Greater Wax Moth Galleria Mellonella Research Articles

Effect of the Biostimulants of Microbiological Origin on the Entomopathogenic and Plant Parasitic Nematodes from Miscanthus × Giganteus Plantations

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

Insect immunity - adaptive mechanisms and survival strategies

Host-pathogen interactions play an important role in understanding the dynamics of the insect immune system. The analysis of environmental modulators, both biotic and abiotic, directs our attention to the impact of the surroundings on the effectiveness of immunological responses. This knowledge is essential for the comprehensive understanding of insect’s immune reaction after infection with a pathogen. This article discusses the role of the immune system in insect, with a special emphasis on greater wax moth Galleria mellonella and highlights its adaptive capabilities. The processes are not only extremely interesting area of scientific research but also indicate potential practical applications in the context of plan protection, pest population control and medicine.

Phenotypic and genotypic characterization of Campylobacter coli isolates from the Vietnamese poultry production network; a pilot study

IntroductionChanging farming practices and the associated increase in the use of antibiotics are amongst the main drivers shaping the global increase of Campylobacter infections. The effects farming practices have on Campylobacter species, need to be studied at the global scale, particularly in emerging middle-income countries, where the demand for low-cost poultry meat is rising. While Campylobacter jejuni causes the majority of poultry associated diarrhoea, Campylobacter coli causes a significant amount of disease but are relatively understudied. In this study we characterised seven C. coli strains isolated from chicken faeces and chicken meat in Thai Nguyen province, Vietnam.MethodsWhole Genome Sequencing and phenotypic assays (growth, motility, antimicrobial resistance testing, virulence assay) were performed to reveal the genetic relatedness and pathophysiological characteristics of the isolates. ResultsAll isolates were resistant to ciprofloxacin and nalidixic acid but susceptible to phenicols. Three were resistant to macrolides azithromycin and erythromycin. Six isolates were classified as multi-drug resistant. All isolates had similar growth rates in laboratory culture media, while five were hyper-motile. Lethality towards a tractable host-model system, larvae of the greater wax moth Galleria mellonella, often used to determine Campylobacter virulence, was demonstrated for the first time for C. coli. DiscussionMultilocus sequence typing data identified five ST’s all within the C. coli ST-828 clonal complex and were previously reported in North American (ST-829), European (ST-1586), and Asia (ST-5511) from patients suffering from gastroenteritis, emphasising the global spread of these strains. This work highlights the importance of further research into this understudied global threat.

Immunophenotyping of hemocytes from infected Galleria mellonella larvae as an innovative tool for immune profiling, infection studies and drug screening

In recent years, there has been a considerable increasing interest in the use of the greater wax moth Galleria mellonella as an animal model. In vivo pharmacological tests, concerning the efficacy and the toxicity of novel compounds are typically performed in mammalian models. However, the use of the latter is costly, laborious and requires ethical approval. In this context, G. mellonella larvae can be considered a valid option due to their greater ease of use and the absence of ethical rules. Furthermore, it has been demonstrated that the immune system of these invertebrates has similarity with the one of mammals, thus guaranteeing the reliability of this in vivo model, mainly in the microbiological field. To better develop the full potential of this model, we present a novel approach to characterize the hemocyte population from G. mellonella larvae and to highlight the immuno modulation upon infection and treatments. Our approach is based on the detection in isolated hemocytes from G. mellonella hemolymph of cell membrane markers typically expressed by human immune cells upon inflammation and infection, for instance CD14, CD44, CD80, CD163 and CD200. This method highlights the analogies between G. mellonella larvae and humans. Furthermore, we provide an innovative tool to perform pre-clinical evaluations of the efficacy of antimicrobial compounds in vivo to further proceed with clinical trials and support drug discovery campaigns.

Genomics, transcriptomics, and peptidomics of the greater wax moth Galleria mellonella neuropeptides and their expression in response to lead stress.

