Galleria Mellonella Larvae Research Articles

Biofilm Inhibition of Inula viscosa (L.) Aiton and Globularia alypum L. Extracts Against Candida Infectious Pathogens and In Vivo Action on Galleria mellonella Model.

The increasing importance of fungal infections has fueled the search for new beneficial alternatives substance from plant extracts. The current study investigates the antifungal and antibiofilm activity of Inula viscosa (L.) Aiton and Globularia alypum (L.) leaves extracts against Candida both in vitro and in vivo. The inhibition of planktonic and sessile Candida albicans and Candida glabrata growth using both leaf extracts are evaluated. Moreover; an in vivo infection model using Galleria mellonella larvae; infected and treated with the extracts are performed. All extracts show fungicidal activity; with a minimum fungicidal concentration (MFC) ranging from 128 to 512µgmL-1 against the two selected strains of Candida. In particular, the best results are obtained with methanolic extract of I. viscosa and G. alypum with an MFC value of 128µgmL-1 . The extracts are capable to prevent 90% of biofilm development at minor concentrations ranging from 100.71 ± 2.49µgmL-1 to 380.4 ± 0.92µgmL-1 . In vivo, tests on Galleria mellonella larvae show that the extracts increase the survival of the larvae infected with Candida. The attained results reveal that I. viscosa and G. alypum extracts may be considered as new antifungal agents and biofilm inhibiting agents for the pharmaceutical and agro-food field.

Dual RNA Sequencing of Beauveria bassiana-Infected Spodoptera frugiperda Reveals a Fungal Protease with Entomopathogenic and Antiphytopathogenic Activities.

Insect pests and phytopathogens significantly impact crop yield and quality. The fall armyworm (FAW) Spodoptera frugiperda and the phytopathogen Fusarium graminearum cause substantial economic losses in crops like barley and wheat. However, the entomopathogen Beauveria bassiana shows limited efficacy against FAW, and its antiphytopathogenic activities against F. graminearum remain unclear. Here, dual RNA sequencing was performed to identify differentially expressed genes in B. bassiana-infected FAW larvae. We found that the BbAorsin gene was significantly upregulated at 36 and 48 h post-infection. BbAorsin encodes a serine-carboxyl protease and is mainly expressed in blastospores and hyphae. Overexpression of BbAorsin in B. bassiana ARSEF2860 enhanced virulence against Galleria mellonella and FAW larvae and inhibited F. graminearum growth. The recombinant BbAorsin protein induced apoptosis and necrosis in FAW hemocytes and inhibited F. graminearum spore germination. These findings shed light on transcriptomic mechanisms governing insect-pathogen interactions, which could aid in developing dual-functional entomopathogens and anti-phytopathogens.

Isolation and Identification of Entomopathogenic Fungi from Soils of Manipur (N-E India)

Background: Entomopathogenic fungi (EPF) are natural adversaries of insects, serving a crucial role in the regulation of insect pest populations. In response to the growing demand for sustainable agricultural practices that prioritize environmental protection, human safety and animal welfare, the utilization of bio-control agents like entomopathogenic fungi offers a superior and safe alternative to chemical pesticides. Entomopathogenic fungi effectively infect and eliminate insects, thereby contributing to the control of insect populations through the induction of lethal infections known as epizootics. Isolating EPF from the soil is an effective method as they naturally inhabit soil ecosystems. The north-eastern region of India possesses a forest cover exceeding 80%, with Manipur alone accounting for nearly 75% of forest cover in its total geographical area. This abundant forest cover, along with undisturbed land, contributes to the region’s wealth of micro flora and fauna, including a thriving population of entomopathogenic fungi. However, the potential of these fungi in pest population management remains largely unexplored. Therefore, this study was conducted to investigate the diversity of these promising entomopathogenic fungi. Methods: In this study, we isolated fungi from the soils of ten districts of Manipur and identified several isolates with entomopathogenic properties. Soil bating using Galleria mellonella larvae was employed for the isolation of entomopathogenic fungi. Result: A total of 73 fungal isolates were obtained from 100 soil samples, out of which 54 were identified as entomopathogenic fungi. The genus Aspergillus constituted the most commonly isolated entomopathogenic fungi, followed by isolates of Beauveria, Clonostachys, Talaromyces, Trichoderma, Fusarium, Aspergillus, Candida and Meyerozyma genera. Diversity studies revealed variations in the types and proportions of fungi among different regions of Manipur. Pathogenicity tests confirmed the virulence of the isolated entomopathogenic fungi, with 14 isolates of Beauveria bassiana and two isolates of Talaromyces purpureogenus causing 100% mortality of the test insects. The isolated fungi exhibited excellent performance in insect control and could be further mass-produced for effective pest management.

