Mellonella Larvae Model Research Articles

Doxifluridine effectively kills antibiotic-resistant Staphylococcus aureus in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality globally, often exacerbated by infections such as methicillin-resistant Staphylococcus aureus (MRSA). The rise in antibiotic-resistant strains complicates treatment and underscores the need for novel therapeutic drugs. In this paper, we further investigated the antimicrobial potential of a fluoropyrimidine anticancer drug doxifluridine against multidrug-resistant S. aureus. Determination of minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC), monitoring of growth curve, time-kill assays, biofilm bactericidal assays, and chequerboard studies were conducted to evaluate the antibacterial efficacy of doxifluridine. Safety was assessed via hemolysis and cytotoxicity assays, and an in vivo Galleria mellonella larvae model was employed to test protective effects. Doxifluridine demonstrated significant antibacterial activity against clinical multidrug resistance (MDR) S. aureus isolates, with MIC and MBC values ranging from 0.5 to 2 µg/mL and 1 to 4 µg/mL, respectively. The results revealed doxifluridine's potent bactericidal effects within 8 hours. Moreover, doxifluridine-treated bacteria showed a substantial reduction in biofilm mass and viability. Furthermore, chequerboard assays indicated synergistic interactions between doxifluridine and other antibiotics, reducing MIC values by two- to eightfold. More importantly, safety evaluations confirmed that doxifluridine did not exhibit hemolytic toxicity or cytotoxicity. Finally, doxifluridine significantly increased the survival rate of MRSA-infected G. mellonella larvae in vivo. In brief, doxifluridine exhibited promising in vitro and in vivo antibacterial activity against MRSA, suggesting its potential as a repurposed drug for treating resistant bacterial infections in COPD patients.IMPORTANCEThe study provides robust evidence for the antibacterial efficacy of doxifluridine against Methicillin-resistant Staphylococcus aureus in chronic obstructive pulmonary disease (COPD) patients. Its rapid action, ability to disrupt biofilms, and synergistic effects with other antibiotics, combined with a favorable safety profile, highlight its potential as a repurposed therapeutic agent. Future clinical trials will be essential to confirm these findings and pave the way for its integration into clinical practice. This work not only provides candidate for tackling the management of bacterial infections in COPD but also exemplifies the potential of drug repurposing in combating antibiotic-resistant infections.

Exploring the presence, genomic traits, and pathogenic potential of extended-spectrum β-lactamase Escherichia coli in freshwater, wastewater, and hospital effluents.

The purpose of this work was to study extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) in freshwaters, hospital effluents, and wastewaters during two sampling campaigns in 2021. Water sampling was performed at 24 stations in the Ourthe watershed in Belgium. A total of 644 ESBL (n=642) and AmpC (n=2) E. coli strains were isolated. Disk-diffusion assays were performed following the EUCAST's recommendations. All strains were tested for the presence of blaCTX-M-1, blaCTX-M-2, and blaCTX-M-9 gene groups by PCR. Genes belonging to blaCTX-M-1 and blaCTX-M-9 groups were detected, respectively, in 73.6% and 14.9% of the strains. No blaCTX-M-2 group's gene was found. A subset of strains (n=40) was selected for whole genome sequencing. Escherichia coli serotype O18: H7 ST 1463 was predominant (n=14) in the sequenced strains and showed pathogenicity in the Galleria mellonella larvae model. β-lactamase genes identified were blaCTX-M (n=21), with blaCTX-M-15 mostly represented (n=15), as well as blaTEM (n=11), blaOXA (n=7), blaSHV (n=9), and carbapenemase (CP) genes were observed in several strains-blaKPC-3 (n=19), blaNDM-1 (n=1), blaVIM-1 (n=2), and blaOXA-244 (n=2)-even from freshwaters. ESBL-EC are widely distributed in the aquatic environment in Belgium and contain a variety of ESBL and CP genes.

K1 capsule-dependent phage-driven evolution in Escherichia coli leading to phage resistance and biofilm production.

