INTRODUCTION:

Gram-negative bacteria (GNB) infections including urinary tract, respiratory tract, bloodstream infections and surgical site or wound have been reported to increase significantly worldwide in recent years, they are major contributors to morbidity, mortality, and escalating healthcare costs in patients admitted to intensive care units.^1,2 Over recent years, they have become endemic in many tertiary healthcare units and hospitals.³Multidrug-resistant Gram-negative bacteria (MDR-GNB) are microorganisms that have acquired resistance to broad-spectrum antibacterial agents, for instance, third-generation cephalosporins, through a variety of mechanisms.⁴ They have dramatically increased in all parts of the world committed to the excessive use of antibiotics including the use without treatment indication causing an amplified risk of treatment failure for serious infections and high medical costs.^5,6 The mechanisms of the antimicrobial resistance in GNB may be divided into four categories: drug inactivation; drug uptake limitation; drug efflux and drug target modification. Owing to the structural differences and others, Gram-negative bacteria can use all four mechanisms, while Gram-positive bacteria are less likely to use limiting the uptake of a drug (the lipopolysaccharide in the outer membrane is absent) and drug efflux mechanisms. ⁷ However, plants are a source of a wide range of bioactive substances including, among others, alkaloids, flavonoids, terpenoids, phenolics, and essential oils that possess various therapeutic properties, such as antibacterial, antioxidant and immunomodulatory actions which could be an effective alternative source for many therapeutics. Therefore, they are continuously explored to develop novel drugs.^8,9,10 Polygonum aviculare is a plant with a global distribution, commonly found in temperate regions. Its various parts exhibit antifungal, antibacterial, antioxidant, anthelmintic, antidiabetic, and anticancer activities, and are also used in the treatment of kidney disorders and dermatological problemes.^11,12 Zygophyllum album L. is a shrubby plant that belongs to the Zygophyllaceae. It includes approximately 27 genera and 285 species, which are frequently restricted to arid and semiarid regions.¹³ The plant is used as a diuretic, antihistaminic, local anesthetic, antidiabetic, antihyperlipidemic, antispasmolytic, antidiarrheal, carminative, anti-inflammatory, antiseptic and stimulant agent for the treatment of skin diseases, diarrhea and typhoid.^14,15

MATERIAL AND METHODS:

Collection of plant material:

P. aviculare and Z. album were collected from Khenchela and Biskra (Algeria) respectively in December 2023. The taxonomic identification of the plant material was confirmed by Dr: Zeraib A, department of Agronomy, Abbes Laghrour University, Khenchela (Algeria).

Extraction method:

P. aviculare and Z. album samples (20g) were macerated with 70% aqueous-methanol (MeOH) and 70% aqueous-ethanol (EtOH) (200Ml) separately for 72 hours at room temperature. The extracts were filtered and concentrated with a vacuum evaporator at 40°C. The crude extracts were collected and allowed to completely dry at room temperature.¹⁶The obtained extracts were stored in the refrigerator until use.

Determination of Total Phenol Content:

The total soluble phenolic compounds in the different plant extracts were determined with Folin-Ciocalteu reagent using gallic acid as a standard. Extracts were diluted to the concentration of 1mg/Ml in methanol and 0.5 Ml of the resulting solution extract was mixed with 2.5 Ml of Folin Ciocalteu reagent and 2 Ml of Na₂HCO₃ (7.5%). After 15min at 45°C, the absorbance was measured at 765nm.¹⁷

Determination of Total flavonoid content:

The total flavonoid content of plants extracts was performed by the Aluminum Chloride colorimetric method. A volume of 1 Ml of 2% AlCl₃ ethanol solution was mixed with 1 Ml of sample solution (1mg/Ml). After incubation for 10 min at room temperature, the absorbance was measured with a spectrophotometer at 415nm. Quercetin was used as a standard for the calibration curve.¹⁸

Microorganisms:

The test organisms included 3 Gram-negative reference strains: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 700603 and 04 MDR-GN strains isolated from urine samples of persons suffering from urinary-tract infections. The isolates were identified by standard microbiological methods.¹⁹ Their susceptibilities were tested using the Muller Hinton Agar containing a range of antibiotics. Selected multidrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli 1, and Escherichia coli 2 were screened for their high level of antibiotic resistance following the disk diffusion method as per CLSI guidelines.²⁰

The resistance patterns of selected Gram-negative bacteria are given as following:

E. coli 1

· Resistant to: Amoxicillin, Amoxicillin+ Clavulanic acid, Ticarcillin, Piperacillin, Cefazolin, Cefotaxime, Gentamicin, Nalidixic acid and Trimethoprim/ Sulfamethoxazole.

· Intermediate to Ciprofloxacin.

E. coli 2

· Resistant to: Amoxicillin, Amoxicillin+ Clavulanic acid, Ticarcillin, Piperacillin and Cefazolin.

K. pneumoniae

· Resistant to: Amoxicillin, Amoxicillin+ Clavulanic acid, Ticarcillin, Piperacillin, Cefazolin, Cefotaxime and Trimethoprim/ Sulfamethoxazole.

