In vitro and in vivo toxicity and antibacterial efficacy of melittin against clinical extensively drug-resistant bacteria

Abstract

Background: Melittin is one of the most studied antimicrobial peptides, and several in vitro experiments have demonstrated its antibacterial efficacy. However, there is evidence showing melittin has non-promising effects such as cytotoxicity and hemolysis. Therefore, concerns about unwanted collateral toxicity of melittin lie ahead in the path toward its clinical development. With these considerations, the present study aimed to fill the gap between in vitro and in vivo studies.

Methods: In the first step, in vitro toxicity profile of melittin was assessed using cytotoxicity and hemolysis tests. Next, a maximum intraperitoneal (i.p.) sub-lethal dose was determined using BALB/c mice. Besides toxicity, antimicrobial efficacy of melittin against extensively drug-resistant (XDR) Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and KPC-producing Klebsiella pneumonia (KPC-KP) pathogens were tested using both in vitro and in vivo methods.

Results: Melittin showed extensive hemolysis (HD₅₀ = 0.44 µg/mL), and cytotoxicity (IC₅₀ = 6.45 µg/mL) activities with i.p. LD₅₀ value of 4.98 mg/kg in BALB/c mice. In vitro antimicrobial evaluation showed melittin MIC range from 8 to 32 µg/mL for the studied pathogens. Treatment of infected mice with repeated sub-lethal doses of melittin (2.4 mg/kg) displayed no beneficial effect on their survival and peritoneal bacterial loads.

Conclusions: These results indicate that melittin at its safe dose could not exhibit antimicrobial activity, which hinders its application in clinical practice.

Keywords: Animal model; Antimicrobial Drug Resistance; Antimicrobial Peptides; Melitten; Sepsis; Toxic Potential.

Fig. 1

In vitro and in vivo toxicity results of melittin peptide. (A) Cytotoxicity of melittin peptide (0.625-10 µg/mL) on human primary fibroblast cells by the MTT assay. The half-maximal inhibitory concentration (IC₅₀) of melittin to fibroblast cells was 6.45 µg/mL. (B) Human blood hemolysis assay for melittin peptide (0.061–15.6 µg/mL) in which Triton X-100 and phosphate buffered saline (PBS) were used as positive and negative controls, respectively. Results (cytotoxicity and hemolysis) are expressed as mean ± SD. Statistical differences in relation to the control group (100 %) are represented as * p < 0.05, ** p < 0.01 and ***p < 0.001. (C) In vivo intraperitoneal LD₅₀ (median lethal dose) determination for melittin using BALB-c mice (n = 5)

Fig. 2

Photomicrographs of liver and kidney sections stained with hematoxylin and eosin dyes (400X, scale-bars = 50 μm). (A) Liver micrograph from an untreated mouse, displaying no damage. (B) Liver micrograph from multi-dose administration (16/8 h intervals) of melittin (2.4 mg/kg), displaying no damage. (C) Kidney micrographs from an untreated mouse showing no damage. (D) Kidney micrograph from multi-dose administration (16/8 h intervals) of melittin (2.4 mg/kg), also indicating no damage

Fig. 3

Time-kill curves performed on A. baumannii (A), S. aureus (B), and K. pneumonia (C) treated with 1×MIC (green line), 2×MIC (yellow line), and 4×MIC (red line) doses of melittin peptide

Fig. 4

The results of the survival experiment determined the efficacy of melittin, along with untreated and colistin/vancomycin controls. The neutropenic mice (n = 10 each) were inoculated with a lethal amount of A. baumannii (A-10⁷ CFU/mouse), S. aureus (B-10⁷ CFU/mouse), and K. pneumonia (C-10⁸ CFU/mouse) and treated with phosphate buffered saline (PBS), melittin (2.4 mg/kg) and colistin/vancomycin as described in methods. The difference was defined as significant as *p < 0.5

Fig. 5

Results of peritoneal CFU assessment to determine the efficacy of melittin along with untreated and colistin/vancomycin controls. The neutropenic mice (n = 15 each group) were inoculated with a sub-lethal amount of A. baumannii (A-10⁶ CFU/mouse), S. aureus (B-10⁶ CFU/mouse), and K. pneumonia (C-10⁷ CFU/mouse) and treated with phosphate buffered saline (PBS), melittin (2.4 mg/kg) and colistin/vancomycin as described in methods. Results are expressed as mean ± SD. (n = 5 each studied time point). Difference were defined as significant as *p < 0.5, **p < 0.01, and ***p < 0.001 between groups