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SHORT COMMUNICATION
Year : 2020  |  Volume : 24  |  Issue : 6  |  Page : 593-596  

Galleria mellonella as an experimental model for studying periodontopathogens


Department of Bioscience and Oral Diagnosis, Institute of Science and Technology, São Paulo State University (Unesp), São José dos Campos, SP, Brazil

Date of Submission20-Dec-2019
Date of Decision29-May-2020
Date of Acceptance11-Jun-2020
Date of Web Publication21-Sep-2020

Correspondence Address:
Dr. Ana Lia Anbinder
Av Engenheiro Francisco Jose Longo, 777, Jardim Sao Dimas, Sao Jose dos Campos, SP
Brazil
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jisp.jisp_631_19

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   Abstract 


In the present study, Galleria mellonella was evaluated as a potential infection model for periodontal bacteria, more specifically, Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. All the bacteria evaluated were pathogenic to G. mellonella, causing their death in a concentration-dependent manner, and a decrease in their hemocyte count. Moreover, it was possible to recover the bacteria from the larvae hemolymph and determine the colony-forming units per larvae. G. mellonella is an effective model that may help to better understand the host-microbe interactions in periodontics.

Keywords: Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Galleria mellonella, periodontal diseases, Porphyromonas gingivalis


How to cite this article:
Santos TA, Scorzoni L, Santos Ad, Junqueira JC, Anbinder AL. Galleria mellonella as an experimental model for studying periodontopathogens. J Indian Soc Periodontol 2020;24:593-6

How to cite this URL:
Santos TA, Scorzoni L, Santos Ad, Junqueira JC, Anbinder AL. Galleria mellonella as an experimental model for studying periodontopathogens. J Indian Soc Periodontol [serial online] 2020 [cited 2020 Nov 26];24:593-6. Available from: https://www.jisponline.com/text.asp?2020/24/6/593/295648




   Introduction Top


Periodontitis is a multifactorial inflammatory disease associated with dysbiotic biofilms[1] and caused mainly by Gram-negative, anaerobic, and proteolytic bacteria, such as Aggregatibacteractinomycetemcomitans, Porphyromonasgingivalis, and Fusobacteriumnucleatum.

Invivo studies are extremely important not only for the analysis of periodontal disease pathogenesis but also for the development of innovative treatments. However, due to ethical issues, reducing the number of animals used in studies by employing alternative techniques and refined experimental protocols should be considered.[2] In this context, invertebrate animal models are one alternative for screening host-pathogen interaction that could reduce the use of vertebrates and add information to thein vitro studies. Galleriamellonella is an insect frequently used as an experimental model to evaluate microbial virulence, compound toxicity, and antimicrobial effectiveness. These larvae exhibit an immune response analogous to the vertebrates innate immune response.[3] Similar to mammalian blood, the larvae hemolymph contains hemocytes, which are immune cells compared in terms of function to mammalian neutrophils.[3] Through a similar mechanism via superoxide production used by human neutrophils, hemocytes have the ability to phagocytize and kill bacterial cells, in addition to having homologous proteins essential for the production of superoxide.[4] It is possible to evaluate the host response to the pathogen through hemocyte density.[5] The pathogenicity of the microorganism correlates inversely with the number of hemocytes in the larvae; the higher the pathogenicity of the microorganism, the lower was the number of hemocytes. Few reports describe the use of G.mellonella as an infection model for periodontal pathogens.

In this study, G.mellonella was evaluated as an animal model for periodontopathogens infection [Figure 1]. For this purpose, determination of survival curves and hemocyte concentration, and colony-forming units (CFU) recovery from the larvae hemolymph were performed after infection with P.gingivalis (ATCC 33277), F.nucleatum (ATCC 25586), and A.actinomycetemcomitans (ATCC 29522).
Figure 1: Galleria mellonella experimental model; (a) Inoculation of periodontopathogen in the larvae last left proleg; (b) Alive larvae after 1 h of PBS inoculation; (c) Melanized alive larvae after 1 h of infection with Aggregatibacter actinomycetemcomitans; (d) Melanized dead larvae after 24 h of infection with Aggregatibacter actinomycetemcomitans

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The bacteria were grown on  Brucella More Details agar supplemented with 5% defibrinated sheep blood and 1% solution of hemin and menadione for 5 days in an anaerobic chamber at 37°C. For the assays, suspensions were prepared at concentrations of 107, 108, and 109 cells/mL, using a spectrophotometer at 660 nm, 550 nm, and 600 nm for P.gingivalis, F.nucleatum, and A.actinomycetemcomitans, respectively.

