Subinhibitory concentrations of nisin enhance virulence gene expression in Staphylococcus aureus and increase mortality in Galleria mellonella.

Alternative strategies for controlling Staphylococcus aureus and other pathogens have been continuously investigated, with nisin, a bacteriocin widely used in the food industry as a biopreservative, gaining increasing attention. In addition to its antimicrobial properties, bacteriocins have significant effects on genome functionality even at inhibitory concentrations. This study investigated the impact of subinhibitory concentrations of nisin on S. aureus. Culturing in the presence of 0.625 µmol l-1 nisin, led to the increased relative expression of hla, saeR, and sarA, genes associated with virulence while expression of the sea gene, encoding staphylococcal enterotoxin A (SEA), decreased. In an in vivo experiment, Galleria mellonella larvae inoculated with S. aureus cultured in the presence of nisin exhibited 97% mortality at 72 h post-infection, compared to over 40% of larvae mortality in larvae infected with S. aureus. A comprehensive understanding of the effect of nisin on the transcriptional response of virulence genes and the impact of these changes on the virulence of S. aureus can contribute to assessing the application of this bacteriocin in food and medical contexts.

Suppressing the virulence factors of Candida auris with baicalein through multifaceted mechanisms.

Candida auris, a rapidly spreading multi-drug-resistant fungus, is causing lethal infections under certain conditions globally. Baicalin (BE), an active ingredient extracted from the dried root of Scutellaria baicalensis Georgi, exhibits antifungal activity. However, studies have shown the distinctive advantages of Traditional Chinese medicine in combating fungal infections, while the effect of BE, an active ingredient extracted from the dried roots of Scutellaria baicalensis Georgi, on C. auris, remains unknown. Therefore, this study aims to evaluate the potential of BE as an antifungal agent against the emerging multidrug-resistant C. auris. Various assays and models, including microbroth dilution, time growth curve analysis, spot assays, adhesion tests, flocculation test, cell surface hydrophobicity assay, hydrolase activity assays, XTT assay, violet crystal assay, scanning electron microscope (SEM), confocal laser scanning microscope (CLSM), flow cytometry, Live/dead fluorescent staining, reactive oxygen species (ROS), cell wall assay, aggregation assay, porcine skin model, Galleria mellonella larvae (G. mellonella larvae) infection model, and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were utilized to investigate how baicalein suppresses C. auris through possible multifaceted mechanisms. The findings indicate that BE strongly inhibited C. auris growth, adhesion, and biofilm formation. It also effectively reduced drug resistance and aggregation by disrupting the cell membrane and cell wall while reducing colonization and invasion of the host. Transcriptome analysis showed significant modulation in gene expression related to different virulence factors post-BE treatment. In conclusion, BE exhibits significant effectiveness against C. auris, suggesting its potential as a viable treatment option due to its multifaceted suppression mechanisms.

Temperatures above 37°C increase virulence of a convergent Klebsiella pneumoniae sequence type 307 strain.

Background: Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Methods: Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Results: Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. Conclusion: This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.

Azole resistance in Aspergillus flavus and associated fitness cost.

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

Protease S of entomopathogenic bacterium Photorhabdus laumondii: expression, purification and effect on greater wax moth Galleria mellonella.

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

Transfection of entomopathogenic Metarhizium species with a mycovirus confers hypervirulence against two lepidopteran pests.