Understanding bacterial phage resistance mechanisms has implications for developing phage-based therapies. This study aimed to explore the development of phage resistance in Escherichia coli K1 isolates' to K1-ULINTec4, a K1-dependent bacteriophage. Resistant colonies were isolated from two different strains (APEC 45 and C5), both previously exposed to K1-ULINTec4. Genome analysis and several parameters were assessed, including growth capacity, phage adsorption, phenotypic impact at capsular level, biofilm production, and virulence in the in vivo Galleria mellonella larvae model. One out of the six resistant isolates exhibited a significantly slower growth rate, suggesting the presence of a resistance mechanism altering its fitness. Comparative genomic analysis revealed insertion sequences in the region 2 of the kps gene cluster involved in the capsule biosynthesis. In addition, an immunoassay targeting the K1 capsule showed a very low positive reaction compared to the control. Nevertheless, microscopic images of resistant strains revealed the presence of capsules with a clustered organization of bacterial cells and biofilm assessment showed an increased biofilm production compared to the sensitive strains. In the G. mellonella model, larvae infected with phage-resistant isolates showed better survival rates than larvae infected with phage-sensitive strains. A phage resistance mechanism was identified at the genomic level and had a negative impact on the K1 capsule production. The resistant isolates showed an increased biofilm production and a decreased virulence in vivo.

Inhibition of the blaOXA-48 gene expression in Klebsiella pneumoniae by a plasmid carrying CRISPRi-Cas9 system

Antibiotic resistance is an increasing concern that threatens the effectiveness of treating bacterial infections. The spread of carbapenem resistant Klebsiella pneumoniae poses a significant threat to global public health. To combat this issue, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system is being developed. This system includes a single guide RNA (sgRNA) and a nuclease dead Cas9 (dCas9), which work together to downregulate gene expression. Our project involved the use of the CRISPRi system to reduce gene expression of the beta-lactamase oxacillin-48 (blaOXA-48) gene in K. pneumoniae. We designed a sgRNA and cloned it into pJMP1363 plasmid harboring the CRISPRi system. The pJMP1363-sgRNA construct was transformed in K. pneumoniae harboring the blaOXA-48 gene. The MIC test was used to evaluate the antimicrobial resistance, and quantitative real-time RT-PCR was used to confirm the inhibition of the OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA construct expression. The Galleria mellonella larvae model was also utilized for in vivo assay. Following the transformation, the MIC test indicated a 4-fold reduction in meropenem resistance, and qRT-PCR analysis revealed a 60-fold decrease in the mRNA OXA-48 harboring the pJMP1363-sgRNA construct expression. Additionally, G. mellonella larvae infected with OXA-48 producing K. pneumoniae harboring the pJMP1363-sgRNA showed higher survival rates. Based on the findings, it can be concluded that the CRISPR interference technique has successfully reduced antibiotic resistance and virulence in the K. pneumoniae harboring the blaOXA-48 gene.

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.

Genome sequence of a virulent and hypermucoviscous-like Klebsiella michiganensis clinical isolate

ObjectivesThe hypermucoviscous-like phenotype has been described in Klebsiella pneumoniae species complex (KpSC) and was described as a contributor of increased virulence. This study described the characterization and whole-genome sequencing of an antibiotic susceptible and hypermucoviscous-like Klebsiella michiganensis 9273 clinical isolate.Data descriptionHere, we report the genome sequence of a K. michiganensis clinical isolate obtained from a urinary tract infection exhibiting the hypermucoviscous-like phenotype. The draft genome sequence consisted of 145 contigs and ~ 6.6 Mb genome size. The annotation revealed 6648 coding DNA sequences and 56 tRNA genes. The strain belongs to the sequence type (ST) 50, and the OXY-1 beta-lactam resistance gene, aph(3′)-Ia gene for aminoglycoside resistance and multidrug efflux pumps were identified. The fyuA siderophore receptor of yersiniabactin siderophore was identified. Increased virulence was observed in Galleria mellonella larvae model and increased capsule production was determined by uronic acid quantification. The clinical implications of this phenotype are unknown, but the patient outcome might worsen compared to susceptible- or MDR-classical K. michiganensis isolates.