P. aeruginosa

· Resistant to: Imipenem, Gentamicin, Amikacin, Ciprofloxacin, Chloramphenicol and trimethoprim+ sulfamethoxazole.

Antibacterial Assay:

The disc diffusion method for antibacterial susceptibility testing was carried out to investigate the potential antibacterial activity of the plant extracts against selected strains. Each bacteria was inoculated into 0.9% sterile saline solution, adjusted to 0.5 Mc Farland standard and used to lawn Muller Hinton agar plates evenly. A stock solution of extracts was prepared by dissolving 0.1g and 0.2g of extract with 1 Ml of DMSO to produce final concentrations of 100mg/Ml and 200mg/Ml, then 10𝜇L of each concentration was impregnated into a sterilized disc (Whatman, N°1, 06 mm in diameter). DMSO was used as a negative control. Finally, the plates were incubated at 37°C for 24 hours. The inhibition zones were measured in millimeters.²¹

Determination of minimum inhibitory concentration:

Double-fold dilution was made to get seven different concentrations of the plant extracts (200, 100, 50, 25, 12.5, 6.25, and 3.125mg/Ml). 100µl of each standardized suspension of the test organisms were inoculated into a series of sterile tubes containing 5Ml of nutrient broth. Thereafter, 0.1Ml of the prepared successive two-fold serial dilutions of the plant extracts concentrations were added and incubated at 37°C for 24 hours. The MICs were read as the least concentration that inhibited any visible growth (absence of turbidity) of the test organisms.²²

Statistical analysis:

for the antibacterial activity, total phenol content and flavonoid content, the experiments were conducted in triplicate. The results were expressed as an arithmetic mean±standard deviation using Excel (2016).

Antibacterial and CMI assay:

The disk diffusion method for antimicrobial susceptibility testing was performed to determine the antibacterial activity of Z. album and P. aviculare extracts against selected MDR-GNB. The results showed that the plant extracts exhibited antibacterial activity at varying levels against multidrug-resistant (MDR) bacteria. (Table 1). Ethanol extract of P. aviculare recorded activity against the test pathogens with inhibition zones in the range of 9.66 -10.66 mm against MDR-GNB and 10-11.66 mm against E. coli ATCC 25922 and K. pneumoniae ATCC 700603 while methanol extract exhibited high activity against E. coli ATCC 25922, K. pneumoniae ATCC 700603 and P. aeruginosa ATCC 27853 with inhibition zones varying between 15.33 to 19.00 mm and 7.00 to 9.66 mm against MDR-GNB at the concentrations of 200 mg/Ml. However, methanol extract of Z. album was shown to have activity against E. coli 1, P. aeruginosa, E. coli ATCC 25922 and K. pneumoniae ATCC 70060327853 with inhibition zones within the range 9.66 -10.66 mm while E. coli 2, K. pneumoniae and P. aeruginosa ATCC 27853 were resistant. Dissimilarly, ethanol extract exhibited activity against, E. coli 1 and P. aeruginosa with inhibition zones corresponding to 9.00 and 9.66 mm respectively. The data obtained through MIC revealed that the MIC values of different plants extracts were found in the range of 3.125‑200 mg/Ml (Figure 01).

Table 1. Antibacterial activity of different plants extracts (diameter of inhibition zone in mm*) (Means ± SD).

Bacterial strain	Concentration	*Z. album*		*P. aviculare*		DMSO
Bacterial strain	Concentration	MeOH extract	EtOH extract	MeOH extract	EtOH extract
*E. coli* 1	200mg/Ml	10.33±1.15	9.00±00	7.00±00	10.66±0.47	00.00
*E. coli* 1	100mg/Ml	8.00±1.00	9.00±00	7.00±00	10.33±0.47	00.00
*E. coli* 2	200mg/Ml	00.00	8.00±00	8.00±00	9.66±0.47	00.00
*E. coli* 2	100mg/Ml	00.00	00.00	7.00±00	10.33±0.47	00.00
*K. pneumoniae*	200mg/Ml	7.00±00	00.00	9.66±0.94	9.66±0.47	00.00
*K. pneumoniae*	100mg/Ml	7.00±00	00.00	9.66±0.94	9.00±0.81	00.00
*P. aeruginosa*	200mg/Ml	10.66±0.94	9.66±0.47	14.33±0.47	9.00±00	00.00
*P. aeruginosa*	100mg/Ml	10.33±1.88	9.00±00	14.33±1.24	8.00±00	00.00
*E. coli* ATCC 25922	200mg/Ml	9.66±0.94	00.00	15.33±2.05	10.00±0.81	00.00
*E. coli* ATCC 25922	100mg/Ml	7.33±0.47	00.00	11.33±1.24	9.33±1.24	00.00
*K. pneumoniae* ATCC 700603	200mg/Ml	9.66±1.69	00.00	19.00±1.47	11.66±0.47	00.00
*K. pneumoniae* ATCC 700603	100mg/Ml	8.66±0.47	00.00	11.33±1.69	9.66±1.24	00.00
*P. aeruginosa* ATCC 27853	200mg/ml	8.00±00	7.00±00	19.00±1.47	00.00	00.00
*P. aeruginosa* ATCC 27853	100mg/ml	7.00±00	7.00±00	15.00±0.81	00.00	00.00