Light-colored G.mellonella larvae (Lepidoptera: Pyralidae), with no spots on their cuticles and weighting 190–230 mg, were selected for the study. The survival curves were obtained by inoculating 10 μL of periodontopathogens in the larvae last left proleg at the concentrations of 107, 108, and 109 cells/mL, and phosphate-buffered saline for the control group, using micro syringes. Twelve larvae were used for each periodontopathogen concentration evaluation. After inoculation, the animals were incubated at 37°C in the dark, without food. The survival was evaluated daily for 7 days. Sub-lethal concentrations of P.gingivalis (107 cells/mL), F.nucleatum (108 cells/mL), and A.actinomycetemcomitans (108 cells/mL) were used for the evaluation of the hemocyte concentration and CFUs recovery from the hemolymph. After infection, the larvae were incubated at 37°C for 3 h. For hemocyte concentration determination, a hemolymph sample of 3 larvae was collected and diluted with an anticoagulant solution (2% NaCl, 0.1 M glucose, 30 mM sodium citrate, 26 mM citric acid, and 10 mM EDTA). The hemocyte concentration was estimated using a Neubauer chamber. For each treatment, four groups of 3 larvae each were analyzed (a total of 12 animals per treatment). For the determination of periodontopathogens CFUs recovered from the larvae, the collected hemolymph was diluted in PBS, plated on Brucella blood agar supplemented with 1% hemin and menadione and incubated under anaerobic conditions (37°C for 5 days). The statistical analysis was performed by the log-rank method (Mantel-Cox), ANOVA, or Student's t-test (α = 5%; GraphPad Prism 6, La Jolla, California, USA).

The larvae survival rates inversely correlate with microorganism pathogenicity. All bacteria evaluated were able to kill G.mellonella in a concentration-dependent manner. The infection with P.gingivalis at 107 cells/mL resulted in 50% of larvae survival after 7 days, but when infected with the concentrations of 108 and 109 cells/mL of P.gingivalis, all larvae were dead within 24 h [Figure 2]a. When the larvae were infected with 107, 108, and 109 cells/mL of F.nucleatum, survival rates of 66.67%, 13.33%, and 6.67%, respectively, were observed after 7 days [Figure 2]b. Infection with A.actinomycetemcomitans at 109 cells/mL caused 100% caterpillar mortality within 48 h of inoculation; this was significantly higher than the mortality observed at other concentrations. Infections with 107 and 108 cells/mL led to similar results (P = 0.0645) after 7 days of infection [Figure 2]c. P.gingivalis was the most pathogenic bacteria for G.mellonella, causing 100% mortality within 24 h at lower concentrations than the other bacteria.
Figure 2: Survival curve of Galleria mellonella after infection with different concentrations of periodontopathogens; (a) Porphyromonas gingivalis. There was a statistical difference between the concentrations of 107and 108cells/mL (P<0.0001) and 107and 109cells/mL (P<0.0001); (b) Fusobacterium nucleatum. There was a statistical difference between the concentrations of 107and 108cells/mL (P=0.0022), and 107and 109cells/mL (P=0.0003); (c) Aggregatibacter actinomycetemcomitans. There was a statistical difference between the concentrations of 107and 109cells/mL (P=0.0023), and 108and 109cells/mL (P=0.0362). Same letters over the lines indicate the absence of statistical difference after Log-Rank test (Mantel Cox)