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

Coxiella co-opts the Glutathione Peroxidase 4 to protect the host cell from oxidative stress–induced cell death

The causative agent of human Q fever, Coxiella burnetii, is highly adapted to infect alveolar macrophages by inhibiting a range of host responses to infection. Despite the clinical and biological importance of this pathogen, the challenges related to genetic manipulation of both C. burnetii and macrophages have limited our knowledge of the mechanisms by which C. burnetii subverts macrophages functions. Here, we used the related bacterium Legionella pneumophila to perform a comprehensive screen of C. burnetii effectors that interfere with innate immune responses and host death using the greater wax moth Galleria mellonella and mouse bone marrow-derived macrophages. We identified MceF (Mitochondrial Coxiella effector protein F), a C. burnetii effector protein that localizes to mitochondria and contributes to host cell survival. MceF was shown to enhance mitochondrial function, delay membrane damage, and decrease mitochondrial ROS production induced by rotenone. Mechanistically, MceF recruits the host antioxidant protein Glutathione Peroxidase 4 (GPX4) to the mitochondria. The protective functions of MceF were absent in primary macrophages lacking GPX4, while overexpression of MceF in human cells protected against oxidative stress-induced cell death. C. burnetii lacking MceF was replication competent in mammalian cells but induced higher mortality in G. mellonella, indicating that MceF modulates the host response to infection. This study reveals an important C. burnetii strategy to subvert macrophage cell death and host immunity and demonstrates that modulation of the host antioxidant system is a viable strategy to promote the success of intracellular bacteria.

Efficiency of the Iraqi Isolates of Beauveria Bassiana & Metarhizium Anisopliae in Controlling the Greater Wax Moth Galleria Mellonella L.

The current study was conducted in the laboratories of the Plant Protection Department/College of Agriculture and Forestry/University of Mosul during the year 2019-2020. The aim of this study was to evaluate the efficiency of the Iraqi isolate of the biological control fungi Beauveria bassian and Metarhizium anisopliaeas chemical pesticides alternatives in controlling different stages of the greater wax moth Galleria mellonella. The results of the biological effect of the fungi Beauveria bassiana and Metarhizium anisopliae on the greater wax moth life stages showed that the cumulative mortality of eggs increased gradually with increasing concentration used. However, Beauveria bassiana was more efficient in achieving higher mortality rates than M. anisopliae in general. The highest cumulative mortality rate for eggs was at the highest concentration (7.5g/l) for both fungi. On the other hand, it was found that the highest mortality rate in wax moth larvae was inB. bassianacombined treatmentwhere spraying the larvae and the feed. The third and fourth larval instars were more sensitive than the sixth and seventh larval instars, which were not affected by the lower concentrations of the pesticide (2.5 and 5 g/L), regardless of pesticide type. The results also showed that the mortality rate in all phases increases with the increase in the post-treatment period. The results of the study indicated the superiority of B. bassiana in all treatments, concentrations and periods after treatment and led to the highest mortality rate compared with using M. anisopliae. The cumulative mortality rate in the greater wax moth was directly proportional to the increase in treatment methods and the increase in concentration and the time period after treatment.

Debilitation of Galleria mellonella hemocytes using CytCo a cytolytic-like protein derived from the entomopathogen Conidiobolus obscurus

Cytolytic (Cyt)-like genes are present in both pathogenic bacteria and fungi. Bacterial Cyt proteins can destroy insect midgut epithelial cells after ingestion by hosts and some of them have been developed as biopesticides; however, few studies have investigated their functions in fungal pathogens. This study investigated the effects of a Cyt-like protein (CytCo) derived from Conidiobolus obscurus (Entomophthoromycotina) on the hemocytes of the greater wax moth Galleria mellonella larvae. The results showed a significant decline in hemocyte viability after treatment with CytCo in vivo or in vitro. The hemocyte density in the hemolymph was reduced by 65.2% and 50.2% after 12 h in vivo and 6 h in vitro treatments, respectively. Apoptosis/necrosis tests using fluorescence microscopy demonstrated that CytCo-treated hemocytes displayed apoptosis, and many of them also showed necrosis after 6 h in vitro treatment. Based on transcriptome analysis, several genes involved in the programmed cell death signaling pathway were upregulated in the CytCo-treated hemocytes. Meanwhile, the differentially expressed genes related to energy production, signal transduction, transcription regulation, and melanization were upregulated, demonstrating activated immune responses; those putatively related to hemocyte adhesion were downregulated, possibly in response to the reduction of hemocytes in hemolymph. In conclusion, CytCo as a virulence factor, could irreversibly incapacitate host hemocytes, playing an important role in debilitating insect immunity. This novel insecticidal protein holds a potential to develop biopesticide for controlling agroforestry pests.

