Search Results (152)

Entomopathogenic fungi (EPF) are one of the most environmentally friendly ways to control a plethora of chewing insects such as T. pityocampa, G. mellonella, and A. grisella. Bioassay of EPF on these highly damaging pests is considered important in the face of climate change in order to research alternative solutions that are capable of limiting chemical control, the overuse of which increases insects’ resistance to chemical compounds. In this study, the insecticidal virulence of Metarhizium robertsii isolates, retrieved from forest ecosystems, was tested on second-instar larvae of T. pityocampa, G. mellonella, and A. grisella. Bioassays were carried out in the laboratory, where experimental larvae were sprayed with 2 mL of a six-conidial suspension from each isolate. Mortality was recorded for 144 h after exposure. Mean mortality, lethal concentrations, sporulation percentage, and sporulation time were estimated for each isolate. Metarhizium isolates resulted in the highest mortality (89.2% for G. mellonella and 90.2% for A. grisella). Based on the LC₅₀ estimates determined by the concentration–mortality relationships for the tested fungal isolates, we demonstrated significant virulence on larvae of G. mellonella, A. grisella, and T. pityocampa. Our results indicate that entomopathogenic fungi have the potential to become a very useful tool in reducing chemical applications. Full article

Background: There is an urgent need for novel treatment options for Neisseria gonorrhoeae. Methenamine is an interesting urinary antiseptic with a very low propensity to induce antimicrobial resistance. Methods: We assessed the MICs of methenamine-hippurate for 18 N. gonorrhoeae isolates. We then assessed the in vivo efficacy of methenamine-hippurate against N. gonorrhoeae using the Galleria mellonella infection model. Results: We found that all the gonococcal isolates had a methenamine-hippurate MIC of 300 mg/L. This MIC was not higher in isolates with higher ceftriaxone MICs. No toxicity of methenamine at the doses tested was found, and doses as low as 200 mg/kg were effective in the G. mellonella model. Conclusions: Further studies in mice and humans are required to assess if methenamine-hippurate could be used to treat gonococcal urethritis alone or in combination with other agents such as ceftriaxone. Full article

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen responsible for a range of severe infections and nosocomial outbreaks. Phage-based therapy and biocontrol represent effective strategies to combat the prevalence of A. baumannii. This study reports a novel phage, BUCT775, capable of specifically lysing A. baumannii, and investigates its physiological properties, genomic characteristics, in vivo therapeutic efficacy, and environmental disinfection performance. Phage BUCT775 is a podovirus that forms clear, well-defined plaques with an average diameter of 2.5 ± 0.52 mm. It exhibits a broad range of temperature stability (4–55 °C) and pH stability (pH 3–12). The optimal multiplicity of infection (MOI) for phage BUCT775 is 0.01. At an MOI of 0.01, it demonstrates a latent period of approximately 10 min and exhibits a high burst size. Genomic sequencing and bioinformatics analysis revealed that phage BUCT775 belongs to the order Caudoviricetes and the family Autographiviridae. Its genome has a G + C content of 39.3% and is not known to contain virulence genes or antibiotic resistance genes. Phage BUCT775 exhibited significant therapeutic effects on A. baumannii-infected G. mellonella larvae, increasing the 120 h survival rate of the larvae by 20%. Additionally, phage BUCT775 efficiently eliminated A. baumannii in the environment, with an average clearance rate exceeding 98% within 3 h. These studies suggest that phage BUCT775 holds significant potential for application in phage therapy and environmental disinfection. Full article