Enteropathogenic Escherichia coli is a predominant pathotype in healthy pigs in Hubei Province of China.

This study aims to investigate the prevalence of intestinal pathogenic Escherichia coli (InPEC) in healthy pig-related samples and evaluate the potential virulence of the InPEC strains. A multiplex PCR method was established to identify different pathotypes of InPEC. A total of 800 rectal swab samples and 296 pork samples were collected from pig farms and slaughterhouses in Hubei province, China. From these samples, a total of 21 InPEC strains were isolated, including 19 enteropathogenic E. coli (EPEC) and 2 shiga toxin-producing E. coli (STEC) strains. By whole-genome sequencing and in silico typing, it was shown that the sequence types and serotypes were diverse among the strains. Antimicrobial susceptibility assays showed that 90.48% of the strains were multi-drug resistant. The virulence of the strains was first evaluated using the Galleria mellonella larvae model, which showed that most of the strains possessed medium to high pathogenicity. A moderately virulent EPEC isolate was further selected to characterize its pathogenicity using a mouse model, which suggested that it could cause significant diarrhea. Bioluminescence imaging (BLI) was then used to investigate the colonization dynamics of this EPEC isolate, which showed that the EPEC strain could colonize the mouse cecum for up to 5 days.

Emergence of colistin-heteroresistant and carbapenem-resistant hypervirulent Klebsiella pneumoniae

To investigate the clinical emergence of colistin-heteroresistant, hypervirulent, and multidrug-resistant Klebsiella pneumoniae, and characterize the underlying molecular mechanisms. The population analysis profiles (PAPs) method was used to detect colistin heteroresistance. The time-killing assay was used to examine the effect of colistin on carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro. Galleria mellonella larvae infection model was used to test the potential virulence. qRT-PCR assay was conducted to compare the expression levels of efflux pump genes. Next and third-generation sequencing were conducted to analyse the genomic features. Two colistin-heteroresistant isolates were detected from a multi-center carbapenem-resistant Enterobacterales (CRE) surveillance study in China, which exhibited similar survival rates as the K2 hypervirulent reference strain ATCC 43816 in a G. mellonella larvae model. The two isolates belonged to ST11, harbouring the iucABCD, iutA, iroBCD, and rpmA2 hypervirulent genes and pLVPK-like virulence plasmids. Colistin showed a weak effect on the heteroresistant strains in vitro. The efflux pump genes acrA, acrB, tolC, oqxA, and oqxB were upregulated in this subpopulation compared to the parental strains. This study showed the clinical emergence of colistin-heteroresistant, hypervirulent, and multidrug-resistant Klebsiella pneumoniae. AcrAB-TolC and OqxAB efflux overexpression were involved in mediating colistin heteroresistance.

A dextrorotatory residues-incorporated bioactive dodecapeptide against enterohemorrhagic Escherichia coli

Aim: This study aims to report an engineered peptide zp39 with favorable bioactivity against enterohemorrhagic Escherichia coli (E. coli, EHEC). Its antibacterial mechanisms and application in a real food system are assessed. Methods: Spatial conformation of synthetic peptide zp39 (GIIAGIIiKIKk-NH2, lowercase letters indicate dextrorotatory amino acids) was predicted by PEPstrMOD and its secondary structure was further determined by circular dichroism (CD) spectroscopy. Then, standard E. coli O157:H7 strain ATCC 43888 was used to evaluate the bioactivity of zp39. A double dilution method was applied to investigate its efficacy in normal broth medium, serum, and highly saline conditions. Its effects on cell membrane permeability and potential were measured by fluorescent assays. Thereafter, morphological changes of E. coli O157:H7 cells were monitored by electron microscopy technologies. Finally, the potential application of zp39 in controlling EHEC in food was tested with spinach juice and the Galleria mellonella larvae model was employed to assess the in vivo efficacy. Results: Peptide zp39 presented an amphiphilic helical structure. It effectively inhibited the growth of E. coli O157:H7 at a concentration of 4 μmol/L in a bactericidal mode. Mechanistic studies revealed that it affected membrane permeability and potential in a dose-dependent manner. Moreover, zp39 maintained satisfactory bioactivity against E. coli O157:H7 even in the presence of 70% serum or 1,000 μmol/L chloride salts. In spinach juice application, > 90% E. coli O157:H7 cells were killed within 2 h after exposure to 64 μmol/L zp39. In vivo study proved that treatment with 64 μmol/L zp39 could effectively boost the survival ratio of infected larvae by 50%. Conclusions: This study depicts a synthetic dodecapeptide that shows the potential application in controlling EHEC. This molecule may be developed into a highly effective antimicrobial agent applied to prevent food contamination and associated infections.

