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Colour patterns of G. mellonella larvae at 24 h after intrahemocoelic injection. Untreated larvae and control larvae showed no melanization (a). Larvae injected with EPEC showed no (a), slight (b), moderate (c), to severe (d) melanization depending on EPEC dose. Larval appearance was photographed with a Ricoh R10 digital camera.
Source publication
The use of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae), an economical insect model, for the study of enteropathogenic Escherichia coli (Migula) (EPEC), a diarrheagenic human pathogen, has been demonstrated previously but remains poorly understood. The present study characterizes the Galleria-EPEC system extensively for future studies us...
Contexts in source publication
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... significant differences in circulating hemocyte count between treatments (Quasi-Poisson GLM, F = 14.8, df = 3 and 156, Larvae that displayed moderate to severe melanization ( Fig. 3c and d) showed (a) significantly higher mortality (by 89%, z = 13.6, P < 0.0001), (b) lower survival time (by 15 d on average, t = −9.44, P < 0.0001), and (c) longer time to pupation (by 2. 6 d on average, z = 3.77, P = 0.0002) compared to insects that showed slight to no melanization ( Fig. 3a and b). P < 0.0001) and between time points (F = ...
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... that displayed moderate to severe melanization ( Fig. 3c and d) showed (a) significantly higher mortality (by 89%, z = 13.6, P < 0.0001), (b) lower survival time (by 15 d on average, t = −9.44, P < 0.0001), and (c) longer time to pupation (by 2. 6 d on average, z = 3.77, P = 0.0002) compared to insects that showed slight to no melanization ( Fig. 3a and b). P < 0.0001) and between time points (F = 2.77, df = 4 and 152, P = 0.03) were found (Fig. 9). No differences were detected in hemocyte count between treatments over time (i.e., no significant interaction between treatment and time; F = 1.32, df = 12 and 140, P = 0.21). Larvae injected with EPEC showed a significantly reduced number of ...
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... (immunopathology). The mechanism of EPEC pathogenesis in G. mellonella is currently unknown but previous research suggested that EPEC virulence in the hemolymph is reduced by the inactivation of the T3SS and both inactivation and constitutive activation of the Cpx envelope stress response ( Leuko and Raivio 2012). In this study, melanization (Fig. 3) was the first visible sign of an immune response against EPEC post-injection, indicating the activation of the PPO cascade. Melanization first occurred at the dorsal vessel, around which melanin can be seen through the cuticle (Fig. 3b and c). This was likely the result of melanized particle accumulation around the ostia (valves) of ...
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... activation of the Cpx envelope stress response ( Leuko and Raivio 2012). In this study, melanization (Fig. 3) was the first visible sign of an immune response against EPEC post-injection, indicating the activation of the PPO cascade. Melanization first occurred at the dorsal vessel, around which melanin can be seen through the cuticle (Fig. 3b and c). This was likely the result of melanized particle accumulation around the ostia (valves) of the dorsal vessel by hemolymph movement and subsequent phagocytosis by sessile periostial hemocytes (Sigle and Hillyer 2016). The severity of melanization was dose-dependent ( Fig. 2c), indicating increasing activation of the PPO cascade with ...
Citations
... Larvae receiving the medium and high infection doses were essentially unsuccessful, and those few larvae that were successful required a longer time period to make the transition. Many major microbial studies using G. mellonella to assess pathogenicity measure only the larval stage [71,82,89,94,[113][114][115][116]. This is likely due to the increased and rapid mortality of larvae in these studies, with death occurring in a matter of hours with some pathogens. ...
Most mycoplasmal infections result in chronic, clinically silent disease. In direct contrast, Mycoplasma alligatoris elicits a fulminant, multisystem disease in the natural host, Alligator mississippiensis (American alligator). The goals of the study were to better understand the disease in the natural host and to determine if the invertebrate model G. mellonella could serve as a surrogate alternate host. The survival of alligators infected intratracheally was dose dependent (p=0.0003), ranging from no mortality (10² CFU) to 100% mortality (10⁸ CFU), with 60% mortality at the 10⁴ and 10⁵ CFU infectious dose. Microbial load in blood, joints, and brain was dose dependent, regardless of whether alligators were infected intratracheally or intravenously (p <0.002). Weight loss was similarly impacted (p <0.001). Experimental infection of the invertebrate Galleria mellonella mirrored the result in the natural host. In a dose response infection study, both larval survival curves and successful pupation curves were significantly different (p≤0.0001) and dose dependent. Infected insects did not emerge as moths (p <0.0001). Here, we describe the first study investigating G. mellonella as a surrogate model to assess the pathogenic potential of M. alligatoris. G. mellonella survival was dose dependent and impacted life stage outcome.
