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Acinetobacter baumannii, a top‐priority WHO pathogen, causes life‐threatening infections in immunocompromised patients, leading to prolonged hospitalisation and high mortality. Here, we used the Galleria mellonella model to investigate community strain C98 (Ab‐C98) virulence via transcriptomic analysis. Ab‐C98 showed greater killing and faster colonisation in larvae than the clinical reference strain (ATCC BAA1605). Genes in three iron clusters, acinetobactin, baumannoferrin and the Feo system, were significantly up‐regulated. Targeted knockout of siderophore genes (basC, bfnD, and the gene encoding isochorismatase) significantly increased the survival of infected larvae by at least 35.16%, identifying these genes as potential targets for developing anti‐virulence agents against A. baumannii. [ABSTRACT FROM AUTHOR]

Galleria mellonella (L.) is a worldwide major devastating insect pest of honey bee colonies and causes significant damage in the stored beekeeping industry. Mass rearing of this pest is critical where it considered a valuable and a favorable alternative invertebrate model for studies in biology, behavior, ecology, molecular biology, physiology, and control. For this purpose, newly hatched larvae were reared on five different nutritional diets until reaching the full grown 6th instar to evaluate their fitness and quality control parameters. The possibility to reduce insect rearing costs were assessed by replacing skimmed milk powder (added for its protein and energy intake) with ground date seed and replace glycerin with Paraffin wax as alternative and cheaper sources. The results indicated that the pupation percentage recorded 98, 96, 94, 100, and 100% for G. mellonella reared on diets D1, D2, D3, D4, and D5, respectively. Also, survival percentage was unaffected significantly by diet type, it was, 94, 94, 90, 98, and 96% respectively. Larvae fed on D4 and D5 diets recorded an equal value of growth rate which recorded 10.1 mg/day approximately, however, the lowest growth rate was (8.8 mg/day) for D1 (standard diet). After irradiation with 150 Gy, the average number of eggs laid by irradiated females crossed with normal males was 1134.4 and 1124.6 eggs/female for D5 and D1, respectively. Additionally, the average number of eggs laid when irradiated males crossed with normal females was 1278 and 1293 eggs/female for D1 and D5; respectively. Our findings suggest that the lowest cost of alternative artificial diets could be used for G. mellonella rearing protocol by replacing the skimmed milk powder and Paraffin wax with the ground date seed and glycerin. [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]

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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 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]

Oxyclozanide (OXY) is an anthelmintic widely used in the treatment of flatworm infection and fasciolosis. It also has antiadenovirus, antibiofilm, antifungal, and antibacterial activities. Various chemicals have been suggested as alternative chemicals in insect pest management. Here, the oxidative and genotoxic effects of OXY on 7th instars, pupae and adults of the model organism Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) were examined. First-instar larvae were reared on 0.003, 0.03, 0.3, and 1.5 g OXY per 100 g artificial diets. Compared with all tested OXY concentrations and controls without OXY, dietary OXY led to increased antioxidant capacity and genotoxic effects. Concentrations of malondialdehyde, an oxidative stress marker, were significantly increased in adults of larvae reared on OXY-charged diets at 0.3 and 1.5 g/100 g compared to the adult control group. We also recorded a significant increase in the genotoxic test data (Tail length, Tail DNA %, Tail moment) at the same stages and concentrations. We recorded significant increases in glutathione-S-transferase, superoxide dismutase (SOD) and glutathione peroxidase activities in larvae fed high OXY concentrations. SOD and catalase activities were also significantly increased at the concentration of 0.03 g/100 g of OXY in the pupal and adult stages. Cytochrome P450 monooxygenase activity was significantly increased at the highest concentration of OXY in the larval and pupal stages. Also, our regression analysis indicates a correlation between the markers of oxidative stress, antioxidant enzymes and comet parameters. These data indicate that OXY induces oxidative stress and antioxidative enzyme response. [ABSTRACT FROM AUTHOR]

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]

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]

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]

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]