The European honey bee (Apis mellifera) significantly contributes to Australian agriculture, especially in honey production and the pollination of key crops. However, managed bee populations are declining due to pathogens, agrochemicals, poor forage, climate change, and habitat loss. Major threats include bacteria, fungi, mites, and pests. With the increasing demand for pollination and the movement of bee colonies, monitoring these threats is essential. It has been demonstrated that honey constitutes an easily accessible source of environmental DNA. Environmental DNA in honey comes from all organisms that either directly or indirectly aid in its production and those within the hive environments. In this study, we extracted eDNA from 135 honey samples and tested for the presence of DNA for seven key honey bee pathogens and pests—Paenibacillus larvae, Melissococcus plutonius (bacterial pathogens), Nosema apis, Nosema ceranae (microsporidian fungi), Ascosphaera apis (fungal pathogen), Aethina tumida, and Galleria mellonella (arthropod pests) by using end-point singleplex and multiplex PCR assays. N. ceranae emerged as the most prevalent pathogen, present in 57% of the samples. This was followed by the pests A. tumida (40%) and G. mellonella (37%), and the pathogens P. larvae (21%), N. apis (19%), and M. plutonius (18%). A. apis was detected in a smaller proportion of the samples, with a prevalence of 5%. Additionally, 19% of the samples tested negative for all pathogens and pests analysed. The data outlines essential information about the prevalence of significant arthropod, fungal, and bacterial pathogens and pests affecting honey bees in Australia, which is crucial for protecting the nation’s beekeeping industry. Full article

The pleuromutilin derivative, the compound PL-W, was synthesized by introducing a 4-fluorophenyl group at the C21 position and selected for comprehensive antibacterial evaluation. PL-W demonstrated notable antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with a minimum inhibitory concentration (MIC) of 0.03125 µg/mL, which is significantly lower than that of tiamulin (0.5 µg/mL). Crystal violet (CV) staining revealed that it inhibited MRSA biofilm formation and electron microscopy revealed that it disrupted bacterial cell division and, possibly, the synthesis of essential cell wall proteins. In both in vivo models, PL-W exhibited excellent performance. In the Galleria mellonella infection model, treatment with different concentrations of PL-W increased the survival rate from 20% to 90% and significantly reduced the bacterial load. In the mouse model of MRSA pneumonia, a 10 mg/kg dose of PL-W increased the survival rate to 70%, decreased the bacterial load in the lungs, and alleviated inflammatory damage. Molecular docking studies indicated that PL-W had a similar docking pose and comparable binding affinity to that of lefamulin, with hydrogen bond interactions that are crucial for binding to the peptidyl transferase center (PTC). Moreover, it demonstrated no significant reduction in cell viability in HepG2 and HEK293 cells, even at high concentrations (≤50 µg/mL). Overall, PL-W shows significant potential as a novel anti-MRSA agent owing to its potent in vitro and in vivo activities and low cytotoxicity. Full article

Citrobacter braakii can colonize the intestinal tract of humans and animals and occasionally act as opportunistic pathogen. Although isolated from food and the environment, its potential as a foodborne pathogen remains uncertain. Twenty C. braakii isolates were previously collected from salami and soft cheese artisanal productions. In the present study, the potentialities of C. braakii as a food safety hazard were explored by a genomic comparison of C. braakii newly sequenced genomes with publicly available genomes, including those of clinical relevance, and a pathogenicity assessment in Galleria mellonella as an in vivo infection model. Phylogenomic reconstruction revealed that one salami clone and two C. braakii genomes of the soft cheese production were closely related (from 11 to 28 core SNP differences) to C. braakii publicly available clinical genomes. All genomes carried the chromosomally located bla_CMY and/or qnrB genes and were resistant to cephalosporins and/or had reduced susceptibility to ciprofloxacin. G. mellonella larvae showed 90% mortality after challenge with C. braakii strains carrying the vex and tvi operons coding for the capsular polysaccharide (Vi antigen), in comparison to 40% of strains lacking these two operons. The high mortality rate of vex- and tvi-positive C. braakii isolated from food processing plants suggests C. braakii to be a possible foodborne hazard. Full article

The novel pathogen Candida auris has rapidly become a major health threat due to its high virulence, resistance to multiple antifungal agents, and remarkable environmental persistence. This study evaluated the influence of phenotypic traits and antifungal minimum inhibitory concentrations (MICs) on C. auris virulence using a Galleria mellonella infection model. Ten clinical strains, categorized as aggregative or non-aggregative, were analyzed for antifungal susceptibility and survival outcomes. All strains exhibited fluconazole resistance, with variable susceptibilities to other antifungals. Survival analysis revealed that the non-aggregative phenotype was independently associated with reduced survival in G. mellonella (HR = 2.418, p = 0.015), while antifungal MICs and invasive origin were not significant independent predictors of mortality in an elastic net-adjusted multivariable model. Strong correlations were observed between certain antifungal MICs, suggesting potential cross-resistance patterns; however, no independent association with virulence was identified. These results suggest that C. auris possesses not only an enhanced ability to develop antifungal resistance but also the capacity to do so without incurring fitness costs that could attenuate its virulence. Full article