In Vitro Effect on Piglet Gut Microbiota and In Vivo Assessment of Newly Isolated Bacteriophages against F18 Enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) causing post-weaning diarrhea (PWD) in piglets have a detrimental impact on animal health and economy in pig production. ETEC strains can adhere to the host's small intestinal epithelial cells using fimbriae such as F4 and F18. Phage therapy could represent an interesting alternative to antimicrobial resistance against ETEC infections. In this study, four bacteriophages, named vB_EcoS_ULIM2, vB_EcoM_ULIM3, vB_EcoM_ULIM8 and vB_EcoM_ULIM9, were isolated against an O8:F18 E. coli strain (A-I-210) and selected based on their host range. These phages were characterized in vitro, showing a lytic activity over a pH (4-10) and temperature (25-45 °C) range. According to genomic analysis, these bacteriophages belong to the Caudoviricetes class. No gene related to lysogeny was identified. The in vivo Galleria mellonella larvae model suggested the therapeutic potential of one selected phage, vB_EcoS_ULIM2, with a statistically significant increase in survival compared to non-treated larvae. To assess the effect of this phage on the piglet gut microbiota, vB_EcoS_ULIM2 was inoculated in a static model simulating the piglet intestinal microbial ecosystem for 72 h. This study shows that this phage replicates efficiently both in vitro and in vivo in a Galleria mellonella model and reveals the safety of the phage-based treatment on the piglet microbiota.

Staphylococcus aureus antibiofilm agents from Combretum species (Combretaceae) by metabolomics profiling

About 80 % of all chronic bacterial infections are related to biofilms. Among several bacteria capable of developing biofilms, Staphylococcus aureus and Pseudomonas aeruginosa are recognized as two major opportunistic pathogens associated with biofilm infections, especially those related to medical devices. In light of the exciting progress of metabolomics within the field of natural products, and given the pressing need for new alternatives to control bacterial pathogens, this study applied metabolomic profiling and statistical analyses to identify S. aureus or P. aeruginosa biofilm inhibitors from three Brazilian Combretum species. The extracts were obtained from leaves and bark of C. leprosum, C. laxum, and C. lanceolatum, by solvent extraction with hexane (Hx), dichloromethane (Dcm), and hydromethanolic (Me70). Me70 extracts from leaves of all tested species were active against S. aureus biofilm formation (inhibition rate of 92 % for C. lanceolatum, 80 % for C. leprosum, and 65 % for C. laxum), but they did not inhibit bacterial growth. Other extracts from C. leprosum also presented similar results, decreasing S. aureus biofilm formation without affecting cell growth (inhibition of 90 % for bark Me70 extract, 72 % for bark Dcm extract, and 69 % for leaf Hx extract). Dcm extract from C. leprosum leaves decreased biofilm formation in 69 % while having antibacterial activity (85 % inhibition of bacterial growth). None of the extracts were active against P. aeruginosa. Metabolomic analysis by LC-MS followed by statistical evaluation clearly evidenced chemical differences associated with the activity level of the extracts. Univariate analysis by Pearson's r correlation suggested thirty-five metabolites with positive correlation with S. aureus antibiofilm activity, including alkaloids, tannins, phenolic acids, sugars, and glycosylated and non-glycosylated flavonoids. We obtained in vitro confirmation that ellagic acid is a key component for antibiofilm activity by Combretum extracts against S. aureus, showing circa 70% inhibition. It is likely that ellagic acid prevents bacterial-material surfaces interaction, since it led to increased hydrophobicity in S. aureus surface at the same concentrations that affected biofilm formation (up to 0.1563 µM). Importantly, ellagic acid, and the 4 most active Combretum crude extracts, are not toxic as determined using Galleria mellonella larvae model. The lower selective pressure toward bacterial resistance is an attractive feature of antibiofilm agents compared with antibiotics. Their identification will contribute to the development of novel anti-infective strategies, including less prone adherent surfaces, with a direct impact on the reduction of infections associated with medical devices.