Effects of passages through an insect or a plant on virulence and physiological properties of the fungus Metarhizium robertsii.

Species of the genus Metarhizium are characterized by a multitrophic lifestyle of being arthropod parasites, rhizosphere colonizers, endophytes, and saprophytes. The process of adaptation to various organisms and substrates may lead to specific physiological alterations that can be elucidated by passaging through different hosts. Changes in virulence and cultivation properties of entomopathogenic fungi subcultured on different media or passaged through a live insect host are well known. Nevertheless, comparative in-depth physiological studies on fungi after passaging through insect or plant organisms are scarce. Here, virulence, plant colonization, hydrolytic enzymatic activities, toxin production, and antimicrobial action were compared between stable (nondegenerative) parent strain Metarhizium robertsii MB-1 and its reisolates obtained after eight passages through Galleria mellonella larvae or Solanum lycopersicum or after subculturing on the Sabouraud medium. The passaging through the insect caused similar physiological alterations relative to the plant-based passaging: elevation of destruxin A, B, and E production, a decrease in protease and lipase activities, and lowering of virulence toward G. mellonella and Leptinotarsa decemlineata as compared to the parent strain. The reisolates passaged through the insect or plant showed a slight trend toward increased tomato colonization and enhanced antagonistic action on tomato-associated bacterium Bacillus pumilus as compared to the parental strain. Meanwhile, the subculturing of MB-1 on the Sabouraud medium showed stability of the studied parameters, with minimal alterations relative to the parental strain. We propose that the fungal virulence factors are reprioritized during adaptation of M. robertsii to insects, plants, and media.

Lipopeptides from an isolate of Bacillus subtilis complex have inhibitory and antibiofilm effects on Fusarium solani.

Bacillus subtilis species complex is known as lipopeptide-producer with biotechnological potential for pharmaceutical developments. This study aimed to identify lipopeptides from a bacterial isolate and evaluate their antifungal effects. Here, we isolated and identified a lipopeptide-producing bacterium as a species of Bacillus subtilis complex (strain UL-1). Twenty lipopeptides (six iturins, six fengycins, and eight surfactins) were identified in the crude extract (CE) and fractions (F1, F2, F3, and F4), and the highest content of total lipopeptides was observed in CE and F2. The chemical quantification data corroborate with the hemolytic and antifungal activities that CE and F2 were the most hemolytic and inhibited the fungal growth at lower concentrations against Fusarium spp. In addition, they caused morphological changes such as shortening and/or atypical branching of hyphae and induction of chlamydospore-like structure formation, especially in Fusarium solani. CE was the most effective in inhibiting the biofilm formation and in disrupting the mature biofilm of F. solani reducing the total biomass and the metabolic activity at concentrations ≥ 2µg/mL. Moreover, CE significantly inhibited the adherence of F. solani conidia on contact lenses and nails as well as disrupted the pre-formed biofilms on nails. CE at 100mg/kg was nontoxic on Galleria mellonella larvae, and it reduced the fungal burden in larvae previously infected by F. solani. Taken together, the lipopeptides obtained from strain UL-1 demonstrated a potent anti-Fusarium effect inducing morphological alterations and antibiofilm activities. Our data open further studies for the biotechnological application of these lipopeptides as potential antifungal agents. KEY POINTS: • Lipopeptides inhibit Fusarium growth and induce chlamydospore-like structures. • Lipopeptides hamper the adherence of conidia and biofilms of Fusarium solani. • Iturins, fengycins, and surfactins were associated with antifungal effects.

Culture of Steinernema glaseri on three solid media and their virulence against Galleria mellonella larvae

Culture of Steinernema glaseri on three solid media and their virulence against Galleria mellonella larvae

Negative interactions and virulence differences drive the dynamics in multispecies bacterial infections.