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Regarding hemocyte concentration after 3 h of infection, a reduction in the hemocyte count was observed with all evaluated bacteria [Figure 3]. Infection with P.gingivalis led to a reduction of 63.88% in the number of hemocytes, while F.nucleatum and A.actinomycetemcomitans led to a decrease of 72.34% and 60.11%, respectively.
Figure 3: Mean and standard deviations of the hemocyte count from Galleria mellonella hemolymph after inoculation of periodontopathogens. Different letters over the bars indicate significant statistical difference after Student's t-test; (a) 107 cells/mL of Porphyromonas gingivalis; P = 0.0003; (b) 108 cells/mL of Fusobacterium nucleatum; P < 0.0001; (c) 108 cells/mL of Aggregatibacter actinomycetemcomitans; P = 0.0003

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The amount of P.gingivalis recovered from the larvae hemolymph 3 h after infection was lower (2.9 × 104 cells/mL) than the initially inoculated concentration (107 cells/mL), in contrast to the other bacteria tested. In the larvae infected with 108 cells/mL of F.nucleatum and A.actinomycetemcomitans, the microbial recovery was found to be 2.295 × 108 cells/mL and 3.65 × 108 cells/mL, respectively, equivalent to the initial inoculated amount. Oxygen contact may have influenced the recovery of strictly anaerobic microorganisms such as P.gingivalis. Although we have evaluated the bacterial burden only in the hemolymph, bacteria can also invade other tissues of the larvae, such as the gut and fat body. Furthermore, in addition to the hemocytes, the immune response of the larvae is also mediated by proteins, antimicrobial peptides, and melanin,[3] which possibly act against P.gingivalis. Despite the significantly reduced amount of P.gingivalis recovered, the mortality rate was still higher. This could be explained by P.gingivalis virulence factors such as capsule, fimbriae, and lipopolysaccharides, and by the activation of the immune system, which requires a high-energy cost and may not be sustained if the bacterial stimulus is intense, leading to larvae septic death. The specific role of P.gingivalis virulence factors in mortality rates should be investigated further.

This study demonstrated that G.mellonella could be used as a model for infection by the facultative anaerobic bacterium A.actinomycetemcomitans, as well as by strictly anaerobic bacteria P.gingivalis and F.nucleatum. Although it cannot replace vertebrate models of periodontitis induction, this simple model effectively allows the study of the interaction of periodontopathogens and the host. It was possible to evaluate the virulence of the microorganisms by evaluating G.mellonella survival curves, hemocyte counts, and microbial recovery. Different branches of research in periodontics, such as the investigation of alternative treatments and evaluation of the virulence of clinical strains, may benefit from the use of G.mellonella as a model of infection for periodontopathogens.

Financial support and sponsorship

This project was funded by São Paulo Research Foundation FAPESP (2016/06946-1 and 2017/05439-1) and by Coordination of Improvement of Higher Education Personnel CAPES.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Papapanou PN, Sanz M, Buduneli N, Dietrich T, Feres M, Fine DH, et al. Periodontitis: Consensus report of workgroup 2 of the 2017 world workshop on the classification of periodontal and peri-implant diseases and conditions. J Clin Periodontol 2018;45:S162-70.  Back to cited text no. 1
    
2.
Tannenbaum J, Bennett BT. Russell and Burch's 3Rs then and now: The need for clarity in definition and purpose. J Am Assoc Lab Anim Sci 2015;54:120-32.  Back to cited text no. 2
    
3.
Pereira TC, de Barros PP, Fugisaki LRO, Rossoni RD, Ribeiro FC, de Menezes RT, et al. Recent advances in the use of Galleria mellonella model to study immune responses against human pathogens. J Fungi (Basel) 2018;4:128.  Back to cited text no. 3
    
4.
Bergin D, Reeves EP, Renwick J, Wientjes FB, Kavanagh K. Superoxide production in Galleria mellonella hemocytes: Identification of proteins homologous to the NADPH oxidase complex of human neutrophils. Infect Immun 2005;73:4161-70.  Back to cited text no. 4
    
5.
Tsai CJ, Loh JM, Proft T. Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing. Virulence 2016;7:214-29.  Back to cited text no. 5
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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