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Introduction: The capsular polysaccharide (CPS) of pathogenic bacteria is a critical virulence factor, often evading phagocytosis by host immune cells, while also interfering with the contact of the pathogen with host cells and contributing to biofilm formation. Klebsiella pneumoniae , a Gram-negative human pathogen associated with high antimicrobial resistances, produces 77 CPS serotypes. The CPS masks proteinaceous factors but also protects K. pneumoniae from uptake by host phagocytic cells and activation of the complement system. In addition to nosocomial, urinary tract and bloodstream infections or pneumonia hypervirulent strains have a highly mucoid phenotype and can cause soft tissue infections, liver abscesses, and meningitis as well. The CPS is therefore crucial for both escaping detection by the immune system and enhancing the virulence potential. Methods: In this study, we generated a non-encapsulated mutant (Kpn 2146∆ wza) to observe how the CPS interferes with K. pneumoniae adhesion, survival in blood, and invasiveness in an experimental infection model. Results: Infection of A549 lung epithelial cells showed similar adherence levels for the wild-type and non-capsulated strain, while our data showed a moderately higher internalization of Kpn 2146Δ wza when compared to the wild-type. In whole blood killing assays, we demonstrate that the K. pneumoniae capsule is essential for survival in human blood, protecting K. pneumoniae against recognition and clearance by the human immune system, as well as complement-mediated opsonization and killing. The non-encapsulated mutant, in contrast, was unable to survive in either whole blood or human plasma. Infections of Galleria mellonella larvae showed a significantly decreased virulence potential of the CPS-deficient mutant. Discussion: In conclusion, our data indicate a crucial role of CPS in vivo. [ABSTRACT FROM AUTHOR]

The spread of multidrug-resistant microbes has made it necessary and urgent to develop new strategies to deal with the infections they cause. Some of these are based on nanotechnology, which has revolutionized many fields in medicine. Evaluating the safety and efficacy of these new antimicrobial strategies requires testing in animal models before being tested in clinical trials. In this context, Galleria mellonella could represent a valid alternative to traditional mammalian and non-mammalian animal models, due to its low cost, ease of handling, and valuable biological properties to investigate host–pathogen interactions. The purpose of this review is to provide an updated overview of the literature concerning the use of G. mellonella larvae as an animal model to evaluate safety and efficacy of nanoparticles and nanomaterials, particularly, of those that are used or are under investigation to combat microbial pathogens. [ABSTRACT FROM AUTHOR]

An innovative approach to ticks and insect pests management is necessary to mitigate the challenges posed by the indiscriminate use of chemical pesticides, which can lead to resistance development and environmental pollution. Despite their great potential, biological control agents have significant manufacturing, application, and stability limitations. Currently, using phytochemicals, biosynthesized nanoparticles, and bioagents to get rid of arthropods might be a good alternative that would make farmers less worried about residues and resistance. The present investigation was carried out to determine for the first time the in vitro acaricidal and insecticidal efficacies of endogenous two entomopathogenic nematodes (EPNs), Heterorhabditis indica and Steinernema sp. combined with either Proteus mirabilis-synthesized silver nanoparticles or Rosmarinus officinalis essential oil against the camel tick, Hyalomma dromedarii larvae and females, and greater wax moth, Galleria mellonella larvae as well. We also determined the potential effects of these treatments on the biological characteristics of H. dromedarii's engorged females. We further investigated R. officinalis essential oil (EO) profiling and nanoparticle (AgNPs) characterization. All the evaluated combinations demonstrated synergistic effects on the larvae of G. mellonella and H. dromedarii, as well as on engorged females. When H. indica was mixed with EO or AgNPs, it worked well than when Steinernema sp. was mixed with EO or AgNPs. This was shown by the highest number of tick and insect mortalities and the lowest lethal concentration (LC50) values. One day after G. mellonella was exposed to H. indica (1,000 infective juveniles (IJs)) together with EO at 60 or 40 mg/mL, all tested individuals died. We obtained the same results when H. dromedarii females exposed to the same level of EPN with 60 mg/mL EO, and when H. dromedarii larvae treated with H. indica at 500 IJs + EO at 25 mg/mL. Treatments altered all biological parameters of engorged females, revealing extremely noticeable differences between the treated and untreated groups. Gas chromatography–mass spectrometry (GC-MS) analysis identified a total of 28 compounds in the R. officinalis EO. Visual observation showed a color change from yellow to dark brown for AgNPs biosynthesized from P. mirabilis; the transmission electron microscopy (TEM) image and ultraviolet–visible (UV-Vis) spectrum showed well-dispersed particles with a diameter of 5–45 nm; and the greatest surface plasmon peaked at 320 nm. The results demonstrated the high efficacy of combining EPN, H. indica, with EO to control tick and insect pests. This is due to its acaricidal activity on different stages of H. dromedarii, including larvae and engorged females, and its larvicidal effect on G. mellonella. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is one of the most important foodborne pathogens that can cause invasive listeriosis. In this study, the virulence levels of 26 strains of L. monocytogenes isolated from food and clinical samples in Shanghai, China, between 2020 and 2022 were analyzed. There were significant differences among isolates in terms of their mortality rate in Galleria mellonella, cytotoxicity to JEG-3 cells, hemolytic activity, and expression of important virulence genes. Compared with other STs, both the ST121 (food source) and ST1930 (clinic source) strains exhibited higher G. mellonella mortality. The 48 h mortality in G. mellonella of lineage II strains was significantly higher than that in lineage I. Compared with other STs, ST1930, ST3, ST5, and ST1032 exhibited higher cytotoxicity to JEG-3 cells. Based on the classification of sources (food and clinical strains) and serogroups (II a, II b, and II c), there were no significant differences observed in terms of G. mellonella mortality, cytotoxicity, and hemolytic activity. In addition, ST121 exhibited significantly higher hly, inlA, inlB, prfA, plcA, and plcB gene expression compared with other STs. A gray relation analysis showed a high correlation between the toxicity of G. mellonella and the expression of the hly and inlB genes; in addition, L. monocytogenes may have a consistent virulence mechanism involving hemolysis activity and cytotoxicity. Through the integration of in vivo and in vitro infection models with information on the expression of virulence factor genes, the differences in virulence between strains or subtypes can be better understood. [ABSTRACT FROM AUTHOR]

Background: Photodynamic therapy (PDT) is a treatment modality that uses light to activate a photosensitizing agent, destroying target cells. The growing awareness of the necessity to reduce or eliminate the use of mammals in research has prompted the search for safer toxicity testing models aligned with the new global guidelines and compliant with the relevant regulations. Objective: The objective of this study was to assess the impact of PDT on alternative models to mammals, including in vitro three-dimensional (3D) cultures and in vivo, in invertebrate animals, utilizing a potent photosensitizer, 2-hydroxychalcone. Methods: Cytotoxicity was assessed in two cellular models: monolayer (2D) and 3D. For this purpose, spheroids of two cell lines, primary dermal fibroblasts (HDFa) and adult human epidermal cell keratinocytes (HaCat), were developed and characterized following criteria on cell viability, shape, diameter, and number of cells. The survival percentages of Caenorhabditis elegans and Galleria mellonella were evaluated at 1 and 7 days, respectively. Results: The findings indicated that all the assessed platforms are appropriate for investigating PDT toxicity. Furthermore, 2-hydroxychalcone demonstrated low toxicity in the absence of light and when mediated by PDT across a range of in vitro (2D and 3D cultures) and in vivo (invertebrate animal models, including G. mellonella and C. elegans) models. Conclusion: There was a strong correlation between the in vitro and in vivo tests, with similar toxicity results, particularly in the 3D models and C. elegans, where the concentration for 50% viability was approximately 100 µg/mL. [ABSTRACT FROM AUTHOR]

