Galleria mellonella larvae exhibit a weight-dependent lethal median dose when infected with methicillin-resistant Staphylococcus aureus

Abstract

Galleria mellonella is a recognised model to study antimicrobial efficacy; however, standardisation across the scientific field and investigations of methodological components are needed. Here we investigate the impact of weight on mortality following infection with Methicillin-resistant Staphylococcus aureus (MRSA). Larvae were separated into six weight groups (180–300 mg at 20 mg intervals) and infected with a range of doses of MRSA to determine the 50% lethal dose (LD50), and the ‘lipid weight’ of larvae post-infection was quantified. A model of LD50 values correlated with weight was developed. The LD50 values, as estimated by our model, were further tested in vivo to prove our model. We establish a weight-dependent LD50 in larvae against MRSA and demonstrate that G. mellonella is a stable model within 180–260 mg. We present multiple linear models correlating weight with: LD50, lipid weight, and larval length. We demonstrate that the lipid weight is reduced as a result of MRSA infection, identifying a potentially new measure in which to understand the immune response. Finally, we demonstrate that larval length can be a reasonable proxy for weight. Refining the methodologies in which to handle and design experiments involving G. mellonella, we can improve the reliability of this powerful model.

Hospital sink traps as a potential source of the emerging multidrug-resistant pathogen Cupriavidus pauculus: characterization and draft genome sequence of strain MF1

“…Larvae were selected to be 21±1 mm in length and healthy, as determined by a uniform cream colour, with no indications of melanization. Larval length can be correlated to weight according to a linear regression, such that a length of 21±1 mm equates to a weight of 208.2±22.2 mg, which is within the optimum range (180–260 mg) for determination of median lethal dose (LD 50 ) using this model [ 59 ]. To prepare the C. pauculus MF1 inoculum, the isolate was grown overnight in 10 ml LB broth (Fisher Scientific, UK) at 37 °C and collected by centrifugation at 2500 g for 10 min.…”

“…Injected larvae were placed into sterile Petri dishes and incubated at 37 °C in the dark without food or water for 48 h. After 24 and 48 h, the percentage of live larvae in each treatment group was recorded. Larvae were recorded as dead when they met at least two of the following criteria: (i) obvious melanization, (ii) no response to touch, (iii) no correction when rolled on back [ 59 ]. For each time point, the LD 50 was calculated by plotting c.f.u./larva against percentage live larvae and interpolating the curve for 50 %.…”

Antibiofilm Activities of Borneol-Citral-Loaded Pickering Emulsions against Pseudomonas aeruginosa and Staphylococcus aureus in Physiologically Relevant Chronic Infection Models

“…Then 10-μL doses of diluted bacterial suspensions (2 × 10 5 CFU/ G. mellonella ) with treatments together were quickly (less than 2 min per experimental group to avoid bacterial death) injected into the right hind proleg of the larvae using a Hamilton syringe (Hamilton Company, Reno, NV, USA), and the infected larvae were incubated in the dark without food or water at 37°C for up to 72 h. The following control groups were included: larvae inoculated with PS, larvae inoculated with ethanol dissolved in PS, and larvae inoculated with S. aureus P8-AE1 only, respectively. Larvae were recorded as dead when they met the following criteria ( 62 ): (i) complete melanization, (ii) did not respond to touch, and (iii) could not correct itself when rolled onto its back. Experiments were repeated at least 3 times (at least 10 larvae per group).…”

Galleria mellonella—A Model for the Study of aPDT—Prospects and Drawbacks

“…Pathogens can also be administered orally, but commercially available larvae are in the final stage of maturation before pupation, during which time they are almost non-feeding. In this regard, oral administration is carried out using a special probe or at an early stage of maturation [ 52 , 53 , 54 ].…”