Valuation The efficiency of Nanotechnology and biological activity of Nanoparticle for two plant extracts Ricinus communis and Neriun oleander against immature stages of Greater wax moth.Galleria mellonella (Lepidoptera: pyralidae)

The results of present study conducted on the Laboratory of advance entomology \ Department of biology \ Faculty of Education for Girls \ University of Kufa, during the years 2018-2019 for evaluating The efficiency of Nanotechnology and biological control and the effect of two plant extracts on the different immature stages of the greater wax moth Galleria mellonella (Lepidoptera: Pyrallidae). eggs, first and fourth larval instars. The cold aqueous extract of castor Ricinus communis and Nerium oleander leaves ,from which different concentrations ( 1,2,3 and 4) mg \ ml were prepared in the Laboratory. The experiments include the enhancement of these plant extracts of toxic nature with chemically prepared particles, silver nanoparticles, and conducting multiple experiments for the same instars of the insect, which was treated with aqueous plant extracts .The statistical results as well as the observation during different stages of the experiments were superior of these nanoextracts over plant extracts in the mortality of eggs and first and fourth latval instars. Experiments indicated that the mortality percentages when treated with aqueous leaves extract of castor were (76, 91, 96) % respectively while the same instars were treated with nano extract of castor was(83.3 ,92.3 ,98.6 )% respectivly . The leaves extract of N.oleander was (86.6%, 96.6%, 99.4%) compared with (93.3, 100, 100) % respectively of the nanoparticle of the eggs and larvae of the first and fourth phases at 4 mg / ml. It also proved that the first instar was the most sensitive to the extract of the fourth instar, where the highest mortality percentages was (96.6, 100) % compared with (86.6, 93.3)% respectively of the first and fourth larval instars when treated with and nano of N. oleander and R.communis respectively.

UV-induced mutagenesis of Beauveria bassiana (Hypocreales: Clavicipitaceae) yields two hypervirulent isolates with different transcriptomic profiles.

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) can infest over 300 plant species and cause huge economic losses. Beauveria bassiana (Hypocreales: Clavicipitaceae) is one of the most widely used entomopathogenic fungi (EPF). Unfortunately, the efficacy of B. bassiana against S. frugiperda is quite low. Hypervirulent EPF isolates can be obtained by ultraviolet (UV)-irradiation. Here we report on the UV-induced mutagenesis and transcriptomic analysis of B. bassiana. The wild-type (WT) B. bassiana (ARSEF2860) was exposed to UV light to induce mutagenesis. Two mutants (named 6M and 8M) showed higher growth rates, conidial yields, and germination rates compared to the WT strain. The mutants showed higher levels of tolerance to osmotic, oxidative, and UV stresses. The mutants showed higher protease, chitinase, cellulose, and chitinase activities than WT. Both WT and mutants were compatible with the insecticides matrine, spinetoram, and chlorantraniliprole, but incompatible with emamectin benzoate. Insect bioassays showed that both mutants were more virulent against S. frugiperda and the greater wax moth Galleria mellonella. Transcriptomic profiles of the WT and mutants were determined by RNA-sequencing. The differentially expressed genes (DEGs) were identified. The gene set enrichment analysis (GSEA), protein-protein interaction (PPI) network, and hub gene analysis revealed virulence-related genes. Our data demonstrate that UV-irradiation is a very efficient and economical technique to improve the virulence and stress resistance of B. bassiana. Comparative transcriptomic profiles of the mutants provide insights into virulence genes. These results provide new ideas for improving the genetic engineering and field efficacy of EPF. © 2023 Society of Chemical Industry.

