Potentiation of Antibacterial Activity of Cefixime in Synergy with Cirsium arvense (L.) Scop. Against Resistant Bacterial Isolates ^†

Abstract

Antibiotic resistance is a major health priority, with the concern over antibiotic resistance growing. The rise in antibiotic-resistant bacteria coupled with the limited new therapeutics puts pressure on community and hospital healthcare systems and leads to excessive morbidity and mortality. Given this, there has been significant interest into potential new and/or combination antibacterial treatments including certain plant extracts. When combined with resistance medicines, these extracts can re-sensitize their potency. One such plant is Cirsium arvense which has been traditionally used for its anti-inflammatory and antimicrobial properties. Preliminary findings suggest that its extracts may enhance the activity of conventional antibiotics against resistant strains like MRSA. The current study assessed cefixime’s and Cirsium arvense extracts’ synergistic antibacterial efficacy against Escherichia coli, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial activity of extracts and the susceptibility profile of antibiotics were assessed using the disc diffusion and microbroth dilution assays. Chequerboard, time-kill kinetics, and protein content tests were performed to verify synergistic antibacterial effect. Our results demonstrated that when these extracts were applied to clinical strains of bacteria along with the cefixime, there was complete or partial synergy was displayed. Time-kill kinetics demonstrated that synergism was dependent on both concentration and time, and bacterial isolates treated with these exhibited significantly reduced bacterial growth and protein content. Taken together, these results show that Cirsium arvense extracts enhance the efficacy of conventional antibiotics against antibiotic-resistant strains of Escherichia coli, Acinetobacter baumannii, and MRSA and clearly suggest that these plant extracts could be used as additives to current resistant antibiotics for the future management or treatment of resistant bacterial infections.

1. Introduction

Antimicrobial resistance (AMR) has emerged as a significant global health challenge. The growing prevalence of antibiotic-resistant bacteria, along with the slow pace of new drug development, is placing substantial strain on healthcare systems worldwide. AMR is undermining the effective treatment of common bacterial infections, from mild illnesses like pharyngitis to severe diseases such as meningitis, pneumonia, and tuberculosis. As resistance spreads, both first-line and second-line antibiotics are losing their effectiveness, making these infections progressively harder to control. The World Health Organization has recognized AMR as one of the top ten global public health threats facing humanity, and recent studies continue to report alarming rates of resistance in both community and hospital settings [1]. While antibiotics such as fluoroquinolones, macrolides, cephalosporins, and penicillin have revolutionized medicine [1], their overuse and misuse have accelerated the emergence of resistance [2].

Bacteria such as methicillin resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and multidrug-resistant Mycobacterium tuberculosis (MDR-TB), Escherichia coli, and Acinetobacter now pose major treatment challenges [2]. It is estimated that approximately 70% of hospital- or community-acquired bacterial pathogens exhibit resistance to at least one commonly used antibiotic, posing a major challenge to effective clinical management of infectious diseases [3]. The Centers for Disease Control and Prevention (CDC) reports around 2.8 million cases of antibiotic-resistant infections annually in the U.S., resulting in 35,000 deaths [4], while in Asia, antibiotic resistance is estimated to cause 1 child death every nine minutes [5]. This growing threat has led global health bodies such as the European Antimicrobial Resistance Surveillance Network (EARS-Net) to track AMR trends by species and region [6].

Bacterial resistance can be intrinsic or acquired, enabling microbes to evade previously effective treatments through diverse mechanisms such as enzymatic degradation (e.g., β-lactamases), altered drug targets, reduced membrane permeability, or overexpression of efflux pumps [7,8]. Given this, there is an urgent need to enhance the efficacy of existing antibiotics or discover new agents capable of treating resistant infections.

One promising avenue in combating bacterial infections is the use of plant-derived phytochemicals, which are rich in antimicrobial compounds and have been traditionally employed for centuries in ethnomedicine. Recent studies have reaffirmed the antibacterial efficacy of several medicinal plants, including Curcuma longa, Allium sativum, Berberis lyceum, and Artemisia absinthium, highlighting their potential as alternative or adjunct therapies against resistant pathogens [9]. Active phytochemicals such as alkaloids, polyphenols, volatile oils, and tannins exhibit antimicrobial properties by disrupting bacterial cell membranes, inhibiting enzymes, and modulating host immune responses—making them promising resistance-modifying agents [10,11,12]. However, despite growing evidence of their antimicrobial efficacy, the synergistic effects of many traditional medicinal plants when combined with conventional antibiotics remain underexplored—particularly against multidrug-resistant pathogens such as MRSA.

Among these, Cirsium arvense—commonly known as creeping thistle-has gained increasing attention in recent years due to its documented antioxidant, anti-inflammatory, immunomodulatory, and antimicrobial properties [13,14,15,16,17,18]. Its bioactive compounds may be capable of restoring antibiotic potency when used in combination therapies, but this has not been fully explored.

Given the above, in the current study, we aimed to assess if the combination of antibiotics and crude plant extracts of Cirsium arvense could be antimicrobial towards resistant clinical isolates of Escherichia coli, Acinetobacter baumannii, and MRS. We treated antibiotic -resistant strains of E. coli, A. baumanii, and MRSA with isolated extracts from C. arvense and then assessed bactericidal and bacteriostatic effects. This was done with or without cefixime. Our findings revealed a notable synergistic effect between C. arvense extracts and cefixime, significantly inhibiting the growth of cefixime-resistant strains. These results highlight the potential of C. arvense as a valuable source of bioactive compounds with antibacterial properties that could help make current resistant antibiotics usable again.

2. Materials and Methods

2.1. Chemicals

Ethyl acetate (EA), methanol (M), myricetin, quercetin, syringic acid, gallic acid, rutin, catechin, gentisic acid, cinnamic acid, kaempferol, apigenin, and coumarin were purchased from Merck (Darmstadt, Germany). Cefixime, ciprofloxacin dimethyl sulfoxide (DMSO), nutritional broth, foetal bovine serum, nutrient agar, and Coomassie brilliant blue were purchased from Sigma-Aldrich (Darmstadt, Germany).

