Exploring the antibacterial and anti-biofilm activity of two Iranian medical-grade kinds of honey on multidrug-resistant Pseudomonas aeruginosa

Abstract

Introduction

Pseudomonas aeruginosa is a prominent multidrug-resistant and biofilm-forming bacteria. Mono-floral honey, enriched with a variety of biological compounds, can be categorized as medical-grade honey due to its notable pharmacological benefits. In this study, two types of Iranian honey were thoroughly characterized, and the antimicrobial and anti-biofilm properties were examined against three clinical strains of multidrug-resistant P. aeruginosa.

Methods

Citrus and Thyme honey from Alborz were selected based on physicochemical, phytochemical, and melissopalynological tests conducted from a medical perspective. The antibacterial activity of the honey samples against three clinical strains of multidrug-resistant P. aeruginosa isolated from wound infections was evaluated using both the well-diffusion and broth microdilution methods. Additionally, an antibiofilm assay was performed using the crystal violet method in microplates.

Results

Both medical grade honey samples exhibited considerable antibacterial activity against the three P. aeruginosa isolates at 75–100% v/v concentrations with inhibition zones measuring between 15 and 30 mm. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values for both types of honey were 6.25% v/v (final concentration). The antibiofilm assay indicated that both types of honey demonstrated varying levels of antibiofilm activity. Citrus honey at 9% concentration was the most effective, showing an average inhibition rate of 59%, while Citrus honey at 2.3% final concentration exhibited the least effectiveness with an average inhibition rate of 23%.

Discussion

A thorough analysis of the honeys studied confirmed their authenticity and the presence of medicinal compounds. The results of honey tests correspond to the normal range (natural Honey) in the Council of the European Union. Based on the evaluation and compliance with the medical grade criteria including authenticity, health, qualities well botanical origin mentioned honey is classified in medical grade. The antibacterial results indicated that both Thyme and Citrus honeys effectively inhibit the growth and biofilm formation of P. aeruginosa. Therefore, these honeys may serve as natural and safe alternatives or adjuncts to conventional antibiotic therapy for wound healing and infection management.

Introduction

In contemporary healthcare, the emergence of multi-drug resistant (MDR) infections poses a substantial challenge, as traditional antibiotics are increasingly losing their effectiveness [1]. Multidrug resistance (MDR) is defined as the acquisition of resistance to at least one antimicrobial agent from three or more distinct classes of antibiotics complicating treatment options. This resistance arises from mechanisms such as genetic mutations, the acquisition of resistance genes through horizontal gene transfer, and the active efflux of drugs from bacterial cells. The overuse and misuse of antibiotics in both healthcare and agriculture have accelerated the emergence of MDR strains, representing a significant global threat to human health, underscoring the urgent need for novel therapeutic strategies [2, 3]. As the global healthcare community confronts the rising threat of MDR infections [4], the investigation of alternative antimicrobial agents, including natural components like honey and medicinal plants, is gaining traction as a viable approach to combat antibiotic resistance and enhance treatment efficacy.

Pseudomonas aeruginosa is an opportunistic pathogen recognized for its resistance to antimicrobial treatment, particularly in burn and immunocompromised patients [5]. One of the primary challenges associated with P. aeruginosa infections is its ability to form biofilms [6]. Biofilms adhere to surfaces and are encased in a protective matrix of extracellular polymeric substances (EPS). They form through stages of attachment, growth, and dispersal, and can develop in various environments. One significant clinical concern is that biofilms contribute to multidrug resistance (MDR) in P. aeruginosa [7]. The EPS matrix impedes the penetration of antibiotics, while the unique microenvironment within the biofilm allows bacteria to alter their metabolism and express resistance genes. Moreover, P. aeruginosa within biofilms communicate via quorum sensing, enhancing their survival and resistance mechanisms [8]. It is believed that biofilms play a substantial role in over 90% of chronic wound infections in vulnerable patients, which makes these biofilm-associated infections more challenging to treat and highlights the need for novel therapeutic approaches to combat these resilient microbial communities [9].

Honey is a natural product produced by Apis mellifera bees from the secretions of living plant parts or from plant-sucking insects, or flower nectar, resulting in floral honey [10]. Mono-floral honey, derived from the nectar of a single type of flower, is a distinct category of honey [4]. Natural honey along with numerous vitamins as well as proper bioactive additives, is a medicinal drug being prescribed through physicians for a huge sort of human problems from historic instances until today [11]. Its prominent antibacterial properties have been well-documented in both modern and traditional medicine, and it is effective against multi-drug-resistant (MDR) bacteria [4]. Several studies have demonstrated that certain mono-floral honey can be classified as medical-grade honey (MGH), which is considered an alternative or complementary natural product to antibiotics in wound management [12]. Additionally, MGH shows a significant positive pharmacological effect (e.g. anti-inflammatory, immunomodulatory antioxidant, wound healing) properties on human physiology resulting in a high dosage of phytochemical and biological compounds [13–16].