Simple Summary: This study aimed to evaluate the active constituents and fumigation effectiveness of 16 distinct plant essential oils (EOs) on greater wax moth (GWM) eggs and fifth instar larvae. The results show that wintergreen, star anise, and clove oils exerted significant insecticidal effects on both GWM eggs and fifth instar larvae. Methyl salicylate and trans-anethole, which constitute 93.26% and 87.75% of wintergreen and star anise oils, respectively, function as the sole active ingredient of their respective oils. Eugenol, which constitutes 77.75% of clove oil, is primarily responsible for its insecticidal activity, although it is not the sole contributor. All these EO components exhibit significant insecticidal properties, with trans-anethole demonstrating the highest level of toxicity. Our research adds valuable insights to the literature by presenting the first investigation into the effectiveness of EOs for fumigating GWM eggs. Moreover, the results highlight the potential use of anise oil as a novel approach for managing GWM, thus offering a more efficient alternative to traditional methods. The greater wax moth (GWM, Galleria mellonella) is a prevalent pest of the honeybee and a significant risk to both honeybee populations and honeycomb storage. Research on the toxicity of essential oils (EOs) to GWM larvae has provided promising results, although their ovicidal effects and active ingredients require further study. Identifying effective plant compounds is essential for developing insecticides for GWM control. This study assessed the fumigation efficacy of 16 EOs on GWM eggs and fifth instar larvae and determined the effectiveness of these EOs and their primary components for fumigating fifth larvae. Wintergreen, star anise, and clove oils demonstrated significant insecticidal effects on GWM eggs and fifth instar larvae, resulting in a mortality rate exceeding 80% within 48 h. Gas chromatography-mass spectrometry analysis identified methyl salicylate (93.26%), trans-anethole (87.75%), and eugenol (77.75%) as the primary compounds in wintergreen, star anise, and clove oils, respectively. Further toxicity testing confirmed that these compounds were responsible for the observed insecticidal properties of the EOs. Notably, trans-anethole exhibited the lowest LC50 value (25.22 μL/L) against the fifth instar larvae of GWM and significant toxicity against GWM eggs and fifth instar larvae, suggesting its potential as a viable option for the future control of GWM populations. [ABSTRACT FROM AUTHOR]

Simple Summary: Cuticular lipid composition is extensively studied in social insects. Our work is the first to investigate metamorphosis-related changes and the effect of copulation on the cuticular free fatty acid (FFA) profile of male and female nonsocial Galleria mellonella Linnaeus (Lepidoptera: Pyralidae). The radical reconstruction occurring inside the pupal body is accompanied by qualitative and quantitative changes in the cuticular FFA profiles on its surface, which differ between male and female pupae. A GC/MS analysis revealed the presence of 11 FFAs (from C8:0 to C21:1) in male pupae, and 13 FFAs in females. Adult virgin moths possess significantly fewer FFAs than pupae, but demonstrate three FFAs that are absent from pupae. Copulation significantly affected the composition of the FFAs and cholesterol concentration in the cuticle. The majority of insects reproduce sexually. Among the many factors involved in controlling the reproductive process, cuticular lipids play an important role as unique chemical signatures of species, developmental stage, and sex, and participate in mate recognition. An understanding of the sex- and metamorphosis-related fluctuations in the cuticular lipid profiles of harmful insects is necessary to hamper their reproductive process. A GC/MS analysis of the cuticular lipids of the beehive pest Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) revealed 11 FFAs in the male pupae (C8:0, C9:0, C14:0, C15:0, C16:1, C16:0, C17:0, C18:1, C18:0, C20:1, and C21:1) together with another two in the females (C10:0 and C17:1). As metamorphosis progressed, some FFAs disappeared from the pupal cuticle (C8:0 and C17:0 in both sexes, and C10:0, C17:1, and C20:1 only in female pupae) and the levels of the others changed. In adult virgin males and females, C8:0, C17:1, and C17:0 reappeared and two FFAs absent in pupae (C6:0 and C11:0) appeared. In virgin males, C13:0 also appeared (absent in pupae). Copulation resulted in the disappearance of C13:0 and C17:1, decreased the concentrations of C9:0, C11:0, C18:1, and C18:0, and elevated the amounts of C14:0, C16:1, and C16:0 in mated males. In mated females, the concentrations of C11:0, C14:0, C15:0, C16:0, C17:1, and C18:1 increased while C18:1 decreased. Copulation reduced cholesterol levels in mated females, and increased those in males. [ABSTRACT FROM AUTHOR]

Greater wax moth (GWM), Galleria mellonella (Lepidoptera: Pyralidae), is a destructive pest of honeybee hives. They lay eggs in the bee hives; and the hatched larvae feed on beeswax. The unique ability of GWM to digest the beeswax that contains ethene (CH2=CH2) bond has attracted attention because the same bond is also present in plastic polymers. Polymer-digesting ability, being a very uncommon among animals, we suspected a assistive role of gut bacteria of the insect in the process. The present study aimed to isolate and characterize potential polymer-degrading bacteria from GWM gut, following a culture-dependent approach, and to characterize them morphologically, biochemically, and with 16S rDNA sequences. We also intended to study polymer-degrading abilities of those bacteria, and check the presence of esterase, one of the most potent enzymes involved in plastic polymer degradation, using tributyrin agar test. We found nine bacterial isolates from GMW gut. Out of them, six showed positive results for plastic degradation to varying extents (19.3-31.2%), after 30 days of co-culture with plastic sheets. The esterase enzyme was found to be present in all of them in a qualitative test. Through 16S rDNA sequencing, four isolates were identified as gram-negative Serratia marcescens strains, one as gram-negative Ralstonia pickettii and one as gram-positive Bacillus cereus, some of which are known potential polymer degraders. Therefore, our hypothesis of involvement of gut bacteria in the digestion of polymers by Greater wax moth larvae was perhaps correct. There is possibility of exploiting the bacterial isolates for plastic-pollution remediation; after deeper and further experimentation. [ABSTRACT FROM AUTHOR]

Worldwide, invasive candidiasis are a burden for the health system due to difficulties to manage patients, to the increasing of the resistance of the current therapeutics and the emergence of naturally resistant species of Candida. In this context, the development of innovative antifungal drugs is urgently needed. During invasive candidiasis, yeast is submitted to many stresses (oxidative, thermic, osmotic) in the human host. In order to resist in this context, yeast develops different strategy, especially the biosynthesis of trehalose. Starting from the 3D structural data of TPS2, an enzyme implicated in trehalose biosynthesis, we identified hydrazone as an interesting scaffold to design new antifungal drugs. Interestingly, our hydrazone derivatives which demonstrate antifungal and anti-biofilm effects on Candida spp., are non-toxic in in vitro and in vivo models (Galleria mellonella). [ABSTRACT FROM AUTHOR]

This is the report of mixed infection of an entomopathogenic nematode and a free-living bacteriophage nematode both invading Galleria mellonella larvae, apparently showing strong intraguild competition. The entomopathogenic nematodes isolated in this study were classified as Heterorhabditis kankabi n. sp., and Metarhabditis rainai through light and electronic microscopy, sequencing of ITS and 28S ribosomal DNA regions, and phylogenetic analyses. The bacteriophage nematode M. rainai was recognized as a facultative necromenic nematode, as it consumed the food resources obtained by entomopathogenic nematodes, directly affecting the life cycle of the later by stopping their development and forcing them to leave G. mellonella in search of new hosts. [ABSTRACT FROM AUTHOR]

Objective To evaluate the pathogenicity of Exophiala dermatitidis melanin in Galleria mellonella infection model. Methods Melanin of albino strain of Exophiala dermatitidis was restored by co-culture with E. dermatitidis (mel3). The G. mellonella were infected with albino, melanized strtain, and melanin restored albino strain. Survival rate, macromorphological and histopathological changes of larvae were observed under the optimal infection concentration. Results The melanin production was restored in albino strain of Exophiala dermatitidis, and conidia at a concentration of 1×107/mL was the optimal infection concentration. At the end of observation, the albino strain displayed lower survival rate compared to both the melanized stain (χ2=6.11，P=0.013) and melanin restored albino strain (χ2=6.37, P=0.010). However, the survival rates did not differ significantly between melanized and melanin restored albino strain (χ2=0.01，P=0.940). Histopathological examination revealed that the melanized strain and melanin restored albino strain attracted aggregation of more Galleria mellonella hemocytes, forming larger infectious nodules in comparison to albino strains. Conclusions In the infection model of Galleria mellonella, the albino strain shows stronger pathogenicity than the melanized strain. Restoration of melanin of albino strain can reverse the pathogenicity.

