Mellonella Larvae Research Articles

A potent candicidal peptide designed based on an encrypted peptide from a proteinase inhibitor

Antimicrobial peptides (AMP) represent an alternative in the treatment of fungal infections associated with countless deaths. Here, we report a new AMP, named KWI-19, which was designed based on a peptide encrypted in the sequence of an Inga laurina Kunitz-type inhibitor (ILTI). KWI-19 inhibited the growth of Candida species and acted as a fungicidal agent from 2.5 to 20 μmol L−1, also showing synergistic activity with amphotericin B. Kinetic assays showed that KWI-19 killed Candida tropicalis cells within 60 min. We also report the membrane-associated mechanisms of action of KWI-19 and its interaction with ergosterol. KWI-19 was also characterized as a potent antibiofilm peptide, with activity against C. tropicalis. Finally, non-toxicity was reported against Galleria mellonella larvae, thus strengthening the interest in all the bioactivities mentioned above. This study extends our knowledge on how AMPs can be engineered from peptides encrypted in larger proteins and their potential as candicidal agents.

Repurposing Mitomycin C in Combination with Pentamidine or Gentamicin to Treat Infections with Multi-Drug-Resistant (MDR) Pseudomonas aeruginosa.

The aims of this study were (i) to determine if the combination of mitomycin C with pentamidine or existing antibiotics resulted in enhanced efficacy versus infections with MDR P. aeruginosa in vivo; and (ii) to determine if the doses of mitomycin C and pentamidine in combination can be reduced to levels that are non-toxic in humans but still retain antibacterial activity. Resistant clinical isolates of P. aeruginosa, a mutant strain over-expressing the MexAB-OprM resistance nodulation division (RND) efflux pump and a strain with three RND pumps deleted, were used. MIC assays indicated that all strains were sensitive to mitomycin C, but deletion of three RND pumps resulted in hypersensitivity and over-expression of MexAB-OprM caused some resistance. These results imply that mitomycin C is a substrate of the RND efflux pumps. Mitomycin C monotherapy successfully treated infected Galleria mellonella larvae, albeit at doses too high for human administration. Checkerboard and time-kill assays showed that the combination of mitomycin C with pentamidine, or the antibiotic gentamicin, resulted in synergistic inhibition of most P. aeruginosa strains in vitro. In vivo, administration of a combination therapy of mitomycin C with pentamidine, or gentamicin, to G. mellonella larvae infected with P. aeruginosa resulted in enhanced efficacy compared with monotherapies for the majority of MDR clinical isolates. Notably, the therapeutic benefit conferred by the combination therapy occurred with doses of mitomycin C close to those used in human medicine. Thus, repurposing mitomycin C in combination therapies to target MDR P. aeruginosa infections merits further investigation.

A Preliminary Evaluation on the Antifungal Efficacy of VT-1161 against Persister Candida albicans Cells in Vulvovaginal Candidiasis.

Persister cells are a small fraction of the microbial population that survive lethal concentrations of antimicrobial agents. Candida albicans causes vaginal candidiasis, including recurrent vulvovaginal candidiasis, and may survive common antifungal treatments. The triazole VT-1161 is an antifungal agent that specifically targets fungal CYP51, as opposed to the human CYP enzyme. This work illustrates a new role of VT-1161 in eradicating the biofilm created from the persister cells of a primary biofilm of a clinical vaginal isolate of C. albicans. Antifungal activity was determined by the minimum inhibitory concentration (MIC), and the primary biofilm was treated with amphotericin B to obtain persister cells that were able to form a new biofilm. Results obtained using the new azole VT-1161 showed that VT-1161 not only eradicated a secondary biofilm formed from the persister-derived biofilm and counteracted the adhesion of C. albicans in vitro to human cells but also ameliorated C. albicans-induced infection in vivo in Galleria mellonella larvae, suggesting that it could be proposed as an alternative therapeutic strategy for the treatment of recurrent candidiasis.

