Phthalocyanine derivative attenuates TNF-α production in macrophage culture and prevents alveolar bone loss in experimental periodontitis.

AIM: This study investigated the activity and mechanism of action of the iron tetracarboxyphthalocyanine (FeTcPc) on tumor necrosis factor alpha (TNF-α) production and its impact on experimental periodontitis. METHODS: RAW 264.7 macrophages were treated with FeTcPc, activated with lipopolysaccharide (LPS) at 10 ng/mL, and the TNF-α levels were measured, as well as the nuclear factor kappa B (NF-κB) activation. Subsequently, a mouth gel containing 1% FeTcPc was topically administered to the gingival tissue of mice with periodontitis-induced ligatures. Bone loss and the gene expression of Tnfα, p65 (NF-κB), and receptor-activating nuclear factor kappa B ligand (Rankl) were quantified in gingival tissue. Finally, the systemic toxicity of FeTcPc was estimated in Galleria mellonella larvae. RESULTS: In an activated RAW 264.7 macrophage culture, 100 µM FeTcPc reduced TNF-α release and NF-κB activation. Regarding experimental periodontitis, topical application of mouth gel containing 1% FeTcPc blocked alveolar bone loss. Additionally, 1% FeTcPc reduced the expression of Tnfα, p65 (NF-κB), and Rankl in gingival tissue. Finally, administration FeTcPc at doses ranging from 1 to 1000 mg/kg did not cause acute systemic toxicity in G. mellonella. CONCLUSION: Overall, we demonstrated the potential of mouth gel containing FeTcPc as a therapeutic strategy for managing osteolytic inflammatory disorders, such as periodontitis.

Antifungal activity of indolicidin-derived peptide In-58 against Sporothrix globosa in vitro and in vivo.

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.

Consumption and Digestion of Plastics by Greater Hive Moth Larvae.

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.

Human breast milk isolated lactic acid bacteria: antimicrobial and immunomodulatory activity on the Galleria mellonella burn wound model.

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-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.

The dynamics of Staphylococcal infection and their treatment with antibiotics and bacteriophage in the Galleria mellonella model system.

Critical to our understanding of infections and their treatment is the role the innate immune system plays in controlling bacterial pathogens. Nevertheless, many in vivo systems are made or modified such that they do not have an innate immune response. Use of these systems denies the opportunity to examine the synergy between the immune system and antimicrobial agents. In this study we demonstrate that the larva of Galleria mellonella is an effective in vivo model for the study of the population and evolutionary biology of bacterial infections and their treatment. To do this we test three hypotheses concerning the role of the innate immune system during infection. We show: i) sufficiently high densities of bacteria are capable of saturating the innate immune system, ii) bacteriostatic drugs and bacteriophages are as effective as bactericidal antibiotics in preventing mortality and controlling bacterial densities, and iii) minority populations of bacteria resistant to a treating antibiotic will not ascend. Using a highly virulent strain of Staphylococcus aureus and a mathematical computer-simulation model, we further explore how the dynamics of the infection within the short term determine the ultimate infection outcome. We find that excess immune activation in response to high densities of bacteria leads to a strong but short-lived immune response which ultimately results in a high degree of mortality. Overall, our findings illustrate the utility of the G. mellonella model system in conjunction with established in vivo models in studying infectious disease progression and treatment.

Evaluation of Klebsiella pneumoniae pathogenicity through holistic gene content analysis.

Klebsiella pneumoniae is a Gram-negative bacterium that causes both community- and healthcare-associated infections. Although various virulence factors and highly pathogenic phenotypes have been reported, the pathogenicity of K. pneumoniae is still not fully understood. In this study, we utilized whole-genome sequencing data of 168 clinical K. pneumoniae strains to assess pathogenicity. This work was based on the concept that the genetic composition of individual genomes (referred to as holistic gene content) of the strains may contribute to their pathogenicity. Holistic gene content analysis revealed two distinct groups of K. pneumoniae strains ('major group' and 'minor group'). The minor group included strains with known highly pathogenic clones (ST23, ST375, ST65 and ST86). The minor group had higher rates of capsular genotype K1 and presence of nine specific virulence genes (rmpA, iucA, iutA, irp2, fyuA, ybtS, iroN, allS and clbA) compared to the major group. Pathogenicity was assessed using Galleria mellonella larvae. Infection experiments revealed lower survival rates of larvae infected with strains from the minor group, indicating higher virulence. In addition, the minor group had a higher string test positivity rate than the major group. Holistic gene content analysis predicted possession of virulence genes, string test positivity and pathogenicity as observed in the G. mellonella infection model. Moreover, the findings suggested the presence of as yet unrecognized genomic elements that are either involved in the acquisition of virulence genes or associated with pathogenicity.