Abstract Lepidopteran silk is a complex mixture of proteins, consisting mainly of fibroins and sericins. Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for silk fibers. So far, five genes encoding sericin proteins have been identified in Bombyx mori. Having previously identified sericin protein 150 (SP150) as a major sericin-like protein in the cocoons of the pyralid moths Galleria mellonella and Ephestia kuehniella, we describe the identification of its homolog in B. mori. Our refined gene model shows that it consists of four exons and a long open reading frame with a conserved motif, CXCXCX, at the C-terminus, reminiscent of the structure observed in a class of mucin proteins. Notably, despite a similar expression pattern, both mRNA and protein levels of B. mori SP150 were significantly lower than those of its pyralid counterpart. We also discuss the synteny of homologous genes on corresponding chromosomes in different moth species and the possible phylogenetic relationships between SP150 and certain mucin-like proteins. Our results improve our understanding of silk structure and the evolutionary relationships between adhesion proteins in the silk of different lepidopteran species.

Simple Summary: Candida albicans can cause common mucosal and severe systemic infections, especially in immunocompromised patients. Current usage of antifungal drugs often leads to side effects and the development of drug-resistant strains. Cordyceps militaris, a parasitic fungus with various medicinal properties, is known for its immunomodulatory role. This study addresses the need for effective treatments against C. albicans infections by analyzing the inhibitory effects of C. militaris aqueous extract on C. albicans and its impact on the immune response in larvae of the greater wax moth Galleria mellonella. C. militaris extract, at nontoxic concentrations, significantly prolonged the survival of larvae infected with a sublethal dose of C. albicans. Additionally, the extract boosted the immune response by increasing hemocyte count and upregulating genes responsible for producing antimicrobial peptides. These findings suggest that C. militaris extract can effectively modulate the immune system and provide protection against fungal infections. This research highlights the potential of C. militaris as a natural, safe, and effective treatment option for C. albicans infections. Therefore, the aqueous extract of C. militaris is contributing to the development of alternative medicines. In addition, the utilization of G. mellonella larvae in this study can reduce the reliance on mammalian models in medical research, benefiting both human health and scientific research. Cordyceps militaris-derived formulations are currently used for multiple purposes because of their medical properties, especially immune system modulation. This study analyzes the inhibitory effects of C. militaris aqueous extract on Candida albicans infections and the immune response in larvae of the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae). Larvae exhibited melanization within 1 h of being infected with C. albicans inoculum at a concentration of 106 cells/larvae, and died within 24 h from a lethal dose. Aqueous extract of C. militaris proved to be nontoxic at concentrations of 0.25 and 0.125 mg/larvae, and had the greatest ability to prolong the survival of larvae infected with a sublethal dose of C. albicans at a concentration of 105 cells/larvae. Moreover, the number of hemocytes in the hemolymph of G. mellonella increased after infection with C. albicans and treatment with the aqueous extract of C. militaris at 1, 24, and 48 h by 1.21 × 107, 1.23 × 107, and 1.4 × 107 cells/100 µL, respectively. The highest number of hemocytes was recorded after treatment of infected G. mellonella with the extract for 48 h. Transcriptional upregulation of the immune system was observed in certain antimicrobial peptides (AMPs), showing that the relative expression of galiomicin, gallerimycin, and lysozyme genes were upregulated as early as 1 h after infection. Therefore, we conclude that C. militaris aqueous extract can modulate the immune system of G. mellonella and protect against infection from C. albicans. [ABSTRACT FROM AUTHOR]

Benzyl alcohol (E1519) is an aromatic alcohol used in the pharmaceutical and food industry. It is used to protect food products against microorganisms during storage, as a flavoring in the production of chocolate and confectionery products, as an important ingredient in fragrance, and as a preservative in medical products. However, little is known of its effect on insects. The main aim of this study was to determine the influence of benzyl alcohol on the defense systems of the wax moth Galleria mellonella, i.e., its cuticular lipid composition and critical elements of its immune system. A gas chromatography/mass spectrometry (GC/MS) analysis found benzyl alcohol treatment to elicit significant quantitative and qualitative differences in cuticular free fatty acid (FFA) profiles. Our findings indicate that benzyl alcohol treatment increased the levels of HSP70 and HSP90 and decreased those of HSF1, histamine, and cysteinyl leukotriene. Benzyl alcohol application also increased dismutase level in the hemolymph and lowered those of catalase and 8-OHdG. The treatment also had negative effects on G. mellonella hemocytes and a Sf9 cell line in vitro: 48-h treatment resulted in morphological changes, with the remaining cells being clearly spindle-shaped with numerous granules. The high insecticidal activity of compound and its lack of toxicity towards vertebrates suggest it could be an effective insecticide. [ABSTRACT FROM AUTHOR]

Introduction: Recent legislative advancements emphasize the need for alternative methods in assessing efficacy and safety while adhering to the 3Rs principles (reduce, replace, and refine) of animal experimentation. In this context, Galleria mellonella has become a well-established model in safety and efficacy research. However, existing studies predominantly focus on its use in injectable routes concerning pathogens and chemical compounds, leaving a significant gap in understanding other administration pathways, particularly topical application. To address this gap and contribute to the validation of G. mellonella as a model for cutaneous absorption studies, our investigation compares the toxicity data of gallic acid (GA) in mammalian models, alternative animal models such as G. mellonella and Caenorhabditis elegans, as well as monolayer cell cultures, providing insights valuable for future research. Methods: This was an experimental study aimed at evaluating the toxicity and permeation of gallic acid (GA) using different in vivo and in vitro alternative models, including G. mellonella, C. elegans, and monolayer cell cultures. Our study specifically evaluated the toxicity of GA in solution using human keratinocytes (HaCat), human dermal fibroblasts (HDFa), and human liver cell lines (HepG2), in addition to invertebrate animal models of C. elegans and semi-solid formulations in G. mellonella via topical administration. The results demonstrated GA's higher toxicity in C. elegans, followed by sequential susceptibility in HaCat, HepG2, and HDFa cells. Results: Surprisingly, G. mellonella displayed a notably high tolerance to GA, presenting no discernible alterations in cellular immune responses in injectable nor topical administration. Observations of the humoral immune response in G. mellonella larvae showcased melanization both administration conditions, indicating absorption following topical administration of higher GA concentrations. Conclusions: GA demonstrated low toxicity in cellular models, whereas G. mellonella revealed promise as a methodology for topical toxicity testing in cutaneous absorption. However, further research is essential to validate its efficacy. These findings demonstrated the potential to establish correlations in toxicology data between various alternative methodologies and mammals, potentially aiding in the prediction of toxicity in humans. [ABSTRACT FROM AUTHOR]

The ERG6 gene encodes the sterol C24-methyltransferase converting zymosterol to fecosterol in the ergosterol biosynthetic pathway. Here, we extend the results of functional analysis of the CgERG6 gene, which was previously shown to modulate drug susceptibility in Candida glabrata mutant cells, by demonstrating that its deletion leads to increased susceptibility to cycloheximide, 4-nitroquinoline-N-oxide and weak organic acids, and such effects are associated with attenuated virulence. Together with abrogated efflux of drug substrates by CgCdr1p and CgPdr12p, the Cgerg6Δ mutation leads to reduced cell surface hydrophobicity and decreased virulence of the mutant cells of C. glabrata. The absence of CgErg6p impacts the lipid organization and function of the plasma membrane, resulting in non-specific permeability and abrogation of normal function of membrane-bound proteins accompanied by decreased virulence in Cgerg6Δ cells. Galleria mellonella larvae were used as a non-vertebrate animal host model to determine differences in the virulence potential of C. glabrata strains (parental strain and the Cgerg6Δ deletion mutant). We found that Cgerg6Δ mutant strain attenuated in virulence caused 25–30% survival of larvae compared with parental strain. [ABSTRACT FROM AUTHOR]