NEW EXTRACTION METHOD OF ENTOMOPATHOGENIC NEMATODES USING THE BASIC FLOTAC AND THE MINI-FLOTAC TECHNIQUES

The FLOTAC basic technique is a recognised method used mainly in the veterinary field to extract parasites from the faeces of animals found in the stomach and intestine. This study is the first application of the FLOTAC and Mini-FLOTAC techniques to extract infective juveniles of entomopathogenic nematodes from infested Galleria mellonella. The EPN used for this purpose was an endemic Italian nematode belonging to the Rhabitidae family Steinernema carpocapsae. Galleria mellonella larvae were infected and extraction was performed a few days after infection using both techniques. A comparison was then made between the two methods. The results showed that infective juveniles could be extracted from the infected larva as early as three days after infection. The difference between the FLOTAC basic technique and the Mini-FLOTAC technique was not significant in the extraction of entomopathogenic nematodes. Due to its sensitivity and operating easiness the Mini-FLOTAC technique is a promising method for the extraction of entomopathogenic nematodes. It allows faster extraction of nematodes from the inhabited soil and insect host than the conventional White trap method. Key Words: Microbial biocontrol, Entomopathogenic nematodes, Nematode extraction, FLOTAC, Mini-FLOTAC

The chronic wound milieu changes essential oils' antibiofilm activity—an in vitro and larval model study

Essential Oils (EOs) are currently being researched as potential antibiofilm agents to combat infections related to chronic wound biofilms. As documented in the literature, EOs’ in vitro antibacterial properties are often assessed using standard microbiological media and conditions that do not accurately reflect the actual environment of a chronic wound. To address this issue, In vitro Wound Milieu (IVWM) medium, which closely resembles the environment of a chronic wound, was applied for culturing S. aureus biofilms (n = 12) in this research. Biofilms cultivated in the standard Tryptic Soy Broth (TSB) medium served as a control for the experiment. Key biofilm features were analyzed and compared. Subsequently, staphylococci were exposed to the activity of thyme or rosemary EOs (T-EO and R-EO, respectively). As proof of concept, the cytotoxicity of T-EO and its antimicrobial in vivo activity were assessed using a G. mellonella larvae model. Key features of biofilm-forming cells were lower in the IVWM than in the TSB medium: biomass (up to 8 times), metabolic activity (up to 9 times), cell number (up to 100 times), and the live/dead cells ratio. Conversely, biofilm thickness was higher (up to 25%) in IVWM. These differences translated into varied responses of the biofilms to EOs exposure. The application of T-EO led to a greater reduction (up to 2 times) in 67% of biofilm-forming strains in IVWM compared to the TSB medium. Conversely, exposure to R-EO resulted in a higher reduction (up to 2.6 times) of 83% of biofilm-forming strains in TSB than in IVWM. The application of T-EO was not only non-toxic to G. mellonella larvae but also increased the survival of larvae infected with staphylococci (from 48 to 85%). Our findings suggest that EOs not only show promise as agents for treating biofilm-related wound infections but also that providing conditions reflecting the specific niche of the human body is of paramount importance in influencing the results obtained. However, before clinical application, challenges related to the methods of assessing their activity, microbial intra-species variability, and different levels of activity of various EOs should be analyzed and standardized.

Abietic Acid as a Novel Agent against Ocular Biofilms: An In Vitro and Preliminary In Vivo Investigation.

Biofilm-related ocular infections can lead to vision loss and are difficult to treat with antibiotics due to challenges with application and increasing microbial resistance. In turn, the design and testing of new synthetic drugs is a time- and cost-consuming process. Therefore, in this work, for the first time, we assessed the in vitro efficacy of the plant-based abietic acid molecule, both alone and when introduced to a polymeric cellulose carrier, against biofilms formed by Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans in standard laboratory settings as well as in a self-designed setting using the topologically challenging surface of the artificial eye. These analyses were performed using the standard microdilution method, the biofilm-oriented antiseptic test (BOAT), a modified disk-diffusion method, and eyeball models. Additionally, we assessed the cytotoxicity of abietic acid against eukaryotic cell lines and its anti-staphylococcal efficacy in an in vivo model using Galleria mellonella larvae. We found that abietic acid was more effective against Staphylococcus than Pseudomonas (from two to four times, depending on the test applied) and that it was generally more effective against the tested bacteria (up to four times) than against the fungus C. albicans at concentrations non-cytotoxic to the eukaryotic cell lines and to G. mellonella (256 and 512 µg/mL, respectively). In the in vivo infection model, abietic acid effectively prevented the spread of staphylococcus throughout the larvae organisms, decreasing their lethality by up to 50%. These initial results obtained indicate promising features of abietic acid, which may potentially be applied to treat ocular infections caused by pathogenic biofilms, with higher efficiency manifested against bacterial than fungal biofilms.

