Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line.

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.

Third generation cephalosporin-resistant Klebsiella pneumoniae thriving in patients and in wastewater: what do they have in common?

BACKGROUND: Klebsiella pneumoniae are ubiquitous bacteria and recognized multidrug-resistant opportunistic pathogens that can be released into the environment, mainly through sewage, where they can survive even after wastewater treatment. A major question is if once released into wastewater, the selection of lineages missing clinically-relevant traits may occur. Wastewater (n = 25) and clinical (n = 34) 3rd generation cephalosporin-resistant K. pneumoniae isolates were compared based on phenotypic, genotypic and genomic analyses. RESULTS: Clinical and wastewater isolates were indistinguishable based on phenotypic and genotypic characterization. The analysis of whole genome sequences of 22 isolates showed that antibiotic and metal resistance or virulence genes, were associated with mobile genetic elements, mostly transposons, insertion sequences or integrative and conjugative elements. These features were variable among isolates, according to the respective genetic lineage rather than the origin. CONCLUSIONS: It is suggested that once acquired, clinically relevant features of K. pneumoniae may be preserved in wastewater, even after treatment. This evidence highlights the high capacity of K. pneumoniae for spreading through wastewater, enhancing the risks of transmission back to humans.

Burkholderia cenocepacia BCAM2418-induced antibody inhibits bacterial adhesion, confers protection to infection and enables identification of host glycans as adhesin targets.

Trimeric Autotransporter Adhesins (TAA) found in Gram-negative bacteria play a key role in virulence. This is the case of Burkholderia cepacia complex (Bcc), a group of related bacteria able to cause infections in patients with cystic fibrosis. These bacteria use TAAs, among other virulence factors, to bind to host protein receptors and their carbohydrate ligands. Blocking such contacts is an attractive approach to inhibit Bcc infections. In this study, using an antibody produced against the TAA BCAM2418 from the epidemic strain Burkholderia cenocepacia K56-2, we were able to uncover its roles as an adhesin and the type of host glycan structures that serve as recognition targets. The neutralisation of BCAM2418 was found to cause a reduction in the adhesion of the bacteria to bronchial cells and mucins. Moreover, in vivo studies have shown that the anti-BCAM2418 antibody exerted an inhibitory effect during infection in Galleria mellonella. Finally, inferred by glycan arrays, we were able to predict for the first time, host glycan epitopes for a TAA. We show that BCAM2418 favoured binding to 3'sialyl-3-fucosyllactose, histo-blood group A, α-(1,2)-linked Fuc-containing structures, Lewis structures and GM1 gangliosides. In addition, the glycan microarrays demonstrated similar specificities of Burkholderia species for their most intensely binding carbohydrates.

Cationic lipid-based formulations for encapsulation and delivery of anti-EFG1 2' OMethylRNA oligomer.

The effective protection and delivery of antisense oligomers to its site of action is a challenge without an optimal strategy. Some of the most promising approaches encompass the complexation of nucleic acids, which are anionic, with liposomes of fixed or ionizable cationic charge. Thus, the main purpose of this work was to study the complexation of cationic liposomes with anti-EFG1 2'OMe oligomers and evaluate the complex efficacy to control Candida albicans filamentation in vitro and in vivo using a Galleria mellonella model. To accomplish this, cationic dioleoyl-trimethylammoniumpropane (DOTAP) was mixed with three different neutral lipids dioleoyl-phosphocholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) and monoolein (MO) and used as delivery vectors. Fluorescence Cross Correlation Spectroscopy measurements revealed a high association between antisense oligomers (ASO) and cationic liposomes confirming the formation of lipoplexes. In vitro, all cationic liposome-ASO complexes were able to release the anti-EFG1 2'OMe oligomers and consequently inhibit C. albicans filamentation up to 60% after 72 h. In vivo, from all formulations the DOTAP/DOPC 80/20 ρchg = 3 formulation proved to be the most effective, enhancing the G. mellonella survival by 40% within 48 h and by 25% after 72 h of infection. In this sense, our findings show that DOTAP-based lipoplexes are very good candidates for nano-carriers of anti-EFG1 2'OMe oligomers.

