Efficiency of bacteriophage therapy against Cronobacter sakazakii in Galleria mellonella (greater wax moth) larvae

Abstract

Cronobacter sakazakii, an opportunistic pathogen found in milk-based powdered infant formulae, has been linked to meningitis in infants, with high fatality rates. A set of phages from various environments were purified and tested in vitro against strains of C. sakazakii. Based on host range and lytic activity, the T4-like phage vB_CsaM_GAP161, which belongs to the family Myoviridae, was selected for evaluation of its efficacy against C. sakazakii. Galleria mellonella larvae were used as a whole-animal model for pre-clinical testing of phage efficiency. Twenty-one Cronobacter strains were evaluated for lethality in G. mellonella larvae. Different strains of C. sakazakii caused 0 to 98% mortality. C. sakazakii 3253, with an LD50 dose of ~2.0×10(5) CFU/larva (24 h, 37 °C) was selected for this study. Larvae infected with a dose of 5×LD50 were treated with phage GAP161 (MOI=8) at various time intervals. The mortality rates were as high as 100% in the groups injected with bacteria only, compared to 16.6% in the group infected with bacteria and treated with phage. Phage GAP161 showed the best protective activity against C. sakazakii when the larvae were treated prior to or immediately after infection. The results obtained with heat-inactivated phage proved that the survival of the larvae is not due to host immune stimulation. These results suggest that phage GAP161 is potentially a useful control agent against C. sakazakii. In addition, G. mellonella may be a useful whole-animal model for pre-screening phages for efficacy and safety prior to clinical evaluation in mammalian models.

‘Get in Early’; Biofilm and Wax Moth (Galleria mellonella) Models Reveal New Insights into the Therapeutic Potential of Clostridium difficile Bacteriophages

“…Work is ongoing to examine other MOIs. Although previous work reported the inoculation of bacteria/phages by intrahaemocoelic injection (Abbasifar et al, 2014; Beeton et al, 2015), we found ingestion via the oral route more efficient at inducing colonization and producing reproducible therapy results. The observed higher efficacy of the phages when used prophylactically rather than with remedial treatment is consistent with a previous report (Beeton et al, 2015).…”

“…Although the insect is used as an infection model for various pathogens (Mukherjee et al, 2010; Ramarao et al, 2012; Hill et al, 2014; Beeton et al, 2015) and antimicrobial testing (Seed and Dennis, 2009; Abbasifar et al, 2014; Beeton et al, 2015; Olszak et al, 2015), to our knowledge this report is the first published data to describe its use to study C. difficile colonization and phage treatment. We established that 10 5 C. difficile cells were sufficient for colonization to occur in the larvae and to cause the first and total mortality at 24 and 60 h respectively post-infection.…”

“…On arrival, the larvae were stored immediately at 4°C and used within 1 week. Larvae with approximate weight of 0.25–0.30 g were selected for in vivo analysis (Abbasifar et al, 2014). The larvae were surface-sterilized with cotton swaps dipped in 70% ethanol.…”

Assessing phage therapy against Pseudomonas aeruginosa using a Galleria mellonella infection model

“…Mammalian models are crucial for testing the efficacy of phages prior to human trials, but drawbacks include the need for sufficient infrastructure, substantial costs as well as the need for ethical approval. G. mellonella larvae have been used to examine numerous host-pathogen interactions ranging from studies of pathogenicity to antimicrobial activity, with a small number of these examining the potential for phage therapy [12][13][14].…”

Cronobacter sakazakii: stress survival and virulence potential in an opportunistic foodborne pathogen

“…Two studies in particular have characterized the ability of bacteriophages to reduce and control growth of C. sakazakii in PIF. 34,35 However, the application of bacteriophages is not limited to environmental control but can also be used in vivo, as has been demonstrated in insect and mouse infection models, where bacteriophages were shown to protect insect larvae 36 or reduce bacterial load in urinary tract infections in mice. 37 Bacteriophages have furthermore been successfully used as immuno-stimulants to increase expression of pro-inflammatory host factors.…”