2.2. Plant Extraction

Fresh leaves and stems of C. arvense free of disease were collected from Lasdanna, Bagh Azad Kashmir. Plant species were identified and verified by Prof. Mushtaq Ahmad, Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, Pakistan. A verified specimen was kept on file at the herbarium of medicinal plants under voucher number PHM-578. After collection, plant material was thoroughly washed with distilled water to remove dust and debris, then air-dried under shade at room temperature for several days. The powdered plant material was subjected to maceration using three analytical-grade solvents—ethyl acetate (EA), methanol (M), and distilled water (aqueous, Aq)—ranging from non-polar to polar. A solvent-to-powder ratio of 4:1 was maintained, and the mixture was allowed to stand at 25 °C for 72 h. During this period, the samples were sonicated for 10 min daily to enhance extraction efficiency. After the extraction period, the mixtures were filtered, and the resulting filtrates were concentrated under reduced pressure using a rotary evaporator (Bibby, Stone, Staffordshire, UK) set at 45 °C. The dried extracts were then transferred into labelled containers and stored at −80 °C until further analysis.

Percent Extract Recovery

The percentage recovery obtained from the dried extract was calculated using the following formula. Extract recovery (%) = C/D × 100

D = weight (g) of powdered plant.

C = weight (g) of dried extract.

2.3. Phytochemical Analysis

Quantification of Polyphenols

Polyphenols in crude extracts were quantified by RP-HPLC as outlined previously [19], using an Agilent Chem Station and a Zorbex C8 analytical column ChemStation Rev. B.03.01 (4.6 × 250 nm and 5 µm particle size) equipped with a DAD detector (Agilent Technologies, Waldbronn, Germany). Mobile phases A and B contained: methanol/water/acetic acid in 5:10:85:1 and 40:60:0:1 ratios, respectively. The mobile phases were run at a flow rate of 1 mL/min. The mobile phase was eluted at different gradients. In terms of mobile phase B, the concentration was changed from 0 to 50% B over a period of 0–20 min. This was followed by a gradient of 50–100% B for; 20–25 min, and lastly, 100% B was run for 25–30 min. Extracts (20 μL; 10 mg/mL) were filtered using 0.45 µm membrane filters and injected into the column with a 10 min reconditioning phase between the two samples. Phenolic standards including vanilliv acid, rutin, gallic acid, catechin, syringic acid, apocyanin, coumaric acid, gentisitc acid, apigenin, myricetin, quercetin, and kaempferol were prepared in concentrations of 10, 20, 50, 100, and 200 μg/mL in methanol. The UV absorption spectra of the samples were recorded at 368 nm (myricetin, kaempferol, and quercetin), 325 nm (gentisic acid, apigenin, and caffeic acid), 279 nm (coumaric acid, catechin, and gallic acid), and 257 nm (rutin and vanillic acid). Polyphenols were quantified as μg/mg of sample from the calibration curve. They were calculated using the formula 3.3 × (σ/b), where σ and b stand for the standard deviation of the response and the slope of the calibration curve, respectively.

2.4. Antimicrobial Assessment

2.4.1. Cultures

Clinically isolated strains of resistant Gram-positive S. aureus (S.A.) (MIC-104) and Gram-negative E. coli (MIC-102) and Acinetobacter (ATCC-19606) bacteria were provided by the Armed Forces Institute of Pathology (AFIP), Combined Military Hospital (CMH).

2.4.2. Primary Resistance Characterization of Antibiotics by DISC Diffusion Method

The disc diffusion method was used to examine the resistance profiles of five antibiotics—ciprofloxacin, lincomycin, clarithromycin, doxycycline, and cefixime—against resistant clinical isolates. Turbidity was confirmed according to the McFarland 0.5 turbidity standard after the bacterial culture was renewed by incubating the inoculum at 37 °C for 24 h.

Bacterial cultures were swabbed onto sterile nutrient agar plates. Nutrient agar plates were covered with discs soaked in 5 µL of antibiotic solution (4 mg/mL in DMSO), which were then incubated for 24 h at 37 °C. Each disc’s zone of inhibition (ZOI) was measured using a vernier calliper. The antibiotics chosen for additional testing were those that demonstrated <14 or no ZOI against the majority of isolates, indicating resistance.

2.5. Antibacterial Activity of Crude Extracts

2.5.1. Disc Diffusion Method

The antibacterial activity of crude extracts of selected plants was evaluated by the disc diffusion method. Extract (5 µL in 20 mg/mL) soaked filter paper discs were placed onto sterile agar plates for 24 h at 37 °C and the ZOI was measured. DMSO and ciprofloxacin (4 mg/mL) served as positive and negative controls, respectively.

2.5.2. Microbroth Dilution Method

Based on the result from the disc diffusion assay, extracts exhibiting ZOI ≥ 12 mm were further screened to find the minimum inhibitory concentration (MIC) by the microbroth dilution method [19]. The 12 mm threshold was chosen as a practical indicator of moderate-to-strong antibacterial activity, in line with the published literature [19] where ZOI values ≥ 12 mm are often considered biologically significant for crude plant extracts. Bacterial inoculums (5 × 10⁴ CFU/mL) were put into each well of a 96-well plate containing twofold serial dilutions of extracts (100, 50, 25, and 12.5 μg/mL) and antibiotics (10, 5, 2.25, and 1.125 µg/mL). A zero-hour reading was taken by measuring the absorbance (600 nm) after 30 min of incubation. Later, the plate was incubated for 24 h at 37 °C, and absorbance was recorded again.

2.6. Assessment of Synergism Between Test Extracts and Antibiotics

The checkerboard technique was used to assess the synergistic activity of C. arvense extracts and antibiotics as previously described [19]. The experiment was carried out in 96-well microtiter plates. Cefixime and plant extracts were prepared in two dilutions: 2.5 µL of each dilution was placed vertically and 2.5 µL of each cefixime dilution was added horizontally, resulting in a fixed amount of the first sample and a decreased amount of the second in each row or column. Bacterial inoculum with5 × 10⁴ CFU/mL was added to the wells and the plates were subjected to incubation at 37 °C for a duration of 24 h and a combinational MIC, defined as the lowest concentration of combination that resulted in no visible growth. The interaction between the plant extract and the antibiotic was assessed using the fractional inhibitory concentration index (FICI), calculated as follows:

Furthermore, absorbance was measured at 0 and 24 h for additional calculations.

$${\displaystyle \raisebox{1ex}{${\mathit{M}\mathit{I}\mathit{C}}_{\mathit{A}/\mathit{B}}$}\!\left/ \!\raisebox{-1ex}{${\mathit{M}\mathit{I}\mathit{C}}_{\mathit{A}}$}\right.}\mathbf{+}{\displaystyle \frac{{\mathit{M}\mathit{I}\mathit{C}}_{\mathit{B}/\mathit{A}}}{{\mathit{M}\mathit{I}\mathit{C}}_{\mathit{B}}}}$$

(1)

where

MICA = MIC of extract;

MICB = MIC of antibiotic;

MICIA/B = MIC of extract in combination with antibiotic;

MICB/A = MIC of antibiotic in combination with extract.