Antisense locked nucleic acid gapmers to control Candida albicans filamentation.

Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNA-ASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2'-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field.

Insights into the Antimicrobial Activities and Metabolomes of Aquimarina (Flavobacteriaceae, Bacteroidetes) Species from the Rare Marine Biosphere.

Two novel natural products, the polyketide cuniculene and the peptide antibiotic aquimarin, were recently discovered from the marine bacterial genus Aquimarina. However, the diversity of the secondary metabolite biosynthetic gene clusters (SM-BGCs) in Aquimarina genomes indicates a far greater biosynthetic potential. In this study, nine representative Aquimarina strains were tested for antimicrobial activity against diverse human-pathogenic and marine microorganisms and subjected to metabolomic and genomic profiling. We found an inhibitory activity of most Aquimarina strains against Candida glabrata and marine Vibrio and Alphaproteobacteria species. Aquimarina sp. Aq135 and Aquimarina muelleri crude extracts showed particularly promising antimicrobial activities, amongst others against methicillin-resistant Staphylococcus aureus. The metabolomic and functional genomic profiles of Aquimarina spp. followed similar patterns and were shaped by phylogeny. SM-BGC and metabolomics networks suggest the presence of novel polyketides and peptides, including cyclic depsipeptide-related compounds. Moreover, exploration of the 'Sponge Microbiome Project' dataset revealed that Aquimarina spp. possess low-abundance distributions worldwide across multiple marine biotopes. Our study emphasizes the relevance of this member of the microbial rare biosphere as a promising source of novel natural products. We predict that future metabologenomics studies of Aquimarina species will expand the spectrum of known secondary metabolites and bioactivities from marine ecosystems.

Marine Sponge and Octocoral-Associated Bacteria Show Versatile Secondary Metabolite Biosynthesis Potential and Antimicrobial Activities against Human Pathogens.

Marine microbiomes are prolific sources of bioactive natural products of potential pharmaceutical value. This study inspected two culture collections comprising 919 host-associated marine bacteria belonging to 55 genera and several thus-far unclassified lineages to identify isolates with potentially rich secondary metabolism and antimicrobial activities. Seventy representative isolates had their genomes mined for secondary metabolite biosynthetic gene clusters (SM-BGCs) and were screened for antimicrobial activities against four pathogenic bacteria and five pathogenic Candida strains. In total, 466 SM-BGCs were identified, with antimicrobial peptide- and polyketide synthase-related SM-BGCs being frequently detected. Only 38 SM-BGCs had similarities greater than 70% to SM-BGCs encoding known compounds, highlighting the potential biosynthetic novelty encoded by these genomes. Cross-streak assays showed that 33 of the 70 genome-sequenced isolates were active against at least one Candida species, while 44 isolates showed activity against at least one bacterial pathogen. Taxon-specific differences in antimicrobial activity among isolates suggested distinct molecules involved in antagonism against bacterial versus Candida pathogens. The here reported culture collections and genome-sequenced isolates constitute a valuable resource of understudied marine bacteria displaying antimicrobial activities and potential for the biosynthesis of novel secondary metabolites, holding promise for a future sustainable production of marine drug leads.

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata.

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.

A Transcriptomics Approach To Unveiling the Mechanisms of In Vitro Evolution towards Fluconazole Resistance of a Candida glabrata Clinical Isolate.

Candida glabrata is an emerging fungal pathogen. Its increased prevalence is associated with its ability to rapidly develop antifungal drug resistance, particularly to azoles. In order to unravel new molecular mechanisms behind azole resistance, a transcriptomics analysis of the evolution of a C. glabrata clinical isolate (isolate 044) from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day), and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. All the evolved strains were found to accumulate lower concentrations of azole drugs than the parental strain, while the ergosterol concentration remained mostly constant. However, only the population displaying resistance to all azoles was found to have a gain-of-function mutation in the C. glabrataPDR1 gene, leading to the upregulation of genes encoding multidrug resistance transporters. Intermediate strains, exhibiting posaconazole/clotrimazole resistance and increased fluconazole/voriconazole MIC levels, were found to display alternative ways to resist azole drugs. Particularly, posaconazole/clotrimazole resistance after 31 days was correlated with increased expression of adhesin genes. This finding led us to identify the Epa3 adhesin as a new determinant of azole resistance. Besides being required for biofilm formation, Epa3 expression was found to decrease the intracellular accumulation of azole antifungal drugs. Altogether, this work provides a glimpse of the transcriptomics evolution of a C. glabrata population toward multiazole resistance, highlighting the multifactorial nature of the acquisition of azole resistance and pointing out a new player in azole resistance.

