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

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.

Keywords: alternative model; greater wax moth; host-pathogen interactions; infectivity; insect model.

FIGURE 1

Melanisation scoring system adapted from Senior et al. (2011).

FIGURE 2

(A) Schematic of the imaging setup used to record time-lapse photography of larvae 1: Black felt shroud to reduce the effects of natural light on quality of images; 2: Camera: Logitek C100 5 mp; 3: table lamp for consistent lighting; 4: perspex incubator: Stuart Scientific SI140; 5: Greaseproof paper for better contrast in images. (B) Automated syringe pump setup used for inoculating larva. (C) Injections were administered through the rear pro-leg. (D) Example of image output from SkyStudio Pro time-lapse software.

FIGURE 3

Average survival percentage at 72 h incubation for G. mellonella infected with 10 μL of 10⁷ CFU/mL of respective isolates. Error bars indicate standard error. Results indicate a variance in lethality of C. perfringens infection toward G. mellonella. ^∗ = Mann–Whitney U-test comparing larval survival to uninfected control.

FIGURE 4

Mean melanisation score of pooled data, 72 h post inoculation of 10 μL of a range of dilutions from 10⁷ to 10³ CFU/mL of Clostridium perfringens JBCNJ055 and JBFR014. Scoring system is as follows: 1 = healthy, 2 = < 50% melanisation, 3 = > 50% melanisation, 4 = dead. Data shown is pooled from three distinct repeats and error bars represent standard deviation. Injection with 10 μl of 10⁷ was required to cause potent melanisation resulting in scores higher than 3. Survival percentages of G. mellonella after inoculation with 10 μL of a range of dilutions 10⁵–10 CFU/mL of C. perfringens.

FIGURE 5

Kaplan–Meier survival distributions for dose dependent challenges of JBCNJ055 and JBFR014. ATCC 13124 is avirulent at all dosage levels. Three repeats of each experiment were pooled. Results were collated as % survival. Strikethrough indicates censored data (unaffected larvae). All levels of infection data are significant (P < 0.001) [Mantel (cox) log rank test] for both isolates investigated indicating larval survival is based upon the number of bacteria injected.

FIGURE 6

Comparison of the pathogenicity of cell-free supernatants and washed cultures toward G. mellonella of three distinct C. perfringens isolates with differing virulence gene composition. Cell-free supernatants were able to cause disease but with varying degrees of potency. Error bars represent standard deviation.

FIGURE 7

Comparison of G. mellonella histopathology of control and infected groups after 72 h incubation after inoculation with 10⁵ CFU of JBCNJ055. Sections were stained with H+E, Gram-stain and Masson Fontana (highlighting melanin deposition). Infected groups show distinct loss of tissue structure, systemic proliferation of Gram-positive bacteria and a large production of melanin within peripheral tissues indicative of systemic pathogenicity. Scale bars represent 1 mm. C, Cuticle; M, Muscle layer; N/Nd, Nodules; Fb, Fat body; Nc, Necrosis.

FIGURE 8

Average melanisation score for larvae infected with 10 μL of 10⁷ JBCNJ055 with a further injection of either penicillin G (2 mg/kg), bacitracin (64 mg/kg), tetracycline (64 mg/kg), or neomycin (2400 mg/kg). Healthy larvae with no signs of infection were scored 1. Larvae showing nodulation and lateral line melanisation scored 2. Larvae exhibiting systemic melanisation scored 3. Dead, fully pigmented larvae scored 4. There is a potent increase in melanisation score when penicillin G is used in response to the infection.