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Introduction: Managing burn injuries is a challenge in healthcare. Due to the alarming increase in antibiotic resistance, new prophylactic and therapeutic strategies are being sought. This study aimed to evaluate the potential of live Lactic Acid Bacteria for managing burn infections, using Galleria mellonella larvae as an alternative preclinical animal model and comparing the outcomes with a common antibiotic. Methods: The antimicrobial activity of LAB isolated from human breast milk was assessed in vitro against Pseudomonas aeruginosa ATCC 27853. Additionally, the immunomodulatory effects of LAB were evaluated in vivo using the G. mellonella burn wound infection model. Results and discussion: In vitro results demonstrated the antimicrobial activity of Lactic Acid Bacteria against P. aeruginosa. In vivo results show that their prophylactic treatment improves, statistically significant, larval survival and modulates the expression of immunity-related genes, Gallerimycin and Relish/NF-kB, strain-dependently. These findings lay the foundation and suggest a promising alternative for burn wound prevention and management, reducing the risk of antibiotic resistance, enhancing immune modulation, and validating the potential G. mellonella as a skin burn wound model. [ABSTRACT FROM AUTHOR]

Enterotoxigenic Escherichia coli (ETEC) is the major bacterial cause of diarrheal diseases in pigs, particularly at young ages, resulting in significant costs to swine farming. The pathogenicity of ETEC is largely dependent on the presence of fimbriae and the ability to produce toxins. Fimbriae are responsible for their initial adhesion to the intestinal epithelial cells, leading to the onset of infection. In particular, the F4 type (K88) fimbriae are often attributed to neonatal infections and have also been associated with post-weaning diarrheal infections. This disease is traditionally prevented or treated with antibiotics, but their use is being severely restricted due to the emergence of resistant bacteria and their impact on human health. Emerging approaches such as aptamers that target the F4-type fimbriae and block the initial ETEC adhesion are a promising alternative. The aim of this study is to assess the effectiveness of two aptamers, Apt31 and Apt37, in controlling ETEC infection in the G. mellonella in vivo model. Initially, the dissociation constant (KD) of each aptamer against ETEC was established using real-time quantitative PCR methodology. Subsequently, different concentrations of the aptamers were injected into Galleria mellonella to study their toxicity. Afterwards, the anti-ETEC potential of Apt31 and Apt37 was assessed in the larvae model. The determined KD was 81.79 nM (95% CI: 31.21-199.4 nM) and 50.71 nM (95% CI: 26.52-96.15 nM) for the Apt31 and Apt37, respectively, showing no statistical difference. No toxicity was observed in G. mellonella following injection with both aptamers at any concentration. However, the administration of Apt31 together with ETEC-F4+ in G. mellonella resulted in a significant improvement of approximately 30% in both larvae survival and health index compared to ETEC-F4+ alone. These findings suggest that aptamers have promising inhibitory effect against ETEC infections and pave the way for additional in vivo studies. [ABSTRACT FROM AUTHOR]

Lepidopteran silk is a complex mixture of proteins, consisting mainly of fibroins and sericins. Sericins are a small family of highly divergent proteins that serve as adhesives and coatings for silk fibers. So far, five genes encoding sericin proteins have been identified in Bombyx mori. Having previously identified sericin protein 150 (SP150) as a major sericin-like protein in the cocoons of the pyralid moths Galleria mellonella and Ephestia kuehniella, we describe the identification of its homolog in B. mori. Our refined gene model shows that it consists of four exons and a long open reading frame with a conserved motif, CXCXCX, at the C-terminus, reminiscent of the structure observed in a class of mucin proteins. Notably, despite a similar expression pattern, both mRNA and protein levels of B. mori SP150 were significantly lower than those of its pyralid counterpart. We also discuss the synteny of homologous genes on corresponding chromosomes in different moth species and the possible phylogenetic relationships between SP150 and certain mucin-like proteins. Our results improve our understanding of silk structure and the evolutionary relationships between adhesion proteins in the silk of different lepidopteran species. [ABSTRACT FROM AUTHOR]

Mycetoma, a neglected infection of subcutaneous tissues, poses a significant health burden, especially in tropical regions. It is caused by fungal (eumycetoma) and bacterial (actinomycetoma) pathogens, with current treatments often providing unsatisfactory outcomes. This study aims to discover novel broad-spectrum antimicrobial agents to circumvent the lengthy and costly diagnostic procedures. Eight essential oils (EOs) from the roots and aerial parts of Geigeria alata, Lavandula angustifolia, Melaleuca alternifolia, Myristica fragrans, Pimpinella anisum, Syzigum aromaticum, and Thymus vulgaris were prepared using steam distillation. The in vitro antimycetomal activity against Madurella mycetomatis and Actinomadura madurae strains was assessed using resazurin assays. The chemical compositions of the EOs were analyzed using gas chromatography and mass spectrometry (GC–MS). Promising EOs underwent further in vivo toxicity and efficacy testing in Galleria mellonella larvae models. EOs of G. alata roots, M. fragrans, P. anisum, S. aromaticum, and T. vulgaris showed wide-spectrum dual in vitro antimycetomal activity against all tested strains, with minimum inhibitory concentrations (MICs) ranging from 0.004 to 0.125% v/v. G. alata aerial parts and L. angustifolia EOs demonstrated activity predominantly against A. madurae, while M. alternifolia EO did not inhibit any tested strains. M. fragrans and P. anisum EOs significantly enhanced the survival of M. mycetomatis-infected larvae without inducing toxicity in uninfected larvae. Notably, P. anisum EO tended to enhance the survival of A. madurae-infected larvae, ranking it as the most promising EO among those tested. The investigated EOs, particularly P. anisum, exhibited promising broad-spectrum antimycetomal activity against fungal and bacterial pathogens responsible for mycetoma. These findings highlight the potential of essential oils as a basis for developing novel antimycetomal agents, offering hope for improved treatment strategies for this neglected disease. [ABSTRACT FROM AUTHOR]

Simple Summary: This study tested the use of Galleria mellonella larvae as a biodegradation strategy for various types of plastics that were provided to larvae as their sole food source. The physical and enzymatic action of the larvae fragmented and consumed the plastics in various proportions, but none were digested. This was confirmed by the presence of microplastics in excreta and in larval digestive tracts, which affected their life cycle continuity by inducing the early formation of pupae and reducing the number of eggs produced. The accumulation and unsustainable management of plastic waste generate environmental pollution that affects ecosystems, wildlife, and human health. We studied the possibility of using the consumption and digestion of oxo-biodegradable, compostable plastics and polypropylene from face masks by the fifth-instar larvae of G. mellonella as a strategy for the sustainable management of plastic waste. We used Fourier transform infrared spectrophotometry (FTIR) to determine the percentage of consumption and presence of microplastics in the digestive tract and excreta for 10 treatments evaluated for 135 h. The effects of plastics on the continuity of the life cycle of the greater hive moth were also determined. We established that the larvae fragmented and consumed 35.2 ± 23% of the plastics evaluated, with significant differences between treatments. Larvae were able to consume more of the intermediate layers of masks (86.31%) than the other plastics. However, none of the plastics were digested. Instead, microplastics accumulated in the excreta, resulting in nutritional deficits that affected the continuity of the life cycle, including the induction of the early formation of pupae after 24 h and a reduction in the number of eggs laid by the females. [ABSTRACT FROM AUTHOR]