... Besides their low cost and the possibility of testing multiple groups of larvae in a short time, G. mellonella larvae possess an innate immune system similar to mammals and can be maintained at 37 • C, like the bacterial pathogens of mammals [25,29]. So, it was recently observed that the STX2d phage and the type 3 secretion system (T3SS) contribute to the virulence of AE-STEC O80:H2 and EPEC O127:H6, respectively, in G. mellonella larvae [25,30,31], while the contribution of the T3SS to the virulence of AE-STEC O157:H7 is less clear [32,33]. However, no study has been performed yet to assess the role of the pS88 plasmids of AE-STEC and EPEC O80:H2. ...
... The pS88 plasmid conjugation strategy was based on the presence of the ColVencoding cva operon on the pS88 plasmid of bovine AE-STEC and EPEC O80:H2 [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. The recipient strain was the E. coli K12 DH10B strain harboring the recombinant plasmid pAuto-ColV-Switch1.0 ...
... Two other specific properties of the AE-STEC and EPEC O80:H2 are the production of the AE lesion and the invasiveness properties encoded by pS88-like plasmid-located genes [15,18,19,21]. The role of the T3SS responsible for the development of the AE lesion was not assessed in his study but is indeed partially responsible for the lethality of the human EPEC E2348/69 (serotype O127:H6) in G. mellonella larvae compared to a mutant in one of the encoding genes, as observed by others [31]. However, the situation seems different for AE-STEC O157:H7 [32,33], possibly because the Stx toxins play a more important role ( [30], this study). ...
The invasiveness properties of Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) O80:H2 in humans and calves are encoded by genes located on a pS88-like ColV conjugative plasmid. The main objectives of this study in larvae of the Galleria mellonella moth were therefore to compare the virulence of eight bovine STEC and EPEC O80:H2, of two E. coli pS88 plasmid transconjugant and STX2d phage transductant K12 DH10B, of four E. coli O80:non-H2, and of the laboratory E. coli K12 DH10B strains. Thirty larvae per strain were inoculated in the last proleg with 10 μL of tenfold dilutions of each bacterial culture corresponding to 10 to 106 colony-forming units (CFUs). The larvae were kept at 37 °C and their mortality rate was followed daily for four days. The main results were that: (i) not only the STEC and EPEC O80:H2, but also different E. coli O80:non-H2 were lethal for the larvae at high concentrations (from 104 to 106 CFU) with some variation according to the strain; (ii) the Stx2d toxin and partially the pS88 plasmid were responsible for the lethality caused by the E. coli O80:H2; (iii) the virulence factors of E. coli O80:non-H2 were not identified. The general conclusions are that, although the Galleria mellonella larvae represent a useful first-line model to study the virulence of bacterial pathogens, they are more limited in identifying their actual virulence properties.
... Among gram-positive microorganisms, Staphylococcus aureus [71][72][73][74][75][76][77], Streptococcus pyogenes [78][79][80], Streptococcus pneumoniae [81][82][83], Streptococcus mutans [19,20,[84][85][86], L. monocytogenes [4,44,65,66,87,88], Enterococus faecalis [89][90][91][92], Enterococcus faecium [93][94][95][96], L. rhamnosus GG [63,64], C. butyricum Miyairi 588 [63], C. perfringens [67,68], Mycobacterium bovis [23], Mycobacterium abscessus [97,98], and Mycobacterium tuberculosis [99][100][101][102] were mentioned. Among gram-negative bacteria, E. coli [103][104][105][106], S. enterica Typhimurium [107][108][109], K. pneumonia [110][111][112][113], A. baumanii [39,[114][115][116][117], Francisella tularensis [118][119][120], P. aeruginosa [60,121,122], and P. gingivalis [70,123,124] have been involved. G. mellonella was also used for testing representatives of fungal pathogens, such as Candida albicans [21,25,[125][126][127][128], Candida dubliniensis [21,25,129,130], Aspergillus fumigatus [38,131,132], Cryptococcus neoformans [57,[133][134][135], and Madurella mycetomatis [45,[136][137][138], and also viruses [139][140][141][142] and bacteriophages [143][144][145][146][147][148][149][150]. ...
