PMC8382527]. https://doi.org/10.1155/2021/5548404.

  • 34.

    Bajes HR, Oran SA, Bustanji YK. Chemical Composition and Antiproliferative and Antioxidant Activities of Methanolic Extract of Alcea setosa A. Malvaceae. Res J Pharm Technol. 2021:6447-54. https://doi.org/10.52711/0974-360x.2021.01115.

  • 35.

    Elbitar H, Alhage J. In vitro screening for antioxidant and antimicrobial properties of three lebanese medicinal plants crude extracts. Pharmacognosy Res. 2019;11(2):127. https://doi.org/10.4103/pr.pr_171_18.