2.7. Time-Kill Kinetics

Time-kill kinetics was evaluated as described previously [20]. All resistant bacterial isolates were grown and diluted to 4 × 10⁴ CFU/mL into mid-logarithmic phase. Then, the diluted bacterial suspension was incubated at 37 °C with MIC, 2MIC, FICI, and 2FICI concentrations of the test extract alone and in combination with cefixime. Growth was measured by absorbance at the time intervals of 0, 3, 6, 9, 12, 24, and 48 h.

2.8. Estimation of Bacterial Protein

The protein content of the bacterial samples was quantified by the Bradford method after treatment with extracts alone or in combination with cefixime to assess the possible mechanism of bacterial growth inhibition [21]. Bacterial inoculum was treated with MIC, 2MIC, FICI, and 2FICI values of the extract alone or in combination with cefixime and incubated at 37 °C for 24 h. Each sample was centrifuged for 5 min at 1006× g, and the bacterial pellet was separated and placed at −4 °C for 48 h. Phosphate buffer, BSA (1 mg/mL; 0–50 µg/mL), distilled water, and inoculum were used as diluent, positive control, negative control, and blank, respectively.

A bacterial pellet of all isolates was washed three times with phosphate buffer, suspended in 20 µL of the buffer, and sonicated for 10 min. An aliquot of 5 µL of suspension and 195 µL of the Bradford reagent (1:4) were added into a 96-well plate. The plate was incubated at room temperature for 5 min with continuous sonication, and absorbance was measured. The protein content of all samples was determined from the calibration curve.

2.9. Statistical Analysis

Data are presented as mean ± SEM (n = 3) of the respective values. All statistical tests are listed in the figure legends, and significance was determined as p < 0.05.

3. Results

3.1. Extract Recovery Varies with Solvent Polarity

Three solvents, ranging from polar to nonpolar, were used to prepare crude extracts of C. arvense. The percentage of extract recovery was determined by weighing the dried crude extracts obtained after solvent evaporation. The aqueous (Aq) extract had the highest percentage of extract recovery (7.33% w/w), which progressively decreased as solvent polarity decreased. The phytoconstituents from C. arvense were extracted using the M, EA, and water solvents, yielding values of 7.33, 4.86, and 6.33%, respectively, of the 450 g of dry plant used for extraction (

Table 1. Percentage extract recovery of Cirsium arvense.

Extract Name	Extract Weight (g)	Extract Recovery (w/w%)
CAL-(M)	14.5 ± 0.03	4.86 ± 0.05
CAL-(EA)	20 ± 0.05	6.33 ± 0.03
CAL-(Aq)	22 ± 0.06	7.33 ± 0.02

Note: CAL(M) = methanolic leaf extract of Cirsium arvense, CAL (EA) = ethyl acetate leaf extract of Cirsium arvense, CAL (Aq) = aqueous leaf extract of Cirsium arvense.

).

3.2. Aqueous Extract Exhibits Highest Total Flavonoid Content

We next wanted to quantify the total flavonoid content (TFC) in Cirsium arvense extracts to better understand the distribution of these bioactive compounds across different solvents. To isolate the flavonoids, dried extracts obtained from maceration with various solvents were first dissolved in their respective solvents. The total flavonoid content was then measured using the aluminium chloride colorimetric method. A standard calibration curve was generated using quercetin (y = 0.0649x − 0.043, R² = 0.9927), and the absorbance was recorded at 415 nm. The TFC was expressed as milligrams of quercetin equivalents per gram of extract (mg QE/g). As shown in

Table 2. Total flavonoid content determination of C. arvense extracts.

Extract Name	(µg QE/mg of Extract) ± S.D.
CAL-(M)	0.2 ± 0.00
CAL-(EA)	0.4 ± 0.00
CAL-(Aq)	3.1 ± 0.01

Note: CAL(M) = methanolic leaf extract of Cirsium arvense, CAL (EA) = ethyl acetate leaf extract of Cirsium arvense, CAL (Aq) = aqueous leaf extract of Cirsium arvense.

, the aqueous extract exhibited the highest flavonoid content, indicating that polar solvents—particularly water—are more effective in extracting flavonoids from C. arvense.

3.3. Aqueous Extract Contains Highest Total Phenolic Content

Next, we determined the total phenolic content of the extracts using a similar method as above. The total phenolic content (TPC) of Cirsium arvense extracts was determined using a calibration curve (y = 0.0915x − 0.098, R² = 0.9939), and the results were expressed as µg gallic acid equivalents per mg of extract (µg GAE/mgE). As presented in

Table 3. Total phenolic content determination of C. arvense extracts.

Extract Name	(µg GAE/mg of Extract) ± S.D.
CAL-(M)	3.5 ± 0.01
CAL-(EA)	13.7 ± 0.02
CAL-(Aq)	14.3 ± 0.01

Note: CAL(M) = methanolic leaf extract of Cirsium arvense, CAL (EA) = ethyl acetate leaf extract of Cirsium arvense, CAL (Aq) = aqueous leaf extract of Cirsium arvense.

, the aqueous (Aq) extract showed the highest phenolic content, followed by the ethyl acetate (EA) extract, with the methanolic (M) extract having the lowest. This trend reinforces the efficiency of polar solvents—particularly water—in extracting phenolic compounds from C. arvense.

3.4. RP-HPLC Reveals Apigenin, Catechin, and Rutin as Major Polyphenols in C. arvense Extracts

Next, we aimed to identify and quantify specific polyphenolic compounds present in Cirsium arvense extracts to gain deeper insight into the phytochemical constituents that may contribute to its antibacterial activity. To do this, we employed reverse-phase high-performance liquid chromatography with diode array detection (RP-HPLC-DAD). Extracts were first reconstituted in appropriate solvents and filtered before injection into the HPLC system. The identification of compounds was achieved by comparing the retention times and UV absorption spectra of peaks in the sample chromatograms with those of twelve authenticated polyphenolic standards. As detailed in

Table 4. RP-HPLC-based quantification of polyphenols (µg/mg of extract) in C. arvense.

