Infection Model Research Articles

A scalable and universal strategy for constructing long-term antibacterial coatings with lubricant property on medical catheters

Medical catheters might cause a high morbidity of catheter-associated urinary tract infections (CAUTIs) because of the bacterial adhesion onto the surface. In addition, the inherent hydrophobicity of catheter materials exacerbates mechanical friction, leading to tissue damage. Therefore, coatings with integrated antibacterial and lubricative properties are appealing approaches to mitigate these challenges. In this study, a kind of polyurethane (PU)-based coatings were developed and prepared on medical catheters, which were modified with a cationic antibacterial agent, quaternized ammonium-modified methyldiethanolamines (QMDEAs). Due to the reaction among castor oil tertiary alcohols, isocyanates and QMDEAs, a polymer cross-linked network was fabricated, in which a hydrophilic polymer, polyvinylpyrrolidone (PVP), was added to form a semi-interpenetrating network. The structures of the coatings were regulated by adjusting the lengths of alkyl chain of QMDEAs, concentrations of pre-coated solutions, ratios of QMDEA:isocyanate, and parameters of preparation process. Among the coatings we prepared, PU-C10–46 possessed the optimized performances, which reduced the dynamic friction coefficient by 91.38 % and had the antibacterial efficiency of 99.9 % with good biocompatibility. The in vivo anti-infective properties of PU-C10–46 were also demonstrated by an infected animal model. Furthermore, PU-C10–46 was produced in an industrial equipment, and the large-scaled industrial products also had the same performances. This work could provide a promising strategy to deal with the challenges of medical catheter-associated infection.

Gelatin/Dopamine/Zinc-Doped Ceria/Curcumin nanocomposite hydrogels for repair of chronic refractory wounds

Chronic wound healing is a common clinical challenge, characterized by bacterial infection, protracted inflammatory response, oxidative stress, and insufficient neovascularization. Nanozymes have emerged as a promising solution for treating skin wounds due to their antioxidant, antibacterial, and angiogenic properties. In recent years, combining nanozymes with hydrogels to jointly promote wound healing has attracted increasing research interest. However, most of the current nanocomposite hydrogels are still not effective in simultaneously controlling inflammatory, oxidative stress and bacterial invasion in wound healing. Improving the therapeutic functional diversity and efficacy of nanocomposite hydrogels remains a problem that needs to be addressed. In this study, we prepared nanocomposite hydrogels (GelMD-Cur@ZHMCe) by combining methylacrylated gelatin modified with dopamine (GelMD) with Zinc-doped hollow mesoporous cerium oxide nanoparticles loaded with curcumin (Cur@ZHMCe). The resulting hydrogels exhibited excellent water absorption, adhesion, and biocompatibility. In vitro and in vivo studies have demonstrated that GelMD-Cur@ZHMCe has excellent antioxidant, antibacterial, anti-inflammatory and vasculature-promoting properties, which enable it to rapidly promote wound repair. The wound healing rate of the rat total skin defect infection model treated with GelMD-Cur@ZHMCe reached 98.5±4.9 % after 14 days of treatment. It was demonstrated that this multifunctional nanocomposite hydrogel provides a promising therapeutic strategy for skin repair.

Antifungal Tetrahydrocarbazole Compound CAR-8 Induces Endoplasmic Reticulum Stress in Candida albicans.