Genomic analysis of vB_PaS-HSN4 bacteriophage and its antibacterial activity (in vivo and in vitro) against Pseudomonas aeruginosa isolated from burn

The most frequent infections caused by Pseudomonas aeruginosa are local infections in soft tissues, including burns. Today, phage use is considered a suitable alternative to cure infections caused by multi-drug-resistant (MDR) and extensively drug-resistant (XDR) bacteria. We investigated the potential of a novel phage (vB_PaS-HSN4) belonging to Caudoviricetes class, against XDR and MDR P. aeruginosa strains in vivo and in vitro. Its biological and genetic characteristics were investigated. The phage burst size and latent were 119 and 20 min, respectively. It could tolerate a broad range of salt concentrations, pH values, and temperatures. The combination with ciprofloxacin significantly enhanced biofilm removal after 24 h. The genome was dsDNA with a size of 44,534 bp and encoded 61 ORFs with 3 tRNA and 5 promoters. No virulence factor was observed in the phage genome. In the in vivo infection model, treatment with vB_PaS-HSN4 increased Galleria mellonella larvae survival (80%, 66%, and 60%) (MOI 100) and (60%, 40%, and 26%) (MOI 1) in the pre-treatment, co-treatment, and post-treatment experiments, respectively. Based on these characteristics, it can be considered for the cure of infections of burns caused by P. aeruginosa.

Identification of Virulence Factors in Isolates of Candida haemulonii, Candida albicans and Clavispora lusitaniae with Low Susceptibility and Resistance to Fluconazole and Amphotericin B.

Emerging life-threatening multidrug-resistant (MDR) species such as the C. haemulonii species complex, Clavispora lusitaniae (sin. C. lusitaniae), and other Candida species are considered as an increasing risk for human health in the near future. (1) Background: Many studies have emphasized that the increase in drug resistance can be associated with several virulence factors in Candida and its knowledge is also essential in developing new antifungal strategies. (2) Methods: Hydrophobicity, adherence, biofilm formation, lipase activity, resistance to osmotic stress, and virulence 'in vivo' on G. mellonella larvae were studied in isolates of C. haemulonii, C. albicans, and C. lusitaniae with low susceptibility and resistance to fluconazole and amphotericin B. (3) Results: Intra- and interspecies variability were observed. C. haemulonii showed high hydrophobicity and the ability to adhere to and form biofilm. C. lusitaniae was less hydrophobic, was biofilm-formation-strain-dependent, and did not show lipase activity. Larvae inoculated with C. albicans isolates displayed significantly higher mortality rates than those infected with C. haemulonii and C. lusitaniae. (4) Conclusions: The ability to adhere to and form biofilms associated with their hydrophobic capacity, to adapt to stress, and to infect within an in vivo model, observed in these non-wild-type Candida and Clavispora isolates, shows their marked virulence features. Since factors that define virulence are related to the development of the resistance of these fungi to the few antifungals available for clinical use, differences in the physiology of these cells must be considered to develop new antifungal therapies.

The active ingredients in Chinese peony pods synergize with antibiotics to inhibit MRSA growth and biofilm formation

Staphylococcus aureus (S. aureus) is a zoonotic pathogen that infects both humans and animals. The rapid spread of methicillin-resistant S. aureus (MRSA) and its resistance to antibiotics, along with its ability to form biofilms, poses a serious challenge to the clinical application of traditional antibiotics. Peony (Paeonia lactiflora Pall.) is a traditional Chinese medicine with multiple pharmacological effects. This study observed the strong antibacterial and antibiofilm activity of the water extract (WE) and ethyl acetate extract (EA) of Chinese peony pods against MRSA. The combination of EA and vancomycin, cefotaxime, penicillin G or methicillin showed a synergistic or additive antibacterial and antibiofilm effects on MRSA, which is closely related to the interaction of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PG) and methyl gallate (MG). The active ingredients in peony pods have been found to increase the sensitivity of MRSA to antibiotics and demonstrate antibiofilm activity, which is mainly related to the down-regulation of global regulatory factors sarA and sigB, extracellular PIA and eDNA encoding genes icaA and cdiA, quorum sensing related genes agrA, luxS, rnaIII, hld, biofilm virulence genes psma and sspA, and genes encoding clotting factors coa and vwb, but is not related to genes that inhibit cell wall anchoring. In vivo test showed that both WE and EA were non-toxic and significantly prolonged the lifespan of G. mellonella larvae infected with MRSA. This study provides a theoretical basis for further exploration of the combined use of PG, MG and antibiotics to combat MRSA infections.