Standard Polyphenols	Wavelength	CAL-(EA)	CAL-(M)	CAL-(Aq)
Vanillic acid	257	0.11 ± 0.01	0.10 ± 0.01	0.04 ± 0.00
Rutin		1.33 ± 0.02	0.42 ± 0.02	0.23 ± 0.03
Gallic acid	279	ND	ND	0.02 ± 0.00
Catechin		ND	2.09 ± 0.02	1.81 ± 0.003
Syringic acid		0.10 ± 0.01	ND	ND
Apocyanin		0.02 ± 0.00	0.06 ± 0.00	0.03 ± 0.00
Coumaric acid		0.02 ± 0.00	0.02 ± 0.00	0.01 ± 0.00
Gentisic acid	325	0.04 ± 0.01	0.05 ± 0.00	0.04 ± 0.00
Apigenin		11.70 ± 0.02	1.31 ± 0.01	0.06 ± 0.00
Myricetin	368	0.19 ± 0.01	0.38 ± 0.02	0.27 ± 0.02
Quercetin		0.10 ± 0.02	0.07 ± 0.00	ND
Kaempferol		0.10 ± 0.03	0.01 ± 0.00	0.01 ± 0.00

Note: CAL(M) = methanolic leaf extract of Cirsium arvense; CAL (EA) = ethyl acetate leaf extract of Cirsium arvense; CAL (Aq) = aqueous leaf extract of Cirsium arvense.

, several compounds—including rutin, catechin, syringic acid, gentisic acid, myricetin, and quercetin—were detected in notable quantities.

The polyphenols that were present in the greatest quantities were apigenin (11.70 ± 0.02 μg/mg of extract), rutin (2.5 ± 0.20 μg/mg of extract) in the ethyl acetate extract of the leaves as shown on Figure 1, and catechin (4.03 ± 0.04 μg/mg of extract) in the methanol extract of the stem as shown in the Figure 2. Coumaric acid, kaempferol, myricetin, and gentisic acid were detected in smaller quantities. The chromatographic profile of the aqueous (C) extracts are presented in Figure 3, illustrating the distribution and intensity of the detected polyphenolic compounds.

3.5. Clinical Bacterial Isolates Exhibited Cefixime Resistance

Before beginning to work with our clinical strains, we determined their overall resistance profile. Antibiotic susceptibility testing was performed on clinical isolates of E. coli, Acinetobacter baumannii, and Staphylococcus aureus using the disc diffusion method, with zones of inhibition (ZOI) compared with CLSI guidelines [22].

Our results (

Table 5. Antibacterial susceptibility testing of antibiotics from major antibiotic classses.

Antibiotics	Antibacterial Activity (ZOI mm ± S.D.)
	R. E. coli	MRSA	R. A. baumannii
Ciprofloxacin	18 ± 0.16	23 ± 0.12	15 ± 0.21
Doxycycline	33 ± 0.12	24 ± 0.11	25 ± 0.15
Cefixime	NA	NA	NA
Lincomycin	22 ± 0.2	25 ± 0.11	14 ± 0.17
Clarithromycin	33 ± 0.1	37 ± 0.10	19 ± 0.16
DMSO	NA	NA	NA

) showed that ciprofloxacin, lincomycin, clarithromycin, and doxycycline inhibited the growth of the clinical isolates to varying extents, producing ZOIs ranging from 19 to 31 mm for E. coli, 18–36 mm for A. baumannii, and 14–25 mm for S. aureus, but this did not correlate with resistance. However, none of the tested isolates produced a measurable zone of inhibition in response to cefixime. This complete lack of inhibition confirms that all three clinical isolates were resistant to cefixime.

Based on these findings, cefixime was selected for combination (synergistic) testing with C. arvense extracts to evaluate whether the plant-derived compounds could potentiate its antibacterial activity against these resistant pathogens.

3.6. Cirsium arvense Extracts Exhibited Mild-to-Moderate Antibacterial Activity Against Cefixime-Resistant Isolates

Next, we wanted to determine the overall effect of the C. arvense extracts on these bacteria. To achieve this, we once again used the disc diffusion method. Our results showed that when compared to ciprofloxacin, all extracts demonstrated mild-to-moderate growth inhibition of clinical isolates. The extract from C. arvense (EA) demonstrated a ZOI of 11 mm against MRSA, A. baumannii, and E. coli that were resistant to cefixime. Similarly, C. arvense (M) extract showed a maximum of 12 mm ZOI against MRSA and E. coli that were resistant to cefixime.

Finally, in the presence of Aq extract, significant growth inhibition was seen in cefixime-resistant E. coli (ZOI 12 ± 0.7 mm). These results suggest that C. arvense extracts are capable of inhibiting the growth of antibiotic-resistant bacteria, as evidenced by measurable zones of inhibition against multiple clinical isolates. The activity observed, particularly with the methanolic and aqueous extracts, suggests that these plant-derived compounds interfere with bacterial proliferation.

3.7. Minimum Inhibitory Concentration (MIC) of Cirsium arvense Extracts Demonstrates Significant Antibacterial Activity

Next, we wanted to confirm the results above. This was achieved through the determination of minimum inhibitory concentration (MIC) values using the broth dilution method (

Table 6. Minimum inhibitory concentration of C. arvense extracts against resistant bacterial isolates.

Extracts	MICs of the Extracts and Drugs Against Test Bacterial Isolates µg/mL ± S.D.
	MRSA	R. E. coli	R. A. baumannii
CAL-(EA)	200 ± 0.13	NA	94 ± 0.08
CAL-(M)	150 ± 0.11	200 ± 0.13	90.2 ± 0.16
CAL-(Aq)	100 ± 0.12	150 ± 0.22	86.4 ± 0.13
Cefixime	100 ± 0.14	100 ± 0.11	100 ± 0.07
Ciprofloxacin	3.33 ± 0.06	1.11 ± 0.12	1.11 ± 0.12
DMSO	NA	NA	NA

Note: CAL(M) = methanolic leaf extract of Cirsium arvense, CAL (EA) = ethyl acetate leaf extract of Cirsium arvense, CAL (Aq) = Aqueous leaf extract of Cirsium arvense, R. E. coli = resistant Escherichia coli, MRSA = methicillin-resistant Staphylococcus aureus, A. baumannii = Acinetobacter baumannii, NA = not applicable.

