Galleria mellonella as an Infection and Antibiotic Treatment Model for Acinetobacter baumannii

Summary

The Galleria mellonella insect infection model provides an easy, affordable, and relevant platform to inform on bacterial virulence and screen antimicrobial compounds. Using Acinetobacter baumannii, we show how antibiotic minimum inhibitory concentration data and a direct treatment approach can guide more advanced testing in a meaningful way.

Abstract

Galleria mellonella, commonly known as the greater wax moth or waxworm, is an insect infection model that provides researchers with an informative, simple, and economically feasible way to test the virulence of bacterial pathogens and potential treatment regimens against them. One such pathogen is Acinetobacter baumannii, a World Health Organization top-priority pathogen and a global health threat. The prevalence of deadly, multidrug-resistant A. baumannii in hospital settings, causing > 100,000 deaths in 2021, makes finding new treatment options paramount. A crucial piece of information needed to help eradicate a bacterial infection using antimicrobial compounds is the minimum inhibitory concentration-the lowest dose of a compound that can clear the infection. This value can be determined initially in vitro but then must be tested in a relevant infection model in vivo.

Using the waxworm infection model and three different strains of A. baumannii-a virulent type strain, a hypervirulent clinical strain, and a virulent environmental strain-we demonstrate how to use minimum inhibitory concentration data to guide initial antibiotic treatment testing. We also compare two assay styles: infection followed by treatment (infect-wait-treat) and infection and treatment together (infect-and-treat). The results, showing similar trends in waxworm survival between both methods, demonstrate that the infect-and-treat protocol can be as informative as the more traditional infect-wait-treat method, with the benefit of saving valuable time and resources.

Introduction

The Galleria mellonella (greater wax moth or waxworm) invertebrate animal infection model is an increasingly popular choice among researchers to gain insight into the virulence of a bacterial pathogen, test antimicrobial efficacy, and unravel the intricate cellular processes that confer multidrug resistance. Numerous studies have been conducted on a variety of Gram-positive and Gram-negative pathogenic bacteria^1,2,3,4, as well as intracellular pathogens^5,6, enteric bacteria<....

Protocol

Cultures of pathogenic bacteria and waxworms that received the pathogenic bacteria must be disposed of as biohazardous waste according to institutional procedures.

1. Experimental preparation

1. Prepare autoclaved solutions of saline (0.85% w/v sodium chloride, 100 mL) and ultrapure water (100 mL). Prepare 70% ethanol (25 mL), diluting from a 95% or 100% stock as needed with sterile ultrapure water (see Table of Materials).
2. Prepare 1 mL of a concentrated stock solution of each antimicrobial compound of interest in the appropriate solvent (organic or aqueous) and store according to the pro....

Results

It is very important to take the time to sort, weigh, and clean the G. mellonella larvae prior to their use as indicated here and elsewhere^4,16,27,28 to ensure comparative results (Figure 1A). This process can take many hours, so depending on the scale of the experiment, it may be best to undertake the task the day before, although it can be done on the da.......

Discussion

The G. mellonella infection model is an evolving method to study the virulence of pathogenic microbes and discover new treatments or therapeutic regimens for them. Researchers generally agree on the many advantages this system offers versus mammalian models (e.g., inexpensive, technically simple, similar immune response)^4,5,15,16; they also agree on the pitfalls. The greatest concern i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microfuge tubes	Sarstedt	72.690.301	General use; autoclaved for sterility
95% ethanol	Fisher	01-337-539	Diluted to 70% using sterile ultrapure water; for work surface and syringe/needle cleaning
Analytical balance	Sartorius	ENTRIS641-1SUS	Weighing reagents
Balance	Denver Instrument	SI-402	Weighing larvae and reagents
Clindamycin hydrochloride	Bio Basic	CB0312	Antibiotic (lincoamide class)
Colistin sulphate	Gold Bio	C-921-1	Antibiotic (polymyxin class)
Cotton-tipped applicators	MedPro	018432	6-inch, sterile, single use; for cleaning waxworms
Culture tube caps	Bellco Glass	2005-00016	16 mm, autoclaved for sterility
Culture tubes	Pyrex	9820	6 inch, 13 mL, autoclaved for sterility; for liquid culture growth
DensiCheck densitometer	BioMerieux	21255	Densitometer to standardize bacterial cultures
DMSO	Bio Basic	D0231	Preparation of antibiotic stock solutions
Galleria mellonella	Super Cricket Farms		Canadian source for waxworms
Glass tubes	Fisher	14-961-26	12 x 75 mm; autoclaved for sterility; for use with the densitometer
Hamilton syringes with needle	Millipore Sigma	20734	10 µL volume, 26 G needle, bevel tip; for waxworm injections
Incubator (shaking)	New Brunswick Scientific	M1352-0000	Excella E24 Incubator Shaker; for liquid culture incubation
Incubator (static)	Fisher	11-690-550D	Isotemp Incubator Oven Model 550D; for solid (LB agar) culture incubation and treated waxworm incubation
Incubator (static)	Thermo Fisher Scientific	PR505750R-CN	15 °C; for waxworm storage
LB agar, Lennox	BD Difco	240110	Solid growth media (20 g/L: 5 g/L sodium chloride, 10 g/L tryptone, 5 g/L yeast extract, 15 g/L agar)
LB broth, Lennox	BD Difco	240230	Liquid growth media (20 g/L: 5 g/L sodium chloride, 10 g/L tryptone, 5 g/L yeast extract)
Meropenem trihydrate	Gold Bio	M-820-10	Antibiotic (carbapenem class)
Micropipettes	Mandel	Various	Gilson single channel pipettes (P10, P20, P200, P1000)
Petri dishes, sterile, disposable	SPL Life Sciences	10090	90 x 15 mm, sterile; for solid growth media (agar plates) and waxworm segregation
Sodium chloride	Fisher	BP358	Preparation of 0.85 % sodium chloride in MilliQ water; autoclaved for sterility
Ultrapure water	Millipore	ZLXLSD51040	MilliQ water purification system; ultra pure water for media and solution preparation; autoclaved for sterility
Wood stick applicators	MedPro	018472	6 inch, sterile, single use; for culture inoculation and streak plating