Nematodes from the genus Meloidogyne are extremely harmful to agricultural crops due to their wide geographic distribution, variety of hosts, and strong reproductive capacity. The most used phytonematoid control method is the application of nematicides, which can also affect human health and the environment. Selenol ester derivatives have pharmaceutical applications, but little is known about their biological activities. We examined whether six selenol ester derivatives were nematicidal and ovicidal to the nematodes Caenorhabditis elegans and Meloidogyne incognita, and determined their toxicity to non-target organisms. The compound Se-p-methyl phenylselenobenzoate (2e) exhibited the lowest nematicidal and ovicidal Lethal concentration (LC50) to C. elegans (4.42 and 0.56 mg/L, respectively) and M. incognita (4.6 and 2.77 mg/L, respectively), which were similar or lower than the LC50 values for the control treatment with fluensulfone in C. elegans (4.07 and 6.05 mg/L, respectively) and M. incognita (43.07 and 49.63 mg/L, respectively). Compound 2e efficiently controlled the phytoparasite in greenhouse and reduced the number of galls and eggs present in tomato roots. Compared with the negative control, compound 2e was not toxic to other organisms such as Chlorella vulgaris algae, Galleria mellonella and Aedes aegypti larvae, and the aquatic protozoan Tetrahymena pyriformis. Our findings indicate that the selenol ester derivatives are potent and effective nematicides with no toxicity to the non-target organisms tested. [ABSTRACT FROM AUTHOR]

Nowadays, particularly metallic, and polymeric nanoparticles (NPs) are widely produced and used in many fields. Due to the increase in both their usage and diversity, their release and accumulation in the environment are also accelerating. Therefore, their interactions with cells, especially immune cells, and their health risks are not fully understood. The impacts of metallic alumina (Al) NPs and polystyrene (PS) NPs obtained after the polymerization of carcinogenic styrene on living organisms have not yet been elucidated. Galleria mellonella larvae can biodegrade plastics. While biodegradation and solving the waste problem have attracted much attention, the interactions of this distinctive property of G. mellonella larvae in the immune system and ecosystem are not yet completely understood. Al and PS NPs were applied to G. mellonella separately. Al NPs were purchased and PS NPs were prepared from PS by single-emulsion technique and characterized. Then LC50 values of these NPs on G. mellonella were determined. The interactions of these NPs with encapsulation, melanization, and phenoloxidase activity, which express innate immune responses in G. mellonella larvae, were revealed. NP exposure resulted in suppression of the immune response, probably because it affects the functions of hemocytes such as enzymatic activation, hemocyte division, and populations. In this context, our data suggest that Al and PS NPs induce toxic impacts and negatively alter the physiological status of G. mellonella. It is also shown that G. mellonella has the potential to be an impactful alternative model for biosafety and nanotoxicology studies. [ABSTRACT FROM AUTHOR]

There is abundant evidence that parasitoids manipulate their hosts by envenomation to support the development and survival of their progeny before oviposition. However, the specific mechanism underlying host nutritional manipulation remains largely unclear. To gain a more comprehensive insight into the effects induced by the gregarious ectoparasitoid Iseropus kuwanae (Hymenoptera: Ichneumonidae) on the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) larvae, we sequenced the transcriptome of both non‐envenomed and envenomed G. mellonella larvae, specifically targeting genes related to lipid metabolism. The present study revealed that 202 differentially expressed genes (DEGs) were identified and 9 DEGs were involved in lipid metabolism. The expression levels of these 9 DEGs relied on envenomation and the duration post‐envenomation. Further, envenomation by I. kuwanae induced an increase in triglyceride (TG) level in the hemolymph of G. mellonella larvae. Furthermore, silencing GmPLA2 in G. mellonella larvae 24 h post‐envenomation significantly decreased the content of 4 unsaturated fatty acids and TG levels in the hemolymph. The content of linoleic acid and α‐linoleic acid were significantly decreased and the content of oleic acid was significantly increased by exogenous supplement of arachidonic acid. Meanwhile, the reduction in host lipid levels impairs the growth and development of wasp offspring. The present study provides valuable knowledge about the molecular mechanism of the nutritional interaction between parasitoids and their hosts and sheds light on the coevolution between parasitoids and host insects. [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.