). As shown in Table 6, the aqueous extract exhibited the strongest activity against MRSA (MIC 100 ± 0.12 µg/mL), resistant E. coli (150 ± 0.22 µg/mL), and A. baumannii (86.4 ± 0.13 µg/mL). The methanolic extract showed moderate activity, with MICs of 150 ± 0.11 µg/mL against MRSA, 200 ± 0.13 µg/mL against resistant E. coli, and 90.2 ± 0.16 µg/mL against A. baumannii. The ethyl acetate extract demonstrated activity against MRSA (200 ± 0.13 µg/mL) and A. baumannii (94 ± 0.08 µg/mL), but was inactive against resistant E. coli. Compared to the standard antibiotic cefixime, which showed MICs around 100 µg/mL for all strains, the extracts showed comparable or slightly higher MICs, while ciprofloxacin exhibited much lower MIC values, reflecting its higher potency. All results were statistically significant (p < 0.05). These results suggest that C. arvense extracts, particularly the aqueous and methanolic fractions, possess antibacterial activity against clinically significant antibiotic-resistant bacteria.

3.8. Evaluation of Synergistic Interactions

To further explore the potential of Cirsium arvense extracts as adjuncts to conventional antibiotics, we investigated whether these extracts could enhance the antibacterial effect of cefixime against resistant bacterial strains. The goal was to determine if combining the plant extracts with cefixime could not only reduce the antibiotic dose needed but also result in bactericidal activity, effectively killing the bacteria rather than just inhibiting their growth.

The synergistic activity was assessed using the checkerboard broth dilution method to calculate the fractional inhibitory concentration index (FICI), and confirmed by time-kill assays to distinguish bacteriostatic from bactericidal effects. The combination treatments targeted resistant E. coli, MRSA, and A. baumannii isolates.

As demonstrated in

Table 7. Minimum inhibitory concentration of C. arvense extracts alone and in combination with cefixime, showing synergistic interaction against resistant bacterial isolates.

Test Samples	MIC of Extracts Alone	MIC Extract + Cefixime	Fold Reduction	FICI	Interpretation
R. E. coli
CAL-EA	200	25	8	0.6	Additive
Cef	100	12.5	8
CAL-Aq	150	62.5	2.4	0.6	Synergistic
Cef	100	12.5	8
R. A. baumannii
CAL-EA	86	21.5	4	0.6	Additive
Cef	100	25	4
CAL-M	94	11.75	8	0.3	Synergistic
Cef	100	25	4
CAL-Aq	90	22.5	4	0.2	Synergistic
Cef	100	12.5	8
MRSA
CAL-EA	150	62.5	2	0.1	Synergistic
Cef	100	6.25	16
CAL-M	200	100	2	0.6	Additive
Cef	100	12.5	8
CAL-Aq	100	12.5	8	0.2	Synergistic
Cef	100	12.5	8

Note: ≤0.5 FICI = Synergism, 0.5–1 FICI = Additive, 1–4 FICI = Indifference, ≥4 FICI = Antagonism, CAL-EA = Cirsium arvense leaf ethyl acetate extract, CAL-(M) = Cirsium arvense leaf methanol extract, CAL-Aq = Cirsium arvense leaf aqueous extract.

, combining C. arvense extracts with cefixime significantly reduced the MIC of cefixime by 8- to 16-fold. Importantly, the results suggest that these combinations achieved bactericidal effects, resulting in a ≥3 log₁₀ reduction in viable bacterial counts compared to the antibiotic alone. The results were statistically significant (p < 0.005), confirming strong synergy and bactericidal activity. These findings suggest that C. arvense extracts can potentiate cefixime’s efficacy.

3.9. Time-Kill Kinetics

Next, we wanted to determine the effect of concentration and time on the above results. A time-kill assay was conducted to determine whether the antibacterial activity of Cirsium arvense extract was concentration-dependent or time-dependent. This method provides insight into the bactericidal dynamics of the extract and its potential synergy with antibiotics. Three bacterial strains were tested to evaluate the spectrum and behviour of the extract over time.

Each strain was exposed to the extract at four concentrations: MIC, 2 × MIC, FICI, and 2 × FICI. Bacterial growth was monitored at regular intervals over 48 h by measuring absorbance at 600 nm. Data were plotted using Origin 2D software.

Our results indicated that there was a concentration-dependent killing effect, with higher concentrations of the extract (2MIC and 2FICI) showing a more rapid and sustained reduction in bacterial growth. These results begin to suggest that the combination of C. arvense extract and cefixime exerts a bactericidal effect, particularly at higher concentrations.

3.10. Synergistic Time–Kill Effect of Cirsium arvense Extract and Cefixime Against R. E. coli

Next, we wanted to assess these results in the context of cefixime. A time-kill kinetics assay was employed over a 48 h period to dynamically evaluate the antibacterial efficacy of cefixime alone and in combination with C. arvense methanol extract against resistant E. coli. As shown in Figure 4A, the combination treatment showed an initial increase in bacterial growth during the first 3 h, followed by a sustained reduction in bacterial counts from 3 to 48 h. We did however observe an unexpected resurgence in bacterial growth after 36 h at the MIC and 2 × MIC of the methanol extract alone, suggesting potential bacterial regrowth or tolerance. However, the decline in antibiotic concentration over time may account for the regrowth observed. To summarise the results:

A (Methanol extract): Initial bacterial growth was observed in the first 3 h, followed by a decline up to 36 h. Regrowth after 36 h in the MIC and 2 × MIC treatments suggests limited bactericidal activity when used alone. Combination with cefixime led to greater suppression, approximately 70–80%.

B (Ethyl acetate extract): The combination treatment resulted in rapid and sustained bacterial reduction (80–85%), outperforming either agent alone, indicating enhanced bactericidal effect.

C (Aqueous extract): The combination closely mirrored ciprofloxacin’s activity, showing strong and prolonged inhibition, suggesting potent synergy (90–95%).

Taken together, these results suggest a bactericidal effect of the combination treatment overall. Although these results show potential, the regrowth after prolonged exposure indicates the need for optimized dosing strategies to maintain bacterial suppression.

3.11. Synergistic Time–Kill Effect of Cirsium arvense Extract and Cefixime Against MRSA

Next, a time-kill kinetics assay was conducted to assess the dynamic antibacterial effects of Cirsium arvense extracts alone and in combination with cefixime against MRSA over a defined time period. This approach was selected to capture changes in bacterial viability over time, providing more detailed insight into the rate and extent of bacterial killing than static MIC measurements. The methanol extract (A) showed initial bacterial growth with a decline from 3 to 36 h, followed by slight regrowth, indicating a transient bactericidal effect as shown in the Figure 5A. The ethyl acetate extract (B) maintained consistent bacterial inhibition throughout the 48 h period. The aqueous extract (C) exhibited the most pronounced bactericidal activity, closely mimicking the effect of ciprofloxacin, suggesting its strong potential as an adjunct antibacterial agent. These results suggest that the combination these agents not only inhibit bacterial growth but actively kills the bacteria over time.