Role of paracoccin on Paracoccidioides brasiliensis virulence and susceptibility to antifungal drugs in the Galleria mellonella larvae model

ABSTRACT Paracoccin (PCN), a Paracoccidioides brasiliensis glycoprotein, has been reported to play roles in fungal biology and paracoccidioidomycosis pathogenesis. Lectin and chitinase domains account for the PCN’s dual roles as an immunomodulatory agent and virulence factor. Soluble PCN injected in P. brasiliensis infected mice, by interacting with TLRs’ N-glycans, drives the host immune response toward a protective Th1 axis. Otherwise, mice infection with yeasts overexpressing PCN (ov-PCN) revealed that PCN acts as a fungal virulence factor, thanks to its chitinase activity on the cell wall, resulting in resistance to phagocytes’ fungicidal activity and development of severe paracoccidioidomycosis. Because antifungal drug administration follows the disease diagnosis, we studied the PCN effect on yeast resistance or susceptibility to antifungal agents. Using a paracoccidioidomycosis model developed in Galleria mellonella larvae, we confirmed the observation, in the murine host, that ov-PCN yeasts display maximum virulence compared to wild-type (wt-PCN) or PCN-silenced (kd-PCN) yeasts. PCN overexpression accounted for the highest susceptibility of P. brasiliensis to antifungal and reduced relative mRNA expression of genes encoding proteins related to cell wall remodeling. The lowest virulence, detected in infection with kd-PCN yeasts, correlated with the lowest susceptibility to antifungals and impact on genes for cell wall remodeling. So, we defined that the grade of endogenous PCN production influences the P. brasiliensis virulence and susceptibility to antifungal drugs, as well as the expression of genes related to cell wall remodeling. We postulate that this variable gene expression is mechanistically associated with P. brasiliensis virulence changes.

Assessment of synergistic activity of rhamnolipid and linezolid against methicillin-resistant Staphylococcus aureus in-vitro and in-vivo with Galleria mellonella larvae model.

Antibiotic resistance, one of the most crucial public health problems, has increased the interest in synergy studies of antibiotics with existing antibiotics and natural compounds to make current treatment more effective in addition to new drug development. In this study, the effectiveness of rhamnolipid and linezolid on the Galleria mellonella larvae model in-vitro and in-vivo against Methicillin-resistant Staphylococcus aureus isolates, which are problematic in treatment, were investigated. Four S.aureus (One ATCC 29213 strain and three methicillin-resistant strains) were used in the study. Two MRSA isolates were resistant to linezolid, and one was susceptible. Partial synergy was observed in one resistant strain, and although no synergy was observed in the other resistant strain, the minimum inhibitory concentration of the resistant strain decreased from 16 to 4μg/mL with a four-fold decrease and reached the susceptibility limit. No change was observed in the MIC of linezolid-susceptible strains. The G.mellonella larval model demonstrated that combined therapy was more effective than monotherapy by survival function tests and CFU determination. RML/LNZ combination improved survival compared to monotherapy and decreased the bacterial burden from 108 to 103.

Correlation Between Drug Resistance and Virulence of Candida Isolates from Patients with Candidiasis.