Low dose γ-radiation induced effects on wax moth (Galleria mellonella) larvae

Larvae of the greater wax moth Galleria mellonella are common pests of beehives and commercial apiaries, and in more applied settings, these insects act as alternative in vivo bioassays to rodents for studying microbial virulence, antibiotic development, and toxicology. In the current study, our aim was to assess the putative adverse effects of background gamma radiation levels on G. mellonella. To achieve this, we exposed larvae to low (0.014 mGy/h), medium (0.056 mGy/h), and high (1.33 mGy/h) doses of caesium-137 and measured larval pupation events, weight, faecal discharge, susceptibility to bacterial and fungal challenges, immune cell counts, activity, and viability (i.e., haemocyte encapsulation) and melanisation levels. The effects of low and medium levels of radiation were distinguishable from the highest dose rates used – the latter insects weighed the least and pupated earlier. In general, radiation exposure modulated cellular and humoral immunity over time, with larvae showing heightened encapsulation/melanisation levels at the higher dose rates but were more susceptible to bacterial (Photorhabdus luminescens) infection. There were few signs of radiation impacts after 7 days exposure, whereas marked changes were recorded between 14 and 28 days. Our data suggest that G. mellonella demonstrates plasticity at the whole organism and cellular levels when irradiated and offers insight into how such animals may cope in radiologically contaminated environments (e.g. Chornobyl Exclusion Zone).

Pseudomonas aeruginosa exotoxin A induces apoptosis in Galleria mellonella hemocytes

The cellular immune response of the greater wax moth Galleria mellonella to Pseudomonas aeruginosa exotoxin A was investigated for the first time. The insects were challenged with a sublethal dose of exoA, and then hemocyte parameters were assessed. The analysis showed a statistically significant decrease in the total hemocyte count (THC), which was associated with significant decreases in the number of granulocytes and plasmatocytes. In turn, no statistically significant changes were observed in the number of spherulocytes and oenocytoides. Fluorescent staining indicated that cells collected from the exoA-challenged larvae exhibited features characteristic for apoptotic and autophagic cell death, e.g. cytoplasm vacuolization and chromatin condensation. The flow cytometry analysis revealed a significant increase in the number of phosphatidylserine- and active caspase 3-positive hemocytes challenged with exoA, which proved apoptosis induction. Our results will help in understanding the role of exotoxin A during P. aeruginosa infections not only in insects but also in mammals, including humans.

Efficacy of gamma radiation on the greater wax moth <i>Galleria mellonella</i> L.

The effect of ionizing radiation from 50 to 450 Gy on the eggs and larvae 100 to 1500 Gy. on larvae of greater wax moth Galleria mellonella L. The results revealed that the egg hatchability reduced to 50% at 102.70 Gy. In the most radiation resistant eggs, 350 Gy resulted in no pupal formation while a dose of 250 Gy resulted in no adult formation. Within 51.53 hr, a dose of 500 Gy reduced the larval survival to 50%. With 148, 574 and 680 Gy, the proportion of larvae that matured into adults reduced to 50%, 10%, and 0%, respectively. A radiation dose between 300 and 400 Gy was found adequate for egg sterilization and larval mortality.

Bioactivity and Prospects of Using Ethanolic Extracts of Some Plants and Bee Glue (Propolis) to Control the Greater Wax Moth Galleria mellonella (L) (Lepidoptera: Pyralidae)