The development of new effective antifungal agents is essential to combat fungal infections. Tetrahydrocarbazole has been exploited as a promising skeleton against various pathogenic microorganisms and is used to search for novel active antifungal compounds. In this study, a library composed of small tetrahydrocarbazole compounds was screened, and a potent antifungal agent, CAR-8, was identified with a minimum inhibitory concentration of 2-4 μg/mL against Candida albicans. CAR-8 showed strong fungicidal activities and killed almost all C. albicans within 3 h at a concentration of 16 μg/mL. At concentrations of 2 and 8 μg/mL, CAR-8 significantly inhibited the formation of hyphae and biofilms. Moreover, CAR-8 at 10 and 20 mg/kg reduced the fungal load and improved the survival in the C. albicans infection model in the invertebrate Galleria mellonella. Transcriptome analysis revealed significant changes in the expression of genes associated with protein processing in the endoplasmic reticulum (ER), ER-associated degradation, and unfolded protein response (UPR), which suggested that CAR-8 treatment induced ER stress. Moreover, CAR-8 treatment resulted in various phenotypes similar to tunicamycin, a classical ER stress inducer. These included nonconventional splicing of HAC1 mRNA, the fragmented morphology of ER, the distribution changes of GFP-Snc1 in Saccharomyces cerevisiae, and cell apoptosis probably caused by ER stress. More importantly, the disruption of IRE1 or HAC1 increased the sensitivity of C. albicans to CAR-8, confirming that the UPR signaling pathway was critical for CAR-8 resistance. Overall, our study identifies a potent ER stress-induced antifungal compound that will help the discovery of new antifungal drugs.

Disruption to de novo uridine biosynthesis alters β-1,3-glucan masking in Candida albicans.

The uridine derivatives UDP-glucose and UDP-N-acetylglucosamine are important for cell wall construction as they are the precursors for the synthesis of β-1,3-glucan and chitin, respectively. Previous studies have demonstrated attenuated virulence of uridine auxotrophs in mice, which has been attributed to insufficient uridine levels for growth in the host. We have discovered that uridine deprivation in the uridine auxotroph ura3ΔΔ disrupts cell wall architecture by increasing surface mannans, exposing β-1,3-glucan and chitin, and decreasing UDP-sugar levels. Cell wall architecture and UDP-sugars can be rescued with uridine supplementation. The cell wall architectural disruptions in the ura3ΔΔ mutant also impact immune activation since the mutant elicited greater TNFα secretion from RAW264.7 macrophages than wild type. To determine if cell wall defects contributed to decreased virulence in the ura3ΔΔ mutant, we used a murine model of systemic infection. Mice infected with the ura3ΔΔ mutant exhibited increased survival and reduced kidney fungal burden compared with mice infected with wild type. However, suppression of the immune response with cyclophosphamide did not rescue virulence in mice infected with the ura3ΔΔ mutant, indicating the attenuation in virulence of uridine auxotrophs can be attributed to decreased growth in the host but not increased exposure of β-1,3-glucan. Moreover, the ura3ΔΔ mutant is unable to grow on ex vivo kidney agar, which demonstrates its inability to colonize the kidneys due to poor growth. Thus, although uridine auxotrophy elicits changes to cell wall architecture that increase the exposure of immunogenic polymers, metabolic fitness costs more strongly drive the observed virulence attenuation.IMPORTANCECandida albicans is a common cause of bloodstream infections (candidemia). Treatment of these bloodstream infections is made difficult because of increasing antifungal resistance and drug toxicity. Thus, new tactics are needed for antifungal drug development, with immunotherapy being of particular interest. The cell wall of C. albicans is composed of highly immunogenic polymers, particularly β-1,3-glucan. However, β-1,3-glucan is naturally masked by an outer layer of mannoproteins, which hampers the detection of the fungus by the host immune system. Alteration in cell wall components has been shown to increase β-1,3-glucan exposure; however, it is unknown how the inability to synthesize precursors to cell wall components affects unmasking. Here, we demonstrate how cell wall architecture is altered in response to a deficit in precursors for cell wall synthesis and how uridine is a crucial component of these precursors.

A First-in-Class Pyrazole-isoxazole Enhanced Antifungal Activity of Voriconazole: Synergy Studies in an Azole-Resistant Candida albicans Strain, Computational Investigation and in Vivo Validation in a Galleria mellonella Fungal Infection Model.