The present study was initiated to validate whether the artificial or natural diets are the best for Galleria mellonella and Plodia interpunctella hosts for rearing Trichogramma turkestanica Meyer and to assess the effect of reared host eggs on the morpho-biological features of Trichogramma turkesta, nica and their suitability for parasitism. Results for the life tables parameters indicate that the shortest generation time and the lowest doubling time (DT) were recorded Plodia interpunctella which reared artificially (Plodia A), while the highest net reproductive rate (R0) was recorded for Galleria mellonella which reared artificially (Galleria A). The artificial diets are better than those for the natural diet for rearing G. mellonella and P. interpunctella and their generation periods in terms of the measured parameters. Results concerning the morphological features of Trichogramma females that were reared on the eggs of Galleria fed on artificial and natural diets were hosts better than Plodia A., Sitotroga N., and Plodia N under laboratory conditions. Also, the obtained data prove that the maximum mean percentage of successful parasitism and the maximum number of emerged female parasitoids were for Galleria A. In comparison, the rate of emerged adults from parasitized eggs was for Plodia A. It is of interest to point herein that the role of the host diet leads to improving the morpho-biological parameters of Trichogramma which can enhance the success of biological control programs as an essential part of Integrated Pest Management (IPM). This contributes to achieving the Sustainable Development Goals (SDGs). [ABSTRACT FROM AUTHOR]

IntroductionThe capsular polysaccharide (CPS) of pathogenic bacteria is a critical virulence factor, often evading phagocytosis by host immune cells, while also interfering with the contact of the pathogen with host cells and contributing to biofilm formation. Klebsiella pneumoniae, a Gram-negative human pathogen associated with high antimicrobial resistances, produces 77 CPS serotypes. The CPS masks proteinaceous factors but also protects K. pneumoniae from uptake by host phagocytic cells and activation of the complement system. In addition to nosocomial, urinary tract and bloodstream infections or pneumonia hypervirulent strains have a highly mucoid phenotype and can cause soft tissue infections, liver abscesses, and meningitis as well. The CPS is therefore crucial for both escaping detection by the immune system and enhancing the virulence potential.MethodsIn this study, we generated a non-encapsulated mutant (Kpn2146∆wza) to observe how the CPS interferes with K. pneumoniae adhesion, survival in blood, and invasiveness in an experimental infection model.ResultsInfection of A549 lung epithelial cells showed similar adherence levels for the wild-type and non-capsulated strain, while our data showed a moderately higher internalization of Kpn2146Δwza when compared to the wild-type. In whole blood killing assays, we demonstrate that the K. pneumoniae capsule is essential for survival in human blood, protecting K. pneumoniae against recognition and clearance by the human immune system, as well as complement-mediated opsonization and killing. The non-encapsulated mutant, in contrast, was unable to survive in either whole blood or human plasma. Infections of Galleria mellonella larvae showed a significantly decreased virulence potential of the CPS-deficient mutant.DiscussionIn conclusion, our data indicate a crucial role of CPS in vivo.

ABSTRACT Klebsiella pneumoniae is a significant healthcare-associated pathogen, notable for its diverse virulence and antibiotic resistance profiles. This study aimed to characterize the genotypic and phenotypic diversity of K. pneumoniae isolates and evaluate their virulence using the Galleria mellonella model. Biomass production, metabolic activity, capsule formation, and siderophore production were assessed in 27 K. pneumoniae isolates from hospital-associated infections. Lethality curves were generated using the G. mellonella model, with survival monitored hourly from 16 to 48 hours. The most common sequence types (ST) identified were the high-risk clones ST307 (N = 10), ST512 (N = 8), ST101 (N = 7), and ST661 (N = 2). These STs were associated with distinct K-locus, including KL102, KL107, KL17, and KL39. Most isolates belonged to the O2afg locus (N = 18), with the K. pneumoniae carbapenemase genotype detected in 96.3% of strains. None of the isolates were classified as hypervirulent. Phenotypically, ST661 exhibited the highest biomass production despite showing similar metabolic activity to other STs. A positive correlation was observed between biomass and siderophore production, while capsule production was inversely correlated with biomass. In the G. mellonella model, ST661 demonstrated the highest virulence, resulting in 100% mortality by 48 hours, compared to survival rates of 21.4% for ST101, 38.0% for ST307, and 31.2% for ST512. These findings underscore the pathogenic potential of ST661 isolates with enhanced biofilm production. The G. mellonella model may serve as an effective in vivo system for evaluating the virulence of emerging K. pneumoniae lineages.IMPORTANCEWe demonstrate that the Galleria mellonella model is a useful tool to analyze the virulence of carbapenem-resistant Klebsiella pneumoniae strains. Our findings highlight the pathogenicity of carbapenem-resistant K pneumoniae isolates, particularly the role of the ST661 that, despite being a rare lineage, harbors the blaVIM gene and is associated with high biofilm production and the highest mortality rates.

ABSTRACT Larvae of Galleria mellonella (the greater wax moth) are being increasingly used as a model to study microbial pathogenesis. In this model, bacterial virulence is typically measured by determining the 50% lethal dose (LD50) of a bacterial strain or mutant. The use of G. mellonella to study Pseudomonas aeruginosa pathogenesis, however, is challenging because of the extreme sensitivity of larvae to this bacterium. For some P. aeruginosa strains, as few as 1–5 colony-forming units are sufficient to kill G. mellonella, which poses challenges for determining LD50 values. For this reason, some groups have used time-to-death as a measure of P. aeruginosa virulence, but methodologies have not been standardized. We provide a detailed protocol for using the time at which 50% of larvae have died (LT50) at a particular inoculum as a measure of P. aeruginosa virulence. We also describe a quality control metric for enhancing the reproducibility of LT50 values. This approach provides an accurate and reproducible methodology for using G. mellonella larvae to measure and compare the virulence of P. aeruginosa strains.IMPORTANCEPseudomonas aeruginosa is a significant cause of morbidity and mortality. The invertebrate Galleria mellonella is used as a model to determine the virulence of P. aeruginosa strains. We provide a protocol and analytical approach for using a time-to-death metric to accurately quantify the virulence of P. aeruginosa strains in G. mellonella larvae. This methodology, which has several advantages over 50% lethal dose approaches, is a useful resource for the study of P. aeruginosa pathogenicity.

An innovative approach to ticks and insect pests management is necessary to mitigate the challenges posed by the indiscriminate use of chemical pesticides, which can lead to resistance development and environmental pollution. Despite their great potential, biological control agents have significant manufacturing, application, and stability limitations. Currently, using phytochemicals, biosynthesized nanoparticles, and bioagents to get rid of arthropods might be a good alternative that would make farmers less worried about residues and resistance. The present investigation was carried out to determine for the first time the in vitro acaricidal and insecticidal efficacies of endogenous two entomopathogenic nematodes (EPNs), Heterorhabditis indica and Steinernema sp. combined with either Proteus mirabilis-synthesized silver nanoparticles or Rosmarinus officinalis essential oil against the camel tick, Hyalomma dromedarii larvae and females, and greater wax moth, Galleria mellonella larvae as well. We also determined the potential effects of these treatments on the biological characteristics of H. dromedarii’s engorged females. We further investigated R. officinalis essential oil (EO) profiling and nanoparticle (AgNPs) characterization. All the evaluated combinations demonstrated synergistic effects on the larvae of G. mellonella and H. dromedarii, as well as on engorged females. When H. indica was mixed with EO or AgNPs, it worked well than when Steinernema sp. was mixed with EO or AgNPs. This was shown by the highest number of tick and insect mortalities and the lowest lethal concentration (LC50) values. One day after G. mellonella was exposed to H. indica (1,000 infective juveniles (IJs)) together with EO at 60 or 40 mg/mL, all tested individuals died. We obtained the same results when H. dromedarii females exposed to the same level of EPN with 60 mg/mL EO, and when H. dromedarii larvae treated with H. indica at 500 IJs + EO at 25 mg/mL. Treatments altered all biological parameters of engorged females, revealing extremely noticeable differences between the treated and untreated groups. Gas chromatography–mass spectrometry (GC-MS) analysis identified a total of 28 compounds in the R. officinalis EO. Visual observation showed a color change from yellow to dark brown for AgNPs biosynthesized from P. mirabilis; the transmission electron microscopy (TEM) image and ultraviolet–visible (UV-Vis) spectrum showed well-dispersed particles with a diameter of 5–45 nm; and the greatest surface plasmon peaked at 320 nm. The results demonstrated the high efficacy of combining EPN, H. indica, with EO to control tick and insect pests. This is due to its acaricidal activity on different stages of H. dromedarii, including larvae and engorged females, and its larvicidal effect on G. mellonella.