The greater wax moth (Galleria mellonella) is one of the most important pests of stored or unattended combs that cause severe damage to bee broods, threatening the development of apiculture in various countries, especially Sudan. A laboratory experiment was conducted in the College of Agricultural Studies, Sudan University of Science and Technology, Shambat, Sudan, to explore the potency of ethanolic extract (EE) of different parts of three different plants and bee glue against the third larval instar of greater wax moth. The experiment was laid out in Randomized Complete Block Design and replicated five times. The percentage of mortality of GWM was recorded after 24, 48, 72, and 96 hours post-treatment. Analysis of the variance of the obtained data revealed significant differences regarding insecticidal effect between the EEs of the four test products and the control. The EE of Eucalyptus camaldulensis, Propolis, Nigella sativa, and Carum carvi have bioactivity action against the third instar larvae of GWM with an increased percentage of mortality according to the increase of concentration as well as elapse of time. Hence, the highest concentration (15%) caused 86.7%, 83.3%, 73.3 and 66.7% larval mortality after 96 hours for EE of leaves of E. camaldulensis, powder of bee glue (Propolis), seeds of N. sativa and seeds of C. carvi respectively. The results also clearly demonstrated that the EE of the powder of Propolis is significantly more toxic than E. camaldulensis, N. sativa, and C. carvi, where the LC50 values were 3.1% for Propolis, 5.0% for E. camaldulensis, 7.0 % for N. sativa and 7.7% for C. carvi. The products mentioned above at the mentioned concentration are environmental friendly, safe for honeybees, economically feasible, and affordable for small beekeepers.

Influence of zinc oxide nanoparticles (ZnO NPs) on the hemocyte count and hemocyte-mediated immune responses of the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae)

In this study, we examined the effects of different doses (100, 500, 1000, 3000, and 5000 ppm) of zinc oxide nanoparticles (ZnO NPs) on the total hemocyte count and hemocyte-mediated immune responses of the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae). The results showed that NPs caused a decrease in hemocyte count at 1000, 3000, and 5000 ppm doses. To investigate the effects of ZnO NPs on the encapsulation and melanization response of G. mellonella, the pre-dyed Sephadex chromatography beads were injected into the hemolymph of each last-instar larva. Larvae were dissected in the 4th and 24th hours after the injection. The level of the encapsulation response and melanization status around the beads were determined under microscopy. The analyses of the beads injected into the insects as encapsulation targets revealed that the number of weakly encapsulated beads increased significantly at 100, 1000, 3000, and 5000 ppm doses when compared to the control group after a short (4-h) post-injection. The number of melanized beads increased significantly at 100, 1000, and 3000 ppm doses in comparison to the control group after the short (4-h) post-injection. Finally, the number of melanized beads decreased significantly at 1000 and 5000 ppm doses when compared to the control group after the long-term (24-h) post-injection.

Enzymatic and insecticidal activities of the Amsacta moorei entomopoxvirus glycosyltransferase, AMV248

AbstractThe family Poxviridae is divided into two subfamilies, the Chordopoxvirinae of vertebrates and the Entomopoxvirinae of invertebrates. The Amsacta moorei entomopoxvirus (AMEV, Entomopoxvirinae) has the potential to be used in gene therapy, as an expression vector, and as a biopesticide. It was suggested that AMV248 protein is a putative glycosyltransferase (GT) but was also shown to be an attachment protein to host receptors. GTs encoded by some other viruses catalyse the binding of sugars molecules to growth hormones of the host insects rendering the hormones inactive. Consequently, larval development is arrested and frequently results in larval mortality. In this study, AMV248 protein was shown to be a GT and the purified enzyme catalysed the production of uridine diphosphate (UDP) from the substrates UDP‐glucose and UDP‐N‐acetylglucosamine. This AMEV enzyme may behave much like the ecdysteroid UDP‐glucosyltransferase of baculoviruses. Various concentrations of the GT enzyme were tested for its insecticidal activity against gypsy moth Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Lymantriidae), lackey moth Malacosoma neustria (Linnaeus, 1758) (Lepidoptera: Lasiocampidae), cotton bollworm Helicoverpa armigera (Hübner, 1808) (Lepidoptera: Noctuidae) and the greater wax moth Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae) larvae. It had varying deleterious effects on all test larvae but L. dispar was the most sensitive to the enzyme. While this enzyme exhibits properties with potential to be developed as an insecticide in biocontrol strategies, investigations are needed to ascertain its value in pest management.

