Galleria mellonella Infection Model Identifies Both High and Low Lethality of Clostridium perfringens Toxigenic Strains and Their Response to Antimicrobials.

Sammy Kay,Ronald Dixon,Joseph Edwards,Joseph Brown

doi:10.3389/fmicb.2019.01281

Sammy Kay, Ronald Dixon + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2019.01281

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Abstract

Research progress into mechanisms of the anaerobe Clostridium perfringens and associated diseases has been frustrated by the lack of reliable infection models. Wax moth larvae (Galleria mellonella) have emerged as a viable alternative to other models of infection since they are economic, survive at 37°C and require no specialist equipment. This study aims to establish to what extent G. mellonella larvae can be used to study the virulence of C. perfringens strains and its suitability for studying novel treatment strategies by an improved time-lapse approach to data collection. Mortality and morbidity rates of larvae challenged with 105 CFU of C. perfringens isolates from various sources were observed over 72 h and dose response data obtained. Phenoloxidase enzyme activity was investigated as a marker for immune response and tissue burden assessed by histopathological techniques. Results demonstrate that C. perfringens is pathogenic toward G. mellonella although potency varies dramatically between C. perfringens isolates and the reference strain ATCC 13124 was shown to be avirulent. Infection with C. perfringens strains activated the melanisation pathway resulting in melanin deposition but no increase in enzyme activity was observed. Efficacy of antibiotic therapy (penicillin G, bacitracin, neomycin, and tetracycline) administered parenterally to some extent correlates with that of in vitro analysis. The findings suggest G. mellonella might be a useful in vivo model of infection and convenient as a pre-screening assay for virulence of C. perfringens strains or as a simple, cheap and rapid in vivo assay in the first stage development of novel therapeutics against anaerobes.HIGHLIGHTS–Potential novel in vivo model for the study of Clostridium perfringens infection.–Novel time-lapse approach to data collection.–First report of the pathogenicity of C. perfringens toward G. mellonella.–First report of the efficacy of antibiotic therapy in response to C. perfringens infection in G. mellonella.

Highlights

Insect models have been shown to be helpful in our understanding of the virulence of bacterial pathogens in humans (Tsai et al, 2016)
The larval stage of the Greater Wax moth (Galleria mellonella) has emerged as an insect model of particular value since it survives at 37◦C, which is essential for demonstration of specific microbial virulence factors (Smoot et al, 2001)
We have investigated the pathogenicity of C. perfringens in G. mellonella larvae

Summary

Introduction

Insect models have been shown to be helpful in our understanding of the virulence of bacterial pathogens in humans (Tsai et al, 2016). Insects share some similarities with mammalian processes and possess a basic innate immune system (Ramarao et al, 2012). Alternative model organisms such as the zebra fish (Danio rerio), silkworm (Bombyx mori), tobacco hornworm (Manduca sexta), fruit fly (Drosophila melanogaster), and nematodes (Caenorhabditis elegans) (Browne et al, 2013) are not associated with the same ethical considerations as in the use of higher vertebrates (Peterson et al, 2008). Whilst G. mellonella has not evolved an adaptive immune response, they possess a semi-complex cellular and humoral innate immunity. This innate system, in insects, shares remarkable similarities to that of mammals (Jander et al, 2000). The humoral response consists of various antimicrobial peptides, opsonins, extracellular nucleic acid traps and the phenol-oxidase pathway as described recently in detail by Tsai et al (2016)

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