Antibacterial Activity of Jelleine-I, a Peptide Isolated from Royal Jelly of Apis mellifera, Against Colistin-Resistant Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae can acquire resistance mechanisms to colistin and present a pan-resistant phenotype. Therefore, new alternative agents are imperative to control this pathogen, and the peptide Jelleine-I stands out as a promising prototype. Here, the antibacterial activity of Jelleine-I against clinical isolates of colistin-resistant K. pneumoniae (CRKP) was investigated. Antimicrobial activity was assessed by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time kill-curve assay. The release of 260 nm-absorbing materials (DNA/RNA) and the release of proteins were used in the lysis assay. Anti-biofilm activity was studied in microplates. In vivo activity was determined by the lethality assay using Tenebrio molitor larvae. The results show that the MIC of Jelleine-I ranged from 16 to 128 µM and the MBC was on average 128 µM. Jelleine-I at 200 µM killed all CRKP cells in suspension (10⁶ colony-forming units (CFU)/mL) after 150 min of incubation. Jelleine-I acts on the CRKP cell membrane inducing lysis. Biomass and viability of CRKP-induced biofilms are reduced after treatment with Jelleine-I, and the use of this peptide in T. molitor larvae infected with CRKP reduces lethality and improves overall larval health. In conclusion, Jelleine-I is a potential prototype for the development of new antimicrobial agents.

Keywords: Jelleine-I; antimicrobial peptide; antimicrobial pharmacology; colistin-resistant Klebsiella pneumoniae; drug development; pandrug-resistant bacteria.

Figure 1

Time-kill curve with multiple concentrations of Jelleine-I (50, 100, and 200 µM) against a colistin-resistant Klebsiella pneumoniae lineage (isolate 699). The plot shows the number of logarithmic colony-forming units per milliliter (Log₁₀ CFU/mL). Bacterial cells untreated were used as negative control (black circles).

Figure 2

Determination of bacteriolysis through of assay of overflow of 260 nm-absorbing materials (DNA/RNA) (A) and through a test of release of proteins (B). The experiment was done in triplicate for statistical significance. One asterisk (*) indicates statistically different compared to the control with 0.05 < p-value < 0.01. Two asterisks (**) indicate statistically different compared to the control with 0.01 < p-value < 0.001. Three asterisks (***) indicate statistically different compared to the control with 0.001 < p-value < 0.0001. Four asterisks (****) indicate statistically different compared to the control with p-value < 0.0001. The results were analyzed by one-way variance analysis (ANOVA) with Dunnett post-hoc.

Figure 3

Effect of different concentrations of Jelleine-I (32, 80, and 160 µM) on biomass (A) and viability (B) of biofilms formed by a hypervirulent colistin-resistant Klebsiella pneumoniae (isolate 699). The experiment was done in sextuplicate for statistical significance. Two asterisks (**) indicate statistically different compared to the control with 0.01 < p-value < 0.001. Four asterisks (****) indicate statistically different compared to the control with p-value < 0.0001. The results were analyzed by one-way variance analysis (ANOVA) with Dunnett post-hoc.

Figure 4

Survival curves and clinical score of Tenebrio molitor larvae. (A) Survival of larvae after administration of different doses of Jelleine-I (1, 5, 10, 50, and 100 µg) to assess the in vivo toxicity of the peptide. (B) Survival curve of larvae infected with a hypervirulent clinical isolate of colistin-resistant Klebsiella pneumoniae (CRKP; isolate 699). Groups (n = 10) infected with CRKP and treated with Jelleine-I (10, 50, and 100 µg) after 2 h post-infection. Larvae (n = 10) infected and injected with sterile saline (0.9% NaCl) were used as the negative control. (C) Clinical score of larvae infected and treated with Jelleine-I (10, 50, and 100 µg) or saline. One asterisk (*) indicates statistically different compared to the control with 0.05 < p < 0.01. The lethality curves were constructed using the Kaplan–Meier estimator and statistically analyzed using the log-rank test. The clinical score results were analyzed using the Kruskal–Wallis test with Dunn’s post-test. The results are expressed as median and interquartile range (P₂₅–P₇₅).