Development and characterization of potential larvicidal nanoemulsions against Aedes aegypti.

Plant-based insecticides offer advantages such as negligible residual effects, reduced risks to both humans and the environment, and immunity to resistance issues that plague conventional chemicals. However, the practical use of monoterpenes in insect control has been hampered by challenges including their poor solubility and stability in aqueous environments. In recent years, the application of nanotechnology-based formulations, specifically nanoemulsions, has emerged as a prospective strategy to surmount these obstacles. In this study, we developed and characterized nanoemulsions based on cymene and myrcene and assessed their toxicity both in vitro using human keratinocytes (HaCAT) cells and in an in vivo model involving Galleria mellonella larvae. Additionally, we investigated the insecticidal efficacy of monoterpenes against the mosquito Aedes aegypti, the primary dengue vector, via larval bioassay. Employing a low-energy approach, we successfully generated nanoemulsions. The cymene-based nanoemulsion exhibited a hydrodynamic diameter of approximately 98 nm and a zeta potential of -25 mV. The myrcene-based nanoemulsion displayed a hydrodynamic diameter of 118 nm and a zeta potential of -20 mV. Notably, both nanoemulsions demonstrated stability over 60 days, accompanied by controlled release properties and low toxicity towards HaCAT cells and Galleria mellonella larvae. Moreover, the nanoemulsions exhibited significant lethality against third-instar Aedes aegypti larvae at a concentration of 50 mg/L. In conclusion, the utilization of nanoemulsions encapsulating cymene and myrcene presents a promising avenue for overcoming the limitations associated with poor solubility and stability of monoterpenes. This study sheds light on the potential of the nanoemulsions as effective and environmentally friendly insecticides in the ongoing battle against mosquito-borne diseases.

Emergence of an ST1934:KL121 hypervirulent Klebsiella pneumoniae carrying a novel virulence-resistance hybrid plasmid with chromosomal integration of ICEKp1.

To identify the phenotypic and genomic characteristics of K. pneumoniae KP43 from bloodstream infection. KP43 was resistant to ticarcillin and tetracycline and was hypervirulent in the Galleria mellonella larvae infection model, positive for string test, and possessed high-level macrophage killing resistance. The hypervirulence phenotype was associated with the chromosome integration of ICEKp1 carrying iroBCDN-iroP, rmpADC, and peg-344, and a novel plasmid pKP43_vir_amr harboring iutAiucABCD. pKP43_vir_amr was an IncFIBκ/FII virulence-resistance hybrid conjugative plasmid which also carried antibiotic resistance genes. The emergence of such a strain and the spread of the novel virulence-resistance plasmid might pose a potential threat to public health.

Evaluation of Antibacterial and Antibiofilm Activity of Rice Husk Extract against Staphylococcus aureus.

Infections caused by Staphylococcus aureus are particularly difficult to treat due to the high rate of antibiotic resistance. S. aureus also forms biofilms that reduce the effects of antibiotics and disinfectants. Therefore, new therapeutic approaches are increasingly required. In this scenario, plant waste products represent a source of bioactive molecules. In this study, we evaluated the antimicrobial and antibiofilm activity of the rice husk extract (RHE) on S. aureus clinical isolates. In a biofilm inhibition assay, high concentrations of RHE counteracted the formation of biofilm by S. aureus isolates, both methicillin-resistant (MRSA) and -sensitive (MSSA). The observation of the MRSA biofilm by confocal laser scanning microscopy using live/dead cell viability staining confirmed that the bacterial viability in the RHE-treated biofilm was reduced. However, the extract showed no or little biofilm disaggregation ability. An additive effect was observed when treating S. aureus with a combination of RHE and oxacillin/cefoxitin. In Galleria mellonella larvae treated with RHE, the extract showed no toxicity even at high concentrations. Our results support that the rice husk has antimicrobial and antibiofilm properties and could potentially be used in the future in topical solutions or on medical devices to prevent biofilm formation.

