Antimicrobial Efficacy of Indolicidin Against Multi-Drug Resistant Enteroaggregative Escherichia coli in a Galleria mellonella Model

Abstract

Antimicrobial resistance against enteroaggregative Escherichia coli (EAEC), an emerging food-borne pathogen, has been observed in an increasing trend recently. In the recent wake of antimicrobial resistance, alternate strategies especially, cationic antimicrobial peptides (AMPs) have attracted considerable attention to source antimicrobial technology solutions. This study evaluated the in vitro antimicrobial efficacy of Indolicidin against multi-drug resistant enteroaggregative Escherichia coli (MDR-EAEC) strains and further to assess its in vivo antimicrobial efficacy in Galleria mellonella larval model. The minimum inhibitory concentration (MIC; 32 μM) and minimum bactericidal concentration (MBC; 64 μM) of Indolicidin against MDR-EAEC was determined by micro broth dilution method. Indolicidin was also tested for its stability (high-end temperatures, physiological concentration of salts and proteases); safety (sheep RBCs; HEp-2 and RAW 264.7 cell lines); effect on beneficial microflora (Lactobacillus rhamnosus and Lactobacillus acidophilus) and its mode of action (flow cytometry; nitrocefin and ONPG uptake). In vitro time-kill kinetic assay of MDR-EAEC treated with Indolicidin was performed. Further, survival rate, MDR-EAEC count, melanization rate, hemocyte enumeration, cytotoxicity assay and histopathological examination were carried out in G. mellonella model to assess in vivo antimicrobial efficacy of Indolicidin against MDR-EAEC strains. Indolicidin was tested stable at high temperatures (70°C; 90°C), physiological concentration of cationic salts (NaCl; MgCl2) and proteases, except for trypsin and tested safe with sheep RBCs and cell lines (RAW 264.7; HEp-2) at MIC (1X and 2X); the beneficial flora was not inhibited. Indolicidin exhibited outer membrane permeabilization in a concentration- and time-dependent manner. In vitro time-kill assay revealed concentration-cum-time dependent clearance of MDR-EAEC in Indolicidin-treated groups at 120 min, while, in G. mellonella, the infected group treated with Indolicidin revealed an increased survival rate, immunomodulatory effect, reduced MDR-EAEC counts and were tested safe to the larval cells which was concurred histopathologically. To conclude, the results suggests Indolicidin as an effective antimicrobial candidate against MDR-EAEC and we recommend its further investigation in appropriate animal models (mice/piglets) before its application in the target host.

Study on the Structure-Activity Relationship of an Antimicrobial Peptide, Brevinin-2GUb, from the Skin Secretion of Hylarana guentheri

“…Consistent with the haemolysis assay and cytotoxicity assay results, Brevinin-2GUb is much harmful to the wax moth than tB2U-K and tB2U-6K. A previous study demonstrated that the Indolicidin could significantly improve the survival rate, up to 95%, of Galleria mellonella infected by the enteroaggregative E. coli [ 42 ]. In our study, with the increase of the number of net charges, the survival rate of E. coli -infected wax moth is enhanced and the ability of tB2U-6K with the highest test concentration in vivo, whose mortality rate is just 11.11% within five days, is similar to the effect of norfloxacin with the concentration of 20 mg/kg, which makes tB2U-6K more potential to exert equal activity against pathogens in the physiological environment.…”

Synthetic Mimics of Antimicrobial Peptides for the Targeted Therapy of Multidrug‐Resistant Bacterial Infection

“…Indolicidin, a natural antimicrobial peptide, was compared. [24] The establishment of the wound model and the following MRSA inoculation are shown in Figure 7a. Firstly, the SD rats were randomly divided into three groups (control; PLL-POIM-CA; indolicidin).…”

Exploiting Lactoferricin (17–30) as a Potential Antimicrobial and Antibiofilm Candidate Against Multi-Drug-Resistant Enteroaggregative Escherichia coli

“…Furthermore, in vivo manifestations of EAEC have been established in various mammalian models, which involve logistical, ethical, and budgetary constraints ( Philipson et al, 2013 ; Kumar et al, 2016 ). Therefore, as an alternative in vivo model, Galleria mellonella larvae have recently been explored to evaluate the therapeutic potential of novel candidates including AMPs against various bacterial pathogens, including MDR-EAEC ( McCloskey et al, 2019 ; Vergis et al, 2019 ). Moreover, the short life span of larvae and their ability to simulate humans while exploring pathogens of public health significance ( Wojda, 2017 ; Cutuli et al, 2019 ; Blasco et al, 2020 ) extends their importance as an excellent in vivo model to screen novel therapeutics including AMPs.…”