This article aims to provide a theoretical basis for new or adjuvant strategies to facilitate the early diagnosis and treatment of candidiasis and to determine if drug-resistant Candida would affect virulence. Our strains were collected from patients diagnosed with candidiasis in our hospital. The strains were identified by MALDI-TOF system and ITS sequencing. Antifungal sensitivity testing in vitro was performed to evaluate susceptibility of these isolates to current widely used antifungal drugs. The Galleria mellonella larvae model infected by Candida spp. was used to compare the virulence of drug-resistant and susceptible Candida spp. A total of 206 Candida strains were collected from clinical specimens. Candida albicans was the most common species among them, and was predominantly isolated from male patients aged over 40 years in ICU environments suffering from pulmonary and/or cerebral conditions. The accuracy rate of MALDI TOF-MS identification was 92.72% when compared with ITS sequencing as the standard method. Most Candida species, except for C. tropicalis which showed high resistance to micafungin, showed high susceptibilities to voriconazole, itraconazole, amphotericin B and micafungin but were highly resistant to terbinafine. For each specific Candida species, the G. mellonella larvae model revealed that the virulence of drug-resistant Candida isolates did not markedly differ from that of the drug-susceptible isolates, however, the virulence was dose-dependent on inoculated fungal cells in this model. The possibility of Candida infection should not be neglected in patients at critical care hospital settings and C. albicans is the most common causative agent. MALDI-TOF MS has the advantages of rapidity and high accuracy, and should be a preferred method for identification of Candida spp. in a clinical laboratory. Voriconazole, itraconazole, amphotericin B and micafungin can still be recommended as the first line antifungals to treat candidiasis.

Antimicrobial efficacy of chitosan encapsulated Cecropin- A (1–7)- melittin-cell-penetrating peptide against multi-drug-resistant Salmonella Enteritidis

In wake of antimicrobial resistance (AMR), the present study evaluated the antimicrobial efficacy of chitosan-encapsulated Cecropin-A (1–7)-melittin-cell penetrating peptide (ENC CAMA-CPP) against multi-drug-resistant (MDR) Salmonella Enteritidis. The ENC CAMA-CPP synthesized by ionic gelation technique revealed a particle size of 597.5 ± 13.20 nm and zeta potential of 2.80 ± 0.12 mV. The functional groups including encapsulation confirmation and morphology were evident by Fourier transform infrared spectroscopy and scanning electron microscopy, respectively. On HPLC analysis, an encapsulation efficiency of 75.12 ± 1.52% was observed. Further, a pH-dependent sustained release along with a maximum release of 97% was evident at pH ≥ 8.0 up to 120 h. In vitro, ENC CAMA-CPP was found stable against protease enzymes (trypsin and proteinase K) and biological fluids (simulated gastric fluid and simulated intestinal fluid) and was also tested safe for sheep RBCs, mammalian cells, and beneficial lactobacilli. In the in vitro time-kill assay, ENC CAMA-CPP eliminated intracellular MDR S. Enteritidis at 12 h p.i., while in the in vivo Galleria mellonella larvae model, an improved survival rate, reduced bacterial count, with significant (p < 0.001) immunomodulatory effect and minimal cytotoxicity were evident for ENC CAMA-CPP. Overall, ENC CAMA-CPP appears to be a promising therapeutic candidate against MDR S. Enteritidis and warrants further validation in an appropriate animal model.

Antibiotic combinations reduce Staphylococcus aureus clearance.