Bacterial infections are often polymicrobial, leading to intricate pathogen-pathogen and pathogen-host interactions. There is increasing interest in studying the molecular basis of pathogen interactions and how such mechanisms impact host morbidity. However, much less is known about the ecological dynamics between pathogens and how they affect virulence and host survival. Here we address these open issues by co-infecting larvae of the insect model host Galleria mellonella with one, two, three or four bacterial species, all of which are opportunistic human pathogens. We found that host mortality was always determined by the most virulent species regardless of the number of species and pathogen combinations injected. In certain combinations, the more virulent pathogen simply outgrew the less virulent pathogen. In other combinations, we found evidence for negative interactions between pathogens inside the host, whereby the more virulent pathogen typically won a competition. Taken together, our findings reveal positive associations between a pathogen's growth inside the host, its competitiveness towards other pathogens and its virulence. Beyond being generalizable across species combinations, our findings predict that treatments against polymicrobial infections should first target the most virulent species to reduce host morbidity, a prediction we validated experimentally.

Combination Therapy with Ciprofloxacin and Pentamidine against Multidrug-Resistant Pseudomonas aeruginosa: Assessment of In Vitro and In Vivo Efficacy and the Role of Resistance-Nodulation-Division (RND) Efflux Pumps.

The aim of this work was to (i) evaluate the efficacy of a combination treatment of pentamidine with ciprofloxacin against Galleria mellonella larvae infected with an MDR strain of P. aeruginosa and (ii) determine if pentamidine acts as an efflux-pump inhibitor. Resistant clinical isolates, mutant strains overexpressing one of three RND efflux pumps (MexAB-OprM, MexCD-OprJ, and MexEF-OprN), and a strain with the same three pumps deleted were used. MIC assays confirmed that the clinical isolates and the mutants overexpressing efflux pumps were resistant to ciprofloxacin and pentamidine. The deletion of the three efflux pumps induced sensitivity to both compounds. Exposure to pentamidine and ciprofloxacin in combination resulted in the synergistic inhibition of all resistant strains in vitro, but no synergy was observed versus the efflux-pump deletion strain. The treatment of infected G. mellonella larvae with the combination of pentamidine and ciprofloxacin resulted in enhanced efficacy compared with the monotherapies and significantly reduced the number of proliferating bacteria. Our measurement of efflux activity from cells revealed that pentamidine had a specific inhibitory effect on the MexCD-OprJ and MexEF-OprN efflux pumps. However, the efflux activity and membrane permeability assays revealed that pentamidine also disrupted the membrane of all cells. In conclusion, pentamidine does possess some efflux-pump inhibitory activity, in addition to a more general disruptive effect on membrane integrity that accounts for its ability to potentiate ciprofloxacin activity. Notably, the enhanced efficacy of combination therapy with pentamidine and ciprofloxacin versus MDR P. aeruginosa strains in vivo merits further investigation into its potential to treat infections via this pathogen in patients.

Type VII secretion system and its effect on group B Streptococcus virulence in isolates obtained from newborns with early onset disease and colonized pregnant women.

GBS may cause a devastating disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to better understand T7SS contribution to virulence in these different clinical scenarios. GBS genomes [N=33, 17 EOD isolates (serotype III/ST17) and 16 colonizing isolates (12 serotype VI/ST1, one serotype VI/ST19, one serotype VI/ST6, and two serotype 3/ST19)] were analyzed for presence of T7SS genes and genes encoding WXG100 proteins. We also perform bioinformatic analysis. Galleria mellonella larvae were used to compare virulence between colonizing, EOD, and mutant EOD isolates. The EOD isolate number 118659 (III/ST17) was used for knocking out the essC gene encoding a membrane-bound ATPase, considered the driver of T7SS. Most GBS T7SS loci encoded core component genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030)].Bioinformatic analysis indicated that based on sequence type (ST) the clinicalGBS isolates encode at least three distinct subtypes of T7SS machinery. In all ST1isolates we identified two copies of esxA gene (encoding putative WXG100proteins), when only 23.5% of the ST17 isolates harbored the esxA gene. Five ST17isolates encoded two copies of the essC gene. Orphaned WXG100 molecule(SAG0230), distinct from T7SS locus, were found in all tested strains, except inST17 strains where the locus was found in only 23.5% of the isolates. In ST6 andST19 isolates most of the structure T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella modelcompared to colonizing isolates. The 118659DessC strain was attenuated in itskilling ability, and the larvae were more effective in eradicating 118659DessC. We demonstrated that T7SS plays a role during infection. Knocking out the essC gene, considered the driver of T7SS, decreased the virulence of ST17 responsible for EOD, causing them to be less virulent comparable to the virulence observed in colonizing isolates.