The mechanism of action of pepR, a viral-derived peptide, against Staphylococcus aureus biofilms.

OBJECTIVES: To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus biofilms. METHODS: Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths. RESULTS: Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified. CONCLUSIONS: Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.

K2 Capsule Depolymerase Is Highly Stable, Is Refractory to Resistance, and Protects Larvae and Mice from Acinetobacter baumannii Sepsis.

Acinetobacter baumannii is emerging as a major nosocomial pathogen in intensive care units. The bacterial capsules are considered major virulence factors, and the particular A. baumannii capsular type K2 has been associated with high antibiotic resistance. In this study, we identified a K2 capsule-specific depolymerase in a bacteriophage tail spike C terminus, a fragment that was heterologously expressed, and its antivirulence properties were assessed by in vivo experiments. The K2 depolymerase is active under a broad range of environmental conditions and is highly thermostable, with a melting point (Tm ) at 67°C. In the caterpillar larva model, the K2 depolymerase protects larvae from bacterial infections, using either pretreatments or with single-enzyme injection after bacterial challenge, in a dose-dependent manner. In a mouse sepsis model, a single K2 depolymerase intraperitoneal injection of 50 µg is able to protect 60% of mice from an otherwise deadly infection, with a significant reduction in the proinflammatory cytokine profile. We showed that the enzyme makes bacterial cells fully susceptible to the host complement system killing effect. Moreover, the K2 depolymerase is highly refractory to resistance development, which makes these bacteriophage-derived capsular depolymerases useful antivirulence agents against multidrug-resistant A. baumannii infections.IMPORTANCEAcinetobacter baumannii is an important nosocomial pathogen resistant to many, and sometimes all, antibiotics. The A. baumannii K2 capsular type has been associated with elevated antibiotic resistance. The capsular depolymerase characterized here fits the new trend of alternative antibacterial agents needed against multidrug-resistant pathogens. They are highly specific, stable, and refractory to resistance, as they do not kill bacteria per se; instead, they remove bacterial surface polysaccharides, which diminish the bacterial virulence and expose them to the host immune system.

Stress Response Protein BolA Influences Fitness and Promotes Salmonella enterica Serovar Typhimurium Virulence.

The intracellular pathogen Salmonella enterica serovar Typhimurium has emerged as a major cause of foodborne illness, representing a severe clinical and economic concern worldwide. The capacity of this pathogen to efficiently infect and survive inside the host depends on its ability to synchronize a complex network of virulence mechanisms. Therefore, the identification of new virulence determinants has become of paramount importance in the search of new targets for drug development. BolA-like proteins are widely conserved in all kingdoms of life. In Escherichia coli, this transcription factor has a critical regulatory role in several mechanisms that are tightly related to bacterial virulence. Therefore, in the present work we used the well-established infection model Galleria mellonella to evaluate the role of BolA protein in S Typhimurium virulence. We have shown that BolA is an important player in S Typhimurium pathogenesis. Specifically, the absence of BolA leads to a defective virulence capacity that is most likely related to the remarkable effect of this protein on S Typhimurium evasion of the cellular response. Furthermore, it was demonstrated that BolA has a critical role in bacterial survival under harsh conditions since BolA conferred protection against acidic and oxidative stress. Hence, we provide evidence that BolA is a determining factor in the ability of Salmonella to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence.IMPORTANCE BolA has been described as an important protein for survival in the late stages of bacterial growth and under harsh environmental conditions. High levels of BolA in stationary phase and under stresses have been connected with a plethora of phenotypes, strongly suggesting its important role as a master regulator. Here, we show that BolA is a determining factor in the ability of Salmonella to survive and overcome host defense mechanisms, and this is an important step in progress to an understanding of the pathways underlying bacterial virulence. This work constitutes a relevant step toward an understanding of the role of BolA protein and may have an important impact on future studies in other organisms. Therefore, this study is of utmost importance for understanding the genetic and molecular bases involved in the regulation of Salmonella virulence and may contribute to future industrial and public health care applications.