Although most known viruses infecting fungi pathogenic to higher eukaryotes are asymptomatic or reduce the virulence of their host fungi, those that confer hypervirulence to entomopathogenic fungus still need to be explored. Here, we identified and studied a novel mycovirus in Metarhizium flavoviride, isolated from small brown planthopper (Laodelphax striatellus). Based on molecular analysis, we tentatively designated the mycovirus as Metarhizium flavoviride partitivirus 1 (MfPV1), a species in genus Gammapartitivirus, family Partitiviridae. MfPV1 has two double-stranded RNAs as its genome, 1,775 and 1,575 bp in size respectively, encapsidated in isometric particles. When we transfected commercial strains of Metarhizium anisopliae and Metarhizium pingshaense with MfPV1, conidiation was significantly enhanced (t test; P-value < 0. 01), and the significantly higher mortality rates of the larvae of diamondback moth (Plutella xylostella) and fall armyworm (Spodoptera frugiperda), two important lepidopteran pests were found in virus-transfected strains (ANOVA; P-value < 0.05). Transcriptomic analysis showed that transcript levels of pathogenesis-related genes in MfPV1-infected M. anisopliae were obviously altered, suggesting increased production of metarhizium adhesin-like protein, hydrolyzed protein, and destruxin synthetase. Further studies are required to elucidate the mechanism whereby MfPV1 enhances the expression of pathogenesis-related genes and virulence of Metarhizium to lepidopteran pests. This study presents experimental evidence that the transfection of other entomopathogenic fungal species with a mycovirus can confer significant hypervirulence and provides a good example that mycoviruses could be used as a synergistic agent to enhance the biocontrol activity of entomopathogenic fungi.

Limited impact of bacterial virulence on early mortality risk factors in Acinetobacter baumannii bacteremia observed in a Galleria mellonella model.

Acinetobacter baumannii (AB) has emerged as a major pathogen in vulnerable and severely ill patients. It remains unclear whether early mortality (EM) due to AB bacteremia is because of worse clinical characteristics of the infected patients or the virulence of the pathogen. In this study, we aimed to investigate the effect of AB virulence on EM due to bacteremia. This retrospective study included 138 patients with AB bacteremia (age: ≥ 18 years) who were admitted to a tertiary care teaching hospital in South Korea between 2015 and 2019. EM was defined as death occurring within 7 days of bacteremia onset. The AB clinical isolates obtained from the patients' blood cultures were injected into 15 Galleria mellonella larvae each, which were incubated for 5 days. Clinical isolates were classified into high- and low-virulence groups based on the number of dead larvae. Patients' clinical data were combined and subjected to multivariate Cox regression analyses to identify the risk factors for EM. In total, 48/138 (34.8%) patients died within 7 days of bacteremia onset. The Pitt bacteremia score was the only risk factor associated with EM. In conclusion, AB virulence had no independent effect on EM in patients with AB bacteremia.

Characterization of Aspergillus fumigatus secretome during sublethal infection of Galleria mellonella larvae.

Introduction. The fungal pathogen Aspergillus fumigatus can induce prolonged colonization of the lungs of susceptible patients, resulting in conditions such as allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis.Hypothesis. Analysis of the A. fumigatus secretome released during sub-lethal infection of G. mellonella larvae may give an insight into products released during prolonged human colonisation.Methodology. Galleria mellonella larvae were infected with A. fumigatus, and the metabolism of host carbohydrate and proteins and production of fungal virulence factors were analysed. Label-free qualitative proteomic analysis was performed to identify fungal proteins in larvae at 96 hours post-infection and also to identify changes in the Galleria proteome as a result of infection.Results. Infected larvae demonstrated increasing concentrations of gliotoxin and siderophore and displayed reduced amounts of haemolymph carbohydrate and protein. Fungal proteins (399) were detected by qualitative proteomic analysis in cell-free haemolymph at 96 hours and could be categorized into seven groups, including virulence (n = 25), stress response (n = 34), DNA repair and replication (n = 39), translation (n = 22), metabolism (n = 42), released intracellular (n = 28) and cellular development and cell cycle (n = 53). Analysis of the Gallerial proteome at 96 hours post-infection revealed changes in the abundance of proteins associated with immune function, metabolism, cellular structure, insect development, transcription/translation and detoxification.Conclusion. Characterizing the impact of the fungal secretome on the host may provide an insight into how A. fumigatus damages tissue and suppresses the immune response during long-term pulmonary colonization.

Unraveling the efficacy of verbascoside in thwarting MRSA pathogenicity by targeting sortase A.