Wax moths, Galleria mellonella Linnaeus, 1758 and Achroia grisella Fabricius, 1794 are the key pests of honeybee colonies worldwide. They cause wax comb damages which lead to colony perishing and absconding. To control these pests, their genetic background and evolution should be considered. In this study, genetic diversity and demographic history of 2 species of wax moth were explored based on mitochondrial genes (COI). Wax moths were collected from northeatern part of Thailand for morphological and DNA analyses. G. mellonella and A. grisella sequences from GenBank were included for genetic diversity and demographic history analyses (total of 80 and 26 sequences, respectively). Overall genetic diversity for A. grisella samples from Northeastern Thailand was relatively low (h = 0.467 ± 0.132 and π = 0.00150 ± 0.00042). Likewise, low genetic diversity was also observed in G. mellonella samples (h = 0.280 ± 0.101 and π = 0.00046 ± 0.00017). Among samples from different global regions, the haplotype and nucleotide diversity of A. grisella were 0.834 ± 0.050 and 0.00996 ± 0.00124, respectively. The haplotype and nucleotide diversity of G. mellonella were 0.709 ± 0.034 and 0.01084 ± 0.00279, respectively. The ML tree revealed that these 2 species were separated into 2 major clades (G. mellonella clade and A. grisella clade). All sequences corresponding to G. mellonella formed to a single monophyletic clade, while all the sequences corresponding to A. grisella formed another single monophyletic clade. Clade G. mellonella was divided into 4 subclades designated as Clade Gm1, Gm2, Gm3 and Gm4, whereas A. grisella clade was divided into 3 subclades designated as Clade Ag1, Ag2 and Ag3. The sum of squares deviation (SSD) and Harpending's raggedness index (rg) and the results of Tajima's D and Fu's Fs tests indicated population expansion was not found in either species. [ABSTRACT FROM AUTHOR]

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 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.

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.

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.

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.

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]

The greater wax moth, Galleria mellonella (Lepidoptera, Pyralidae), is a major bee pest that inflicts considerable harm on beehives, leading to economic losses. It also serves as a valuable resource insect and a model organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system plays a crucial role in improving economic insect breeding and developing efficient agricultural pest management systems in Lepidoptera. However, the CRISPR/Cas9 protocols have not been developed for G. mellonella. Here, the Gmebony knockout (KO) strain was established using the CRISPR/Cas9 genome editing system. We obtained Gmebony KO strain in the G4 generation, which took approximately 10 months. When compared with wild‐type, the head, notum, and the terminal abdominal surface of 1st to 4th instar larvae in the KO strain changed from yellow to brown, and these regions of the KO strain gradually transformed into a black color from the 5th instar larvae, and the body color of the adult moth in the KO strain changed to black. The developmental period of the early larval and the following larval instars extended. The embryonic hatchability of the Gmebony KO strain was significantly decreased. The pupal body weight of the Gmebony KO strain was not affected. The feasibility of the CRISPR/Cas9 methodology was validated by single‐target editing of Gmebony. Our findings provide the first evidence that the ebony gene can serve as a pigmentation reference gene for genetic modifications of G. mellonella. Meanwhile, it can be utilized in the development of genome editing control strategies and for gene function analyses in G. mellonella. [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]

Potential probiotic yeast strains isolated from fermented food need to meet safe and beneficial conditions for the host's health. The Pichia kudriavzevii YGM091 strain isolated from fermented goat milk has outstanding probiotic characteristics, including: the high survival percentage in digestive system conditions (reaching up 247.13 ± 0.12 and 145.03 ± 0.06% at pH 3.0 and bile salt 0.5%, respectively); good tolerance to temperature, salt, phenol, ethanol; good surface properties such as high hydrophobicity percentage (> 60%), the high auto-aggregation percentage rate (66.56 ± 1.45% after 45 min of incubation) and the high co-aggregation percentage rate with pathogenic bacteria in a short time (> 40% after 2 h of incubation); biofilm forming after 24 h of incubation on abiotic surfaces; antioxidant activity reached excellent level after only 24 h of incubation (The percentage free radical scavenging and the Trolox equivalent reaching up 79.86 ± 0.70% and 92.09 ± 0.75 µg/mL after 72 h of incubation); extracellular enzymes production protease and cellulase with high activity, amylase and pectinase with moderate activity and non-lipase activity. Simultaneously, the YGM091 strain is the in vitro safety yeast: insensitive to antibiotics and fluconazole, negative for gelatinase, phospholipase, coagulase, and non-hemolysis activities. Furthermore, this strain is in vivo safety yeast with the dosages below 106 CFU/larva in the Galleria mellonella model with over 90% survival larvae and the yeast density reduced to just 102–103 CFU/larva after 72 h post-injection. Research results have demonstrated that the Pichia kudriavzevii YGM091 strain is a safe potential probiotic yeast and could become a candidate probiotic food to be used in the future. [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]

