Honey is a natural product produced by Apis mellifera bees from the nectar of flowers or plant exudates. After collection, it is transformed and combined with specific substances from the bees’ own reserves and stored in honeycombs for maturation [1] . Renowned for its health benefits, honey is considered a complex functional food, primarily due to its high concentration of carbohydrates such as fructose, glucose, and isomaltose [2] . In addition, it contains enzymes (e.g., α-amylase and glucose oxidase), proteins, amino acids, phenolic compounds, minerals, vitamins, and organic acids. Its composition is influenced by factors such as the geographic location and flowering season [3] [4] .

In vivo and in vitro studies have demonstrated that honey contains bioactive compounds with anti-inflammatory, antiproliferative, antimutagenic, antitumoral, antiglycemic, antioxidant, antibacterial, and antifungal properties [4] [5] . These compounds can prevent chain oxidation reactions associated with respiratory, cardiovascular, inflammatory, and gastrointestinal diseases [6] . The antibacterial activity of honey is attributed to factors such as hydrogen peroxide (H₂O₂), methylglyoxal (MGO), bee defensin-1, and phenolic compounds [3] . Its high sugar concentration induces osmotic stress, while its low water activity, acidic pH, and hygroscopic nature inhibit bacterial growth [7] .

Phytochemical factors render honey effective against pathogenic bacteria; however, these factors vary depending on the botanical origin, climate conditions, storage, and preservation methods [3] . The most significant antibacterial factor in honey is non-peroxide inhibitors, including polyphenols and certain antimicrobial peptides (e.g., defensin-1, defensin-2, hymenoptaecin, and apidaecin), which can alter bacterial membranes and penetrate pores in their cell walls [8] .

Membrane pore penetration is one of honey’s mechanisms of action. Substances such as flavonoids and phenolics, which interfere with bacterial growth, contribute to its pharmacological properties and, when combined with drugs, may provide an effective alternative [9] .

According to [10] and [8] , the mechanisms of action of honey (e.g., Manuka and Hungarian honeys) can induce structural and morphological changes in bacteria, alter bacterial membrane potential, disrupt growth and bacterial cell cycles, inhibit metabolism, affect efflux pump activity, disrupt quorum sensing, inhibit biofilm formation, and affect bacterial stress responses.

While many mechanisms remain incompletely described, studies indicate that these mechanisms vary between gram-positive and gram-negative bacteria, targeting specific cellular structures [8] , as shown in Figure 1 .

According to Tortora et al. [11] , drugs generally act on five main targets: inhibition of cell wall biosynthesis, inhibition of protein synthesis, nucleic acid (RNA and DNA) biosynthesis, alterations in cell membrane permeability, and folic acid metabolism. Antibiotics such as clarithromycin and rifampicin act on cell wall biosynthesis by blocking transcription and inhibiting RNA synthesis. Resistance mechanisms may include structural changes in RNA polymerase, reduced cellular permeability to antimicrobials, alterations in the receptor site on the 50S ribosomal subunit, and enzymatic inactivation [12] .

There is a need to study the ability of natural substances such as honey to assist the action of antibiotics, including bacterial membrane permeability. This approach would offer a promising alternative for treating infections caused by multidrug-resistant bacteria, which represent a growing public health threat [13] . Honey can transfer many therapeutic properties of plants and is considered to be an essential natural resource for new treatments, avoiding the side effects often associated with synthetic chemical drugs [14] .

Brazil ranks as the tenth-largest honey producer globally. Its physicochemical characteristics and quality depend on the climate, maturation stage, bee species, processing and storage technologies, and floral origin [15] [16] . For example, the floral origin influences the composition of phenolic compounds, proteins, minerals, sugars, and other components in honey [16] .

Studies conducted in municipalities within Brazil’s Central-West Region have shown that local honey exhibits anti-Helicobacter pylori potential, with up to 94% bacterial growth inhibition [17] [18] . The phytochemical composition of honey largely reflects the secondary metabolites present in the plants of its botanical origin, such as flavonoids and phenolic compounds, which are well-known for their antimicrobial properties, thereby substantiating the observed antibacterial activity. However, the effect of honey combined with antibiotics has not yet been studied.