Burkholderia cenocepacia K56-2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation.

Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence.

The multidrug resistance transporters CgTpo1_1 and CgTpo1_2 play a role in virulence and biofilm formation in the human pathogen Candida glabrata.

The mechanisms of persistence and virulence associated with Candida glabrata infections are poorly understood, limiting the ability to fight this fungal pathogen. In this study, the multidrug resistance transporters CgTpo1_1 and CgTpo1_2 are shown to play a role in C. glabrata virulence. The survival of the infection model Galleria mellonella, infected with C. glabrata, was found to increase upon the deletion of either CgTPO1_1 or CgTPO1_2. The underlying mechanisms were further explored. In the case of CgTpo1_1, this phenotype was found to be consistent with the observation that it confers resistance to antimicrobial peptides (AMP), such as the human AMP histatin-5. The deletion of CgTPO1_2, on the other hand, was found to limit the survival of C. glabrata cells when exposed to phagocytosis and impair biofilm formation. Interestingly, CgTPO1_2 expression was found to be up-regulated during biofilm formation, but and its deletion leads to a decreased expression of adhesin-encoding genes during biofilm formation, which is consistent with a role in biofilm formation. CgTPO1_2 expression was further seen to decrease plasma membrane potential and affect ergosterol and fatty acid content. Altogether, CgTpo1_1 and CgTpo1_2 appear to play an important role in the virulence of C. glabrata infections, being at the cross-road between multidrug resistance and pathogenesis.

Characterization of BCAM0224, a multifunctional trimeric autotransporter from the human pathogen Burkholderia cenocepacia.

Members of the trimeric autotransporter adhesin (TAA) family play a crucial role in adhesion of Gram-negative pathogens to host cells. Moreover, these proteins are multifunctional virulence factors involved in several other biological traits, including invasion into host cells and evasion of the host immune system. In cystic fibrosis epidemic Burkholderia cenocepacia strain J2315, we identified a unique TAA (BCAM0224)-encoding gene, previously described as being implicated in virulence. Here, we characterized this multifunctional protein, trying to establish its role in B. cenocepacia pathogenicity. We show that BCAM0224 occurs on the bacterial surface and adopts a trimeric conformation. Furthermore, we demonstrated that BCAM0224 is needed for earlier stages of biofilm formation and is required for swarming motility. In addition, BCAM0224 plays an important role in evasion of the human innate immune system, providing resistance against the bactericidal activity of serum via the complement classical pathway. Finally, BCAM0224 mediates bacterial adhesion to and invasion of cultured human bronchial epithelial cells. Together, these data reveal the high versatility of the BCAM0224 protein as a virulence factor in the pathogenic bacterium B. cenocepacia.

Single-molecule atomic force microscopy unravels the binding mechanism of a Burkholderia cenocepacia trimeric autotransporter adhesin.

Trimeric autotransporter adhesins (TAAs) are bacterial surface proteins that fulfil important functions in pathogenic Gram-negative bacteria. Prominent examples of TAAs are found in Burkholderia cepacia complex, a group of bacterial species causing severe infections in patients with cystic fibrosis. While there is strong evidence that Burkholderia cenocepacia TAAs mediate adhesion, aggregation and colonization of the respiratory epithelium, we still know very little about the molecular mechanisms behind these interactions. Here, we use single-molecule atomic force microscopy to unravel the binding mechanism of BCAM0224, a prototype TAA from B. cenocepacia K56-2. We show that the adhesin forms homophilic trans-interactions engaged in bacterial aggregation, and that it behaves as a spring capable to withstand high forces. We also find that BCAM0224 binds collagen, a major extracellular component of host epithelia. Both homophilic and heterophilic interactions display low binding affinity, which could be important for epithelium colonization. We then demonstrate that BCAM0224 recognizes receptors on living pneumocytes, and leads to the formation of membrane tethers that may play a role in promoting adhesion. Collectively, our results show that BCAM0224 is a multifunctional adhesin endowed with remarkable binding properties, which may represent a general mechanism among TAAs for strengthening bacterial adhesion.