Powerful and Real-Time Quantification of Antifungal Efficacy against Triazole-Resistant and -Susceptible Aspergillus fumigatus Infections in Galleria mellonella by Longitudinal Bioluminescence Imaging.

Aspergillus fumigatus is an environmental mold that causes life-threatening respiratory infections in immunocompromised patients. The plateaued effectiveness of antifungal therapy and the increasing prevalence of triazole-resistant isolates have led to an urgent need to optimize and expand the current treatment options. For the transition of in vitro research to in vivo models in the time- and resource-consuming preclinical drug development pipeline, Galleria mellonella larvae have been introduced as a valuable in vivo screening intermediate. Despite the high potential of this model, the current readouts of fungal infections in G. mellonella are insensitive, irreproducible, or invasive. To optimize this model, we aimed for the longitudinal quantification of the A. fumigatus burden in G. mellonella using noninvasive bioluminescence imaging (BLI). Larvae were infected with A. fumigatus strains expressing a red-shifted firefly luciferase, and the substrate dosage was optimized for the longitudinal visualization of the fungal burden without affecting larval health. The resulting photon flux was successfully validated for fungal quantification against colony forming units (CFU) analyses, which revealed an increased dynamic range from BLI detection. Comparison of BLI to survival rates and health index scores additionally revealed improved sensitivity for the early discrimination of differences in fungal burdens as early as 1 day after infection. This was confirmed by the improved detection of treatment efficacy against triazole-susceptible and -resistant strains. In conclusion, we established a refined G. mellonella aspergillosis model that enables the noninvasive real-time quantification of A. fumigatus by BLI. This model provides a quick and reproducible in vivo system for the evaluation of treatment options and is in line with 3Rs recommendations. IMPORTANCE Triazole-resistant Aspergillus fumigatus strains are rapidly emerging, and resistant infections are difficult to treat, causing mortality rates of up to 88%. The recent WHO priority list underscores A. fumigatus as one of the most critical fungal pathogens for which innovative antifungal treatment should be (urgently) prioritized. Here, we deliver a Galleria mellonella model for triazole-susceptible and -resistant A. fumigatus infections combined with a statistically powerful quantitative, longitudinal readout of the A. fumigatus burden for optimized preclinical antifungal screening. G. mellonella larvae are a convenient invertebrate model for in vivo antifungal screenings, but so far, the model has been limited by variable and insensitive observational readouts. We show that bioluminescence imaging-based fungal burden quantification outperforms these readouts in reliability, sensitivity, and time to the detection of treatment effects in both triazole-susceptible and -resistant infections and can thus lead to better translatability from in vitro antifungal screening results to in vivo confirmation in mouse and human studies.

Characterization of bacteriophage BUCT631 lytic for K1 Klebsiella pneumoniae and its therapeutic efficacy in Galleria mellonella larvae

Severe infections caused by multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) highlight the need for new therapeutics with activity against this pathogen. Phage therapy is an alternative treatment approach for multidrug-resistant K. pneumoniae infections. Here, we report a novel bacteriophage (phage) BUCT631 that can specifically lyse capsule-type K1 K. pneumoniae. Physiological characterization revealed that phage BUCT631 could rapidly adsorb to the surface of K. pneumoniae and form an obvious halo ring, and it had relatively favorable thermal stability (4–50 ​°C) and pH tolerance (pH ​= ​4–12). In addition, the optimal multiplicity of infection (MOI) of phage BUCT631 was 0.01, and the burst size was approximately 303 ​PFU/cell. Genomic analysis showed that phage BUCT631 has double-stranded DNA (total length of 44,812 bp) with a G ​+ ​C content of 54.1%, and the genome contains 57 open reading frames (ORFs) and no virulence or antibiotic resistance related genes. Based on phylogenetic analysis, phage BUCT631 could be assigned to a new species in the genus Drulisvirus of the subfamily Slopekvirinae. In addition, phage BUCT631 could quickly inhibit the growth of K. pneumoniae within 2 ​h in vitro and significantly elevated the survival rate of K. pneumoniae infected Galleria mellonella larvae from 10% to 90% in vivo. These studies suggest that phage BUCT631 has promising potential for development as a safe alternative for control and treatment of multidrug-resistant K. pneumoniae infection.