In the fight against hospital-acquired infections, the challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) necessitates the development of novel treatment methods. This study focused on undermining the virulence of S. aureus, especially by targeting surface proteins crucial for bacterial adherence and evasion of the immune system. A primary aspect of our approach involves inhibiting sortase A (SrtA), a vital enzyme for attaching microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to the bacterial cell wall, thereby reducing the pathogenicity of S. aureus. Verbascoside, a phenylethanoid glycoside, was found to be an effective SrtA inhibitor in our research. Advanced fluorescence quenching and molecular docking studies revealed a specific interaction between verbascoside and SrtA, pinpointing the critical active sites involved in this interaction. This molecular interaction significantly impedes the SrtA-mediated attachment of MSCRAMMs, resulting in a substantial reduction in bacterial adhesion, invasion, and biofilm formation. The effectiveness of verbascoside has also been demonstrated in vivo, as shown by its considerable protective effects on pneumonia and Galleria mellonella (wax moth) infection models. These findings underscore the potential of verbascoside as a promising component in new antivirulence therapies for S. aureus infections. By targeting crucial virulence factors such as SrtA, agents such as verbascoside constitute a strategic and potent approach for tackling antibiotic resistance worldwide. KEY POINTS: • Verbascoside inhibits SrtA, reducing S. aureus adhesion and biofilm formation. • In vivo studies demonstrated the efficacy of verbascoside against S. aureus infections. • Targeting virulence factors such as SrtA offers new avenues against antibiotic resistance.

Synergistic bactericidal activity of a novel dual ß-lactam combination against methicillin-resistant Staphylococcus aureus.

OBJECTIVES: MRSA is a major cause of hospital-acquired and community-acquired infections. Treatment options for MRSA are limited because of the rapid development of ß-lactam resistance. Combining antibiotics offers an affordable, time-saving, viable and efficient approach for developing novel antimicrobial therapies. Both amoxicillin and cefdinir are oral ß-lactams with indications for a wide range of bacterial infections and mild side effects. This study aimed to investigate the in vitro and in vivo efficacy of combining these two ß-lactams against MRSA strains. METHODS: Fourteen representative prevalent MRSA strains with diverse sequence types (STs) were tested with a combination of amoxicillin and cefdinir, using chequerboard and time-kill assays. The Galleria mellonella larvae infection model was used to evaluate the in vivo efficacy of this dual combination against the community-acquired MRSA (CA-MRSA) strain USA300 and the hospital-acquired MRSA (HA-MRSA) strain COL. RESULTS: The chequerboard assay revealed a synergistic activity of the dual amoxicillin/cefdinir combination against all tested MRSA strains, with fractional inhibitory concentration index (FICI) values below 0.5 and at least a 4-fold reduction in the MICs of both antibiotics. Time-kill assays demonstrated synergistic bactericidal activity of this dual combination against the MRSA strain USA300 and strain COL. Moreover, in vivo studies showed that the administration of amoxicillin/cefdinir combination to G. mellonella larvae infected with MRSA strains significantly improved the survival rate up to 82%, which was comparable to the efficacy of vancomycin. CONCLUSIONS: In vitro and in vivo studies indicate that the dual combination of amoxicillin/cefdinir demonstrates a synergistic bactericidal efficacy against MRSA strains of various STs. Further research is needed to explore its potential as a treatment option for MRSA infections.

Essential roles of Rad6 in conidial property, stress tolerance, and pathogenicity of Beauveria bassiana.

Rad6 functions as a ubiquitin-conjugating protein that regulates cellular processes in many fungal species. However, its role in filamentous entomopathogenic fungi remains poorly understood. This study characterizes Rad6 in Beauveria bassiana, a filamentous fungus widely employed as a critical fungicide globally. The results demonstrate a significant association between Rad6 and conidial properties, heat shock response, and UV-B tolerance. Concurrently, the mutant strain exhibited heightened sensitivity to oxidative stress, cell wall interfering agents, DNA damage stress, and prolonged heat shock. Furthermore, the absence of Rad6 significantly extended the median lethal time (LT50) of Galleria mellonella infected by B. bassiana. This delay could be attributed to reduced Pr1 proteases and extracellular cuticle-degrading enzymes, diminished dimorphic transition rates, and dysregulated antioxidant enzymes. Additionally, the absence of Rad6 had a more pronounced effect on genetic information processing, metabolism, and cellular processes under normal conditions. However, its impact was limited to metabolism in oxidative stress. This study offers a comprehensive understanding of the pivotal roles of Rad6 in conidial and hyphal stress tolerance, environmental adaptation, and the pathogenesis of Beauveria bassiana.