Thyme honey, derived from the nectar of thyme flowers [17], has been shown to possess a wide range of biological activities, including antibacterial, antifungal, antispasmodic, and anti-inflammatory effects [18]. Darkened honey, such as Thyme honey, contain higher levels of antioxidant compounds than other types of honey, including rich phenolic compounds and flavonoids [19]. Thyme honey is rich in vitamins B, A, and E and has been traditionally used to treat various conditions, including intestinal pains, coughs, sore throats, joint pains, menstrual cramps, epilepsy, convulsions, headaches, and migraines. In addition, this honey is one of the few types of honey used for the treatment of diabetic patients [20]. The composition of this honey contains more than 200 compounds, the most important is excessive phenolic content which is responsible for its strong antioxidant and antibacterial properties [21]. Reports proposed that these phenolic compounds disrupt the bacterial cell membrane, inhibit essential enzymes, and disrupt the bacterial DNA causing bacterial death [22].

Citrus honey, derived from citrus flowers, is a highly valued mono-floral honey that is produced and consumed globally [23–27]. Several reports shows that citrus honey has promising antibacterial activity against Staphylococcus aureus, Klebsiella spp., Escherichia coli, P. aeruginosa and Candida albicans [28–30]. This honey is rich in flavonoids and various bioactive compounds contribute to its antimicrobial effect [31]. Citrus flavonoids have been shown to disrupt bacterial membranes and inhibit essential enzymes, thereby effectively slowing bacterial growth [32]. In addition, the acidic pH of citrus honey creates a destructive environment for bacteria to survive, which at the same time enhances its antibacterial effect [33]. These combined factors make Citrus honey a highly recommended option for consumers, increasing its market value and encouraging widespread use [34, 35].

Research has shown that the antimicrobial properties of honey are closely linked to its geographical origin and botanical characteristics, which include the pollen, nectar, resins, and oils collected by bees [36–38].However, there are relatively few systematic studies focusing on the classification of honey based on their origin, compositional profiles, and antimicrobial strengths [39, 40]. While numerous studies have highlighted the effectiveness of medical-grade honeys (MGHs) against different antibiotic-resistant bacteria, there is a significant need for comprehensive investigations that fully characterize these honeys, their geographical origins, and their correlation with antibacterial efficacy. This study is part of a broader research initiative aimed at examining, qualifying, and developing a database of medicinal-grade honey in Iran. Various types of MGHs were collected and subjected to a series of analyses, including physical and chemical assessments, melissopalynology, and phytochemical tests Following the selection of two specific honey varieties, microbial tests were conducted using the well-diffusion method, minimum inhibitory concentration determination, and anti-biofilm assays against three MDR clinical P. aeruginosa.

Results

Honey and authentication analysis

The physicochemical characteristics of the analyzed honey samples are detailed in Tables 1 and 2. All authentication criteria adhered to the standards set by the European Union. Furthermore, melissopalynological analysis indicated that citrus and thyme pollen are the predominant pollen types found in Citrus honey and Thyme honey, respectively, as shown in Tables 3 and 4. Additionally, the profiles of polyphenols and amino acids in the studied honey align with the findings of Kaškonienė and Venskutonis [37] as presented in Tables 5 and 6.

Table 1.

Physiochemical characteristics of citrus honey

Test Title	Result of the Test	Environmental Factorsa		Unit	Test Method	Uncertainty	The Acceptable Limit	The Acceptable Limit	Deficiency Type	Decision Rule		Probability of Compliance (PC)	Compliance with Specifications
		T (°C)	H (%)				Min	Max
Reducing Sugars before Hydrolysis	75.15	26.5	26	gram%	INSO 92 Clause 3–7	0.98%	65	-	major	51%	$\le$ PC	100.0%	conforms
Reducing Sugars after Hydrolysis	77.11	26.5	26	-	INSO 92 Clause 3–7	-	-	-	major	-	-	-	-
Sucrose	1.86	26.5	26	gram%	INSO 92 Clause 2–3-4–7	0.82%	-	5	major	51%	$\le$ PC	100.0%	Conforms
Hydroxymethylfurfural	5.84	26.5	26	mg/kg	INSO 92 Clause 12–7	7.8%	-	40	major	51%	$\le$ PC	100.0%	Conforms
Proline	255.2	26.5	26	mg/kg	ISIRI 11145	3.40%	180	-	major	51%	$\le$ PC	100.0%	Conforms
Diastasis Activity (Quantitative)	7.7	26.5	26	Diastasis unit DN	INSO 92 Clause 8–7	6%	8	-	major	51%	$\le$ PC	-	does not conform
Humidity	14.9	26.5	26	%	INSO 92 Clause 2–7	14%	-	20	major	51%	$\le$ PC	100.0%	Conforms
Total Polyphenol	0.33	26.5	26	mg/ml	ISIRI 8986–1	7.30%	0.03	-	major	51%	$\le$ PC	100.0%	Conforms

^aThe environmental factors are T = temperature (°C) and H = humidity (%)

Table 2.