Novel berberine derivatives as adjuvants in the battle against Acinetobacter baumannii: A promising strategy for combating multi-drug resistance

The development of resistance against most of the available antibiotics has made Acinetobacter baumannii (A. baumannii) a pathogen of high risk. In this study, thirty novel berberine derivatives are rationally designed, synthesized, and evaluated for their synergistic antibacterial activities against A. baumannii. Among them, compound 2d shows the most potent synergetic effect to aztreonam against A. baumannii, including carbapenem-resistant and extended-spectrum β-lactamases-producing strains. Moreover, synergistic effects were observed for the combinations of 2d and different antibacterial used in clinical practices, indicating its potent broad-spectrum antibiotic-sensitizing effects against A. baumannii. The combination of 2d and aztreonam significantly improves the survival rates of G. mellonella larvae compared with aztreonam treatment alone. Mechanism studies indicate that 2d inhibits the drug efflux and iron acquisition of the bacteria by targeting the AdeB transporter protein, thus achieving a synergistic antimicrobial efficacy with different antibacterial agents. Therefore, berberine derivatives represent a new family of antimicrobial adjuvants against A. baumannii, with the advantage of dual-function antibacterial effect, and are worthy of further investigation.

Neem-hypericum-bacterial cellulose wound care paste characterized in vitro and in Galleria mellonella in vivo model

Surgical wound infections represent a global health emergency because of antibiotic-resistant bacteria. Commensal and pathogenic bacteria can grow in the form of biofilms on damaged skin, and often exhibit an antibiotic-resistance profile. For this reason, the development of new strategies is necessary for the management of skin wounds, which are based not only on antimicrobial activity but also on the inhibition of bacterial biofilm formation or biofilm removal. In this study, we show the development and the biofilm-detaching properties of a novel bacterial cellulose formulation for wound healing. This paste is made by mixing bacterial cellulose pulp with neem-hypericum oil/water emulsion. Once its biocompatibility on murine fibroblasts has been determined, its efficacy was evaluated in vitro against Pseudomonas aeruginosa and Staphylococcus aureus biofilms and in vivo by using Galleria mellonella larvae as a burn wound infection model. Unlike pure BC, the paste showed promising biofilm detachment properties in vitro and a good biocompatibility activity in vivo, setting the stage for developing new strategies for the treatment of chronic wound infections.

Bbhox2 is a key regulator for conidiation and virulence in Beauveria bassiana

Beauveria bassiana, a well-known filamentous biocontrol fungus, is the main pathogen of numerous field and forest pests. To explore the potential factors involved in the fungal pathogenicity, Bbhox2, an important and conserved functional transcription factor containing homeodomain was carried out by functional analysis. Homologous recombination was used to disrupt the Bbhox2 gene in B.bassiana. The conidia yield of the deletant fungal strain was significantly reduced. The conidial germination was faster, and stress tolerance to Congo red and high osmotic agents were decreased compared with that in the wildtype. Additionally, ΔBbhox2 showed a dramatic reduction in virulence no matter in topical inoculations or in intra-hemolymph injections against Galleria mellonella larvae, which is likely due to the failure of appressorium formation and the defect in producing hyphal body. These results indicate that the Bbhox2 gene markedly contributes to conidiation and pathogenicity in B. bassiana.

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.

Entomopathogenic pseudomonads can share an insect host with entomopathogenic nematodes and their mutualistic bacteria.

A promising strategy to overcome limitations in biological control of insect pests is the combined application of entomopathogenic pseudomonads (EPPs) and nematodes (EPNs) associated with mutualistic bacteria (NABs). Yet, little is known about interspecies interactions such as competition, coexistence, or even cooperation between these entomopathogens when they infect the same insect host. We investigated the dynamics of bacteria-bacteria interactions between the EPP Pseudomonas protegens CHA0 and the NAB Xenorhabdus bovienii SM5 isolated from the EPN Steinernema feltiae RS5. Bacterial populations were assessed over time in experimental systems of increasing complexity. In vitro, SM5 was outcompeted when CHA0 reached a certain cell density, resulting in the collapse of the SM5 population. In contrast, both bacteria were able to coexist upon haemolymph-injection into Galleria mellonella larvae, as found for three further EPP-NAB combinations. Finally, both bacteria were administered by natural infection routes i.e. orally for CHA0 and nematode-vectored for SM5 resulting in the addition of RS5 to the system. This did not alter bacterial coexistence nor did the presence of the EPP affect nematode reproductive success or progeny virulence. CHA0 benefited from RS5, probably by exploiting access routes formed by the nematodes penetrating the larval gut epithelium. Our results indicate that EPPs are able to share an insect host with EPNs and their mutualistic bacteria without major negative effects on the reproduction of any of the three entomopathogens or the fitness of the nematodes. This suggests that their combination is a promising strategy for biological insect pest control.