NagZ modulates the virulence of E. cloacae by acting through the gene of unknown function, ECL_03795.

ß-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent ß-lactam resistance in Enterobacter cloacae. However, NagZ's role in the virulence of E. cloacae remains unclear. Our study, incorporating data on mouse and Galleria mellonella larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of E. cloacae following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ's function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.

In Vivo and in Vitro activity of colistin-conjugated bimetallic silver-copper oxide nanoparticles against Pandrug-resistant Pseudomonas aeruginosa.

BACKGROUND: Addressing microbial resistance urgently calls for alternative treatment options. This study investigates the impact of a bimetallic formulation containing colistin, silver, and copper oxide on a pandrug-resistant, highly virulent Pseudomonas aeruginosa (P. aeruginosa) isolate from a cancer patient at the National Cancer Institute, Cairo University, Egypt. METHODS: Silver nanoparticles (Ag NPs), copper oxide nanoparticles (CuO NPs), and bimetallic silver-copper oxide nanoparticles (Ag-CuO NPs) were synthesized using gamma rays, combined with colistin (Col), and characterized by various analytical methods. The antimicrobial activity of Col-Ag NPs, Col-CuO NPs, and bimetallic Col-Ag-CuO NPs against P. aeruginosa was evaluated using the agar well diffusion method, and their minimum inhibitory concentration (MIC) was determined using broth microdilution. Virulence factors such as pyocyanin production, swarming motility, and biofilm formation were assessed before and after treatment with bimetallic Col-Ag-CuO NPs. The in vivo efficacy was evaluated using the Galleria mellonella model, and antibacterial mechanism were examined through membrane leakage assay. RESULTS: The optimal synthesis of Ag NPs occurred at a gamma ray dose of 15.0 kGy, with the highest optical density (OD) of 2.4 at 375 nm. Similarly, CuO NPs had an optimal dose of 15.0 kGy, with an OD of 1.5 at 330 nm. Bimetallic Ag-CuO NPs were most potent at 15.0 kGy, yielding an OD of 1.9 at 425 nm. The MIC of colistin was significantly reduced when combined with nanoparticles: 8 µg/mL for colistin alone, 0.046 µg/mL for Col-Ag NPs, and 0.0117 µg/mL for Col-Ag-CuO NPs. Bimetallic Col-Ag-CuO NPs reduced the MIC four-fold compared to Col-Ag NPs. Increasing the sub-inhibitory concentration of bimetallic nanoparticles from 0.29 × 10-2 to 0.58 × 10-2 µg/mL reduced P. aeruginosa swarming by 32-64% and twitching motility by 34-97%. At these concentrations, pyocyanin production decreased by 39-58%, and biofilm formation was inhibited by 33-48%. The nanoparticles were non-toxic to Galleria mellonella, showing 100% survival by day 3, similar to the saline-treated group. CONCLUSIONS: The synthesis of bimetallic Ag-CuO NPs conjugated with colistin presents a promising alternative treatment for combating the challenging P. aeruginosa pathogen in hospital settings. Further research is needed to explore and elucidate the mechanisms underlying the inhibitory effects of colistin-bimetallic Ag-CuO NPs on microbial persistence and dissemination.

Physiological response of Metarhizium rileyi with linoleic acid supplementation.

Metarhizium rileyi has a broad biocontrol spectrum but is highly sensitive to abiotic factors. A Colombian isolate M. rileyi Nm017 has shown notorious potential against Helicoverpa zea. However, it has a loss of up to 22 % of its conidial germination after drying, which limits its potential as a biocontrol agent and further commercialization. Conidial desiccation resistance can be enhanced by nutritional supplements, which promotes field adaptability and facilitates technological development as a biopesticide. In this study, the effect of culture medium supplemented with linoleic acid on desiccation tolerance in Nm017 conidia was evaluated. Results showed that using a 2 % linoleic acid-supplemented medium increased the relative germination after drying by 41 % compared to the control treatment, without affecting insecticidal activity on H. zea. Also, the fungus increased the synthesis of trehalose, glucose, and erythritol during drying, independently of linoleic acid use. Ultrastructural analyses of the cell wall-membrane showed a loss of thickness by 22 % and 25 %, in samples obtained from 2 % linoleic acid supplementation and the control, respectively. Regarding its morphological characteristics, conidia inner area from both treatments did not change after drying. However, conidia from the control had a 24 % decrease in length/width ratio, whereas there was no alteration in conidia from acid linoleic. The average value of dry conidia elasticity coefficient from linoleic acid treatment was 200 % above the control. Medium supplementation with linoleic acid is a promising fermentation strategy for obtaining more tolerant conidia without affecting production and biocontrol parameters, compatible solutes synthesis, or modifying its cell configuration.

Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus.

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins' involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

The role of a novel secretory peptidoglycan recognition protein with antibacterial ability from the Chinese Oak Silkworm Antheraea pernyi in humoral immunity.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play a critical role in the immune response of invertebrates and vertebrates. Herein, the short ApPGRP-D gene was cloned from the model lepidopteran Antheraea pernyi. Quantitative PCR (qPCR) confirmed that ApPGRP-D is an immune-related protein and that the expression of ApPGRP-D can be induced by microorganisms. ApPGRP-D is a broad-spectrum pattern recognition protein that activates the prophenoloxidase cascade activation system and promotes the agglutination of microbial cells. Likely due to its amidase activity, ApPGRP-D can inhibit the growth of E. coli and S. aureus. In addition, we demonstrated for the first time that zinc ions, as important metal coenzymes, could promote multiple functions of ApPGRP-D but not its amidase activity.

Effect of erythromycin residuals in food on the development of resistance in Streptococcus pneumoniae: an in vivo study in Galleria mellonella.

Background: The use of antimicrobials to treat food animals may result in antimicrobial residues in foodstuffs of animal origin. The European Medicines Association (EMA) and World Health Organization (WHO) define safe antimicrobial concentrations in food based on acceptable daily intakes (ADIs). It is unknown if ADI doses of antimicrobials in food could influence the antimicrobial susceptibility of human-associated bacteria. Objectives: This aim of this study was to evaluate if the consumption of ADI doses of erythromycin could select for erythromycin resistance in a Galleria mellonella model of Streptococcus pneumoniae infection. Methods: A chronic model of S. pneumoniae infection in G. mellonella larvae was used for the experiment. Inoculation of larvae with S. pneumoniae was followed by injections of erythromycin ADI doses (0.0875 and 0.012 µg/ml according to EMA and WHO, respectively). Isolation of S. pneumoniae colonies was then performed on selective agar plates. Minimum inhibitory concentrations (MICs) of resistant colonies were measured, and whole genome sequencing (WGS) was performed followed by variant calling to determine the genetic modifications. Results: Exposure to single doses of both EMA and WHO ADI doses of erythromycin resulted in the emergence of erythromycin resistance in S. pneumoniae. Emergent resistance to erythromycin was associated with a mutation in rplA, which codes for the L1 ribosomal protein and has been linked to macrolide resistance in previous studies. Conclusion: In our in vivo model, even single doses of erythromycin that are classified as acceptable by the WHO and EMA induced significant increases in erythromycin MICs in S. pneumoniae. These results suggest the need to include the induction of antimicrobial resistance (AMR) as a significant criterion for determining ADIs.

A Galleria Model of Cryptococcus neoformans Infection.

Galleria mellonella larvae are a popular and simple model organism for infectious disease research. Last instar larvae can be purchased inexpensively from commercial suppliers and infected with Cryptococcus. Injection into the proleg of larvae results in systemic infections. Larvae may then be monitored for survival or homogenized to determine fungal burden. Fixation of infected larvae produces samples suitable for histological staining and analysis.

Baohuoside I inhibits virulence of multidrug-resistant Staphylococcus aureus by targeting the transcription Staphylococcus accessory regulator factor SarZ.