Galleria mellonella is a promising in vivo model insect used for microbiological, medical, and pharmacological research. It provides a platform for testing the biocompatibility of various compounds and the kinetics of survival after an infection followed by subsequent treatment, and for the evaluation of various parameters during treatment, including the host–pathogen interaction. There are some similarities in the development of pathologies with mammals. However, a limitation is the lack of adaptive immune response. Antimicrobial photodynamic therapy (aPDT) is an alternative approach for combating microbial infections, including biofilm-associated ones. aPDT is effective against Gram-positive and Gram-negative bacteria, viruses, fungi, and parasites, regardless of whether they are resistant to conventional treatment. The main idea of this comprehensive review was to collect information on the use of G. mellonella in aPDT. It provides a collection of references published in the last 10 years from this area of research, complemented by some practical experiences of the authors of this review. Additionally, the review summarizes in brief information on the G. mellonella model, its advantages and methods used in the processing of material from these larvae, as well as basic knowledge of the principles of aPDT.
... It is important to mention that G. mellonella in vivo model has been used to study E. coli pathogenicity. Based on this model, it is possible to evaluate the immune innate response of G. mellonella against bacteria, namely, melanization and antimicrobial peptides (AP) production (such as cecropin and gloverin) [45]. To achieve a more close experimental design to the porcine model, pig serum has been also studied, since it contains a variety of AP, inhibiting bacterial cell growth and constituting a host's innate immune system against bacterial infection [46]. ...
Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs’ viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.
... So far, there is no study conducting virulence tests using O. fuscidentalis. Virulence test of Enteropathogenic E. coli (EPEC) using G. mellonella, started at 4x10 4 CFU dosage, which led to 0% survival [44]. The cellular immune system and the humoral immune system are the two main immune systems found in insects. ...
Multi-drug resistant (MDR) bacteria are becoming a worldwide problem due to limited options for treatment. Moreover, patients infected by MDR with highly virulent accessories are worsening the symptoms, even to the point of causing death. In this study, we isolated bacteria from 14 inanimate surfaces that could potentially be reservoirs for the spread of bacterial infections in the medical university. Blood agar media was used for bacterial isolation. The bacterial colony that showed hemolytic activities on each surface was tested for antimicrobial susceptibility against eight different antibiotics. We found that MDR bacterium, namely TB1, which was isolated from a toilet bowl, was non-susceptible to ampicillin, imipenem, chloramphenicol, amoxicillin-clavulanic acid, gentamicin, and tetracycline. Another MDR bacterium isolated from the mobile phone screen of security officers, namely HSO, was resistant to chloramphenicol, gentamicin, tetracycline, and cefixime. An in vivo virulence test of bacterial isolates used Omphisa fuscidentalis larvae as an alternative to Galleria mellonella larvae for the infection model. A virulence test of TB1 in O. fuscidentalis larvae revealed 20% survival in the bacterial density of 104 and 105 CFU/larvae; and 0% survival in the bacterial density of 106 CFU/larvae at 24 h after injection. Bacterial identification was performed for TB1 as a potential virulent isolate. Bacterial identification using partial 16s rRNA gene showed that TB1 exhibited 99.84% identity to Escherichia fergusonii 2611. This study concludes that TB1 is a potentially virulent MDR E. fergusonii isolated from toilet bowls at a medical university.
... These multicellular defence structures have been reported in other Gm-bacteria models (e.g. Mycobacterium abscessus [33], Escherichia coli [53] and Clostridium perfringens [54]), and in Gm-fungal (e.g. Candida albicans [41], Aspergillus fumigatus [55]) models. ...
Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here, we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid, and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable, and advantageous additional model to be used alongside existing models to advance tuberculosis research.