Streptococcus suis subtilisin-like serine proteases SspA-1 and SspA-2 interplay with complement C3a and C5a to facilitate bacterial immune evasion and infection

ABSTRACT Streptococcus suis (S. suis), a significant zoonotic bacterial pathogen impacting swine and human, is associated with severe systemic diseases such as streptococcal toxic shock-like syndrome, meningitis, septicaemia, and abrupt fatality. The multifaceted roles of complement components C5a and C3a extend to orchestrating inflammatory cells recruitment, oxidative burst induction, and cytokines release. Despite the pivotal role of subtilisin-like serine proteases in S. suis pathogenicity, their involvement in immune evasion remains underexplored. In the present study, we identify two cell wall-anchored subtilisin-like serine proteases in S. suis, SspA-1 and SspA-2, as binding partners for C3a and C5a. Through Co-Immunoprecipitation, Enzyme-Linked Immunosorbent and Far-Western Blotting Assays, we validate their interactions with the aforementioned components. However, SspA-1 and SspA-2 have no cleavage activity against complement C3a and C5a performed by Cleavage assay. Chemotaxis assays reveal that recombinant SspA-1 and SspA-2 effectively attenuate monocyte chemotaxis towards C3a and C5a. Notably, the ΔsspA-1, ΔsspA-1, and ΔsspA-1/2 mutant strains exhibit compromised survival in blood, and resistance of opsonophagocytosis, alongside impaired survival in blood and in vivo colonization compared to the parental strain SC-19. Critical insights from the murine and Galleria mellonella larva infection models further underscore the significance of sspA-1 in altering mortality rates. Collectively, our findings indicate that SspA-1 and SspA-2 are novel binding proteins for C3a and C5a, thereby shedding light on their pivotal roles in S. suis immune evasion and the pathogenesis.

Bacteriophage-mediated decolonization of Klebsiella pneumoniae in a novel Galleria mellonella gut colonization model with Enterobacteriaceae

Galleria mellonella larvae have emerged as an invertebrate model for investigating bacterial pathogenesis and potential therapies, addressing ethical concerns related to mammalian models. This model has the advantage of having a simple gut microbiome, which is suitable for gut colonization studies. Intestinal colonization by Enterobacteriaceae significantly contributes to the spread of antibiotic resistance. This study aimed to establish a novel Enterobacteriaceae gut colonization larval model and assess its suitability for evaluating distinct antimicrobial efficacies. Larvae were force-fed sequentially with bacterial doses of K. pneumoniae and E. coli at 0, 24, and 48 h, with survival monitoring at 24 h intervals. Bacterial counts were assessed after 48 h and 120 h of force-feeding. Successfully colonized larvae were subjected to one-time force feeding of a bacteriophage cocktail (107 PFU/larvae) or MIC-based meropenem and ciprofloxacin. The colonized bacterial load was quantified by CFU count. Three doses of 106 CFU/larvae resulted in stable gut colonization, independent of the K. pneumoniae or E. coli strain. Compared with the control, force-feeding of the bacteriophage reduced the colonization of the strain Kp 419614 by 5 log10 CFU/larvae, while antibiotic treatment led to a 3 log10 CFU/larval reduction. This novel G. mellonella model provides a valuable alternative for gut colonization studies, facilitating proof-of-concept investigations and potentially reducing or replacing follow-up experiments in vertebrate models.

Structure-Guided Design, Synthesis, and Antivirulence Assessment of Covalent Staphylococcus aureus Sortase A Inhibitors.

Sortase A (SrtA) is a membrane-associated cysteine transpeptidase required for bacterial virulence regulation and anchors surface proteins to cell wall, thereby assisting biofilm formation. SrtA is targeted in antivirulence treatments against Gram-positive bacterial infections. However, the development of potent small-molecule SrtA inhibitors is constrained owing to the limited understanding of the mode of action of inhibitors in the SrtA binding pocket. Herein, we designed and synthesized a novel class of covalent SrtA inhibitors based on the binding mode detailed in the X-ray crystal structure of the ML346/Streptococcus pyogenes SrtA complex. ML346 analog Y40 exhibited 2-fold increased inhibitory activity on Staphylococcus aureus SrtA and showed superior inhibitory effects on biofilm formation in vitro. Y40 protected Galleria mellonella larvae fromS. aureusinfections in vivo while minimally attenuating staphylococcal growth in vitro. Our study indicates that the covalent SrtA inhibitor Y40 is an antivirulence agent that is effective againstS. aureusinfections.