Refining the Galleria mellonella Model by Using Stress Marker Genes to Assess Clostridioides difficile Infection and Recuperation during Phage Therapy

Abstract

The Galleria mellonella is an effective model for probing Clostridioides difficile interactions with phages. Despite valuable insights from this model, the larvae are not easily amenable to assessing detailed clinical responses to either bacteria or phages. Here, larval survival, colonisation and toxin levels were compared to expression profiles of 17 G. mellonella stress genes to monitor Clostridiodes difficile infection (CDI), and recuperation during phage therapy. The larvae were infected with a ribotype 014/020 isolate and treated with an optimised phage cocktail. Larvae treated prophylactically with phages and the phage-control larval group were protected, showing the highest survival, and low C. difficile colonisation and toxin rates, compared to co-infection, remedial and bacterial-control larval groups. Expression of growth (9) and reproduction (2) genes were enhanced within prophylaxis and phage-control larval groups compared to the co-infection, remedial and bacterial control groups. In contrast, expression of infection (2), humoral (1) and cellular (3) immunity genes declined in the prophylactic and phage-control groups but increased in the co-infection, remedial and bacterial control larvae. The molecular markers augment the survival, colonisation and toxin data and allow detailed monitoring of CDI and recovery. This data support the use of stress marker genes as tools to analyse clinical symptoms in this model.

An Optimized Bacteriophage Cocktail Can Effectively Control Salmonella in vitro and in Galleria mellonella

“…Previous data from our laboratory and others have shown that the Galleria model is useful and that it correlates to large scale animal models. More broadly, the G. mellonella model is cheap and is a valuable biological tool to study the virulence and pathogenicity of pathogens including Salmonella , and pharmacokinetics of anti-infectives including phage therapy ( Thomas et al, 2013 ; Nale et al, 2016a , 2020 ). In this study, G. mellonella larvae were colonized with representative isolates and detail evidence of the efficacy of different phage therapeutic regimens to prevent and reduce colonization in the model was obtained and is presented.…”

Preclinical data and safety assessment of phage therapy in humans

“…They also require less expertise to work with, and their cells are similar enough to human cells to allow comparable impact assessment and inference [ 44 ]. Phages have been shown to be stable in these models and the route of phage administration depends on the pathogen under investigation [ 44 , 59 , 67 ]. Through colonisation, survival and immune response studies, these models have been instrumental in determining phage therapeutic potential [ 44 , 59 , 67 ].…”

“…Phages have been shown to be stable in these models and the route of phage administration depends on the pathogen under investigation [ 44 , 59 , 67 ]. Through colonisation, survival and immune response studies, these models have been instrumental in determining phage therapeutic potential [ 44 , 59 , 67 ]. In a recent phage application study, carbapenem-resistant Acinetobacter baumannii infected Galleria mellonella larvae were treated with phage Bφ-R2096 via the proleg [ 68 • ].…”

“…Histological studies also showed less tissue damage and melanisation in the fat body wall and muscle layer in the phage treated group compared to the controls [ 68 • ]. Similarly, our work showed that phages could prevent C. difficile colonisation in insects when an optimised phage cocktail was administered into the larvae via oral gavage and that multiple doses of phages could be used to successfully remove bacterial colonisation and prolong the life of the larvae [ 44 , 67 ].…”

Special Issue: Bacteriophage Treatment as an Alternative Technology to Inactivate Pathogenic Bacteria: A Generalized Worldwide Growing Acceptance

“…Five of the published papers in this Special Issue refer to some strategies to prevent the development of phage-resistant mutants, namely by the use of combined approaches, such as the use of antibiotics [12,20], fatty acids [18], essential oil [19], and mucin [21] during phage treatment. Four of the papers include in vivo studies in animals [14,15,22] and plants [23].…”