Changes in the apolipophorin III in Galleria mellonella larvae treated with Pseudomonas aeruginosa exotoxin A

In the present study, we have demonstrated a correlation in time between changes in the amount of apolipophorin III (apoLp-III) in the fat body and hemocytes of Galleria mellonella larvae challenged with Pseudomonas aeruginosa exotoxin A (exoA). An increase in the amount of apoLp-III was detected 1–8 h after the challenge; then, a temporary decrease was observed after 15 h followed by an increase in the level of apoLp-III, however to a different extent. The profile of apoLp-III forms in the hemolymph, hemocytes, and fat body of the exoA-challenged larvae was analyzed using two-dimensional electrophoresis (IEF/SDS-PAGE) and immunoblotting with anti-apoLp-III antibodies. Two apoLp-III forms differing in isoelectric point values estimated at ∼ 6.5 and ∼ 6.1 in the hemolymph and ∼ 6.5 and ∼ 5.9 in the hemocytes as well as one isoform with pI ∼ 6.5 in the fat body with an additional apoLp-III-derived polypeptide with estimated pI ∼ 6.9 were detected in the control insects. The injection of exoA caused a significant decrease in the abundance of both apoLp-III isoforms in the insect hemolymph. In the hemocytes, a decrease in the amount of the pI ∼ 5.9 isoform was detected, while the major apoLp-III isoform (pI ∼ 6.5) remained unchanged. In addition, appearance of an additional apoLp-III-derived polypeptide with an estimated pI ∼ 5.2 was observed. Interestingly, there were no statistically significant differences in the amount of the main isoform in the fat body between the control and exoA-challenged insects, but the polypeptide with pI ∼ 6.9 disappeared completely. It should be noted that the decrease in the amount of apoLp-III and other proteins was especially noticeable at the time points when exoA was detected in the studied tissues.

A dextrorotatory residues-incorporated bioactive dodecapeptide against enterohemorrhagic Escherichia coli

Aim: This study aims to report an engineered peptide zp39 with favorable bioactivity against enterohemorrhagic Escherichia coli (E. coli, EHEC). Its antibacterial mechanisms and application in a real food system are assessed. Methods: Spatial conformation of synthetic peptide zp39 (GIIAGIIiKIKk-NH2, lowercase letters indicate dextrorotatory amino acids) was predicted by PEPstrMOD and its secondary structure was further determined by circular dichroism (CD) spectroscopy. Then, standard E. coli O157:H7 strain ATCC 43888 was used to evaluate the bioactivity of zp39. A double dilution method was applied to investigate its efficacy in normal broth medium, serum, and highly saline conditions. Its effects on cell membrane permeability and potential were measured by fluorescent assays. Thereafter, morphological changes of E. coli O157:H7 cells were monitored by electron microscopy technologies. Finally, the potential application of zp39 in controlling EHEC in food was tested with spinach juice and the Galleria mellonella larvae model was employed to assess the in vivo efficacy. Results: Peptide zp39 presented an amphiphilic helical structure. It effectively inhibited the growth of E. coli O157:H7 at a concentration of 4 μmol/L in a bactericidal mode. Mechanistic studies revealed that it affected membrane permeability and potential in a dose-dependent manner. Moreover, zp39 maintained satisfactory bioactivity against E. coli O157:H7 even in the presence of 70% serum or 1,000 μmol/L chloride salts. In spinach juice application, > 90% E. coli O157:H7 cells were killed within 2 h after exposure to 64 μmol/L zp39. In vivo study proved that treatment with 64 μmol/L zp39 could effectively boost the survival ratio of infected larvae by 50%. Conclusions: This study depicts a synthetic dodecapeptide that shows the potential application in controlling EHEC. This molecule may be developed into a highly effective antimicrobial agent applied to prevent food contamination and associated infections.