Biofilm plays a crucial role in the pathogenesis and chronicity of urinary tract infections (UTIs). The present work aimed to evaluate the anti-biofilm effects of Formulation (DIF17BRO^® plus NAC) in combination with ciprofloxacin (CPX) on Escherichia coli strains. The antimicrobial activity of ciprofloxacin was evaluated by minimum inhibitory concentration (MIC) determination, and the antibiofilm effects of ciprofloxacin alone and combined with Formulation were evaluated on E. coli ATCC700926, E. coli ATCC10536, E. coli PNT, and E. coli PCA mature biofilms in terms of CFU/mL and biomass quantifications. Moreover, the potential protective effects of Formulation plus ciprofloxacin was tested in a Galleria mellonella in vivo infection assay. Our results underlined the increased microbial reduction in the mature biofilm in the presence of the combination Formulation and CPX, even at a lower concentration of CPX. Formulation increased the percentage of biofilm biomass reduction, inducing a disruption of the biofilm structure itself. Our present findings confirm that MIC CPX combined with Formulation also induced an antimicrobial effect in the G. mellonella assay. Formulation facilitated the perturbation of the biofilm polymeric matrix, enhancing the antibiotic penetration and its antimicrobial action on bacteria, underlining Formulation’s role as an enhancer of ciprofloxacin antibacterial action. Full article

Background: Fungal infections pose an increasingly predominant threat to human and animal health. Modified compounds derived from chemo-diverse natural products offer enhanced therapeutic efficacies and promising approaches to combat life-threatening fungal pathogens. Methods: We performed biosynthetic gene clusters analysis of 2,4-diacetylchloroglucoside (DAPG) in 4292 shotgun metagenomes samples from the healthy and diseased skin. Then, we assessed the antifungal activity of DAPG and the derivative 2,4-diproylphloroglucinol (DPPG) against pathogenic fungi by minimum inhibitory concentrations. The inhibitory effects of DPPG were measured using hyphal growth assay and spore germination assay. Concurrently, the mechanism of DPPG on Aspergillus fumigatus was investigated in membrane permeability and fluidity. The therapeutic efficacy was evaluated in a Galleria mellonella infection model. Results: We observed a significantly higher abundance of bacteria harboring DAPG biosynthetic clusters on healthy skin compared to diseased skin. Further, we designed and synthesized a series of phloroglucinol derivatives based on DAPG and obtained an antifungal candidate DPPG. DPPG not only exhibited robust antifungal activity against Aspergillus spp. and Candida spp. but also impaired hyphal growth and spore germination of A. fumigatus in vitro. A mechanism study showed that DPPG reduced membrane fluidity and increased the leakage of cellular contents, resulting in membrane perturbation and fungal death. Lastly, the therapeutic efficacy of DPPG was confirmed in a G. mellonella infection model. Conclusions: Our study demonstrates that DPPG is a potent scaffold to combat invasive fungal infections. Full article

Lactic acid bacteria (LAB) are gaining increasing attention as functional ingredients in the cosmetic industry, particularly those derived from natural plant sources. Although various LAB strains have been widely applied in cosmetic formulations, studies investigating the effects of naturally derived LAB on the skin remain limited. In this study, we isolated an LAB strain from ginseng and evaluated its potential as a functional cosmetic ingredient. The antimicrobial activity of the strain was assessed against skin-associated pathogens Staphylococcus aureus and Staphylococcus epidermidis, while cytotoxicity was evaluated using HaCaT and Caco-2 cells. Considering the limitations of vertebrate animal testing, infection and survival assays were conducted using Galleria mellonella larvae as an alternative in vivo model. The ginseng-derived strain exhibited 99.93% similarity to Pediococcus pentosaceus and was designated P. pentosaceus THG-219. It exhibited an MIC of 0.625 mg/mL and 1.25 mg/mL against S. aureus KCTC 3881 and S. epidermidis KCTC 1917, respectively. Its antimicrobial activity was further enhanced following ethyl acetate fractionation. P. pentosaceus THG-219 showed no toxicity in G. mellonella larvae and exerted antibacterial effects in this model. No cytotoxicity was observed in HaCaT and Caco-2 cells. Furthermore, P. pentosaceus THG-219 promoted host cell adhesion while inhibiting pathogen adhesion. It also exhibited excellent acid, bile, and heat tolerance, suggesting strong survivability under harsh conditions. Collectively, these results indicate that P. pentosaceus THG-219, isolated from ginseng, is a promising, safe, and stable candidate for development as a functional cosmetic ingredient. Full article