The spread of antibiotic resistance is attracting increased attention to combination-based treatments. Although drug combinations have been studied extensively for their effects on bacterial growth1–11, much less is known about their effects on bacterial long-term clearance, especially at cidal, clinically relevant concentrations12–14. Here, using en masse microplating and automated image analysis, we systematically quantify Staphylococcus aureus survival during prolonged exposure to pairwise and higher-order cidal drug combinations. By quantifying growth inhibition, early killing and longer-term population clearance by all pairs of 14 antibiotics, we find that clearance interactions are qualitatively different, often showing reciprocal suppression whereby the efficacy of the drug mixture is weaker than any of the individual drugs alone. Furthermore, in contrast to growth inhibition6–10 and early killing, clearance efficacy decreases rather than increases as more drugs are added. However, specific drugs targeting non-growing persisters15–17 circumvent these suppressive effects. Competition experiments show that reciprocal suppressive drug combinations select against resistance to any of the individual drugs, even counteracting methicillin-resistant Staphylococcus aureus both in vitro and in a Galleria mellonella larva model. As a consequence, adding a β-lactamase inhibitor that is commonly used to potentiate treatment against β-lactam-resistant strains can reduce rather than increase treatment efficacy. Together, these results underscore the importance of systematic mapping the long-term clearance efficacy of drug combinations for designing more-effective, resistance-proof multidrug regimes.

A Study on the Anti-NF-κB, Anti-Candida, and Antioxidant Activities of Two Natural Plant Hormones: Gibberellin A4 and A7.

Introduction: Gibberellins (GA) are terpenoids that serve as important plant hormones by acting as growth and response modulators against injuries and parasitism. In this study, we investigated the in vitro anti-NF-κB, anti-Candida, and antioxidant activity of gibberellin A4 (GA4) and A7 (GA7) compounds, and further determined their toxicity in vivo. Methods: GA4 and GA7 in vitro toxicity was determined by MTT method, and nontoxic concentrations were then tested to evaluate the GA4 and GA7 anti-NF-κB activity in LPS-activated RAW-luc macrophage cell culture (luminescence assay). GA4 in silico anti-NF-κB activity was evaluated by molecular docking with the software “AutoDock Vina”, “MGLTools”, “Pymol”, and “LigPlot+”, based on data obtained from “The Uniprot database”, “Protein Data Bank”, and “PubChem database”. The GA4 and GA7 in vitro anti-Candida effects against Candida albicans (MYA 2876) were determined (MIC and MFC). GA7 was also evaluated regarding the viability of C. albicans preformed biofilm (microplate assay). In vitro antioxidant activity of GA4 and GA7 was evaluated against peroxyl radicals, superoxide anions, hypochlorous acid, and reactive nitrogen species. GA4 and GA7 in vivo toxicity was determined on the invertebrate Galleria mellonella larvae model. Results: Our data show that GA4 at 30 µM is nontoxic and capable of reducing 32% of the NF-κB activation on RAW-luc macrophages in vitro. In vitro results were confirmed via molecular docking assay (in silico), since GA4 presented binding affinity to NF-κB p65 and p50 subunits. GA7 did not present anti-NF-κB effects, but exhibited anti-Candida activity with low MIC (94 mM) and MFC (188 mM) values. GA7 also presented antibiofilm properties at 940 mM concentration. GA4 did not present anti-Candida effects. Moreover, GA4 and GA7 showed antioxidant activity against peroxyl radicals, but did not show scavenging activity against the other tested radicals. Both compounds did not affect the survival of G. mellonella larvae, even at extremely high doses (10 g/Kg). Conclusion: Our study provides preclinical evidence indicating that GA4 and GA7 have a favorable low toxicity profile. The study also points to GA4 and GA7 interference with the NF-κB via, anti-Candida activity, and a peroxyl radical scavenger, which we argue are relevant biological effects.

Iron chelation and inhibition of metallopeptidases mediate anti-Trichomonas vaginalis activity by a novel 8-hydroxyquinoline derivative

Trichomoniasis is a neglected parasitic infection, with no oral therapeutic alternatives to overcome the pitfalls of currently approved drugs. In this context, the search for new anti-Trichomonas vaginalis drugs is imperative. Here we report the selective anti-T. vaginalis activity of a substituted 8-hydroxyquinoline-5-sulfonamide derivative. Six different derivatives were evaluated for anti-T. vaginalis. In vitro and in vivo toxicity methods, association with metal ions, and investigation on the mechanism of action were performed with the most active derivative, PH 152. Cytotoxicity assays showed selectivity for the parasite and the low toxicity was confirmed in G. mellonella larvae model. The mode of action is related to iron chelation by disrupting Fe-S clusters-dependent enzyme activities in the parasite. Proteomic analysis indicated inhibition of metallopeptidases related to T. vaginalis virulence mechanisms and metabolic pathways. PH 152 presented selective trichomonacidal activity through multitarget action.