Transcriptome analysis and functional study of phospholipase A2 in Galleria mellonella larvae lipid metabolism in response to envenomation by an ectoparasitoid, Iseropus kuwanae.

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.

Application of DNA aptamers to block enterotoxigenic Escherichia coli toxicity in a Galleria mellonella larval model.

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.

Characterization and comparative analysis of sericin protein 150 in Bombyx mori.

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.

Functional genomic analysis of genes important for Candida albicans fitness in diverse environmental conditions.

Fungal pathogens such as Candida albicans pose a significant threat to human health with limited treatment options available. One strategy to expand the therapeutic target space is to identify genes important for pathogen growth in host-relevant environments. Here, we leverage a pooled functional genomic screening strategy to identify genes important for fitness of C. albicans in diverse conditions. We identify an essential gene with no known Saccharomyces cerevisiae homolog, C1_09670C, and demonstrate that it encodes subunit 3 of replication factor A (Rfa3). Furthermore, we apply computational analyses to identify functionally coherent gene clusters and predict gene function. Through this approach, we predict the cell-cycle-associated function of C3_06880W, a previously uncharacterized gene required for fitness specifically at elevated temperatures, and follow-up assays confirm that C3_06880W encodes Iml3, a component of the C. albicans kinetochore with roles in virulence in vivo. Overall, this work reveals insights into the vulnerabilities of C. albicans.

The ER-Resident Ras Inhibitor 1 (Eri1) of Candida albicans Inhibits Hyphal Morphogenesis via the Ras-Independent cAMP-PKA Pathway.

Ras signaling and glycosylphosphatidylinositol (GPI) biosynthesis are mutually inhibitory in S. cerevisiae (Sc). The inhibition is mediated via an interaction of yeast Ras2 with the Eri1 subunit of its GPI-N-acetylglucosaminyl transferase (GPI-GnT), the enzyme catalyzing the very first GPI biosynthetic step. In contrast, Ras signaling and GPI biosynthesis in C. albicans (Ca) are mutually activated and together control the virulence traits of the human fungal pathogen. What might be the role of Eri1 in this pathogen? The present manuscript addresses this question while simultaneously characterizing the cellular role of CaEri1. It is either nonessential or required at very low levels for cell viability in C. albicans. Severe depletion of CaEri1 results in reduced GPI biosynthesis and cell wall defects. It also produces hyperfilamentation phenotypes in Spider medium as well as in bicarbonate medium containing 5% CO2, suggesting that both the Ras-dependent and Ras-independent cAMP-PKA pathways for hyphal morphogenesis are activated in these cells. Pull-down and acceptor-photobleaching FRET experiments suggest that CaEri1 does not directly interact with CaRas1 but does so through CaGpi2, another GPI-GnT subunit. We showed previously that CaGpi2 is downstream of CaEri1 in cross talk with CaRas1 and for Ras-dependent hyphal morphogenesis. Here we show that CaEri1 is downstream of all GPI-GnT subunits in inhibiting Ras-independent filamentation. CaERI1 also participates in intersubunit transcriptional cross talk within the GPI-GnT, a feature unique to C. albicans. Virulence studies using G. mellonella larvae show that a heterozygous strain of CaERI1 is better cleared by the host and is attenuated in virulence.

Dietary oxyclozanide influences antioxidant enzyme activities and damages DNA in Galleria mellonella (Lepidoptera: Pyralidae).

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.

Characterization of novel double-reporter strains of Mycobacterium abscessus for drug discovery: a study in mScarlet.

