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 bacteria⁶, and fungi^2,4, covering a range of human ailments from wound infections to tuberculosis to fungal diseases⁴. G. mellonella is also used for testing the efficacy^1,2,3,6 and toxicity^1,4,7 of therapeutic drugs, adjuvants and combination therapies^4,8,9, and new therapies exploring bacteriophages^3,4,6, nanomaterials¹⁰, and light therapy^4,6. Factors associated with the innate infective characteristics of bacteria can also be studied via genetic manipulation of strains and growth under triggering conditions: genes associated with virulence^5,6,11,12 and that respond to stressors such as oxidative stress, temperature, and metal ions^13,14 have been successfully explored. This versatility is a key advantage of the infection model in addition to its low cost, ease of use, and relevant immune response^4,5,15,16. Overall, G. mellonella is a valuable alternative to mammalian animal infection models or a fantastic first step to study known and emerging human pathogens.

A top-priority pathogen for which new therapies are desperately needed is Acinetobacter baumannii¹⁷. Found primarily in nosocomial settings¹⁸, multidrug-resistant (MDR) A. baumannii is a causative agent of serious infections that are directly related to patient mortality-a staggering 15% of 1.14 million antimicrobial resistance-attributable deaths in 2021¹⁹. A critical component of combatting bacterial pathogens is understanding the minimum inhibitory concentration (MIC) of antimicrobial agents that can work against it. The MIC value can guide in vitro dosing experiments, which can then be tested in an in vivo model. This is a critical step -- especially for a new antimicrobial compound under development -- as a candidate compound that works well in an assay may not work as well when introduced into a complex, living system^7,20,21,22.

We present a method for the use of G. mellonella as an infection model using A. baumannii ATCC 17978-VU (a model laboratory MDR strain)²³, AB030 (a hypervirulent extremely drug-resistant (XDR) clinical strain)²⁴, and AB341-IK15 (a virulent environmental strain)²⁵. At the core of this set of protocols is a method using A. baumannii originally published by Peleg et al.²⁶, which we have modified and extended. Many resources already exist to guide researchers through the handling and maintenance of G. mellonella^{27,28,29,30,31,32}. Additionally, standard infection assay parameters are generally agreed upon but are flexible depending on the specific circumstances and experimental design: waxworm weight is typically 250-350 mg¹⁶; experimental sets usually comprise 10 waxworms; injection volume is commonly 10 µL; administration of a treatment occurs within 0.5-3 h (here, called infect-wait-treat); incubation temperature is almost always 37 °C; and assay duration can range from 4 h to 192 h¹³. Here, we demonstrate an infection protocol coupled with MIC data as a way to screen antibiotic efficacy and inform on therapeutic dose, which can then guide treatment regimens when advancing to mammalian models.This method also outlines a 24 h assay and the combined administration of pathogenic bacteria with a treatment compound (here, called infect-and-treat) to allow for the faster screening of many antimicrobial compounds and/or bacterial isolates.

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 product sheet. To follow this protocol, prepare clindamycin at 20 mg/mL in dimethyl sulfoxide (DMSO), meropenem at 30 mg/mL in DMSO, and colistin at 50 mg/mL in sterile ultrapure water. Store all antibiotics at -20 °C until needed (see Table of Materials).
   NOTE: Always consult the safety data sheet when handling antibiotics and DMSO and follow the recommended precautions.
3. Prepare media for the propagation of the bacterial strains. Here, prepare three lysogeny (LB) agar plates for generating fresh streak plates of the bacteria, and 30 mL of LB broth for culturing the bacteria. Add antibiotics to the solid and liquid media if required for different bacterial strains.
   NOTE: Here, no antibiotics will be added (see Table of Materials). Reagent and media preparation must be performed in a sterile environment, either at the bench with a Bunsen burner or in a biological safety cabinet. All consumables (microfuge tubes, pipette tips, petri dishes, etc.) must be sterile.

2. G. mellonella preparation

1. Hold G. mellonella waxworms in an incubator set to 15 °C and remove as required for up to 2 weeks. They do not need to be fed.
2. The day before beginning the G. mellonella infection assay, prepare the waxworms-sort, weigh, and clean (Figure 1A).
   1. Remove waxworms from the bedding material used for shipping and place them into sterile, plastic Petri dishes until the required number is reached. Choose waxworms that are active and have a cream-coloured and blemish-free cuticle.
      NOTE: Remove any dead waxworms that you may find. It has been suggested that dead waxworms contribute to a faster decline in the others' health²⁷.
   2. Weigh each waxworm and further sort them into Petri dishes based on weight range.
      NOTE: The larvae weight range may differ from the typical (250-350 mg)¹⁶ based on the waxworms provided. Waxworms supplied for this protocol weighed from ≥120 mg to ≤200 mg.
   3. Gently clean the underside of each waxworm, particularly the lower proleg region, using a sterile cotton-tipped swab soaked in 70% ethanol.
3. Place the waxworms in Petri dishes in sets of 10, with each Petri dish representing one treatment.
   NOTE: Do not forget to account for control treatments (no injection, saline-only injection). Additionally, prepare a set of at least 10 waxworms that can be used as substitutes in case of waxworm loss ahead of the experiment or mistakes made with injections during the experiment.
4. Keep the waxworms at room temperature in the dark overnight.