Physiochemical characteristics of thyme honey

Test Title	Result of the Test	Environmental Factorsa		Unit	Test Method	Uncertainty	The Acceptable Limit	The Acceptable Limit	Deficiency Type	Decision Rule		Probability of Compliance (PC)	Compliance with Specifications
		T (°C)	H (%)				Min	Max
Reducing Sugars before Hydrolysis	73.85	26.3	27	gram%	INSO 92 Clause 1–7	0.98%	65	-	major	51%	$\le$ PC	100.0%	conforms
Reducing Sugars after Hydrolysis	76.95	26.3	27	-	INSO 92 Clause 1–7	-	-	-	major	-	-	-	-
Sucrose	2.94	26.3	27	gram%	INSO 92 Clause 1–7	0.82%	-	5	major	51%	$\le$ PC	100.0%	Conform
Hydroxymethylfurfural	25.75	26.3	27	mg/kg	INSO 92 Clause 5–7	7.8%	-	40	major	51%	$\le$ PC	100.0%	Conform
Proline	651.4	26.3	27	mg/kg	ISIRI 11145	3.40%	180	-	major	51%	$\le$ PC	100.0%	Conform
Diastasis Activity (Quantitative)	17.9	26.3	27	Diastasis unit DN	INSO 92 Clause 3–7	6%	8	-	major	51%	$\le$ PC	100%	Conform
Humidity	16.98	27	27	%	INSO 92 Clause 7–12	14%	-	20	major	51%	$\le$ PC	99.5%	Conforms
Total Polyphenol	0.58	26.3	27	mg/ml	ISIRI 8986–1	7.30%	0.03	-	major	51%	$\le$ PC	100.0%	Conforms

^aThe environmental factors are T = temperature (°C) and H = humidity (%)

Table 3.

The Melissoplynological characteristics of thyme honey

	Quantitative Specifications		Quantity (number-Percent)	Volume Unit	Pollen Type
1	Numbers	Minimum Number of Pollens	420	10 mg	Total
2	FrequencyClasses(percent)	Predominant Pollen	> 45%	10 mg	Thyme
3		Secondary Pollen	16–45%	10 mg	-
4		Important Minor Pollen	3–15%	10 mg	Astragalus Type, Asteraceae Type
5		Minor Pollen	1–3%	10 mg	-
6	Pollen Frequency	Very Frequent	> 45%	10 mg	Thyme
7		Frequent	16–45%	10 mg	Astragalus Type, Asteraceae Type
8		Rare	3–45%	10 mg	-
9		Sporadic	< 3%	10 mg	-
	Uni-floral Honey (Relative level of abundance of Thyme)		> 45%	10 mg	Thyme

Table 4.

The Melissoplynological characteristics of citrus honey

	Quantitative Specifications		Quantity (number-Percent)	Volume Unit	Pollen Type
1	Numbers	Minimum Number of Pollens	420	10 mg	Total
2	Frequency classes(percent)	Predominant Pollen	> 45%	10 mg	-
3		Secondary Pollen	16–45%	10 mg	Citrus
4		Important Minor Pollen	3–15%	10 mg	Asteraceae, Lamiaceae, Boraginaceae type
5		Minor Pollen	1–3%	10 mg	-
6	Pollen Frequency	Very Frequent	> 45%	10 mg	-
7		Frequent	16–45%	10 mg	Citrus
8		Rare	3–45%	10 mg	Asteraceae, Lamiaceae, Boraginaceae Type
9		Sporadic	< 3%	10 mg	-
	Uni-floral Honey (Relative level of abundance of citrus)		> 45%	10 mg	Citrus

Table 5.

Polyphenol components (phytochemical characteristics) and the determination of sugars of thyme and citrus honey

Polyphenol Components (μg/g) and Sugar (%)	Citrus	Thyme
3,4-hydroxybenzoic acid (μg/g)	32.44	29.7
2,4-dihdroxybenzoic acid (μg/g)	2.77	2.56
Ferulic Acid (μg/g)	13.70	14.24
Ellagic Acid (μg/g)	92.3	89.49
Trans-cinnamic Acid (μg/g)	0.00	0.00
P-coumaric Acid (μg/g)	6.12	6.13
Syringic Acid (μg/g)	95.24	95.56
Total Content (μg/g)	242.67	237.66
Myricetin (μg/g)	0.00	0.00
Quercetin (μg/g)	0.01	0.01
Catechin (μg/g)	1.21	1.11
Rutin (μg/g)	1.00	1.00
Kaempferol (μg/g)	1.49	1.45
Hesperetin (μg/g)	0.03	0.04
Naringenin (μg/g)	3.45	3.30
Chrysin (μg/g)	0.28	0.31
Fructose (%)	36.23	36.29
Glucose (%)	30.53	33.60
Sucrose (1%)	4.68	4.88
Maltose (%)	1.57	1.67
Reduction sugar (%)	66.77	69.92
Total Sugar (%)	68.34	71.70
F/G ratio	1.18	1.078
Erlose (%)	3.01	3.23
Melezitose (%)	3.16	3.37

Table 6.