The widespread and irrational use of azole antifungal agents has led to an increase of azole-resistant Candida albicans strains with an urgent need for combination drug therapy, enhancing the treatment efficacy. Here, we report the discovery of a first-in-class pyrazole-isoxazole, namely, 5b, that showed remarkable growth inhibition against the C. albicans ATCC 10231 strain in combination with voriconazole, acting as a downregulator of ERG 11 (Cyp51) gene expression with a significant reduction of the yeast-to-hypha morphological transition. Furthermore, C. albicans CYP51 enzyme assay and in-depth molecular docking studies unveiled the unique ability of the combination of 5b and voriconazole to completely fill the CYP51 binding sites. In vivo studies using a Galleria mellonella model confirmed the previously in vitro observed synergistic effect of 5b with voriconazole. Also considering its biocompatibility in a cellular model of human keratinocytes, these results indicate that 5b represents a promising compound for a further optimization campaign.

A Targeted Click-to-Release Activation of the Last-Resort Antibiotic Colistin Reduces its Renal Cell Toxicity.

The use of highly potent but very toxic antibiotics such as colistin has become inevitable due to the rise of antimicrobial resistance. We aimed for a chemically-triggered, controlled release of colistin at the infection site to lower its systemic toxicity by harnessing the power of click-to-release reactions. Kinetic experiments with nine tetrazines and three dienophiles demonstrated a fast release via an inverse-electron-demand Diels-Alder reaction between trans-cyclooctenes (TCO) and the amine-functionalized tetrazine Tz7. The antibiotic activity of colistin against Escherichia coli was masked by TCO units, but restored upon reaction with d-Ubi-Tz, a tetrazine functionalised with the bacterial binding peptide d-Ubi29-41. While standard TCO did not improve toxicity against human proximal tubular kidney HK-2 cells, the installation of an aspartic acid-modified TCO masking group reduced the overall charge of the peptide and entry to the kidney cells, thereby dramatically lowering its toxicity. The analog Col-(TCO-Asp)1 had favourable pharmacokinetic properties in mice and was successfully activated locally in the lung by d-Ubi-Tz in an in vivo infection model, whereas it remained inactive and non-harmful without the chemical trigger. This study constitutes the first example of a systemically acting two-component antibiotic with improved drug tolerability.

Engineered fungus containing a caterpillar gene kills insects rapidly by disrupting their ecto- and endo-microbiomes

Similar to the physiological importance of gut microbiomes, recent works have shown that insect ectomicrobiotas can mediate defensive colonization resistance against fungal parasites that infect via cuticle penetration. Here we show that engineering the entomopathogenic fungus Metarhizium robertsii with a potent antibacterial moricin gene from silkworms substantially enhances the ability of the fungus to kill mosquitos, locusts, and two Drosophila species. Further use of Drosophila melanogaster as an infection model, quantitative microbiome analysis reveals that engineered strains designed to suppress insect cuticular bacteria additionally disrupt gut microbiomes. An overgrowth of harmful bacteria such as the opportunistic pathogens of Providencia species is detected that can accelerate insect death. In support, quantitative analysis of antimicrobial genes in fly fat bodies and guts indicates that topical fungal infections result in the compromise of intestinal immune responses. In addition to providing an innovative strategy for improving the potency of mycoinsecticides, our data solidify the importance of both the ecto- and endo-microbiomes in maintaining insect wellbeing.

An optimized cyclophosphamide-treated mouse model of Mycobacterium abscessus pulmonary infection.

Mycobacterium abscessus pulmonary infections are increasingly problematic, especially for immunocompromised individuals and those with underlying lung conditions. Currently, there is no reliable standardized treatment, underscoring the need for improved preclinical drug testing. We present a simplified immunosuppressed mouse model using only four injections of cyclophosphamide, which allows for sustained M. abscessus lung burden for up to 16 days. This model proved effective for antibiotic efficacy evaluation, as demonstrated with imipenem or amikacin.