3.12. Synergistic Time–Kill Effect of Cirsium arvense Extract and Cefixime Against R. A. baumannii

Next, we repeated the above experiment using A. baumanii. The time-kill assay, which measures bacterial viability at multiple time points to capture the rate and extent of bacterial killing over time, was performed to evaluate the antibacterial effect of Cirsium arvense aqueous extract combined with cefixime against resistant Acinetobacter baumannii (R. A. baumannii). This method allows a dynamic assessment of antimicrobial activity beyond static MIC values. The results (Figure 6) showed bacterial growth inhibition during the first 3 h, followed by a sustained reduction up to 40 h. The killing pattern closely mirrored that of ciprofloxacin, suggesting a strong bactericidal effect of the combination treatment over time.

3.13. Enhanced Bacterial Membrane Disruption Confirmed by Protein Leakage in Cirsium arvense–Cefixime Treatment

Finally, we decided to determine how the extracts could be inhibiting the growth of the bacteria. Our initial prediction was that this was via membrane damage based on previous work. To assess membrane damage, total protein leakage was quantified using the Bradford assay. Bovine serum albumin (BSA) served as the positive control, and a standard curve (y = 0.0096x + 0.0071, R² = 0.9736) was constructed to estimate protein concentrations in bacterial lysates at MIC and FICI concentrations. Absorbance was measured at 595 nm. The results are summarized in

Table 8. Protein estimation of bacterial samples to calculate the inhibitory potential of the test samples.

Test Samples	MIC or FICI (µg/mL)	Concentration of Protein (µg/mL)	Protein Inhibition (%)
MRSA
CAL-(M)	150	34.7	68.1 ± 0.05
CAL-(EA)	200	42.7	60.7 ± 0.01
CAL-(Aq)	100	32.3	70.3 ± 0.01
CAL-(EA) + Cefixime	150 + 12.5	54.0	50.4 ± 0.04
CAL-(M) + Cefixime	200 + 12.5	14.6	86.5 ± 0.02
CAL-(Aq) + Cefixime	100 + 6.25	45.3	58.4 ± 0.02
Control	---	100	---
R. E. coli
CAL-(M)	200	44.1	64.9 ± 0.03
CAL-(Aq)	150	33.0	73.8 ± 0.02
CAL-(M) + Cefixime	62.5 + 6.25	23.1	81.6 ± 0.01
CAL-(Aq) + Cefixime	62.5 + 12.5	21.5	82.9 ± 0.06
Cefixime	100	32.36	61.6 ± 0.02
Control	---	100	---
R. A. baumannii
CAL-(EA)	94	62.0	44.6 ± 0.01
CAL-(M)	90	28.3	74.6 ± 0.06
CAL-(Aq)	86	44.1	60.5 ± 0.04
CAL-(EA) + Cefixime	125 + 25	26.0	76.7 ± 0.03
CAL-(M) + Cefixime	62.5 + 12.5	31	72.3 ± 0.04
CAL-(Aq) + Cefixime	125 + 12.5	13.6	87.8 ± 0.05
Cefixime	100	47.2	57.8 ± 0.01
Control	----	100	---

Note: CAL-EA = Cirsium arvense leaf ethyl acetate extract, CAL-(M) = Cirsium arvense leaf methanol extract, CAL-Aq = Cirsium arvense leaf aqueous extract.

and indicate significant differences in protein leakage across treatments and bacterial strains. To summarise the results:

1. Resistant E. coli

Methanolic extract alone caused 64.9 ± 0.03% leakage, while the aqueous extract showed slightly higher leakage (73.8 ± 0.02%), suggesting that both extracts disrupt the membrane to a considerable extent. Combination treatments further increased leakage: CAL-(M) + cefixime caused 81.6 ± 0.01%, and CAL-(Aq) + cefixime led to 82.9 ± 0.06% leakage—indicating a synergistic effect on membrane damage. Cefixime alone induced 61.6 ± 0.02% leakage, lower than the combinations, confirming the enhancing role of the extracts.

2. MRSA

Methanolic, ethyl acetate, and aqueous extracts caused 68.1 ± 0.05%, 60.7 ± 0.01%, and 70.3 ± 0.01% leakage, respectively—indicating intrinsic membrane-disrupting activity of all extracts. When combined with cefixime, CAL-(M) + cefixime exhibited the most pronounced effect (86.5 ± 0.02%), while CAL-(EA) + cefixime and CAL-(Aq) + cefixime caused 50.4 ± 0.04% and 58.4 ± 0.02%, respectively. This suggests that the methanolic extract is the most effective partner with cefixime against MRSA.

3. Resistant A. baumannii

The methanolic extract alone yielded 74.6 ± 0.06% leakage, the highest among the single-extract treatments, while ethyl acetate and aqueous extracts produced 44.6 ± 0.01% and 60.5 ± 0.04%, respectively. In combination with cefixime, all treatments showed enhanced leakage: CAL-(EA) + cefixime: 76.7 ± 0.03%, CAL-(M) + cefixime: 72.3 ± 0.04%, and CAL-(Aq) + cefixime: 87.8 ± 0.05%. Notably, CAL-(Aq) + cefixime resulted in the highest protein leakage across all treatments and strains, suggesting a strong disruptive effect on the A. baumannii membrane.

These findings support the hypothesis that C. arvense extracts exert antibacterial activity at least partly through membrane disruption, as evidenced by the elevated protein leakage. When combined with cefixime, especially at FICI concentrations, this effect is significantly amplified, suggesting a synergistic mechanism likely involving increased membrane permeability that facilitates antibiotic entry.

4. Discussion

This study demonstrated that Cirsium arvense extracts, particularly the methanolic and aqueous fractions, exhibit significant antibacterial activity against resistant strains of E. coli, MRSA, and A. baumannii. When combined with cefixime, these extracts enhanced antibacterial effects as shown by increased zones of inhibition, reduced MICs, and significantly higher protein leakage, indicating membrane disruption. The aqueous and methanolic extracts, in particular, showed the most potent synergistic interaction with cefixime across multiple assays. Taken together, these findings support the potential use of C. arvense as an adjuvant therapy alongside conventional antibiotics.