Despite this honey antibacterial potential, studies exploring the combined effects of honey and antibiotics remain scarce. In this way, this study aimed to evaluate the bacterial outer membrane permeability mechanisms, by proceeding the synergistic effects of Apis mellifera honey from Brazil’s Central-West Region and with known antibiotics, as well as the bioactive compounds in the honey samples and its botanical origin. By testing the hypothesis that honey enhances antibiotic efficacy through membrane disruption, this research seeks to propose an innovative strategy for combating multidrug-resistant bacteria. For sure, other mechanisms of action must be evaluated later.

Our results provide insights into the effects of honey from Mato Grosso, Central-West Brazil, on the membrane permeability of S. epidermidis, H. pylori, and E. faecalis. Honey 1 from Cáceres, honey 3 from Cuiabá, honey 6 from Porto dos Gaúchos, and honey 5 from Santo Antônio do Leverger, when combined with rifampicin, demonstrated an effect on the membrane permeability of S. epidermidis, H. pylori, and E. faecalis, respectively.

Honey 1 presented two unique secondary pollen types: M. pudica (16.5%) and Protium sp. (24.4%) [28] . Both species are rich in bioactive compounds that contribute to their antibacterial activities ( Figure 4 ).

The resin of Protium sp. contains triterpenes such as α-amyrin and β-amyrin, triterpenic acids (ursolic and oleanolic), coumarins, and essential oils, which can destabilize cellular membranes and interfere with protein synthesis in bacteria such as Staphylococcus aureus and Escherichia coli [29] .

Mimosa pudica contains alkaloids, steroids, flavonoids, tannins, and saponins, which affect bacterial membrane permeability, leading to a loss of cellular integrity. These compounds also interfere with nucleic acid and protein synthesis, disrupting bacterial energy metabolism and inhibiting bacterial growth and survival [30] .

These effects are consistent with findings from research on Manuka honey, renowned for its high methylglyoxal (MGO) content, which exhibits comparable impacts on bacterial membrane permeability through depolarization and the inhibition of energy metabolism [7] . Similarly, studies on Hungarian honey emphasize the role of phenolic compounds in destabilizing bacterial membranes and disrupting biofilm formation, particularly in gram-positive bacteria [10] . Collectively, these findings underscore the universal potential of bioactive compounds derived from diverse botanical and natural sources to enhance bacterial membrane permeability and inhibit critical metabolic pathways.

This activity may be justified by the presence of these important minor pollen types, which, although not predominant, are sources of compounds that can act synergistically to enhance antibacterial activity. For example, flavonoids play a crucial role by inhibiting essential bacterial enzymes such as DNA gyrase, preventing bacterial DNA replication. Furthermore, these compounds increase bacterial cell membrane permeability, allowing toxic substances to enter and leading to cell death [31] .

Saponins interact directly with the lipids of the cell membrane, resulting in destabilization and eventual cell lysis. Terpenoids interfere with denosine triphosphate (ATP) production, essential for bacterial metabolism, while tannins form complexes with bacterial proteins, destabilizing the cell wall. According to De-Melo et al. [15] , this diversity in honey composition can influence its chemical profile, enabling various activities based on the predominance of specific individual components. The synergistic action of these compounds aligns with a broader pattern observed in the studies of De-Melo et al. [15] , demonstrating that the diversity of bioactive constituents in honey and plants directly enhances their capacity to increase bacterial membrane permeability, highlighting their potential as effective antimicrobial agents.

Fresh Honey 3, fresh Honey 6 and its ethyl acetate fraction exhibited an effect on the membrane permeability of H. pylori when combined with rifampicin and clarithromycin, respectively. Honey 3 and 6 presented as important minor pollen, Senna rugosa and Byrsonima sp. with frequencies of 13.7% and 10.3%, respectively [28] . The ability of these honey to affect H. pylori membrane permeability may be related to the presence of Senna rugosa and Byrsonima sp. ( Figure 5 ).