Virulence of Shigatoxigenic and Enteropathogenic Escherichia coli O80:H2 in Galleria mellonella Larvae: Comparison of the Roles of the pS88 Plasmids and STX2d Phage.

The invasiveness properties of Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) O80:H2 in humans and calves are encoded by genes located on a pS88-like ColV conjugative plasmid. The main objectives of this study in larvae of the Galleria mellonella moth were therefore to compare the virulence of eight bovine STEC and EPEC O80:H2, of two E. coli pS88 plasmid transconjugant and STX2d phage transductant K12 DH10B, of four E. coli O80:non-H2, and of the laboratory E. coli K12 DH10B strains. Thirty larvae per strain were inoculated in the last proleg with 10 μL of tenfold dilutions of each bacterial culture corresponding to 10 to 106 colony-forming units (CFUs). The larvae were kept at 37 °C and their mortality rate was followed daily for four days. The main results were that: (i) not only the STEC and EPEC O80:H2, but also different E. coli O80:non-H2 were lethal for the larvae at high concentrations (from 104 to 106 CFU) with some variation according to the strain; (ii) the Stx2d toxin and partially the pS88 plasmid were responsible for the lethality caused by the E. coli O80:H2; (iii) the virulence factors of E. coli O80:non-H2 were not identified. The general conclusions are that, although the Galleria mellonella larvae represent a useful first-line model to study the virulence of bacterial pathogens, they are more limited in identifying their actual virulence properties.

Treatment of Colistin Dependence-Developing Acinetobacter baumannii with Antibiotic Combinations at Subinhibitory Concentrations.

Recent studies have revealed that colistin dependence frequently develops in colistin-susceptible Acinetobacter baumannii isolates. Despite resistance in parental strains, colistin-dependent mutants showed increased susceptibility to several antibiotics, which suggests the possibility of developing strategies to eliminate multidrug-resistant (MDR) A. baumannii. We investigated in vitro and in vivo efficacy of combinations of colistin and other antibiotics using MDR A. baumannii strains H08-391, H06-855, and H09-94, which are colistin-susceptible but develops colistin dependence upon exposure to colistin. An in vitro time-killing assay, a checkerboard assay, and an antibiotic treatment assay using Galleria mellonella larvae were performed. Although a single treatment of colistin at a high concentration did not prevent colistin dependence, combinations of colistin with other antibiotics at subinhibitory concentrations, especially amikacin, eradicated the strains by inhibiting the development of colistin dependence, in the in vitro time-killing assay. Only 40% of G. mellonella larvae infected by A. baumannii survived with colistin treatment alone; however, all or most of them survived following treatment with the combination of colistin and other antibiotics (amikacin, ceftriaxone, and tetracycline). Our results suggest the possibility of the combination of colistin and amikacin or other antibiotics as one of therapeutic options against A. baumannii infections by eliminating colistin-dependent mutants.

Combining bacteriophage and vancomycin is efficacious against MRSA biofilm-like aggregates formed in synovial fluid.