The significance of our findings lies in the urgent global threat posed by the rapid emergence of antibiotic resistance, which has led to the rise of multidrug-resistant (MDR) pathogens that compromise the efficacy of existing therapies. Recent studies underscore this growing concern and highlight the need for novel antimicrobial strategies [23]. In this context, combining plant-based extracts with conventional antibiotics represents a promising approach to restore antibiotic efficacy and overcome resistance mechanisms [24].

The phytochemical analysis performed here revealed that C. arvense contains numerous bioactive secondary metabolites, including quinoline alkaloids, coumarins, flavonoids such as apigenin and catechin, and triterpenes. Compounds like isopimpinellin and heraclenol, previously reported in C. arvense, have known antimicrobial properties [24], which may contribute to the observed synergistic effects with cefixime. Given our findings and this data, the potential therapeutic potential of C. arvense as a natural antimicrobial enhancer is further underlined.

To begin, aqueous and organic solvents (ethyl acetate and methanol) were used to extract the C. arvense (stem and leaves). The results showed that there was an increase in extract recovery with the increase in solvent polarity. Consistent with previous studies, our results demonstrated that extract recovery increased with solvent polarity, with the aqueous extract yielding the highest recovery. This supports earlier findings [25], which showed that higher solvent polarity enhances extraction efficiency. Aqueous extraction could be the method of choice for C. arvense as the phytochemical analysis also showed a better value of phenolic content for the aqueous solvent, as previously reported [26]. Previous research on extraction efficiency employing a variety of polarity solvents demonstrated that elevating the polarity of the solvent strengthens extraction efficiency tremendously [27].

Following extraction, we found that solvent polarity directly influenced the yield of phenols and flavonoids, reinforcing previous trends [28]. The biological significance of this is underscored by the fact that the hydroxylation pattern of phenolics, for example, contributes to their antibacterial potency, as increased hydroxyl groups are associated with enhanced bacterial cell wall disruption [29]. These results support the suitability of water as a preferred solvent for extracting both phenolics and flavonoids from C. arvense.

RP-HPLC-DAD analysis confirmed the presence of several key polyphenolic compounds in different solvent extracts. Notably, apigenin (11.70 ± 0.02 µg/mg) and rutin (1.33 ± 0.02 µg/mg) were most abundant in the ethyl acetate extract, while catechin (4.2 ± 0.02 µg/mg) was dominant in the methanolic extract. These compounds are known for their diverse biological activities, suggesting that C. arvense extracts possess significant therapeutic potential due to their specific composition of antibacterial compounds.

Correlating with this, our study revealed that Cirsium arvense extracts exhibited notable antibacterial activity against multiple resistant bacterial isolates. Notably, the ethyl acetate extract demonstrated strong inhibitory effects against several Gram-positive and Gram-negative strains, although it was ineffective against resistant Escherichia coli. These findings are consistent with recent reports highlighting the selective efficacy of C. arvense extracts against various multidrug-resistant pathogens [30], which attributed the antibacterial effects of C. arvense to lipophilic phytoconstituents such as luteolin, quercetin, rutin, apigenin, linalool, and 1,8-cineole. Our RP-HPLC results confirmed the presence of several of these bioactives, particularly apigenin and rutin, reinforcing their role in the observed antimicrobial activity.

Despite the work above, our findings demonstrated that while the ethyl acetate extract was selective, the cold-water extract exhibited a more broad-spectrum activity against E. coli, Staphylococcus aureus, and Bacillus subtilis. This suggests that aqueous extracts, do indeed retain synergistic or complementary antibacterial compounds. Overall, these results support and expands on earlier findings by demonstrating both the specificity and spectrum of activity of different solvent extracts, emphasizing the potential of C. arvense as a source of effective antimicrobial agents, especially in combination therapies targeting resistant pathogens.

The checkerboard assay revealed a pronounced synergistic interaction between Cirsium arvense extracts and cefixime, with 8- to 16-fold reductions in cefixime’s MIC against resistant bacterial strains. Specifically, the aqueous extract reduced the MIC by 16-fold against resistant E. coli and by 8-fold against MRSA and A. baumannii, while the ethyl acetate extract showed a 16-fold MIC reduction against A. baumannii and an 8-fold reduction against MRSA. These substantial decreases suggest that C. arvense extracts can significantly enhance the efficacy of cefixime, potentially enabling lower therapeutic doses.

Such synergistic effects are critical in the fight against multidrug-resistant pathogens, as they may reduce the selective pressure for resistance development and lower adverse side effects linked to high antibiotic dosages. Recent studies report that ethyl acetate fractions rich in flavonoids have been shown to enhance the activity of β-lactam antibiotics against MRSA and Pseudomonas aeruginosa. These combinations improved antibiotic efficacy by disrupting bacterial membranes, increasing permeability and drug uptake, leading to synergistic effects in checkerboard assays (FICI ≤ 0.5) [26]. The high concentration of bioactive secondary metabolites in C. arvense, including flavonoids, alkaloids, and coumarins, likely underpins this synergy by compromising membrane integrity, as supported by our protein leakage assays. Furthermore, the pronounced effect observed against A. baumannii, a pathogen notorious for its multidrug resistance and clinical challenge, emphasizes the therapeutic potential of combining cefixime with C. arvense extracts. This combination could offer a novel strategy to overcome resistance mechanisms that limit current treatment options. Overall, our findings not only demonstrate the promising antibacterial potentiation by C. arvense but also highlight the importance of exploring plant-based adjuvants to revitalize existing antibiotics in the context of rising antimicrobial resistance.

Recent phytochemical studies of Cirsium arvense confirm the presence of secondary metabolites like rutin, myricetin, catechins, apigenin, and kaempferol. Newer analyses also reveal flavonoid glycosides and triterpenoids such as lupeol. These compounds, already recognized for their antibacterial properties, contribute to its antimicrobial potential through multiple mechanisms [31]. Interestingly, the crude extracts did not exhibit specificity toward a single class of compounds, suggesting that the observed synergistic effect may be due to a complex interplay of multiple constituents but specifically requires the metabolites to be active and present. These findings align with previous reports indicating that plant-derived compounds can enhance antibiotic performance [32].

In the present study, time-kill assays were performed over a 48 h period to assess the dynamic antibacterial effects of Cirsium arvense aqueous extract in combination with cefixime. The aqueous extract displayed a concentration-dependent killing pattern, with greater bacterial inhibition observed at higher concentrations and longer exposure times. Notably, the bacterial reduction pattern of the C. arvense aqueous extract closely mirrored that of ciprofloxacin, a standard fluoroquinolone antibiotic, indicating a strong antibacterial effect even in the absence of cefixime. When used in combination with cefixime, the time-kill curves indicated an additive effect rather than true synergy, despite the fractional inhibitory concentration index (FICI) from the checkerboard assay suggesting a synergistic interaction.