The virulence of Salmonella enterica Serovar Typhimurium in the insect model galleria mellonella is impaired by mutations in RNase E and RNase III.

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium able to invade and replicate inside eukaryotic cells. To cope with the host defense mechanisms, the bacterium has to rapidly remodel its transcriptional status. Regulatory RNAs and ribonucleases are the factors that ultimately control the fate of mRNAs and final protein levels in the cell. There is growing evidence of the direct involvement of these factors in bacterial pathogenicity. In this report, we validate the use of a Galleria mellonela model in S. Typhimurium pathogenicity studies through the parallel analysis of a mutant with a mutation in hfq, a well-established Salmonella virulence gene. The results obtained with this mutant are similar to the ones reported in a mouse model. Through the use of this insect model, we demonstrate a role for the main endoribonucleases RNase E and RNase III in Salmonella virulence. These ribonuclease mutants show an attenuated virulence phenotype, impairment in motility, and reduced proliferation inside the host. Interestingly, the two mutants trigger a distinct immune response in the host, and the two mutations seem to have an impact on distinct bacterial functions.

In-Silico Analysis Highlights the Existence in Members of Burkholderia cepacia Complex of a New Class of Adhesins Possessing Collagen-like Domains.

Burkholderia cenocepacia is a multi-drug-resistant lung pathogen. This species synthesizes various virulence factors, among which cell-surface components (adhesins) are critical for establishing the contact with host cells. This work in the first part focuses on the current knowledge about the adhesion molecules described in this species. In the second part, through in silico approaches, we perform a comprehensive analysis of a group of unique bacterial proteins possessing collagen-like domains (CLDs) that are strikingly overrepresented in the Burkholderia species, representing a new putative class of adhesins. We identified 75 CLD-containing proteins in Burkholderia cepacia complex (Bcc) members (Bcc-CLPs). The phylogenetic analysis of Bcc-CLPs revealed the evolution of the core domain denominated "Bacterial collagen-like, middle region". Our analysis remarkably shows that these proteins are formed by extensive sets of compositionally biased residues located within intrinsically disordered regions (IDR). Here, we discuss how IDR functions may increase their efficiency as adhesion factors. Finally, we provided an analysis of a set of five homologs identified in B. cenocepacia J2315. Thus, we propose the existence in Bcc of a new type of adhesion factors distinct from the described collagen-like proteins (CLPs) found in Gram-positive bacteria.

Antibacterial Activity of ZnO Nanoparticles in a Staphylococcus-aureus-Infected Galleria mellonella Model Is Tuned by Different Apple-Derived Phytocargos.

This research investigates pH changes during the green synthesis of ZnO nanoparticles (NPs) and emphasises its importance in their physicochemical, antibacterial, and biological properties. Varying the synthesis pH from 8 to 12 using "Bravo de Esmolfe" apple extracts neither affected the morphology nor crystallinity of ZnO but impacted NP phytochemical loads. This difference is because alkaline hydrolysis of phytochemicals occurred with increasing pH, resulting in BE-ZnO with distinct phytocargos. To determine the toxicity of BE-ZnO NPs, Galleria mellonella was used as an alternative to non-rodent models. These assays showed no adverse effects on larvae up to a concentration of 200 mg/kg and that NPs excess was relieved by faeces and silk fibres. This was evaluated by utilising fluorescence-lifetime imaging microscopy (FLIM) to track NPs' intrinsic fluorescence. The antibacterial efficacy against Staphylococcus aureus was higher for BE-ZnO12 than for BE-ZnO8; however, a different trend was attained in an in vivo infection model. This result may be related to NPs' residence in larvae haemocytes, modulated by their phytocargos. This research demonstrates, for the first time, the potential of green synthesis to modulate the biosafety and antibacterial activity of NPs in an advanced G. mellonella infection model. These findings support future strategies to overcome antimicrobial resistance by utilizing distinct phytocargos to modulate NPs' action over time.