Amino acid contents (phytochemical characteristics) of thyme and citrus honey

Amino Acid(mg / 100 g dry matter)	Citrus	Thyme
Aspartic Acid	5.60	5.35
Glutamic Acid	5.28	5.2
Asparagine	4.66	4.70
Serine	1.43	1.42
Glutamine	2.06	2.05
Histidine	0.67	0.66
Glycine	0.35	0.38
Threonine	0.75	0.74
Arginine	1.01	1.03
ß-alanine	0.23	0.23
α-alanine	2.2	2.17
Aminobutyric Acid	0.86	0.85
Tyrosine	6.66	6.56
Valine	1.01	0.97
Tryptophan	0.29	0.305
Phenylalanine	15.36	15.38
Isoleucine	0.72	0.73
Leucine	0.65	0.65
Lysine	1.16	1.18
Proline	44.58	44.77

Susceptibility assay by well-diffusion

The results indicate that both types of honey displayed significant differences in their antibacterial effects across all four concentrations when tested against the three isolates of P. aeruginosa (p- value = 0.001), reaffirming their potent antibacterial properties. However, according to clinical and laboratory standard institute guidelines (CLSI 2020) [41], the isolates only exhibited a high level of susceptibility to both honey types at concentrations between 75–100% (v/v). The mean inhibition zones recorded for Thyme honey were 22 ± 4 mm at 75% (v/v) and 28 ± 2 mm at 100% (v/v). In parallel, Citrus honey demonstrated mean inhibition zones of 24 ± 2 mm and 26 ± 1 mm at the same concentrations. The antibacterial activity was notably reduced at a concentration of 50% (v/v), with mean inhibition zones of 9 ± 3 mm for Thyme honey and 10 ± 2 mm for Citrus honey, indicating a level of intermediate susceptibility, while at a concentration of 25% (v/v), no inhibition zones were detected for either type of honey. All recorded inhibition zones are illustrated in Fig. 1.

Fig. 1.

Inhibition zone diameters (mm) of different concentrations (v/v) of thyme and citrus honey samples against three MDR P. aeruginosa clinical isolates

Susceptibility assay by MIC and MBC

The minimum inhibitory concentration (MIC) for both types of honey against all three bacterial isolates was determined to be 12.5% v/v (final concentration 6.25% v/v). The minimum bactericidal concentration (MBC) values aligned with the MIC results, as the 6.25% v/v of both honey types prevented bacterial growth and colony formation. This implies that the concentration of honey required to inhibit bacterial growth is identical to that needed to eradicate the microorganisms (MIC = MBC).

Antimicrobial time-kill assays

Bacterial colonies were counted to calculate the colony-forming units per mL (CFU/mL), and the log10 of CFU/mL was plotted against time to generate time-kill curves. The time-kill assay was performed for all three isolates, and the results are presented as the mean log10 CFU/mL for the three P. aeruginosa isolates. The assay revealed that the effects of both honey samples on P. aeruginosa were concentration-dependent (Figs. 2 and 3). The time-kill curves for the 0.5 × MIC and control conditions exhibited minimal differences across the honey samples. At the MIC concentration, the bacterial count after 24 h was consistent with the MBC, while a marked reduction in viable bacteria was observed within 2 h at the 2 × MIC concentration for both Citrus and Thyme honey.

Fig. 2.

Time-kill assay results for all three Pseudomonas aeruginosa isolates, presented as the mean log10 CFU/ml, showing the effect of Citrus Honey on P. aeruginosa

Fig. 3.

