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The Galleria mellonella-Enteropathogenic Escherichia coli Model System: Characterization of Pathogen Virulence and Insect Immune Responses.

ABSTRACT: The use of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae), an economical insect model, for the study of enteropathogenic Escherichia coli (Migula) (EPEC), a diarrheagenic human pathogen, has been demonstrated previously but remains poorly understood. The present study characterizes the Galleria-EPEC system extensively for future studies using this system. We found that EPEC causes disease in G. mellonella larvae when injected intrahemocoelically but not orally. Disease manifests as increased mortality, decreased survival time, delayed pupation, decreased pupal mass, increased pupal duration, and hemocytopenia. Disease symptoms are dose-dependent and can be used as metrics for measuring EPEC virulence in future studies. The type III secretion system was only partially responsible for EPEC virulence in G. mellonella while the majority of the virulence remains unknown in origin. EPEC elicits insect anti-bacterial immune responses including melanization, hemolymph coagulation, nodulation, and phagocytosis. The immune responses were unable to control EPEC replication in the early stage of infection (≤3 h post-injection). EPEC clearance from the hemocoel does not guarantee insect survival. Overall, this study provided insights into EPEC virulence and pathogenesis in G. mellonella and identified areas of future research using this system.

SUBMITTER: Chen RY

PROVIDER: S-EPMC8315237 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 ?M) and minimum bactericidal concentration (MBC; 64 ?M) 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.

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The Galleria mellonella-Enteropathogenic Escherichia coli Model System: Characterization of Pathogen Virulence and Insect Immune Responses.

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