Evaluation of greater wax moth larvae, Galleria mellonella, as a novel in vivo model for non-tuberculosis Mycobacteria infections and antibiotic treatments.

PURPOSE: To evaluate the suitability of Galleria mellonella larvae as an in vivo model and drug-screening tool for mycobacteria infections. METHODOLOGY: Larvae were infected using a range of inoculum sizes from a variety of rapid-growing mycobacteria, including strains of M. fortuitum, M. marinum and M. aurum. Larval survival, internal bacterial burden and the effects of amikacin, ciprofloxacin, ethambutol, isoniazid and rifampicin treatment on larval survival were measured over 144 h. The effects of these anti-mycobacterial drugs on phagocytosis and circulating haemocyte numbers were also examined using microscopy. RESULTS: Larval survival decreased after infection with M. fortuitum and M. marinum in a dose-dependent manner, but remained unaffected by M. aurum. Heat-killed bacteria did not cause larval death. Where antibiotic monotherapy was efficacious, larval survival post-infection increased in a dose-dependent fashion. However, efficacy varied between different antibiotics and species of infecting mycobacteria and, apart from rifampicin, efficacy in vivo correlated poorly with the in vitro minimum inhibitory concentrations (MICs). Combinations of antibiotics led to higher survival of infected larvae than antibiotic monotherapy. Selected antibiotic treatments that enhanced larval survival reduced the overall internal burden of infecting mycobacteria, but did not eradicate the pathogens. Administration of amikacin or ethambutol to uninfected larvae induced an initial transient increase in the numbers of circulating haemocytes and reduced the phagocytic rate of haemocytes in larvae infected with M. marinum. CONCLUSIONS: This report demonstrates the potential of employing a wax moth larvae model for studying fast-growing mycobacteria infections, and as a cheap, effective system for initial screening of novel treatments.

Effective immunosuppression with dexamethasone phosphate in the Galleria mellonella larva infection model resulting in enhanced virulence of Escherichia coli and Klebsiella pneumoniae.

The aim was to evaluate whether immunosuppression with dexamethasone 21-phosphate could be applied to the Galleria mellonella in vivo infection model. Characterised clinical isolates of Escherichia coli or Klebsiella pneumoniae were employed, and G. mellonella larvae were infected with increasing doses of each strain to investigate virulence in vivo. Virulence was then compared with larvae exposed to increasing doses of dexamethasone 21-phosphate. The effect of dexamethasone 21-phosphate on larval haemocyte phagocytosis in vitro was determined via fluorescence microscopy and a burden assay measured the growth of infecting bacteria inside the larvae. Finally, the effect of dexamethasone 21-phosphate treatment on the efficacy of ceftazidime after infection was also noted. The pathogenicity of K. pneumoniae or E. coli in G. mellonella larvae was dependent on high inoculum numbers such that virulence could not be attributed specifically to infection by live bacteria but also to factors associated with dead cells. Thus, for these strains, G. mellonella larvae do not constitute an ideal infection model. Treatment of larvae with dexamethasone 21-phosphate enhanced the lethality induced by infection with E. coli or K. pneumoniae in a dose- and inoculum size-dependent manner. This correlated with proliferation of bacteria in the larvae that could be attributed to dexamethasone inhibiting haemocyte phagocytosis and acting as an immunosuppressant. Notably, prior exposure to dexamethasone 21-phosphate reduced the efficacy of ceftazidime in vivo. In conclusion, demonstration of an effective immunosuppressant regimen can improve the specificity and broaden the applications of the G. mellonella model to address key questions regarding infection.