Fresh Honey 5 affected the membrane permeability of E. faecalis when combined with rifampicin, and also presented S. rugosa as an important minor pollen, with frequency of 10.3% [28] ( Figure 6 ).

Indeed, the leaf root extracts of this plant include bioactive compounds such as catechins, rutin, epigallocatechin derivatives, kaempferol glycosides, luteolin, dimetric and trimetric procyanidins, stilbenes, naphthopyranones, and flavanones [32] . Other compounds in the genus Senna include alkaloids (piperidine, cassin, and pyridine), anthraquinones (emodin, chrysophanol, aloe-emodin, rhein, physcion, chrysophanol-8-O-glucoside), flavonoids (kaempferol, quercetin, rutin, rhamnetin, cyanidin, hesperidin, gallocatechin, and luteolin), phenolics (tannins, gallic acid, and chlorogenic acid), glycosides, steroids (stigmasterol, β-sitosterol, and daucosterol), terpenoids (β-sitosterol, triterpenoids, sesquiterpenoids, and terpinolene), saponins, and volatile oils [33] .

These bioactive compounds exert antimicrobial activity. Alkaloids and flavonoids such as quercetin and kaempferol interfere with protein synthesis and compromise cell membrane integrity, while tannins and phenolic acids form complexes with bacterial proteins, inhibiting microbial growth [31] .

Anthraquinones, steroids, and glycosides also affect DNA replication and membrane permeability, while terpenoids and saponins primarily target the bacterial cell membrane. Terpenoids disrupt metabolic processes, and saponins cause membrane rupture, leading to cell death. Additionally, glycosides act as prodrugs activated in bacterial cells, and volatile oils disrupt bacterial membrane permeability and inhibit cellular respiration, enhancing antimicrobial efficacy [34] .

These mechanisms may explain the promising activity of S. rugosa against gram-negative bacteria such as Proteus mirabilis, Salmonella Typhimurium, Klebsiella pneumoniae, E. coli, Pseudomonas aeruginosa, and Shigella flexneri, as well as gram-positive bacteria such as S. aureus, S. epidermidis, Streptococcus agalactiae, and E. faecalis [35] [36] .

Honey 6 is an important minor pollen with a frequency of 10.1%. This effect may be related to the presence of Byrsonima sp. as an important minor pollen with a frequency of 10.1% [28] . Byrsonima sp. contains bioactive compounds such as phenolic acids (gallic, caffeic, coumaric, and chlorogenic), flavonoids (catechin, epicatechin, rutin, taxifolin, quercetin, and kaempferol), carotenoids (lutein, zeaxanthin, β-carotene, and β-cryptoxanthin), and volatile compounds (esters, aldehydes, and ketones) [37] .

These compounds are associated with various biological actions, including central nervous system modulation and wound healing, anti-inflammatory, antidiabetic, anticancer, antioxidant, antiviral, and antibacterial activities against K. pneumoniae, H. pylori, S. aureus, S. epidermidis, E. faecalis, Mycobacterium phlei, Bacillus subtilis, and Bacillus cereus [37] .

The compounds in Byrsonima sp. have shown promising activity against H. pylori through mechanisms such as biofilm disruption, inhibition of urease, DNA damage, and interference with protein synthesis, impairing vital bacterial functions [38] [39] .

The ethyl acetate fraction efficiently extracts intermediates such as flavonoids, tannins, phenolic acids, triterpenes, and proanthocyanidins found in both Byrsonima sp. and honey, which may contribute to the observed membrane permeability effect in H. pylori, highlighting this fraction’s antimicrobial role [38] .

Another factor that influences the effects of honey is the class of compounds in its different fractions. As demonstrated by Sugiyarto et al. [40] , ethyl acetate fractions contain phenolic compounds and alkaloids known for their ability to disrupt bacterial cell membranes and to inactivate enzymes, leading to cell lysis. Hexane fractions contain alkaloids and tannins, which also contribute to antimicrobial activity, albeit less intensely than phenolics. The ethyl acetate fractions, in particular, exhibited pronounced effects, aligning with findings by Sugiyarto et al. [40] which suggest that intermediate-polarity compounds, such as flavonoids and tannins, are especially effective in disrupting bacterial membranes.