ABSTRACTOver the last 20 years, the larva of the greater waxmoth, Galleria mellonella, has rapidly increased in popularity as an in vivo mammalian replacement model organism for the study of human pathogens. Experimental readouts of response to infection are most often limited to observing the melanization cascade and quantifying larval death and, whilst transcriptomic and proteomic approaches, and methods to determine microbial load are also used, a more comprehensive toolkit of profiling infection over time could transform the applicability of this model. As an invertebrate, Galleria harbour an innate immune system comprised of both humoral components and a repertoire of innate immune cells – termed haemocytes. Although information on subtypes of haemocytes exists, there are conflicting reports on their exact number and function. Flow cytometry has previously been used to assay Galleria haemocytes, but protocols include both centrifugation and fixation – physical methods which have the potential to affect haemocyte morphology prior to analysis. Here, we present a method for live haemocyte analysis by flow cytometry, revealing that Galleria haemocytes constitute only a single resolvable population, based on relative size or internal complexity. Using fluorescent zymosan particles, we extend our method to show that up to 80% of the Galleria haemocyte population display phagocytic capability. Finally, we demonstrate that the developed assay reliably replicates in vitro data, showing that cell wall β-1,3-glucan masking by Candida albicans subverts phagocytic responses. As such, our method provides a new tool with which to rapidly assess phagocytosis and understand live infection dynamics in Galleria.

ABSTRACTCryptococcus neoformans is an environmental yeast that primarily affects immunocompromised individuals, causing respiratory infections and life-threatening meningoencephalitis. Treatment is complicated by limited antifungal options, with concerns such as adverse effects, dose-limiting toxicity, blood–brain barrier permeability, and resistance development, emphasizing the critical need to optimize and expand current treatment options against invasive cryptococcosis. Galleria mellonella larvae have been introduced as an ethical intermediate for in vivo testing, bridging the gap between in vitro antifungal screening and mouse studies. However, current infection readouts in G. mellonella are indirect, insensitive, or invasive, which hampers the full potential of the model. To address the absence of a reliable non-invasive method for tracking infection, we longitudinally quantified the cryptococcal burden in G. mellonella using bioluminescence imaging (BLI). After infection with firefly luciferase-expressing C. neoformans, the resulting bioluminescence signal was quantitatively validated using colony-forming unit analysis. Longitudinal comparison of BLI to health and survival analysis revealed increased sensitivity of BLI in discriminating cryptococcal burden during early infection. Furthermore, BLI improved the detection of treatment efficacy using first-line antifungals, thereby benchmarking this model for antifungal testing. In conclusion, we introduced BLI as a real-time, quantitative readout of cryptococcal burden in G. mellonella over time, enabling more sensitive and reliable antifungal screening.

Objective: To investigate the source of infection in a patient with recurrent severe neck infections caused by Klebsiella pneumoniae and to analyze the virulence of isolates obtained from different sites of the patient. Methods: We collected preoperative neck abscess puncture fluid, intraoperative neck drainage fluid, sputum, intestinal fecal specimens, and blood samples from a patient who visited Wuxi Second People's Hospital twice between 2017 and 2018. We conducted isolation, identification, drug sensitivity tests, and string tests on the isolates. Capsule serotyping and virulence gene analysis were performed using PCR. The genetic relationship of different isolates was assessed by Multilocus Sequence Typing and virulence was evaluated using the Galleria mellonella infection model. Additionally, whole-genome sequencing was used to analyze the chromosomal and plasmid genes of one isolate. Results: Klebsiella pneumoniae was detected in the sputum and fecal specimens from both hospitalizations, as well as the preoperative ultrasound-guided puncture fluid and intraoperative drainage fluid from the first hospitalization, resulting in six isolates. These isolates were all K16 serotype, positive in the string test, and identified as ST660 by Multilocus Sequence Typing, indicating they belonged to the same clone. Virulence gene analysis showed that wcaG, iucB, iroNB, rmpA, rmpA2, Aer, kfuBC, ureA, fimH, mrkD, uge, and peg344 were positive, while allS, cf29a, and Wzy_K1 were negative. In the Galleria mellonella virulence assay, the lethality of different isolates was dose-dependent. The K16 group showed significantly higher larval mortality compared to other control groups (including K1, K2, K5, K20, and K57 groups). Genome sequencing revealed that plasmid p17388 carried numerous virulence genes and insertion sequences, particularly ISKPN74, and showed high homology with other Klebsiella plasmids. Conclusion: This study is the first to report severe cervical necrotizing fasciitis caused by the K16-ST660 Klebsiella pneumoniae Isolate. The high virulence of these isolates was confirmed by the Galleria mellonella virulence assay and the detection of numerous virulence genes. In-depth analysis of plasmid p17388 suggests that ISKPN74 may enhance stable integration of the plasmid into the bacterial chromosome through recombinases and transposases, thereby reducing the likelihood of plasmid loss and increasing bacterial virulence. Additionally, IS5 family insertion sequences may carry extra promoters or enhancers that, when inserted upstream of mucoviscosity-associated genes such as rmpA, may increase the transcription levels of downstream genes. This ISKPN74-mediated integration or insertion reveals a complex genetic mechanism that may contribute to the severity of infections caused by ST660 isolates. Our findings offer new insights into the virulence and structure of ST660-K16 Klebsiella pneumoniae, suggesting that further investigation into the specific mechanisms by which these insertion sequences enhance virulence could aid in developing novel infection management strategies.

Abstract Natural products received much attention as an environmentally beneficial solution for pest management. Therefore, the extracts of invasive silverleaf nightshade (Solanum elaeagnifolium Cav.) weeds using their berries parts (seeds, peels and mucilage) supported by bioassay-guided fractionation were tested against both the greater wax moth (Galleria mellonella) and Erwinia carotovora pv. carotovora causes of the blackleg of potatoes. The seeds and peels of S. elaeagnifolium were successively extracted by maceration using dichloromethane (DCM), ethyl acetate (EtOAc), and ethanol (EtOH), respectively. While, its mucilage was extracted using EtOAc. The successive EtOH extract of the plant seeds had promising inhibition efficacy and the best minimal inhibition concentration (MIC) of 50 µg/ml against E. Carotovora amongst other extracts (DCM and EtOAc of the plant berries parts). Depending on dose response activity, EtOH extract had G. mellonella larval mortality and pupal duration rates (LC50; 198.30 and LC95; 1294.73 µg/ml), respectively. Additionally, this EtOH extract of seeds was fractionated using preparative TLC to three characteristic bands. The insecticidal and bacterial activities of these isolated bands (SEA, SEB, and SEC) were evaluated at a dose of 100 µg/ml, causing mortality by 48.48, 62.63 and 92.93% (G. mellonella larvae) and inhibition by 15.22, 0.00 and 31.66 mm (E. carotovora), respectively. Moreover, the separated major three bands were tentatively identified using LC-ESI-MS analysis revealing the presence of two phenolic acids; chlorogenic acid (SEA) and dicaffeoyl quinic acid (SEB) in addition to one steroidal saponin (SEC) annotated as borassoside E or yamoscin. Finally, the plant seeds’ successive EtOH extract as well as its active constituents, exhibited potential broad-spectrum activity and the ability to participate in future pest management initiatives. A field study is also recommended to validate its bio-efficacy against selected pests and to develop its formulations.