Biofilm formation is a major clinical challenge contributing to treatment failure of periprosthetic joint infection (PJI). Lytic bacteriophages (phages) can target biofilm associated bacteria at localized sites of infection. The aim of this study is to investigate whether combination therapy of phage and vancomycin is capable of clearing Staphylococcus aureus biofilm-like aggregates formed in human synovial fluid. In this study, S. aureus BP043, a PJI clinical isolate was utilized. This strain is a methicillin-resistant S. aureus (MRSA) biofilm-former. Phage Remus, known to infect S. aureus, was selected for the treatment protocol. BP043 was grown as aggregates in human synovial fluid. The characterization of S. aureus aggregates was assessed for structure and size using scanning electron microscopy (SEM) and flow cytometry, respectively. Moreover, the formed aggregates were subsequently treated in vitro with: (a) phage Remus [∼108 plaque-forming units (PFU)/ml], (b) vancomycin (500 μg/ml), or (c) phage Remus (∼108 PFU/ml) followed by vancomycin (500 μg/ml), for 48 h. Bacterial survival was quantified by enumeration [colony-forming units (CFU)/ml]. The efficacy of phage and vancomycin against BP043 aggregates was assessed in vivo as individual treatments and in combination. The in vivo model utilized Galleria mellonella larvae which were infected with BP043 aggregates pre-formed in synovial fluid. Scanning electron microscopy (SEM) images and flow cytometry data demonstrated the ability of human synovial fluid to promote formation of S. aureus aggregates. Treatment with Remus resulted in significant reduction in viable S. aureus residing within the synovial fluid aggregates compared to the aggregates that did not receive Remus (p < 0.0001). Remus was more efficient in eliminating viable bacteria within the aggregates compared to vancomycin (p < 0.0001). Combination treatment of Remus followed by vancomycin was more efficacious in reducing bacterial load compared to using either Remus or vancomycin alone (p = 0.0023, p < 0.0001, respectively). When tested in vivo, this combination treatment also resulted in the highest survival rate (37%) 96 h post-treatment, compared to untreated larvae (3%; p < 0.0001). We demonstrate that combining phage Remus and vancomycin led to synergistic interaction against MRSA biofilm-like aggregates in vitro and in vivo.

Interactions of alumina and polystyrene nanoparticles with the innate immune system of Galleria mellonella

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.

Increased ompW and ompA expression and higher virulence of Acinetobacter baumannii persister cells

BackgroundAcinetobacter baumannii is one of the main causes of healthcare-associated infections that threaten public health, and carbapenems, such as meropenem, have been a therapeutic option for these infections. Therapeutic failure is mainly due to the antimicrobial resistance of A. baumannii, as well as the presence of persister cells. Persisters constitute a fraction of the bacterial population that present a transient phenotype capable of tolerating supra-lethal concentrations of antibiotics. Some proteins have been suggested to be involved in the onset and/or maintenance of this phenotype. Thus, we investigated the mRNA levels of the adeB (AdeABC efflux pump component), ompA, and ompW (outer membrane proteins) in A. baumannii cells before and after exposure to meropenem.ResultsWe found a significant increase (p-value < 0.05) in the expression of ompA (> 5.5-fold) and ompW (> 10.5-fold) in persisters. However, adeB did not show significantly different expression levels when comparing treated and untreated cells. Therefore, we suggest that these outer membrane proteins, especially OmpW, could be part of the mechanism of A. baumannii persisters to deal with the presence of high doses of meropenem. We also observed in the Galleria mellonella larvae model that persister cells are more virulent than regular ones, as evidenced by their LD50 values.ConclusionsTaken together, these data contribute to the understanding of the phenotypic features of A. baumannii persisters and their relation to virulence, as well as highlight OmpW and OmpA as potential targets for drug development against A. baumannii persisters.

Physicochemical and Structural Evidence that Bacillus cereus Isolated from the Gut of Waxworms (Galleria mellonella Larvae) Biodegrades Polypropylene Efficiently In Vitro.

The online version contains supplementary material available at 10.1007/s10924-023-02878-y.

Effects of insecticidal proteins of Enterobacter cloacae NK on cellular immunity of Galleria mellonella larvae.

Enterobacter cloacae produces insecticidal proteins capable of causing toxicity in pests, but the insecticidal mechanisms of these proteins for insect control remain unclear. To elucidate the mechanisms, the purified insecticidal protein from E. cloacae NK was administered to Galleria mellonella larvae either by intraperitoneal injection or by feeding. The number of hemocytes, apoptosis in immune cells, and polyphenol oxidase (PO) activity of G. mellonella larvae were detected by hemocytometer, Annexin V-FITC/PI, and UV-vis spectrophotometer, respectively. With the extension of the invasion time of NK insecticidal protein, the number of hemocytes in G. mellonella larvae decreased significantly (p < 0.05), whereas the apoptosis rate of hemocytes increased. The activity of PO showed a trend of rising-peak-sharp decline and the melanization reaction was deepened simultaneously. Moreover, the phagocytosis and coating capabilities of hemocytes decreased, and the intraperitoneal injection method was more effective than the feeding method. Taking together, the insecticidal protein of E. cloacae NK inhibits and destroys the cellular immune response of G. mellonella larvae, which suggests an important role in killing the host insect.