3. Bacteria preparation

1. Up to 1 week before beginning the G. mellonella infection assay, prepare fresh streak plates of the strains of interest on LB agar (Streak, Figure 1B). Here, A. baumannii ATCC 17978-VU, AB030, and AB341-IK15 (Table 1) are being used. Incubate the plates, inverted, at 37 °C for 16-18 h. Store these plates at 4 °C until needed.
2. The afternoon before beginning the G. mellonella infection assay, individually inoculate 3 mL of LB broth in sterile 13 mL glass culture tubes with a single colony of each strain using a sterile wooden inoculating stick or sterile inoculating loop. Grow for 16-18 h at 37 °C with shaking (250 rpm) (Grow, Figure 1B).
3. Standardize the overnight cultures to 0.5 McFarland (approximately equivalent to 1.5 × 10⁸ CFU/mL) using a densitometer (Standardize, Figure 1B) (see Table of Materials).
   1. Add 2 mL of sterile saline to a sterile glass tube that fits in the densitometer.
   2. Blank the instrument with sterile saline.
   3. Remove the tube from the densitometer and set in a tube rack.
   4. Add ~50 µL of the overnight culture and gently swirl to mix.
   5. Place the tube back in the densitometer and note the reading.
      NOTE: The ideal density range is 0.5 ± 0.02.
      1. If the reading is <0.48, add additional small volumes (~5-10 µL) of culture until the reading is in range.
      2. If the reading is >0.52, add additional sterile saline until the reading is in range.
4. Prepare each sample as close to use as possible. Keep at room temperature until needed.

4. Antibiotic preparation

Refer to the Clinical and Laboratory Standards Institute (CLSI) guidelines³³ or similar references to find MIC or breakpoint values (the antimicrobial concentration where the bacteria begin to show resistance) for antimicrobial compounds tested on known pathogens. If you are screening antimicrobial agents against a bacterium for the first time, go through the procedures to determine specific MIC values using E-strips³⁴ or microbroth dilution³³.
We have previously determined MIC values across a range of antibiotics for each of the A. baumannii strains²⁵ (Table 2). Here, we chose the one antibiotic against which all three strains showed resistance (clindamycin), and an antibiotic that the strains were susceptible to (meropenem for ATCC 17978-VU and AB341-IK15; colistin for AB030).

1. Knowing the MIC for the desired antibiotics, calculate the concentration required to deliver 10x the MIC to the waxworms.
   NOTE: A range of doses (2x, 5x, 10x, 20x MIC) can be tested for a more comprehensive survey of antibiotic efficacy.
2. Prepare 1 mL of the 10x working solution of each antibiotic in sterile saline (Figure 1C) on the day of use. Set aside at room temperature until needed.
   NOTE: Antibiotics that required initial solubilization in an organic solvent (e.g., DMSO) should be sufficiently diluted in sterile saline once the desired testing concentration is reached such that the original solvent should no longer be relevant. If not, an extra set of waxworms will be needed as a control for the solvent equivalently diluted in sterile saline as the antibiotic. DMSO has been shown to be non-toxic to G. mellonella at concentrations ≤ 30%³⁵.

