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.

Keywords: Clostridiodes difficile infection; Clostridioides difficile; Clostridium difficile; Galleria mellonella; bacteriophage therapy; qPCR.

Figure 1

Kaplan–Meier survival curve of G. mellonella infected with CD105LC2, a clinically relevant ribotype 014/020 isolate, and treated with a 4-phage cocktail in 3 therapy regimens. The insects were treated prophylactically or remedially with the phage cocktail, or with a co-infection of the bacteria and phages. The treatment regimens were compared to Controls 1–5 for untreated insects, or those treated with empty needles, BHI, bacteria or phage cocktail, respectively, as shown in Figure 1 and Table 1. The prophylactic regimen performed best when compared with the remedial regimen (p < 0.01) and co-infection group (p < 0.01). Four larvae were examined per treatment group and the experiment was repeated on three occasions. Data was analysed using the Log Rank Mantel–Cox test in GraphPad Prism 8.

Figure 2

Bacterial and phage load recovered from G. mellonella larvae at the end of the 72nd hour. Four larvae in each of the eight treatment groups were treated with 10⁵CFU/larva of clinical isolate CD105LC2 culture and/or 10⁶ PFU/larva of phage cocktail (in 10 µL volumes) at time points shown in Table 1. Colonisation was ascertained by recovering (A) C. difficile from the guts of insects on CCEY selective plates and (B) phages using double BHI agar method. The lowest amount of bacteria was recovered from the prophylactic treatment (compared to the bacterial control), indicative of successful phage infection (p < 0.0001). The co-infection (with phage and bacteria) treatment also yielded a reduced bacterial load (p < 0.0001). The experiment was repeated three times and data were analysed using the Shapiro–Wilk normality test and Mann–Whitney–Wilcoxon test. n.s. = no significant difference, **** = significance at p < 0.0001.

Figure 3

Expression of 17 G. mellonella growth markers during C. difficile colonisation and phage therapy regimens. The relative expression levels of (A, B) 9 growth, (C) infection and reproduction and (D) cellular and humoral genes were examined during C. difficile colonisation in G. mellonella larvae and when undergoing different phage therapy regimens. G. mellonella larvae were either untreated (1), treated with empty needles (2), 10 µL of either BHI (3), or 10⁵ CFU/mL of CD105LC2 bacterial culture (4). The larvae were also treated with a co-infection of the phages (10⁶ PFU/mL) and bacteria (5), remedially (6) or prophylactically (7), and compared to a group of larvae treated with just the phage cocktail (8). Experiments were repeated three times. Values were normalised using the housekeeping gene 18S rRNA and analysed using GraphPad Prism 8.