Time-kill assay results for all three Pseudomonas aeruginosa isolates, presented as the mean log10 CFU/ml, showing the effect of Thyme Honey on P. aeruginosa

Antibiofilm assay

Light absorption was measured at a wavelength of 595 nm using a microplate reader (Table 7). The inhibition of biofilm formation was then evaluated using the formula: Inhibitory rate = (1 − S/C) × 100%, where S represents the average absorbance of the sample group and C represents the average absorbance of the control group. The results demonstrate that both types of honey exhibited significant differences in their antibiofilm effects at all four concentrations 25, 50, 75, and 100%v/v (final concentrations 2.3, 4.5, 6.8 and 9%v/v) when tested against the three isolates of P. aeruginosa (p- value = 0.001), confirming their biofilm inhibition properties. However, the inhibition rate was notably lower 2.3% v/v for both types of honey. The most effective treatment was 9% v/v Citrus honey, which produced an average inhibition rate of 59%, while the least effective was 2.3% v/v Citrus honey, showing an average inhibition rate of 23% (Fig. 4, Table 8). To illustrate the effects of honey treatments at the cellular level, scanning electron microscopy (SEM) images were obtained. The SEM image of the untreated control clearly shows the organized structure of the biofilm that developed after 24 h of incubation. In contrast, the treated samples displayed a noticeable reduction in biofilm formation, demonstrating the effectiveness of both types of honey as anti-biofilm agents (Fig. 5).

Table 7.

Optical absorption values obtained after treating of three P. aeruginosa Isolates with different concentrations (v/v) of thyme and citrus honey samples in crystal violet method

Different Concentrations of Thyme and Citrus Honey Samples According to the Final Volume	Isolate 1	Isolate 2	Isolate 3	Mean	± SD
Thyme 9%	1.395	1.825	1.698	1.639	0.180
Thyme 6.8%	1.467	1.903	1.782	1.717	0.184
Thyme 4.5%	1.568	1.98	2.134	1.894	0.239
Thyme 2.3%	2.543	2.674	2.973	2.730	0.180
Citrus 9%	1.604	1.39	1.387	1.460	0.102
Citrus 6.8%	1.712	1.423	1.39	1.508	0.145
Citrus 4.5%	1.785	1.566	1.642	1.664	0.091
Citrus 2.3%	2.486	2.847	2.959	2.764	0.202
Negative Control	3.04	3.588	4.114	3.581	0.438

Fig. 4.

Biofilm inhibitory rates (%) of different concentrations (v/v) of Thyme and Citrus honey samples against three P. aeruginosa isolates

Table 8.

Biofilm inhibitory rates (%) of different final concentrations (v/v) of Thyme and Citrus honey samples against three P. aeruginosa isolates

Different Final Concentration of Thyme and Citrus Honey	Biofilm Inhibitory Rates (%) Against P. aeruginosa Isolates
	Isolate 1	Isolate 2	Isolate 3	Mean
Thyme 9%	55	50	59	55
Thyme 6.8%	52	47	57	52
Thyme 4.5%	48	45	49	47
Thyme 2.3%	17	28	29	25
Citrus 9%	48	62	67	59
Citrus 6.8%	44	61	67	57
Citrus 4.5%	42	57	61	53
Citrus 2.3%	19	21	29	23

Fig. 5.

Scanning Electron Microscopy (SEM) image showing the organized structure of the biofilm after 24 h of incubation. A represents the untreated control, while (B1–C2) display the treated samples: B1 80% v/v Thyme honey (final concentration 7.3% v/v); B2 100% v/v Thyme honey (final concentration 9% v/v); C1 80% v/v Citrus honey (final concentration 7.3% v/v); C2 100% v/v Citrus honey (final concentration 9% v/v)

Discussion

This study investigated the antibacterial properties of mono-floral Thyme and Citrus honey against three clinical multidrug-resistant isolates of P. aeruginosa. The findings highlight the therapeutic potential of these honeys as alternatives to conventional antibiotic therapies, particularly in the context of multidrug-resistant (MDR) infections. The efficacy of both Citrus and Thyme honey aligns with established benchmarks for natural honey quality, as specified by the Council of the European Union (DIRECTIVE 2001/110/EC). Based on the evaluation and compliance with the medical grade criteria [13] including authenticity, health, quality as well as botanical origin of mentioned honey are classified in medical grade. The quality of the honey was validated through various authentication characteristics, including phytochemical, physicochemical, and melissopalynological analyses, supporting their use as alternative medicinal agents for bacterial control [35, 37, 42]. The studied honey contains key flavonoids such as Naringenin, Kaempferol, Catechin, Rutin, Hesperetin, Chrysin, Quercetin, and Myricetin, as well as significant polyphenolic acids like 3,4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, Ferulic acid, Ellagic acid, Trans-cinnamic acid, P-coumaric acid, and Syringic acid. These compounds exhibit various antimicrobial mechanisms, including inhibition of DNA helicase by Myricetin, disruption of cell membranes by Quercetin, and hydrogen peroxide generation by Catechin [36, 43]. The presence of these agents not only indicates the potential effectiveness of these honeys in treating infections but also provides a foundation for exploring novel therapeutic applications.