Aims: Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella. Methods: For the haematogenous infection, G. mellonella larvae were implanted with a Kirschner wire (K-wire), infected with S. aureus, and subsequently phages and/or gentamicin were administered. For the early-stage biofilm implant infection, the K-wires were pre-incubated with S. aureus suspension before implantation. After 24 hours, the larvae received phages and/or gentamicin. In both models, the larvae also received daily doses of phages and/or gentamicin for up to five days. The effect was determined by survival analysis for five days and quantitative culture of bacteria after two days of repetitive doses. Results: In the haematogenous infection, a single combined dose of phages and gentamicin, and repetitive injections with gentamicin or in combination with phages, resulted in significantly improved survival rates. In the early-stage biofilm infection, only repetitive combined administration of phages and gentamicin led to a significantly increased survival. Additionally, a significant reduction in number of bacteria was observed in the larvae after receiving repetitive doses of phages and/or gentamicin in both infection models. Conclusion: Based on our results, a single dose of the combination of phages and gentamicin is sufficient to prevent a haematogenous S. aureus implant-related infection, whereas gentamicin needs to be administered daily for the same effect. To treat early-stage S. aureus implant-related infection, repetitive doses of the combination of phages and gentamicin are required. Cite this article: Bone Joint Res 2024;13(8):383–391.

Abstract Acinetobacter baumannii (AB) has emerged as a major pathogen in vulnerable and severely ill patients. It remains unclear whether early mortality (EM) due to AB bacteremia is because of worse clinical characteristics of the infected patients or the virulence of the pathogen. In this study, we aimed to investigate the effect of AB virulence on EM due to bacteremia. This retrospective study included 138 patients with AB bacteremia (age: ≥ 18 years) who were admitted to a tertiary care teaching hospital in South Korea between 2015 and 2019. EM was defined as death occurring within 7 days of bacteremia onset. The AB clinical isolates obtained from the patients’ blood cultures were injected into 15 Galleria mellonella larvae each, which were incubated for 5 days. Clinical isolates were classified into high- and low-virulence groups based on the number of dead larvae. Patients’ clinical data were combined and subjected to multivariate Cox regression analyses to identify the risk factors for EM. In total, 48/138 (34.8%) patients died within 7 days of bacteremia onset. The Pitt bacteremia score was the only risk factor associated with EM. In conclusion, AB virulence had no independent effect on EM in patients with AB bacteremia.

This study aimed to evaluate the effect of biostimulants derived from Streptomyces avermitilis metabolites on entomopathogenic nematodes: Steinernema carpocapsae, Steinernema feltiae, and Heterorhabditis bacteriophora, obtained from the soil of several Miscanthus × giganteus plantations in 2020–2023. The nematodes were isolated, identified, and cultured using live insects (the greater wax moth Galleria mellonella). Three preparations containing biostimulants – Charkor, Stimpo, and Regoplant, were tested for compatibility with entomopathogenic and plant parasitic nematodes. Their effect on nematode survival was evaluated using the Petri dish test. The study showed that the effect of biostimulants on the survival of nematodes depended on the concentration of aversectin contained in the evaluated preparations. Stimpo and Regoplant had an adverse effect on plant parasitic nematodes. The highest G. mellonella mortality was observed at the higher dose of Charkor (0.4%), and the lowest at the lower dose of Regoplant (1.22%). The study showed that the virulence of the nematodes decreased after 48 hours of incubation in Regoplant and Stimpo solutions containing aversectin. The degree of interaction between nematodes depended on the nematode species, trophic preferences, and the concentration of active ingredients in the preparations. This conclusion is crucial for the selection of appropriate types of entomopathogenic nematodes and the application rate of plant parasitic nematodes in the assessment of their short- and long-term spread, persistence, and recycling in field conditions.

Natural products received much attention as an environmentally beneficial solution for pest management. Therefore, the extracts of invasive silverleaf nightshade (Solanum elaeagnifolium Cav.) weeds using their berries parts (seeds, peels and mucilage) supported by bioassay-guided fractionation were tested against both the greater wax moth (Galleria mellonella) and Erwinia carotovora pv. carotovora causes of the blackleg of potatoes. The seeds and peels of S. elaeagnifolium were successively extracted by maceration using dichloromethane (DCM), ethyl acetate (EtOAc), and ethanol (EtOH), respectively. While, its mucilage was extracted using EtOAc. The successive EtOH extract of the plant seeds had promising inhibition efficacy and the best minimal inhibition concentration (MIC) of 50 µg/ml against E. Carotovora amongst other extracts (DCM and EtOAc of the plant berries parts). Depending on dose response activity, EtOH extract had G. mellonella larval mortality and pupal duration rates (LC50; 198.30 and LC95; 1294.73 µg/ml), respectively. Additionally, this EtOH extract of seeds was fractionated using preparative TLC to three characteristic bands. The insecticidal and bacterial activities of these isolated bands (SEA, SEB, and SEC) were evaluated at a dose of 100 µg/ml, causing mortality by 48.48, 62.63 and 92.93% (G. mellonella larvae) and inhibition by 15.22, 0.00 and 31.66 mm (E. carotovora), respectively. Moreover, the separated major three bands were tentatively identified using LC-ESI-MS analysis revealing the presence of two phenolic acids; chlorogenic acid (SEA) and dicaffeoyl quinic acid (SEB) in addition to one steroidal saponin (SEC) annotated as borassoside E or yamoscin. Finally, the plant seeds' successive EtOH extract as well as its active constituents, exhibited potential broad-spectrum activity and the ability to participate in future pest management initiatives. A field study is also recommended to validate its bio-efficacy against selected pests and to develop its formulations. [ABSTRACT FROM AUTHOR]

Research into novel anti-Helicobacter pylori agents represents an important approach for the identification of new treatments for chronic gastritis and peptic ulcers, which are associated with a high risk of developing gastric carcinoma. In this respect, two series of azobenzenesulfonamides were designed, synthesized, and tested against a large panel of human and bacterial CAs to evaluate their inhibitory activity. In addition, computational studies of the novel primary benzenesulfonamides (4a–j) were performed to predict the putative binding mode to both HpCAs. Then, the antimicrobial activity versus H. pylori of the two series was also studied. The best-in-class compounds were found to be 4c and 4e among the primary azobenzenesulfonamides and 5c and 5f belonging to the secondary azobenzenesulfonamides series, showing themselves to exert a promising anti-H. pylori activity, with MIC values of 4–8 μg/mL and MBCs between 4 and 16 μg/mL. Moreover, the evaluation of their toxicity on a G. mellonella larva in vivo model indicated a safe profile for 4c,e and 5c,f. The collected results warrant considering these azobenzenesulfonamides as an interesting starting point for the development of a new class of anti-H. pylori agents. [ABSTRACT FROM AUTHOR]

Simple Summary: In recent years, the use of alternative animal models to vertebrates for the study of infectious processes and antimicrobial drug development has become a major challenge in experimentation. Insects, in particular Galleria mellonella, may represent a good model for preclinical studies, as their response to infections allows for the preliminary selection of molecules with biological activity in a potential sepsis event. However, discordant data are often reported in the literature, and this is often due to the different methods implemented in many laboratories. The aim of this work was therefore to develop a standard protocol for infection with a Gram-positive bacterium, as we consider it important to apply these unified methodologies in order to obtain reproducible data. Our results made it possible to define a correct growth curve of Micrococcus luteus and, in parallel, an infection methodology that would provide consistent and repeatable data. We are therefore confident that this work can be a support for preclinical studies on model insects, as a link between the development of new drugs and its availability for patients. The aim of this work was to develop an experimental protocol for the infection of Galleria mellonella with Gram-positive bacteria. Some physiological characteristics of these insects are comparable to those of vertebrates, therefore allowing the replacement of mammals in the preclinical phases of drug development. G. mellonella Linnaeus 1758 (Lepidoptera: Pyralidae) is accepted as an alternative model for the study of infectious diseases. Since data on infection procedures with different bacterial strains are scarce and sometimes conflicting, also due to different and non-uniform protocols, we developed an experimental protocol that would allow for controlled and repeatable infections, using the Gram-positive bacterium GRAS (Generally Regarded As Safe) Micrococcus luteus. After analyzing the morphology and defining the growth rate of M. luteus, doses of between 101 and 106 CFU/larvae were administered to late-stage larvae. The survival rate of the larvae was monitored up to 7 days and the LD50 determined. The bacterial clearance capacity of the larvae after injection with 103 and 105 CFU/larvae was assessed by hemolymph bacterial load analysis. The results made it possible to define the growth curve of M. luteus correlated with the CFU count; based on the LD50 (103.8 CFU/larvae) calculated on the survival of G. mellonella, infections were carried out to evaluate the immune efficiency of the larvae in bacterial clearance. This protocol, standardized on G. mellonella larvae, could provide a functional tool to study the course of bacterial infections. [ABSTRACT FROM AUTHOR]