This apparent discrepancy highlights the importance of time-kill assays in capturing the full pharmacodynamic profile of antibacterial agents. Unlike static MIC and FICI values, time-kill studies allow for the observation of bacterial killing kinetics across different concentrations and time points, providing a more nuanced understanding of drug performance. Our findings align with previous reports, such as a study [32], which showed that the antibacterial effect of Acacia nilotica combined with oxacillin depended on both concentration and exposure time. Similarly, our results confirm that C. arvense extract enhances bacterial clearance in a time- and dose-responsive manner, reinforcing the potential of plant-derived compounds as effective adjuncts to conventional antibiotics.

By extending the duration of observation to 48 h, our study contributes additional insight into the sustained antibacterial activity of C. arvense extracts. This prolonged effect could be therapeutically valuable in reducing the frequency of dosing or in improving outcomes for persistent infections. Furthermore, the similarity in bactericidal kinetics between C. arvense and ciprofloxacin underscores the potential of this plant extract as a candidate for further development into resistance-modifying agents or adjunct therapies.

In the present study, time-kill assays over 48 h assessed the dynamic antibacterial effects of Cirsium arvense aqueous extract combined with cefixime. The aqueous extract exhibited a clear concentration-dependent killing pattern, with greater bacterial inhibition observed at higher concentrations and longer exposure times. This dose- and time-dependent bactericidal activity is an important attribute that supports the extract’s therapeutic potential, as sustained antibacterial pressure can improve treatment outcomes and reduce resistance development.

Interestingly, the bacterial reduction profile of the C. arvense aqueous extract alone closely resembled that of ciprofloxacin, a well-established fluoroquinolone antibiotic, indicating a strong intrinsic antibacterial effect independent of cefixime. However, when combined with cefixime, the time-kill curves suggested an additive rather than a fully synergistic effect, despite the checkerboard assay indicating synergy via the fractional inhibitory concentration index (FICI). This discrepancy highlights the limitations of static assays like FICI in capturing the dynamic nature of antimicrobial interactions. Time-kill assays offer deeper insight into pharmacodynamics by revealing bacterial killing patterns, regrowth, and post-antibiotic effects. Recent studies with Acacia nilotica and β-lactams confirm these advantages, showing time- and concentration-dependent activity [32].

Our findings support the increasing evidence that plant extracts can enhance antibiotic efficacy through additive or partially synergistic effects that depend on both concentration and exposure time. These complex interactions are likely due to multiple simultaneous mechanisms, such as membrane disruption that aids antibiotic uptake, along with the action of intrinsic antibacterial compounds affecting intracellular targets [33].

Extending the observation period to 48 h provides valuable insight into the sustained antibacterial activity of C. arvense extracts, which could translate into clinical benefits such as reduced dosing frequency or improved management of persistent infections. Moreover, the similarity in bactericidal kinetics between C. arvense and ciprofloxacin highlights the promise of plant-derived compounds as potential resistance-modifying agents or adjunct therapies, especially in an era of increasing antimicrobial resistance.

To further elucidate the mechanism of action, extracellular protein content was measured to assess membrane integrity and protein synthesis inhibition. Untreated bacterial cultures exhibited higher extracellular protein levels, reflecting normal cell turnover. Treatment with C. arvense extracts alone or combined with cefixime significantly reduced extracellular protein content, suggesting the inhibition of bacterial protein synthesis or enhanced membrane stabilization preventing leakage. The aqueous extract showed the greatest inhibition across resistant isolates, with MRSA treated by the aqueous extract alone showing 60.04 ± 0.03% protein inhibition, increasing markedly to 87 ± 0.04% when combined with cefixime. This substantial increase supports the hypothesis that Cirsium arvense potentiates antibiotic action by compromising bacterial physiology, likely through the disruption of membrane integrity or metabolic interference [14].

While protein estimation cannot confirm the mechanism, it may offer a fundamental description of the molecular impact of the synergism between cefixime and extract, but further investigation is needed to prove it.

While the findings of this study highlight the promising antibacterial potential of Cirsium arvense extracts in combination with cefixime, there are some limitations that must be acknowledged. First, while we conducted chemical characterization confirming cefixime resistance, we were unable to perform genetic characterization of the resistance mechanisms. This remains a critical gap, and future work will aim to identify specific genes and mutations contributing to cefixime resistance. Second, we acknowledge the limitation of using a single bacterial strain, as susceptibility can vary significantly between isolates, including those that exceed CLSI breakpoints or exhibit multidrug resistance. To enhance clinical relevance and generalizability, future studies will incorporate multiple isolates with diverse resistance profiles. Furthermore, although the checkerboard assay indicated synergy, the time-kill study suggested an additive effect, underscoring the need for further investigation into the underlying mechanisms of action. It is also important to consider the possibility of variation in phytochemical composition due to environmental factors, plant part used, or extraction method, which could influence reproducibility.

Future work should focus on isolating and characterizing the active constituents within the C. arvense extracts to better understand their individual and combined roles in antibacterial activity. Mechanistic studies using molecular tools (e.g., gene expression and, membrane permeability assays) would also help clarify how these compounds interact with bacterial targets and with antibiotics like cefixime.

Despite the limitations above, which will be overcome in future work, our work clearly shows the potential of these plant extracts to synergise with antibiotics as a method to overcome antibacterial resistance. Further work will determine the broader profile of this as well as determining the underlying mechanisms.

5. Conclusions

In this research, all C. arvense extracts examined exhibited notable antibacterial activity against resistant clinical strains. The ethyl acetate extract showed significant inhibitory effects against R. E. coli, MRSA, and R. A. baumannii, while the methanolic extract demonstrated additive activity, particularly against R. A. baumannii. Furthermore, the synergistic effects observed with the aqueous extract were both concentration- and time-dependent, with the greatest bactericidal activity occurring at 2 × FICI levels after 6 and 9 h, depending on the bacterial species. This was further supported by time-kill kinetics, which confirmed a concentration-dependent killing pattern, closely resembling the activity of ciprofloxacin. Importantly, our study also found a substantial decrease in the protein content of bacterial cells treated with the C. arvense/cefixime combinations.

Taken together, our results highlight the potential of C. arvense extracts, especially the aqueous fraction, as effective adjuvants to cefixime in the treatment of resistant bacterial infections. These encouraging results warrant further investigation, including compound isolation and mechanistic studies.