... Bacteriamediated delivery of dsRNA was pioneered in C. elegans using the RNase III-deficient Escherichia coli strain HT115(DE3) (Kamath and Ahringer, 2003), and this technique was also applied to successfully induce the silencing of target genes in insects (Tian et al., 2009;Zhu et al., 2011;Zhang et al., 2019b). However, E. coli can act as an invader in insects, and it would be safer to use natural symbionts of the target insects as delivery vectors (Whitten et al., 2016;Chen and Keddie, 2021). Similarly, genetically modified algae and yeast have been used to deliver dsRNA in mosquitoes and Drosophila suzukii, respectively (Murphy et al., 2016). ...
... EPEC was transformed with the plasmid pXG-1, enabling the constitutive expression of green fluorescent protein (GFP) for in situ visualization by fluorescence microscopy and chloramphenicol resistance for isolation by selective media (Urban and Vogel 2007). This bacterium was used since 1) EPEC is virulent in the hemocoel of G. mellonella (Leuko andRaivio 2012, Chen andKeddie 2021) and 2) extracellular DNA was serendipitously discovered in the hemolymph of G. mellonella larvae injected intrahemocoelically with EPEC in a preliminary experiment (personal observation), making EPEC a suitable pathogen to study ETs and their effects in G. mellonella. EPEC was cultured in Luria-Bertani (LB) medium (Supp Appendix B.3 [online only]) in glass culture tubes (KIMAX, 16 mm × 100 mm) on a shaker (Mistral Multi-Mixer Model 4600, Lab-Line) at 30°C to match G. mellonella rearing temperature. ...
... G. mellonella larvae were injected intrahemocoelically with two doses of EPEC (1.6 × 10 4 CFU or 2.2 × 10 4 CFU) and a Ringer's control in the presence or absence of G. mellonella hemocyte DNA (500 ng, dissolved and suspended in Ringer's) (Supp Appendix C [online only]). These doses were selected based on an extensive (n = 349) LD 50 experiment using 18 different EPEC doses spanning 0-4.0 × 10 4 CFU (Chen and Keddie 2021). Doses between 8.4 × 10 3 CFU and 2.9 × 10 4 CFU result in all disease outcomes (i.e., recovery and mortality present in different insects at the same dose). ...
Extracellular traps (ETs) released from vertebrate and invertebrate immune cells consist of chromatin and toxic granule contents that are capable of immobilizing and killing microbes. This recently described innate immune response is not well documented in insects. The present study found that ETs were released by hemocytes of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) in vivo and ex vivo after bacterial stimulation. ET release (ETosis), hemolymph coagulation, and melanization likely contributed to the immobilization and killing of the bacteria. The injection of G. mellonella hemocyte deoxyribonucleic acid (DNA) in the presence of bacteria increased bacterial clearance rate and prolonged insect survival. Taken together, these results indicate the presence of insect hemocyte extracellular traps (IHETs) that protect the insect against microbial infection in the hemocoel and represent the first documentation of ETs in insects in vivo.
Aim
This study aims to investigate the prevalence of intestinal pathogenic Escherichia coli (InPEC) in healthy pig-related samples and evaluate the potential virulence of the InPEC strains.
Methods and results
A multiplex PCR method was established to identify different pathotypes of InPEC. A total of 800 rectal swab samples and 296 pork samples were collected from pig farms and slaughterhouses in Hubei province, China. From these samples, a total of 21 InPEC strains were isolated, including 19 enteropathogenic E. coli (EPEC) and 2 shiga toxin-producing E. coli (STEC) strains. By whole-genome sequencing and in silico typing, it was shown that the sequence types and serotypes were diverse among the strains. Antimicrobial susceptibility assays showed that 90.48% of the strains were multi-drug resistant. The virulence of the strains was first evaluated using the Galleria mellonella larvae model, which showed that most of the strains possessed medium to high pathogenicity. A moderately virulent EPEC isolate was further selected to characterize its pathogenicity using a mouse model, which suggested that it could cause significant diarrhea. Bioluminescence imaging (BLI) was then used to investigate the colonization dynamics of this EPEC isolate, which showed that the EPEC strain could colonize the mouse cecum for up to 5 days.