Mycobacterium abscessus (Mab) is an emerging pathogen that poses a severe health threat, especially in people with cystic fibrosis and other chronic lung diseases. Available drugs are largely ineffective due to an exquisite intrinsic resistance, making Mab infections only comparable to multidrug-resistant tuberculosis. Current treatment is based on lengthy multidrug therapy, complicated by poor outcomes and high rates of treatment failure, recurrence, and mortality. Thus, finding new and more efficient drugs to combat this pathogen is urgent. However, drug discovery efforts targeting Mab have been limited, and traditional drug screening methods are labor-intensive, low-throughput, and do not reflect clinical effectiveness. Therefore, this work aimed to develop a new, efficient, and reliable tool for drug screening against Mab that can be used in vitro for identifying hits in a high-throughput manner and in vivo to select drug candidates for future clinical trials. We engineered two stable double-reporter strains of Mab capable of emitting strong fluorescent and luminescent signals. This is due to the expression of mScarlet protein and luciferase enzyme or the entire lux operon. Importantly, these strains maintain the same ground characteristics as the non-transformed Mab strain. We show that these new strains can be applied to various setups, from MIC determination in broth cultures and macrophage infection assays to in vivo infection (using the Galleria mellonella model). Using these strains enhances the potential for high-throughput screening of thousands of compounds in a fast and reliable way. IMPORTANCE: Mycobacterium abscessus (Mab) is currently considered an "incurable nightmare." Its intrinsic resistance, high toxicity, long duration, and low cure rates of available therapies often lead to the clinical decision not to treat. Moreover, one of the significant drawbacks of anti-Mab drug development is the lack of correlation between in vitro susceptibility and clinical efficacy. Most drug screening assays are performed on Mab growing in liquid cultures. But being an intracellular pathogen, inducing granulomas and biofilm formation, the broth culture is far from ideal as in vitro drug-testing setup. This study presents new double-reporter Mab strains that allow direct real-time bacterial detection and quantification in a non-invasive way. These strains can be applied to an extensive range of experimental settings, far surpassing the utility of single-reporter bacteria. They can be used in all steps of the pre-clinical anti-Mab drug development pipeline, constituting a highly valuable tool to increase its success.

Efficient CRISPR/Cas9-mediated ebony gene editing in the greater wax moth Galleria mellonella.

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.

Bioactivity of silverleaf nightshade (Solanum elaeagnifolium Cav.) berries parts against Galleria mellonella and Erwinia carotovora and LC-MS chemical profile of its potential extract.

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.

Optimization of Experimental Infection of the Animal Model Galleria mellonella Linnaeus 1758 (Lepidoptera: Pyralidae) with the Gram-Positive Bacterium Micrococcus luteus.

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.

A Case of Pseudomonas straminea Blood Stream Infection in an Elderly Woman with Cellulitis.

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.

Bioefficacy and molecular characterization of Bacillus thuringiensis strain NBAIR BtGa against greater wax moth, Galleria mellonella L.

Galleria mellonella, the greater wax moth has always been an important pest against honeybees and has remained a nightmare for beekeeping farmers. Management of G. mellonella in live honeybee colonies is very difficult because most current management practices can destroy whole honeybee colonies. In the present study, experiments were conducted to isolate and characterize Bacillus thuringiensis from infected greater wax moth cadavers and to evaluate their biocontrol ability against G. mellonella. The bioefficacy of these isolates has been evaluated against greater wax moth along with the standard strain HD-1. Among all the strains tested, NBAIR BtGa demonstrated higher efficacy compared to other strains, with an LC50 value of 125.17 µg/ml, whereas HD-1 exhibited a significantly higher LC50 value of 946.61 µg/ml. Considering the economic importance of NBAIR BtGa we performed whole genome sequencing of this strain resulting in the identification of a genome size of 5.96 Mb consisting of 6888 protein-coding genes. Gene ontology analysis categorized these genes into three groups based on their roles, i.e., biological functions (2169 genes), cellular components (1900 genes), and molecular functions (2774 genes). Through insecticidal toxicity-related genes (ITRG) profiling of our strain across the genome by Bt toxin scanner and cry processor resulted in the identification of several Cry proteins namely Cry1Ab11, Cry1Ia44, Cry1Aa2, Cry2Af1, Cry1Da2, Cry1Eb1, Cry1Ab5, Cry1Cb2, Cry1Ac2. Besides Cry proteins, other ITRG genes, viz. Vip3Bb2, Zwittermicin A resistance proteins, Chitinase C, Mpp46Ab1, immune inhibitor A, Bmp1, Vpb4Ca1, and Spp1Aa1 were also reported, which show toxicity against lepidopteran pests. The studies were also conducted to test the biosafety of Bt toxins against honeybee larvae and adults, which showed strain NBAIR BtGa was more than 99% safer for honeybee larvae as well as adults. Thus, the data generated ascertains its effectiveness as a biocontrol agent and it can be used further for the development of bio formulation for the management of G. mellonella in honeybee colonies.

Development of a Galleria mellonella Infection Model to Evaluate the Efficacy of Antibiotic-Loaded Polymethyl Methacrylate (PMMA) Bone Cement.

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.

Azobenzenesulfonamide Carbonic Anhydrase Inhibitors as New Weapons to Fight Helicobacter pylori: Synthesis, Bioactivity Evaluation, In Vivo Toxicity, and Computational Studies.

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.