BACKGROUND: Staphylococcus aureus is a versatile pathogen that can cause a wide range of infections in humans. Biofilms play a crucial role in the pathogenicity of S. aureus and contribute to its ability to cause persistent and chronic infections. Baohuoside I has garnered increasing recognition as a natural flavonol glycoside with a wide spectrum of health-related activities. PURPOSE: The antibacterial and anti-biofilm properties of Baohuoside I have not been extensively investigated. Our study aimed to assess its inhibitory effects and the underlying mechanisms on biofilm formation and hemolytic capacity in S. aureus. STUDY DESIGN/METHODS: The impact of Baohuoside I on the biofilm and virulence of S. aureus was evaluated through in vitro experiments and Galleria mellonella as an in vivo infection model. The mechanisms were explored by Drug affinity responsive target stability (DARTS) and validated in genetic knockout strain and through molecular biological experiments using DARTS, molecular docking, electrophoretic mobility shift assay (EMSA), and bio-layer interferometry (BLI). RESULTS: Baohuoside I significantly inhibits the formation of S. aureus biofilms and hemolytic activity at 6.25 µM. Proteomics analysis revealed that treatment with Baohuoside I led to a reduction in the expression of quorum-sensing system agr-regulated genes. DARTS analysis identified Staphylococcus accessory regulator factor (SarZ), a key regulator involved in the expression of virulence factors in S. aureus by acting as activator of the agr quorum-sensing system, was the direct target of Baohuoside I. Molecular docking, DARTS, BLI and EMSA assays collectively confirmed the direct binding of Baohuoside I to SarZ, inhibiting its binding to downstream promoters. Furthermore, it is found through site-directed protein mutagenesis that the Tyr27 and Phe117 residues are key for Baohuoside I binding to SarZ. Additionally, the knockout of SarZ significantly diminished the hemolytic ability of S. aureus, underscoring its crucial role as a pivotal regulator of virulence. Lastly, in vivo tests utilizing the G. mellonella infection model demonstrated the efficacy of Baohuoside I. CONCLUSION: This study provides valuable insights into the mechanism by which Baohuoside I inhibits the virulence of S. aureus through its interaction with SarZ. These findings highlight the significance of SarZ as an effective target against the virulence of S. aureus.

Synergistic anti-virulence efficacy of citral and carvacrol against mixed vaginitis causing Candida albicans and Gardnerella vaginalis: An in vitro and in vivo study.

Mixed vaginitis due to bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) is the most prevalent form and presents a significant therapeutic challenge globally. Since, the administration of monotherapy leads to subsequent recurrent infections, synergistic therapy that completely eradicates both pathogens is of dire need to manage mixed vaginities scenario and to prevent its recurrence. The current investigation was focused on exploring the synergistic inhibitory efficacy of phytochemicals against the virulence traits of individual and mixed species of C. albicans and G. vaginalis in vitro and in vivo (Galleria mellonella). Out of five phytochemicals (carvacrol, thymol, cinnamaldehyde, eugenol, and borneol) screened for synergism with citral [(Ct) as the prime molecule owing to its myriad therapeutic potential], carvacrol (Ca) in combination with citral exhibited promising synergistic effect. Time-kill kinetics and one-minute contact-killing assays demonstrated the phenomenal microbicidal effect of Ct-Ca combination against both mono and dual-species within 30 min and one-minute time intervals, respectively. Furthermore, the sub-CMICs (synergistic combinatorial MIC) of Ct-Ca have significantly eradicated the mature biofilms and remarkably reduced the virulence attributes of both C. albicans and G. vaginalis (viz., yeast to hyphae transition, filamentation, protease production, and hydrophobicity index), in single and dual species states. The non-toxic nature of Ct-Ca combination was authenticated using in vitro (human erythrocyte cells) and in vivo (Galleria mellonella) models. In addition, the in vivo efficacy evaluation and subsequent histopathological investigation was done using the invertebrate model system G. mellonella, which further ascertained the effectiveness of Ct-Ca combination in fighting off the infection caused by individual and mixed species of C. albicans and G. vaginalis. Concomitantly, the current work is the first of its kind to delineate the in vitro interaction of C. albicans and G. vaginalis mixed species at their growth and biofilm states, together emphasizes the promising therapeutic potential of acclaimed phytochemicals as combinatorial synergistic therapy against mixed vaginitis.