Background: The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals ‐ CYP 51 A, B and C ‐ are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. Objectives: This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. Methods: The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. Results: In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. Conclusions: These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus. [ABSTRACT FROM AUTHOR]

Aims The purpose of this work was to study extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) in freshwaters, hospital effluents, and wastewaters during two sampling campaigns in 2021. Methods and results Water sampling was performed at 24 stations in the Ourthe watershed in Belgium. A total of 644 ESBL (n  = 642) and AmpC (n  = 2) E. coli strains were isolated. Disk-diffusion assays were performed following the EUCAST's recommendations. All strains were tested for the presence of bla CTX-M-1, bla CTX-M-2, and bla CTX-M-9 gene groups by PCR. Genes belonging to bla CTX-M-1 and bla CTX-M-9 groups were detected, respectively, in 73.6% and 14.9% of the strains. No bla CTX-M-2 group's gene was found. A subset of strains (n  = 40) was selected for whole genome sequencing. Escherichia coli serotype O18: H7 ST 1463 was predominant (n  = 14) in the sequenced strains and showed pathogenicity in the Galleria mellonella larvae model. β-lactamase genes identified were bla CTX-M (n  = 21), with bla CTX-M-15 mostly represented (n  = 15), as well as bla TEM (n  = 11), bla OXA (n  = 7), bla SHV (n  = 9), and carbapenemase (CP) genes were observed in several strains— bla KPC-3 (n  = 19), bla NDM-1 (n  = 1), bla VIM-1 (n  = 2), and bla OXA-244 (n  = 2)—even from freshwaters. Conclusions ESBL-EC are widely distributed in the aquatic environment in Belgium and contain a variety of ESBL and CP genes. [ABSTRACT FROM AUTHOR]

Purpose: Enterobacteriaceae carrying mcr-9, in particularly those also co-containing metallo-β-lactamase (MBL) and TEM type β-lactamase, present potential transmission risks and lack adequate clinical response methods, thereby posing a major threat to global public health. The aim of this study was to assess the antimicrobial efficacy of a combined ceftazidime/avibactam (CZA) and aztreonam (ATM) regimen against carbapenem-resistant Enterobacter cloacae complex (CRECC) co-producing mcr-9, MBL and TEM. Methods: The in vitro antibacterial activity of CZA plus ATM was evaluated using a time-kill curve assay. Furthermore, the in vivo interaction between CZA plus ATM was confirmed using a Galleria mellonella (G. mellonella) infection model. Results: All eight clinical strains of CRECC, co-carrying mcr-9, MBL and TEM, exhibited high resistance to CZA and ATM. In vitro time-kill curve analysis demonstrated that the combination therapy of CZA + ATM exerted significant bactericidal activity against mcr-9, MBL and TEM-co-producing Enterobacter cloacae complex (ECC) isolates with a 100% synergy rate observed in our study. Furthermore, in vivo survival assay using Galleria mellonella larvae infected with CRECC strains co-harboring mcr-9, MBL and TEM revealed that the CZA + ATM combination significantly improved the survival rate compared to the drug-treatment alone and untreated control groups. Conclusion: To our knowledge, this study represents the first report on the in vitro and in vivo antibacterial activity of CZA plus ATM against CRECC isolates co-harboring mcr-9, MBL and TEM. Our findings suggest that the combination regimen of CZA + ATM provides a valuable reference for clinicians to address the increasingly complex antibiotic resistance situation observed in clinical microorganisms. [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]

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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]

Neuropeptides are crucial in regulation of a rich variety of developmental, physiological, and behavioral functions throughout the life cycle of insects. Using an integrated approach of multiomics, we identified neuropeptide precursors in the greater wax moth Galleria mellonella, which is a harmful pest of honeybee hives with a worldwide distribution. Here, a total of 63 and 67 neuropeptide precursors were predicted and annotated in the G. mellonella genome and transcriptome, in which 40 neuropeptide precursors were confirmed in the G. mellonella peptidome. Interestingly, we identified 12 neuropeptide precursor genes present in G. mellonella but absent in honeybees, which may be potential novel pesticide target sites. Honeybee hives were contaminated with heavy metals such as lead, enabling its bioaccumulation in G. mellonella bodies through the food chain, we performed transcriptome sequencing to analyze the effects of Pb stress on the mRNA expression level of G. mellonella neuropeptide precursors. After treatment by Pb, the expression of neuropeptide F1 was found to be significantly downregulated, implying that this neuropeptide might be associated with responding to the heavy metal stress in G. mellonella. This study comprehensively identified neuropeptide precursors in G. mellonella, and discussed the effects of heavy metals on insect neuropeptides, with the example of G. mellonella. The results are valuable for future elucidation of how neuropeptides regulate physiological functions in G. mellonella and contribute to our understanding of the insect's environmental plasticity and identify potential new biomarkers to assess heavy metal toxicity in insects. [ABSTRACT FROM AUTHOR]