Recent studies have emphasized that medical-grade honey has both direct antibacterial properties [12] and additional benefits such as immunomodulation and wound healing capabilities [44]. Its ability to promote tissue repair and reduce inflammation makes it a valuable resource for managing P. aeruginosa infections, which complicate wound healing [15, 45]. The microbicidal properties of MGHs arise from various mechanisms. Depending on their characteristics, active compounds, and bacterial type, MGHs exhibit both bacteriostatic and microbicidal effects. Phenolic compounds, including flavonoids and phenolic acids—known for their antioxidant and antibacterial properties—play a crucial role in disrupting bacterial cell membranes, inhibiting essential enzymes, altering bacterial DNA, and ultimately inducing bacterial cell death [46]. Moreover, the high sugar content in MGHs creates an osmotic pressure that dehydrates bacterial cells [47], while the acidic pH (3.2 to 4.5) inhibits bacterial growth [36]. Certain types of honey, such as Thyme and Citrus honey also produce hydrogen peroxide when exposed to moisture, which contributes to their antibacterial activity [48].

The results of this study demonstrated that both Thyme and Citrus honey effectively inhibit the growth of MDR P. aeruginosa, suggesting their potential as viable alternatives to traditional antibiotic therapies. The minimum inhibitory concentration for both types of honey against all three bacterial isolates was established at 12.5% v/v, corresponding to a final concentration of 6.25% v/v. Our findings align with previous studies that have demonstrated the antibacterial effects of mono-floral honey, particularly Thyme and Citrus honey. Benlyas et al., [49] evaluated eleven types of mono-floral honey (including Thyme and Orange) against various Gram-negative and Gram-positive bacteria, revealing that Thyme honey exhibited an inhibitory effect on P. aeruginosa (9.50%). A study conducted in Turkey [50] also confirmed the potent antibacterial effects of Thyme honey against Staphylococcus aureus and Acinetobacter baumannii. Research by Benli et al. [51] also highlighted the significant inhibitory effects of Thyme honey against P. aeruginosa and Masoura et al. [52] demonstrated that the antibacterial efficacy of Thyme honey is comparable to, or even exceeds, that of Manuka honey. Another study [53] has indicated the moderate antibacterial potential of Citrus honey, with an average minimum inhibitory dilution of 17.4% (w/v). Furthermore, a study conducted in India revealed significant antibacterial effects of Citrus honey against multidrug-resistant (MDR) bacteria. It was also found that Citrus honey exhibits synergistic effects when combined with certain antibiotics, including Ceftazidime, Gentamicin, and Amikacin [54]. The notably lower MIC rates observed in our study suggest that the Thyme and Citrus honey tested may have a stronger antibacterial effect compared to those reported in the previous studies, reinforcing their potential role in combating MDR bacterial infections.

In this study, the biofilm inhibitory effects of Thyme and Citrus honeys against multidrug-resistant (MDR) P. aeruginosa was also investigated. Our results showed that 9% v/v Citrus honey (final concentration) exhibited the highest anti-biofilm activity, with an average inhibition rate of 59%. Both honey types demonstrated a concentration-dependent relationship, with a notable decrease in inhibition rates at a 25%. This finding aligns closely with a study by Lu et al. [55] who reported the inhibitory potential of New Zealand honeys against MDR P. aeruginosa biofilm, noting effective results across varying concentrations. Furthermore, the observation of reduced biofilm formation following honey treatment, as seen in SEM imaging, supports the finding by Cooper [56] demonstrated that treatment with Medihoney ™ leads to structural changes in P. aeruginosa biofilms, resulting in a significant difference in both biofilm density and viability due to the exposure to honey.

One of the notable aspects of our study is the comprehensive characterization of Citrus and Thyme honey, which are fully recognized for their medicinal properties. Utilizing physicochemical, phytochemical and melissopalynological analyses, we have detailed data on the composition of these honey, including their bioactive compounds and floral sources. This detailed analysis not only confirms the quality and authenticity of our honey as medical-grade products but also sets the stage for future research into the possible synergistic effects of their individual components. In contrast, many existing studies on honey’s antimicrobial properties often lack such detailed compositional data. While the articles highlight the importance of maintaining a minimal processing standard to preserve the biological activity of honey, they do not provide extensive detail about the specific compounds present in the honey examined or their floral sources [57–59].

Conclusion

Both Thyme and Citrus honey could enhance the effectiveness of standard antibiotic therapies separately by targeting both planktonic bacteria and biofilms. This study supports the potential of Thyme and Citrus honey as natural remedies for tackling complex bacterial infections. Utilizing honey’s healing properties for wound care and infection management could present a safe alternative to traditional antibiotic treatments. Further research and clinical trials are warranted to explore the therapeutic potential of honey and its derivatives against MDR bacteria.

Material and methods

Honey sampling

A variety of mono-floral honey samples were collected from 2023 to 2024 straightly from beekeepers from different ecological regions of Iran, representing a diverse range of eco-regions across the country. The geographic locations of each sample were recorded distinctly by GPS device. The samples including 250–300 g of raw honey from each colony and transported to the laboratory at a temperature of < 20 °C.