The antibacterial mechanism of honey involves several biochemical factors that enhance its efficacy. Defensin-1, an antimicrobial peptide secreted by the bee hypopharyngeal glands, is one of the main compounds responsible for honey’s activity against both gram-positive and gram-negative bacteria [10] .

In addition to defensin-1, enzymes and phenolic compounds play crucial roles in combating bacterial infections. This synergistic action among honey components is particularly significant given the increasing resistance of bacteria to conventional antibiotics, positioning honey as a promising alternative for developing new antibacterial strategies [8] [10] .

The honey samples evaluated in this study had a TPC of 26.8 - 80 mg GAE/100 g and a TFC of 3.4 - 11.0 EQ/100 g of honey. Honey 1, 2, 3, and 6 had the highest TPC (40.4 - 80 mg GAE/100 g), comparable to monofloral honey from Minas Gerais in the Southeast Region of Brazil and various regions in Italy (4.88 - 40.7 mg GAE/100 g) [41] - [43] , and from Turkey and Tunisia (16.02 - 120.04 mg GAE/100 g) [44] .

In contrast, the TFC of the honey samples from the present study is lower than that of honey from the Southeast Region of Brazil and regions in Malaysia (29.17 - 91.25 mg EQ/100 g) [43] [45] .

The botanical origin of most of the analyzed honey samples aligns with honey from other areas of Mato Grosso. The samples that presented the best results shared similar floral diversity, particularly among secondary and important minor pollen species. Climate, biome, and botanical diversity play crucial roles in shaping honey’s composition, chemical profile, and bioactive properties, directly influencing its quality and potential therapeutic benefits [15] .

The bioactive compounds identified in the pollen of the honey samples analyzed in this study are likely to act synergistically, contributing to the observed antibacterial activity. This complex composition underscores the critical role of botanical diversity in shaping the therapeutic properties of honey, emphasizing that its antimicrobial efficacy arises from the integrated action of multiple bioactive constituents rather than the influence of a single compound.

Collectively, these findings strengthen the hypothesis that the unique phytochemical profiles of honey from diverse botanical origins contribute significantly to their antimicrobial potential. Further comparative studies on honey from different regions would provide deeper insights into the role of bioactive compounds in antimicrobial mechanisms, advancing their therapeutic applications.

When combined with rifampicin and clarithromycin, four honey samples from the Central-West Region altered the membrane permeability of S. epidermidis, H. pylori, and E. faecalis, an ability that is associated with the presence of bioactive compounds and the botanical origin of the honey. These results confirmed the hypothesis of this study: Combination of honey with antibiotics enhanced the efficacy of these drugs due to the disruption of the bacterial membrane.

The joint action of these bioactive compounds makes them promising candidates for improving the effectiveness of antimicrobial treatments by facilitating the entry of antibacterial substances into cells. Honey’s composition includes various compounds that act on multiple target sites in an additive or synergistic manner.

Additionally, the presence of bioactive compounds related to the species S. rugosa, Protium sp., M. pudica, and Byrsonima sp., specifically in honeys 1, 3, 5, and 6, contributed to their effects on membrane permeability.

Of note, the honey samples alone did not exhibit antibacterial activity against the three tested bacteria.

The combined use of honey and antibiotics can reduce the concentrations of drugs needed to achieve efficacy, thereby limiting the likelihood of resistance development and potentially reversing the susceptibility of some antibiotic-resistant bacteria.

The effect of honey on the permeability of the outer membrane of bacteria is undoubtedly the most significant finding of this study, but the mechanism underlying this permeability requires further investigation.

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) in the form of scholarship grant to Mariana Lenina Menezes Aleixo (CAPES/Demanda Social, Proc. No 1602288). The authors gratefully financial support for this project, Fundação de Amparo à Pesquisa do Estado de Mato Grosso (FAPEMAT—Proc. No. 62437/2013).