Chemical Characterization and Antibacterial Activity of Royal Jelly Against Multidrug-Resistant Pathogens

Given the known antibacterial properties of royal jelly (RJ), we hypothesize that royal jelly could inhibit priority multidrug-resistant (MDR) bacteria, including different strains of vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Klebsiella pneumoniae (CRKP) and Acinetobacter baumannii (CRAB). We further propose that the antibacterial efficacy of royal jelly may be influenced by its chemical composition and by inter- and intraspecies variability among MDR pathogens.

Royal jelly samples were collected from five beekeepers (RJ1-RJ5) in the Mediterranean and continental regions of Croatia. Chemical profiling was conducted using solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS) and Fourier-transform infrared (FTIR) spectroscopy, together with separate assays to measure antioxidant capacity (ABTS) and quantify the content of bioactive compounds. Antibacterial activity was assessed by agar well diffusion assay and by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against 20 MDR strains of VRE, MRSA, CRKP and CRAB, selected from 85 isolates using repetitive sequence-based PCR (rep-PCR) genotyping. MDR status was confirmed by standard susceptibility testing.

All royal jelly samples showed strong antioxidant activity and high amounts of bioactive compounds, with RJ1 consistently exhibiting the highest contents of ABTS, polyphenols, flavonoids and proteins. FTIR analysis revealed variations in carbohydrate and lipid composition among samples, while protein content remained relatively uniform, and indicated the highest mass fractions of sugars, lipids and proteins in RJ1. GC-MS identified octanoic acid (48.09-83.07 %) as the predominant volatile compound, especially abundant in RJ1 and RJ4. Despite some variability in chemical profiles, both chemical composition and antibacterial activity were comparable between samples from the Mediterranean and continental regions. All royal jelly samples inhibited MDR bacteria, suggesting a potential synergistic effect of crude royal jellies, with inhibition zones ranging from 11.8 (CRKP) to 16.8 mm (MRSA). A. baumannii was most susceptible (MIC/MBC=27.2 µg/mL), while E. faecium was the most resistant (MIC=96.6 µg/mL, MBC=126.4 µg/mL). Beyond interspecies differences, pronounced strain-level variability in antibacterial response was also observed.

This is the first study to simultaneously evaluate the antibacterial activity of royal jelly against multiple strains of clinically relevant MDR pathogens alongside comprehensive chemical profiling. Importantly, it reveals for the first time that the efficacy of royal jelly varies not only between species but also among strains within the same species, emphasizing the need to consider strain-level differences in future assessments.