Authentication analysis

To ensure authenticity, these samples were tested for their physicochemical and phytochemical properties. The quality assessments were reviewed and selected based on medical relevance. The physicochemical factors (e.g. reducing sugars before hydrolysis, reducing sugars after hydrolysis, 5-Hydroxymethylfurfural, proline, diastase activity, pH, sucrose and fructose/glucose) were evaluated according to the International Honey Commission (IHC) 49. The physicochemical characteristics follow Nayik et al. [60], Oroian and Ropciuc’s [61] methodology. In addition to these tests, melissopalynological analysis was conducted following the methodology outlined by Louveaux et al. [62]. This analysis is a crucial tool for the botanical and geographical identification of honey, examining the pollen grains and fungal spores present in the samples. Accordingly, 10 g of honey samples in about 30 mL of distilled water was dissolved on a hot plate at a temperature up to 45 °C. Afterward, the mentioned samples were filtrated. In the next step, the liquid was poured into 50 mL conical centrifuge tubes. Then the samples were centrifuged three times (with 3000 rpm) for a total time of 15m. After the last centrifugation, the last remaining liquid was poured into the watch glass and placed it on the hot plate so that the water evaporated completely and the pollen grains remained at the bottom of the container. For quantitative evaluation, pollen grains were transferred to neobar slide and counted. The pollen grains were mounted on aluminum stubs, and then coated with gold in an Emitech EMK 550 sputter. The Photography was made with Cam Scan Hitachi SU3500 Scanning Electron Microscope. In addition, Image tool ver.30 was used to assess quantitative pollen characters. Terminology followed that according to Punt et al. 2006 [63], Halbritter et al. 2008 [64]. Furthermore, phytochemical tests were performed to assess the profiles of polyphenols, aromatics, amino acids, and sugars, as documented in previous studies [37]. Polyphenols were quantified using the Folin-Ciocalteu assay, which measures the color change resulting from polyphenolic compounds reacting with the reagent. Aromatic compounds were analyzed by gas chromatography-mass spectrometry (GC–MS). Amino acids were determined using high-performance liquid chromatography (HPLC), allowing for the separation and quantification of individual amino acids. Sugars were evaluated through HPLC or enzymatic assays to profile both simple and complex carbohydrates [65, 66]. Concurrently, data regarding the vegetation and variability within the pollen spectrum were compiled, which contributed to the geographical indication of the honey [62]. Based on the findings, two honey samples were selected for further experimentation: Citrus honey sourced from the southern slope of the Alborz mountains and Thyme honey from the same region.

Bacterial strains

Three strains of P. aeruginosa were isolated from the wounds of burnt patients at Imam Reza Hospital in Tehran, Iran. Informed consent was obtained from all patients, and the study received approval from the research ethics committee of Tehran University of Medical Sciences. All procedures followed the appropriate guidelines and were conducted by the Declaration of Helsinki. The recruited isolates were confirmed as P. aeruginosa based on their colony morphology, the presence of gram-negative rod shapes, and positive results from the oxidase, urease, and Simon’s citrate tests, as well as their ability to grow at 42 °C. The antibiotic resistance patterns of the isolates against conventional antibiotics were assessed using the disk diffusion method by the Clinical and Laboratory Standards Institute (CLSI) guidelines. The results demonstrated that these three isolates exhibited resistance to ceftazidime, ciprofloxacin, cefepime, aztreonam, piperacillin, and gentamicin.

Susceptibility test by well-diffusion

An antimicrobial susceptibility test was performed using the well diffusion method. Brain heart infusion (BHI) agar plates were prepared and inoculated with bacterial suspensions equilibrant to 0.5 McFarland standard (1.5 × 10 ⁸ cells/ml). An aseptic technique was employed to create wells with a diameter of 6 mm in the agar, which were then filled with 100 µl of various concentrations of honey (ranging from 25 to 100% v/v). A control plate was prepared with normal saline in one of the wells. After incubation of the plates at 37 °C for 24 h, the plates were observed for the zone of bacterial growth inhibition. The inhibition zones were measured in millimeters (mm) and expressed as mean ± SD. The criteria for resistance, susceptibility, and intermediate susceptibility were established according to CLSI 2020 [41]. Specifically, an inhibition zone diameter of 13 mm or less was categorized as resistant, a diameter ranging from 14 to 21 mm was classified as intermediate susceptible and a diameter of 22 mm or greater was deemed susceptible. The tests were repeated three times to minimize errors.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination

The minimum inhibitory concentration (MIC) of Citrus and Thyme honey against the isolated P. aeruginosa strains was determined using the broth microdilution method in 96-well microplates. Initially, fresh bacterial culture suspensions were diluted to achieve a concentration of 5 × 10^5 CFU/ml. Subsequently, 50 µl of serial dilutions of honey, ranging from 3.125% v/v (final concentration 1.56% v/v) to 100% v/v (final concentration 50% v/v) and 50 µl of the adjusted bacterial suspensions were added to the wells of the microplates. To prevent dehydration, the plates were covered and incubated at 37 °C for 24 h with gentle shaking. A well-containing BHI broth medium and the corresponding bacterial concentrations were included for each bacterial strain to verify bacterial viability. The lowest concentration of each honey that inhibited the bacterial growth was then considered as the MIC. Following the determination of the MIC, aliquots of 50 µl from all wells with no visible bacterial growth were inoculated onto BHI agar plates and incubated for an additional 24 h at 37 °C. After incubation, the plates were inspected for bacterial colony formation, and the lowest concentration of honey where no bacterial growth was observed on the plates was identified as the minimum bactericidal concentration (MBC) endpoint [41].

Antimicrobial time-kill assays

The broth macro dilution method was used to perform a time-kill assay according to Hossain et al. [67]. Different concentrations of honey samples were tested at 0.5 × MIC, MIC, and 2 × MIC. 30 ml of Mueller–Hinton broth was inoculated with fresh P. aeruginosa cultures to achieve a concentration of 5 × 10^5 CFU/mL after adding the honey samples. The mixtures were incubated at 37°C in a shaking incubator, with 1 mL samples taken at 0, 2, 8, 16, and 24 h. Serial tenfold dilutions were made in sterile normal saline, and then spread onto Mueller–Hinton agar plates, which were incubated at 37°C for 24 h for colony counting. The log10 of CFU per mL was plotted against time to create a time-kill curve. A reduction of 99.9% (≥ 3 Log10) in CFU indicated bactericidal activity, while a decrease of less than 99.9% (< 3 Log10) or maintained original counts indicated bacteriostatic activity. Ciprofloxacin, an antibiotic for which P. aeruginosa is known to exhibit resistance, was used as the drug control.

Anti-biofilm assay

To assess the impact of Citrus and Thyme honey on the formation of biofilms by P. aeruginosa, the microtiter plate crystal violet (CV) assay was conducted as detailed in a previously published study [42]. In brief, bacterial cultures were grown overnight in culture tubes with shaking at 200 rpm under aerobic conditions at 37 °C using brain heart infusion. Following this, 10 μL of bacterial suspensions, adjusted to a turbidity of 0.5 McFarland standard (1.5 × 10^8 cells/ml), were combined with 190 μL of BHI culture medium and 20 μL of varying concentrations, 25% v/v (final concentration 2.3% v/v) to 100% v/v (final concentration 9% v/v) of the tested honey in each well of the microtiter plate. The plates were then incubated for 4 to 24 h at 37 °C, allowing sufficient time for the bacteria to adhere to the surface. A bacterial culture not exposed to honey served as the negative control. After the incubation period, the culture medium in each well was discarded to remove non-adherent cells, followed by washing with sterile phosphate-buffered saline (PBS) and allowing the wells to dry at room temperature. Subsequently, 200 μL of 0.2% crystal violet solution was added to each well, and the microplate was incubated at 37 °C for 15 min without agitation. The crystal violet dye binds to negatively charged biofilm components, facilitating the tracking and quantification of biofilm biomass [51]. After the washing steps, to extract the dye bound with biofilm-forming bacteria, 200 μL of 30% acetic acid solution was added to each well. Then, the absorbance of the extracted dye was then measured at a wavelength of 595 nm using a microplate reader. For quantitative analysis, three replicate wells were utilized for each treatment condition.

Scanning Electron Microscopy (SEM)

To illustrate the results, scanning electron microscopy (SEM) images of P. aeruginosa were obtained. These images were captured from samples treated with various concentrations of honey solutions, specifically 80% v/v (final concentration 7.3% v/v) and 100% v/v (final concentration 9% v/v) of Thyme and Citrus honey, as well as a control sample followed by the description of Balázs et al. [68].

Statistical analysis

The data are presented as means ± standard deviation (SD) and analyzed using one-way ANOVA. A significance level of p < 0.05 was used to identify statistically significant results. All statistical analyses were performed with IBM SPSS Statistics Version 22, and the graphs were created using Microsoft Excel (2016).

Authors’ contributions

M. A. contributed in methodology. S. A. contributed in methodology, writing original draft. P. Gh contributed to methodology, writing the original draft, review and editing. A. M. contributed to conceptualization, writing the original draft, and data curation. P. S. contributed to conceptualization, supervision, writing the original draft, review and editing.

Funding

No funding.

Data availability

Data is provided within the manuscript.

Declarations

Competing interests

The authors declare no competing interests.