The Gram-negative genus Aeromonas contains diverse bacterial species that are prevalent in aquatic environments. This present study describes three novel Aeromonas strains: A. ichthyocola sp. nov. A-5^T and A. mytilicola subsp. aquatica subsp. nov. A-8^T isolated from rainbow trout (Oncorhynchus mykiss), and A. mytilicola sp. nov. A-7^T isolated from mussels (Mytilus galloprovincialis), respectively. Genomic analyses revealed that strains A-5^T and A-7^T shared the highest 16S rRNA gene sequence similarity with A. rivipollensis P2G1^T (99.7% and 99.8%, respectively), while strain A-8^T exhibited 99.7% identity with A. media RM^T. Together with morphological, physiological, and biochemical data, genome-based analyses provided additional evidence for species differentiation. Digital DNA–DNA hybridization (dDDH; 56.8−65.9%) and average nucleotide identity (ANI; 94.2–95.7%) values fell below the species delineation thresholds, confirming that these isolates represent distinct taxa. Pathogenicity assays using greater wax moth (Galleria mellonella) larvae demonstrated strain-specific virulence profiles. Further genomic analyses identified biosynthetic gene clusters for nonribosomal peptides (NRPs) and ribosomally synthesized and post-translationally modified peptides (RiPPs), which often have roles in secondary metabolite production. Ecological analyses, based on genomic comparisons and metagenomic database searches, revealed the adaptability of the strains to diverse habitats, including freshwater, wastewater, and activated sludge. Based on the genetic and phenotypic data, the novel taxa Aeromonas ichthyocola sp. nov. A-5ᵀ (LMG 33534ᵀ = DSM 117488ᵀ), Aeromonas mytilicola sp. nov. A-7ᵀ (LMG 33536ᵀ = DSM 117490ᵀ), and Aeromonas mytilicola subsp. aquatica subsp. nov. A-8ᵀ (LMG 33537ᵀ = DSM 117493ᵀ) are proposed. Full article

Background: The fitness costs (FCs) of antimicrobial resistance (AMR) are crucial issues in antimicrobial resistance (AMR) onset, spread, and, consequently, public health. In Staphylococcus aureus, AMR can induce significant FCs due to slow growth, low competitiveness, and virulence. Here, we investigated the genomics and FCs emerging for progressively acquiring daptomycin (DAP) and glycopeptide (GLY) reduced susceptibility in MRSA. Methods: Genomics was carried out using Illumina-MiSeq Whole-genome sequencing and bioinformatics. The biological FCs of isogenic MRSA strain pairs progressively acquiring DAP and GLY-reduced susceptibility, under DAP/GLY mono or combined therapy, were performed by in-vitro independent and competitive mixed growth, phenotypic in-vitro virulence analysis, and in-vivo G. mellonella larvae killing. Results: Genomics evidenced four different extremely resistant high-risk clones, i.e., ST-5 N315 HA-MRSA, ST-398 LA-MRSA, ST-22 USA-100 HA-EMRSA-15, and ST-1 MW2 CA-MRSA. In-vitro fitness assays revealed slow growth, lower competitiveness, and reduced virulence, predominantly in Galleria mellonella killing ability, in DAP-S hGISA, DAP-R GSSA, DAP-R hGISA, and DAP-R GISA strains. Conclusions: The occurrence of glycopeptide and daptomycin reduced susceptibility conferred increasing FCs, paid as a gradual reduction in virulence, competitiveness, and slow growth performance. Full article