* Strains were classified as: not sensitive with (diameter <8 mm), sensitive (diameter of 9–14 mm), very sensitive (diameter of 15–19 mm) and extremely sensitive (diameter >20 mm).²³

Table 2. Total phenolic and total flavonoid contents of Z. album and P. aviculare extracts

	Total phenolic content (µg GAE/mg)		Total flavonoid content (µg QE/mg)
Categories	Z. album	P. aviculare	Z. album	P. aviculare
Methanol extract	19.55±1.79	161.99±11.93	19.14±6.11	30.87±3.76
Ethanol extract	25.40±2.39	158.73±8.36	21.40±0.98	46.43±5.82

Figure 1: Minimal inhibitory concentrations (MIC) of plants extracts against selected strains bacteria

Total Phenolic and total flavonoid contents:

Total phenolic content (TPC) and total flavonoid contents (TFC) in different extracts of Z. album and P. aviculare are presented in Table 2. Our results showed that the highest phenolic contents were found in the methanol and ethanol extracts of P. aviculare (161.99±11.93 and 158.73±8.36µg GAE/mg, respectively), whereas the methanol and ethanol extracts of Z. album contained considerably lower phenolic contents (19.55±1.79 and 25.40±2.39µg GAE/mg, respectively). The TFC values followed a similar trend to the TPC values. The highest TFC values were observed in the P. aviculare extracts (30.87±3.76µg QE/mg for methanol extract and 46.43±5.82µg QE/mg for ethanol extract), while the lowest TFC values were found in the Z. album extracts (19.14±6.11µg QE/mg for methanol extract and 21.40±0.98µg QE/mg for ethanol extract)

DISCUSSION:

The search for substances with high antimicrobial activity has been one of the most intensive areas of research in order to attenuate the risk of infectious diseases caused by bacteria, viruses, parasites and fungi. Plant extracts are still considering as new resources of therapeutic agents including antimicrobial agents that could act as alternatives to antibiotics in the treatment of antibiotic-resistant bacteria.^24,25Numerous plants used in traditional medicine are effective in treating various ailments caused by bacterial infections^26,27,28,29. Research has shown that medicinal plants exhibit antimicrobial activity against bacteria associated with several diseases: skin infections,³⁰ dental caries,³¹urinary-tract infections,³² diarrhea³³ and respiratory infections.³⁴ In this study, the antibacterial activities of both tested medicinal plants against tested bacteria showed different results of inhibition zones with pronounced antibacterial activities of plant extracts observed. These results are consistent with previous studies indicating that acetone, ethanol, chloroform, and aqueous extracts of P. aviculare from Egypt were active against both Gram-positive and Gram-negative bacteria.³⁵The same study revealed that chloroform extract showed excellent antimicrobial activity against all tested bacteria and good activity against all tested fungi except Candida albicans. Furthermore, a previous study showed that the polar fractions (methanol and acetone extracts) of P. aviculare exhibited activity against S. aureus ATCC 25923, with MIC 0.625mg/mL.³⁶ However, Z. album extracts showed antibacterial activity against E. coli 1, P. aeuginosa, E. coli ATCC 25922 and K. pneumoniae ATCC 700603 with inhibitory zones varying between 9-10.66mm. Belmimoun et al.³⁷ reported that Z. album methanolic extract was most effective on the different strains than essential oil and exhibited a stronger antibacterial activity against E. coli and Bacillus subtilis. Moreover, the essential oil of Z. album from Tunisia showed significant anti-bacterial activity especially against Gram (+) bacteria.¹³These variations in the sensitivity of the bacterial species tested on the extracts might be due to the differences in the strains used in the research and local environmental factors that affect the potency of medicinal plants, such as temperature, rainfall, day length, and soil characteristics. Saturated organic or aromatic compounds are often obtained through initial methanol or ethanol extraction.³⁸ Moreover, the variations in the sensitivity of the bacterial strains tested between p. aviculare and Z. album extracts can be explained by the content of polyphenolic and flavonoid compounds in each plant which, since many studies have reported their proven antimicrobial activity through multiple mechanisms, including blocking bacterial DNA replication and energy metabolism, modifying bacterial membrane function, decreasing cell attachment and biofilm formation, inhibiting porin protein on cell membranes modifying permeability, and interacting with other agents.^{39,40,41,42,43}

CONCLUSION:

In the present work, we investigated the total phenol content, total flavonoid content, and the antimicrobial activity of P. aviculare and Z. album extracts from Algeria against MDR-GNB. The results of this study showed that P. aviculare and Z. album extracts exhibit significant antibacterial activity. Although the studied plants have shown that they are potentially rich in antibacterial compounds; these findings underscore the requirement of the investigation of plants to substitute antibiotics for treatment of infections.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS:

The authors are grateful to Mr Hamidi Athir-Eddine for providing bacterial strains.

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Received on 31.03.2024 Revised on 23.07.2024

Accepted on 25.10.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):339-344.

DOI: 10.52711/0974-360X.2025.00053

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