In Vitro and In Vivo Assessments of Two Newly Isolated Bacteriophages against an ST13 Urinary Tract Infection Klebsiella pneumoniae.

Antibiotic resistance represents a major public health concern requiring new alternatives including phage therapy. Klebsiella pneumoniae belongs to the ESKAPE bacteria and can cause urinary tract infections (UTIs). The aims of this study were to isolate and characterize new bacteriophages against a K. pneumoniae strain isolated from UTIs and to assess their efficacy in vitro and in vivo in a Galleria (G.) mellonella larvae model. For this purpose, two bacteriophages were newly isolated against an ST13 K. pneumoniae strain isolated from a UTI and identified as K3 capsular types by wzi gene PCR. Genomic analysis showed that these bacteriophages, named vB_KpnP_K3-ULINTkp1 and vB_KpnP_K3-ULINTkp2, belong to the Drulisvirus genus. Bacteriophage vB_KpnP_K3-ULINTkp1 had the narrowest host spectrum (targeting only K3), while vB_KpnP_K3-ULINTkp2 also infected other Klebsiella types. Short adsorption times and latent periods were observed for both bacteriophages. In vivo experiments showed their ability to replicate in G. mellonella larvae and to decrease host bacterial titers. Moreover, both bacteriophages improved the survival of the infected larvae. In conclusion, these two bacteriophages had different in vitro properties and showed in vivo efficacy in a G. mellonella model with a better efficiency for vB_KpnP_K3-ULINTkp2.

The comparison of lytic activity of isolated phage and commercial Intesti bacteriophage on ESBL producer E. coli and determination of Ec_P6 phage efficacy with in vivo Galleria mellonella larvae model

Antibiotic resistance is one of the crucial public health challenges. As a result of rising resistance, as an alternative to antimicrobials, demands for bacteriophage therapy have increased significantly over the years. The objective of this study was to isolate and characterize potentially therapeutic phages active against Escherichia coli (E. coli) and compare the efficacy with commercial Intesti bacteriophage on the extended-spectrum beta-lactamase (ESBL) positive E. coli (ESBL-EC) and performed the effectiveness of bacteriophage using the Galleria mellonella (G. mellonella) larvae model. Intesti bacteriophage is a polyvalent bacteriophage-based drug. The isolated bacteriophages were obtained from the river and clinical isolates of E. coli were used for the enrichment of bacteriophage isolation. The phages were first screened based on plaque morphology and host ranges determined on clinical strains. The susceptibility of phages was determined against 50 clinical isolates of E. coli and eight different laboratory isolates using the spot test technique. E. coli lytic phage Ec_P6 was used to determine the therapeutic and preventive effects on the G. mellonella larvae model. The slides were prepared by G. mellonella hemolymph for cytologic examination, stained with May Grünwald Giemsa (MGG), and evaluated by light microscopy. The results of the activities revealed lytic spectra ranging from 24% to 97%. Overall strains were susceptible to one or more phages from the panel. It was proved that Intesti bacteriophage is very effective in a wide variety of strains of E. coli including test strains, also showed that isolated Ec_P6 phage is as effective as commercial phage. The best MOI of this phage was 0.01, and infectivity decreased above 60 °C. The results suggest that phage is stable at pH values ranging between 5.0 and 9.0. In vivo study was found that in E. coli infection to achieve a survival high rate the infected larvae should be after 2 h treated with 0.01 MOI phage (10 μL, 106 PFU/mL) and colistin doses (10 μL, 2.5 mg/kg). It also prevented infection, increasing the survival of the larvae compared to the untreated control group. Ec_P6 phage was found to have a potential for the treatment of E. coli infections.