Here, we report the simultaneous isolation of Pseudomonas straminea from blood cultures and from a skin ulcer in an elderly woman who suffered from atopic dermatitis and psoriasis and developed acute cellulitis of both arms requiring hospital treatment. To the best of our knowledge, P. straminea has not been previously reported to cause invasive infections in humans. This case highlights how chronic diseases and older age increase the susceptibility to bacterial infections with environmental bacteria of low virulence. Our study describes the microbiological identification of the blood culture isolate, including morpho-molecular characterization and virulence demonstration in a Galleria mellonella model. [ABSTRACT FROM AUTHOR]

Prosthetic joint infections (PJIs) can have disastrous consequences for patient health, including removal of the device, and placement of cemented implants is often required during surgery to eradicate PJIs. In translational research, in vivo models are widely used to assess the biocompatibility and antimicrobial efficacy of antimicrobial coatings and compounds. Here, we aim to utilize Galleria mellonella implant infection models to assess the antimicrobial activity of antibiotic-loaded bone cement (ALBC) implants. Therefore, we used commercially available bone cement loaded with either gentamicin alone (PALACOS R+G) or with a combination of gentamicin and vancomycin (COPAL G+V), compared to bone cement without antibiotics (PALACOS R). Firstly, the in vitro antimicrobial activity of ALBC was determined against Staphylococcus aureus. Next, the efficacy of ALBC implants was analyzed in both the G. mellonella hematogenous and early-stage biofilm implant infection model, by monitoring the survival of larvae over time. After 24 h, the number of bacteria on the implant surface and in the tissue was determined. Larvae receiving dual-loaded COPAL G+V implants showed higher survival rates compared to implants loaded with only gentamicin (PALACOS R+G) and the control implants without antibiotics (PALACOS R). In conclusion, G. mellonella larvae infection models with antibiotic-loaded bone cements are an excellent option to study (novel) antimicrobial approaches. [ABSTRACT FROM AUTHOR]

Vulvovaginal candidiasis (VVC) remains a prevalent fungal disease, characterized by challenges, such as increased fungal resistance, side effects of current treatments, and the rising prevalence of non-albicans Candida spp. naturally more resistant. This study aimed to propose a novel therapeutic approach by investigating the antifungal properties and toxicity of 2-hydroxychalcone (2-HC) and 3′-hydroxychalcone (3′-HC), both alone and in combination with fluconazole (FCZ) and clotrimazole (CTZ). A lipid carrier (LC) was also developed to deliver these molecules. The study evaluated in vitro anti-Candida activity against five Candida species and assessed cytotoxicity in the C33-A cell line. The safety and therapeutic efficacy of in vivo were tested using an alternative animal model, Galleria mellonella. The results showed antifungal activity of 2-HC and 3′-HC, ranging from 7.8 to 31.2 as fungistatic and 15.6 to 125.0 mg/L as fungicide effect, with cell viability above 80% from a concentration of 9.3 mg/L (2-HC). Synergistic and partially synergistic interactions of these chalcones with FCZ and CTZ demonstrated significant improvement in antifungal activity, with MIC values ranging from 0.06 to 62.5 mg/L. Some combinations reduced cytotoxicity, achieving 100% cell viability in many interactions. Additionally, two LCs with suitable properties for intravaginal application were developed. These formulations demonstrated promising therapeutic efficacy and low toxicity in Galleria mellonella assays. These results suggest the potential of this approach in developing new therapies for VVC. [ABSTRACT FROM AUTHOR]

Aeromonas spp. causes the human diseases including diarrhea, gastroenteritis, and bacteremia. Aeromonas spp. can be found in kitchen sponge, one of the reservoirs for food-borne bacterial pathogens. Virulence study of Aeromonas spp. in vivo in animal model is important since the animal model can mimic manifestasions in human infections. Omphisa fuscidentalis was chosen for alternative virulence model, since they are in the same taxonomical order with the well-known infections model, Galleria mellonella. Bacterial isolation and selection of kitchen sponge used Brain Heart Infusion agar and Endo Agar, respectively. Bacterial virulence of KS-1 was injected into Omphisa fuscidentalis larvae. Survival percentage and melanization score of infected larvae were evaluated. Hemolymph of larvae with melanization score of 1 and 4 were stained with Giemsa method to observe the hemocyte changes. Bacterial identification of isolate KS-1 based on 16S rRNA sequence resulted in 96.9% identity to Aeromonas spp. strain VS7. Isolate KS-1 injection to O. fuscidentalis revealed higher bacterial dosage resulting more severe symptoms to the larvae according to survival percentage and melanization score. However, statistical analysis showed evaluation of melanization score could distinguish larvae with 106 and 107 CFU/larva dosage injection, while evaluation of survival percentage could not. Hemocyte of larvae with melanization score 1 had larger and more cytoplasmic vacuolization than the score 4 (healthy larvae). Omphisa fuscidentalis is an alternative of insect model for bacterial infections with survival percentage and melanization score as the evaluation. Cytoplasmic vacuolization of hemocyte can be used as larvae's health indicator in a cellular level. [ABSTRACT FROM AUTHOR]

Candida auris is an emerging, multidrug-resistant yeast that causes systemic infections, mainly in hospitalized or immunosuppressed patients. This pathogen has a high mortality and morbidity rate. This study aims to evaluate the antifungal potential of micafungin (MICA) encapsulated in a nanoemulsion (NEM) against four clades of C. auris and other non-C. auris species. The antifungal potential of MICA and NEM was evaluated by determining mature biofilm inhibition (0.78–50 µg/mL). The antifungal activities of MICA and NEM (5.92 mg/Kg) were evaluated using an in vivo model of Galleria mellonella. The results showed that NEM intensified the antibiofilm action of MICA, especially in 48 h mature biofilms. In vivo results displayed a higher effectiveness of NEM against all clades of C. auris tested, inhibiting the fungal load in the hemolymph and tissues of G. mellonella with a difference of 3 log10. In addition, C. auris infection caused granulomas surrounded by hemocytes, mainly at the lower and upper ends. Conversely, C. albicans developed pseudohyphae, biofilms, filaments, and chlamydospores. In conclusion, encapsulation of MICA in a nanoemulsion enhances its antifungal activity against mature biofilms of C. auris. This strategy may be considered a therapeutic approach for the control of infections and the dissemination of this new global health threat. [ABSTRACT FROM AUTHOR]

Infections caused by KPC-producing K. pneumoniae continue to pose a significant clinical challenge due to their emerging resistance to new antimicrobials. We investigated the association between two drugs whose roles have been repurposed against multidrug-resistant bacteria: fosfomycin and temocillin. Temocillin exhibits unusual stability against KPC enzymes, while fosfomycin acts as a potent "synergizer". We conducted in vitro antimicrobial activity studies on 100 clinical isolates of KPC-producing K. pneumoniae using a combination of fosfomycin and temocillin. The results demonstrated synergistic activity in 91% of the isolates. Subsequently, we assessed the effect on Galleria mellonella larvae using five genetically different KPC-Kp isolates. The addition of fosfomycin to temocillin increased larvae survival from 73 to 97% (+Δ 32%; isolate 1), from 93 to 100% (+Δ 7%; isolate 2), from 63 to 86% (+Δ 36%; isolate 3), from 63 to 90% (+Δ 42%; isolate 4), and from 93 to 97% (+Δ 4%; isolate 10). Among the temocillin-resistant KPC-producing K. pneumoniae isolates (24 isolates), the addition of fosfomycin reduced temocillin MIC values below the resistance breakpoint in all isolates except one. Temocillin combined with fosfomycin emerges as a promising combination against KPC-producing K. pneumoniae, warranting further clinical evaluation. [ABSTRACT FROM AUTHOR]