The antimicrobial peptide P6.2 was previously de novo designed as an alpha helix cationic amphipathic molecule. In previous work, we have shown that this peptide displayed significant antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. However, while P6.2 lacked biofilm-inhibiting properties against the P. aeruginosa strain PA01, it displayed anti-inflammatory effects in a murine acute lung infection model challenged with this pathogen. In this work, the peptide P6.2 antimicrobial activity and its possible synergy with meropenem were evaluated both in vitro and in vivo using a Galleria mellonella infection model against a carbapenem-resistant KPC-producing clinical isolate of P. aeruginosa. Firstly, the cytotoxic effect of the peptide on A549 and RAW264.7 cell lines was assayed, showing no cytotoxicity at 64 µg/mL and below. Then, the MIC (minimal inhibitory concentration) and bactericidal effect against the carbapenemase-producing strain P. aeruginosa M13513 strain were determined. P6.2 showed a MIC between 32 and 64 µg/mL, and a rapid bactericidal activity against this strain (less than 45 min). The peptide stability at different temperatures and in bovine serum at 37 °C was also analyzed, showing good stability and almost no degradation after 15 min of incubation at 100 °C or 24 h at 37 °C in serum, respectively. The antibiofilm activity was also evaluated, and although the peptide did not show biofilm inhibitory activity, it did demonstrate biofilm disruptive activity, together with bactericidal activity inside the pre-formed biofilm. The possible synergistic effect with the carbapenem meropenem was then analyzed in vitro by killing kinetics, revealing a synergistic interaction between P6.2 and the antibiotic against this strain. Finally, P6.2 was evaluated in vivo in the Galleria mellonella larvae infection model. Interestingly, in G. mellonella, P6.2 alone did not completely clear the infection caused by P. aeruginosa M13513. However, when combined with meropenem, P6.2 demonstrated a synergistic effect, leading to increased survival rates in infected larvae. The results presented here highlight the potential that this peptide displays when used in combination with carbapenems against a clinically relevant KPC-producing P. aeruginosa. Full article

Bioinsecticides based on essential oils (EOs) are promising alternatives for agricultural insect control. In this study, the main compounds of Baccharis macraei EOs from two geographical areas were identified using gas chromatography–mass spectrometry (GC-MS). The insecticidal potential against Drosophila melanogaster, Tenebrio molitor, and Galleria mellonella was evaluated, along with acetylcholinesterase (AChE) inhibition and molecular docking analysis. GC-MS analysis revealed oxygenated sesquiterpenes, such as spathulenol, caryophyllene oxide, and α-cadinol. The EO from Site 1 (S₁) exhibited insecticidal activity with lethal dose 50 (LD₅₀) values of 46.50, 465.76, and 241.2 µg/mL for D. melanogaster, T. molitor, and G. mellonella, respectively. The EO from Site 2 (S₂) showed LD₅₀ values of 74.81, 385.62, and 195.2 µg/mL for the same pests. AChE inhibition yielded inhibitory concentration 50 (IC₅₀) values of 12.8 ± 0.13 µg/mL for S₁ and 11.7 ± 0.07 µg/mL for S₂. Molecular docking analysis revealed a strong binding energy between α-cadinol and AChE. These results highlight that EOs from B. macraei are effective and serve as a natural alternative to traditional insecticides. Full article

The incidence of fungal infections continues to increase and one of the factors responsible for these high rates is the emergence of multi-resistant species, hospitalizations, inappropriate or prolonged use of medications, and pandemics, such as the ongoing HIV/AIDS pandemic. The recent pandemic caused by the severe acute respiratory syndrome virus (SARS-CoV-2) has led to a significant increase in fungal infections, especially systemic mycoses caused by opportunistic fungi. There is a growing and urgent need to better understand how these microorganisms cause infection and develop resistance as well as to develop new therapeutic strategies to combat the diverse diseases caused by fungi. Non-mammalian hosts are increasingly used as alternative models to study microbial infections. Due to their low cost, simplicity of care, conserved innate immunity and reduced ethical issues, the greater wax moth Galleria mellonella is an excellent model host for studying fungal infections and it is currently widely used to study fungal pathogenesis and develop innovative strategies to mitigate the mycoses studied. G. mellonella can grow at 37 °C, which is similar to the mammalian temperature, and the anatomy of the larvae allows researchers to easily deliver pathogens, biological products, compounds and drugs. The aim of this review is to describe how G. mellonella is being used as a model system to study fungal infections as well as the importance of this model in evaluating the antifungal profile of potential drug candidates or new therapies against fungi. Full article