Genomic and Functional Characterization of Vancomycin-Resistant Enterococci-Specific Bacteriophages in the Galleria mellonella Wax Moth Larvae Model.

Phages are naturally occurring viruses that selectively kill bacterial species without disturbing the individual’s normal flora, averting the collateral damage of antimicrobial usage. The safety and the effectiveness of phages have been mainly confirmed in the food industry as well as in animal models. In this study, we report on the successful isolation of phages specific to Vancomycin-resistant Enterococci, including Enterococcus faecium (VREfm) and Enterococcus faecalis from sewage samples, and demonstrate their efficacy and safety for VREfm infection in the greater wax moth Galleria mellonella model. No virulence-associated genes, antibiotic resistance genes or integrases were detected in the phages’ genomes, rendering them safe to be used in an in vivo model. Phages may be considered as potential agents for therapy for bacterial infections secondary to multidrug-resistant organisms such as VREfm.

Connections between Exoproteome Heterogeneity and Virulence in the Oral Pathogen Aggregatibacter actinomycetemcomitans.

ABSTRACTAggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen.IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease.

Antibacterial Activity of the Novel Drug Gepotidacin against Stenotrophomonas maltophilia-An In Vitro and In Vivo Study.

Stenotrophomonas maltophilia is increasingly recognized as a nosocomial bacterial pathogen with a multi-drug resistance profile. In this study, the novel drug gepotidacin, the first compound of the novel triazaacenaphthylene topoisomerase inhibitor antibiotics class, was evaluated on its activity against clinical S. maltophilia isolates. Ninety-nine S. maltophilia isolates plus reference strain K279a (N = 100) were tested on their susceptibility towards gepotidacin in a broth microdilution. Additional susceptibility testing was performed towards the commonly applied combination trimethoprim/sulfamethoxazole (TMP/SXT), moxifloxacin, and levofloxacin. The time–kill kinetic of gepotidacin was observed in a time–kill assay. The greater wax moth Galleria mellonella was used to determine the activity of gepotidacin against S. maltophilia in vivo. Gepotidacin showed minimum inhibitory concentrations (MICs) between 0.25 and 16 mg/L (MIC50: 2 mg/L; MIC90: 8 mg/L), independently of its susceptibility towards TMP/SXT. The five TMP/SXT resistant strains exhibited gepotidacin MICs from 1 to 4 mg/L. The S. maltophilia strains resistant to the assessed fluoroquinolones showed in parts high MICs of gepotidacin. The time–kill assay revealed a time- and strain-dependent killing effect of gepotidacin. In vivo, injection of gepotidacin increased the survival rate of the larvae from 61 % to 90 % after 2 days. This study showed antimicrobial effects of gepotidacin towards S. maltophilia.

An engineered protein-based submicromolar competitive inhibitor of the Staphylococcus aureus virulence factor aureolysin

Aureolysin, a secreted metallopeptidase (MP) from the thermolysin family, functions as a major virulence factor in Staphylococcus aureus. No specific aureolysin inhibitors have yet been described, making this an important target for the development of novel antimicrobial drugs in times of rampant antibiotic resistance. Although small-molecule inhibitors are currently more common in the clinic, therapeutic proteins and peptides (TPs) are favourable due to their high selectivity, which reduces off-target toxicity and allows dosage tuning. The greater wax moth Galleria mellonella produces a unique defensive protein known as the insect metallopeptidase inhibitor (IMPI), which selectively inhibits some thermolysins from pathogenic bacteria. We determined the ability of IMPI to inhibit aureolysin in vitro and used crystal structures to ascertain its mechanism of action. This revealed that IMPI uses the “standard mechanism”, which has been poorly characterised for MPs in general. Accordingly, we designed a cohort of 12 single and multiple point mutants, the best of which (I57F) inhibited aureolysin with an estimated inhibition constant (Ki) of 346 nM. Given that animals lack thermolysins, our strategy may facilitate the development of safe TPs against staphylococcal infections, including strains resistant to conventional antibiotics.