5. Waxworm injection and surveillance

1. Prepare to administer injections to the waxworms (Figure 1D).
   1. Clean the work area (either a bench with a Bunsen burner or a biological safety cabinet) with 70% ethanol.
   2. Ensure that the syringe and needle are clean and work properly.
   3. Label the inside of each Petri dish with the treatment name.
2. Place one set of waxworms in the cleaned work area.
3. Draw up 10 µL of the first test solution into the syringe.
4. Remove one waxworm from the Petri dish and restrain it in a way that is controlled and comfortable
   NOTE: There are many ways the waxworms can be controlled. Consider each technique-between fingers³², platform²⁷, microinjector^6,32, pipette tip^30,36-and choose the one that works best for you.
5. Gently insert the needle into the second last, left proleg of the waxworm. Do not insert the needle too far, only a couple of millimeters.
   NOTE: You will feel the needle breaking through the cuticle of the proleg, but insertion of the needle should not be difficult and should not require excess force (excess force may cause trauma to the waxworm and result in unintended death)³. Often, difficulty in piercing the cuticle suggests that the needle is not aligned with the proleg. Reposition the needle until the injection proceeds easily.
6. Inject the test solution at a moderate speed (not too fast). Wait a second or two before withdrawing the needle.
7. Continue to inject the waxworms until the set is complete.
8. Clean the needle and syringe before the next set of injections.
   1. If using a Bunsen burner, flame the needle to heat-kill any surface bacteria or reagent.
   2. Fill the syringe (draw up) with sterile water. Empty the syringe into a small container (e.g., a Petri dish) to visualize the ejected liquid (see NOTE after step 5.8.5).
   3. Fill the syringe with 70% ethanol. Empty the syringe as before.
   4. Fill the syringe with sterile water. Empty the syringe as before.
   5. Fill the syringe with sterile saline. Empty the syringe as before.
      NOTE: Pay close attention to the liquid as it is being drawn up into the syringe, ensuring that the syringe fills without bubbles. Also, watch as the liquid is being dispensed, ensuring that the volume is correct (based on droplet size).
9. Continue with the next treatment with a new set of waxworms.
10. Repeat until all waxworm samples are finished, with the washing steps between every new test solution.
    NOTE: In addition to the test solutions, be sure to have a set of waxworms that receives no injection and a set of waxworms that receives only sterile saline. These sets control for waxworm health (no injection) and injection technique (sterile saline).
11. When injections are complete, place all waxworm sets into an incubator at 37 °C.
12. Monitor the colour and viability of the waxworms after 24 h.
    NOTE: It may be prudent to assess the waxworms at 12 h, depending on what is being tested.
    1. Healthy waxworms will maintain a cream-colored, blemish-free cuticle and respond to touch.
    2. Declining waxworms will darken in color (melanization) and respond to touch.
    3. Deceased waxworms will be very dark in color and will not respond to touch.
       NOTE: There are several helpful guides for the assessment of the health or decline of the waxworms. Evaluation can be as simple as alive or dead, or as detailed as describing the level of melanization^3,5.
13. Record the number of waxworms that have died.
14. Plot waxworm survival versus time. Most commonly, a Kaplan-Meier curve is used.

6. Determine that the antibiotics are not lethal at the desired dose.

1. Prepare 10x MIC antibiotic solutions starting from the concentrated stock solutions using sterile saline as described in section 4.
2. Prepare the waxworms as described in section 2.
3. Administer the antibiotic solutions to the waxworms as described in section 5.
   NOTE: Include the control sets of waxworms (no injection, sterile saline only) as described in step 5.10.
4. Monitor the waxworms as described in section 5.

7. Determine the best infectious dose.

1. Prepare the waxworms as described in section 2.
2. Prepare the cultures as described in section 3.
3. Knowing that the cultures prepared to 0.5 McFarland are at a concentration of ~1.5 × 10⁸ CFU/mL, prepare stepwise 1:10 dilutions in sterile saline to generate 10⁷, 10⁶, 10⁵, and 10⁴ CFU/mL samples. Prepare 0.2 mL of each dilution.
4. Administer 10 µL of each sample to a set of 10 waxworms as described in section 5.
   NOTE: Include the control sets of waxworms (no injection, sterile saline only) as described in step 5.10.
   As only 10 µL is injected into the waxworms, this translates to the injection of a bacterial load of 10⁶, 10⁵, 10⁴, 10³, and 10² CFU, respectively.
5. Monitor the waxworms as described in section 5.
6. Pick the infectious dose that causes ≥50% mortality within 18 h.
   NOTE: Using a dose that is lethal in a short time frame may be critical for the proper assessment of the efficacy of the antibiotic. A study has shown that the lifetime of selected antibiotics after injection into the waxworms can be as little as 24 h¹⁶.

8. Setting up an infection and treatment experiment

1. Prepare the waxworms as described in section 2.
2. Prepare the cultures as described in section 3.
3. Set up the treatment experiment in one of two ways:
   1. Administer the bacterial strain and antibiotic at the same time in one injection (infect-and-treat).
   2. Administer the bacterial strain, then wait for 30-120 min before administering the antibiotic (infect-wait-treat).
      NOTE: Both protocols are described below.
4. Dilute the culture (if required) to the best infectious dose as determined in section 7.
5. Prepare the antibiotic solutions at 10x the MIC in sterile saline as indicated in section 4.
6. Infect-and-treat: Only immediately before use, combine 100 µL of the culture with 100 µL of the 10x MIC antibiotic solution.
   1. Administer the culture + antibiotic solution to a set of 10 waxworms as described in section 5.
      NOTE: Include the control sets of waxworms (no injection, sterile saline only) as described in step 5.10. Additionally, you should include a sample set with culture only (diluted by half with sterile saline to mimic the addition of antibiotic).
7. Infect-wait-treat: Combine 100 µL of the culture with 100 µL of sterile saline.
   1. Administer the culture to a set of 10 waxworms as described in section 5.
   2. Allow the waxworms to rest.
      NOTE: Common rest times are 30 to 180 min¹³. In this protocol, the wait time was 30 min.
   3. Combine 100 µL of the antibiotic solution with 100 µL of sterile saline.
   4. Administer the antibiotic to the same set of 10 waxworms as described in section 5, ensuring that this second injection goes into the opposite proleg (second last, left proleg).
      NOTE: Include the control sets of waxworms (no injection, sterile saline only) as described in step 5.10. Additionally, you should include a sample set that is first injected with equivalently prepared culture and then injected with sterile saline to control for two injections.
8. Monitor the waxworms as described in section 5.

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