The bethylid wasp Goniozus omanensis has been identified as a key natural enemy of Batrachedra amydraula Meyrick (Lepidoptera: Batrachedridae), an important pest of date palm. Efficient rearing of natural enemies can be an important part of biological pest control programmes but cultures of G. omanensis are challenging to maintain on B. amydraula. We evaluate two readily available species of Pyralid moths, Corcyra cephalonica and Galleria mellonella, as potential factitious hosts. We find that G. omanensis can develop on the larvae of either species, but the probability of failure is circa 75% overall. Both trialled host species were approximately equally suitable, but failures were typically during the early stages of brood production when presented with C. cephalonica and during the later stages when presented with G. mellonella. This suggests that C. cephalonica larvae may be more difficult to suppress and that G. mellonella larvae may be of lower nutritional value. The average production of adult parasitoids per host provided was approximately 1 and, given the observed female biased sex ratio, the average number of females was around 0.8. We suggest potential routes to improving the efficiency of rearing G. omanensis on these factitious hosts. [ABSTRACT FROM AUTHOR]

Sweet sagewort (Artemisia annua) is a species that has been used for many years to treat high fever due to its artemycin content. Artemycin is the active ingredient in antimalarial drugs. Additionally, the plant's leaves are consumed as tea or aqueous extract. Invertebrate model organism Galleria mellonella is preferred in immune studies due to its ease of administration, natural immunity shared with mammals, including humans, and rapid determination of results. In this research, we systematically assessed the impact of A. annua extract on the immune system of animals by employing the G. mellonella model organism. We meticulously examined key facets of innate immune responses, including total hemocyte count, encapsulation-melanization, and phenoloxidase activity. Furthermore, we delved into the effects on antioxidant enzyme activity, specifically superoxide dismutase, catalase, and lipid peroxidase. The hemocyte count showed a statistically significant decrease across all given doses of the A. annua extract when compared to the control groups. The A. annua extract had no effect on the catalase activity, malondialdehyde amount, phenoloxidase activity, or melanization response of G. mellonella larvae, according to the findings of our study. Nonetheless, it enhanced SOD activity. In comparison to the control groups, it decreased the strong encapsulation response, which is a cell-mediated immune response, at all doses. While the A. annua extract had little effect on enzymatic processes in animals, it reduced cell-mediated immune responses by altering cell behaviours and count with cytotoxic and genotoxic effects. [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]

Background: Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts. Methods and results: Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph. Conclusions: The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.IMPORTANCEThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in biomass reduction of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

In-58, a peptide derived from indolicidin, shows extraordinary antibacterial activity and lower toxicity than indolicidin toward mammalian cells. Here, we investigated the antifungal activity of In-58 against the human pathogen Sporothrix globosa in vitro and in vivo. In-58 markedly inhibited the growth of Sporothrix globosa isolates in microdilution assays and showed no antagonism with any tested antifungal agent (itraconazole, terbinafine or amphotericin B). Scanning electron microscopy and propidium iodide staining indicated that In-58 alters the cell wall integrity and interacts with DNA, leading to disruption of S. globosa in a dose-dependent manner. In S. globosa, the mitochondrial membrane potential decreased and reactive oxygen species increased after treatment with In-58. In vivo experiments in the Galleria mellonella (greater wax moth) larval infection model revealed the effectiveness of In-58 against S. globosa infection with low toxicity. Our results indicate that In-58 possesses remarkable antifungal activity against S. globosa in vitro and in vivo. It has potential as a novel drug for the treatment of sporotrichosis.

IntroductionManaging 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.MethodsThe 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 discussionIn 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-κB, 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.