Development and validation of prediction models for nosocomial infection and prognosis in hospitalized patients with cirrhosis

BackgroundNosocomial infections (NIs) frequently occur and adversely impact prognosis for hospitalized patients with cirrhosis. This study aims to develop and validate two machine learning models for NIs and in-hospital mortality risk prediction.MethodsThe Prediction of Nosocomial Infection and Prognosis in Cirrhotic patients (PIPC) study included hospitalized patients with cirrhosis at the Qingchun Campus of the First Affiliated Hospital of Zhejiang University. We then assessed several machine learning algorithms to construct predictive models for NIs and prognosis. We validated the best-performing models with bootstrapping techniques and an external validation dataset. The accuracy of the predictions was evaluated through sensitivity, specificity, predictive values, and likelihood ratios, while predictive robustness was examined through subgroup analyses and comparisons between models.ResultsWe enrolled 1,297 patients into derivation cohort and 496 patients into external validation cohort. Among the six algorithms assessed, the Random Forest algorithm performed best. For NIs, the PIPC-NI model achieved an area under the curve (AUC) of 0.784 (95% confidence interval [CI] 0.741–0.826), a sensitivity of 0.712, and a specificity of 0.702. For in-hospital mortality, the PIPC- mortality model achieved an AUC of 0.793 (95% CI 0.749–0.836), a sensitivity of 0.769, and a specificity of 0.701. Moreover, our PIPC models demonstrated superior predictive performance compared to the existing MELD, MELD-Na, and Child-Pugh scores.ConclusionsThe PIPC models showed good predictive power and may facilitate healthcare providers in easily assessing the risk of NIs and prognosis among hospitalized patients with cirrhosis.

Merkel cell polyomavirus small tumor antigen contributes to immune evasion by interfering with type I interferon signaling.

Merkel cell polyomavirus (MCPyV) is the causative agent of the majority of Merkel cell carcinomas (MCC). The virus has limited coding capacity, with its early viral proteins, large T (LT) and small T (sT), being multifunctional and contributing to infection and transformation. A fundamental difference in early viral gene expression between infection and MCPyV-driven tumorigenesis is the expression of a truncated LT (LTtr) in the tumor. In contrast, sT is expressed in both conditions and contributes significantly to oncogenesis. Here, we identified novel functions of early viral proteins by performing genome-wide transcriptome and chromatin studies in primary human fibroblasts. Due to current limitations in infection and tumorigenesis models, we mimic these conditions by ectopically expressing sT, LT or LTtr, individually or in combination, at different time points. In addition to its known function in cell cycle and inflammation modulation, we reveal a fundamentally new function of sT. We show that sT regulates the type I interferon (IFN) response downstream of the type I interferon receptor (IFNAR) by interfering with the interferon-stimulated gene factor 3 (ISGF3)-induced interferon-stimulated gene (ISG) response. Expression of sT leads to a reduction in the expression of interferon regulatory factor 9 (IRF9) which is a central component of the ISGF3 complex. We further show that this function of sT is conserved in BKPyV. We provide a first mechanistic understanding of which early viral proteins trigger and control the type I IFN response, which may influence MCPyV infection, persistence and, during MCC progression, regulation of the tumor microenvironment.

Restriction of arginine induces antibiotic tolerance in Staphylococcus aureus

Staphylococcus aureus is responsible for a substantial number of invasive infections globally each year. These infections are problematic because they are frequently recalcitrant to antibiotic treatment. Antibiotic tolerance, the ability of bacteria to persist despite normally lethal doses of antibiotics, contributes to antibiotic treatment failure in S. aureus infections. To understand how antibiotic tolerance is induced, S. aureus biofilms exposed to multiple anti-staphylococcal antibiotics are examined using both quantitative proteomics and transposon sequencing. These screens indicate that arginine metabolism is involved in antibiotic tolerance within a biofilm and support the hypothesis that depletion of arginine within S. aureus communities can induce antibiotic tolerance. Consistent with this hypothesis, inactivation of argH, the final gene in the arginine synthesis pathway, induces antibiotic tolerance. Arginine restriction induces antibiotic tolerance via inhibition of protein synthesis. In murine skin and bone infection models, an argH mutant has enhanced ability to survive antibiotic treatment with vancomycin, highlighting the relationship between arginine metabolism and antibiotic tolerance during S. aureus infection. Uncovering this link between arginine metabolism and antibiotic tolerance has the potential to open new therapeutic avenues targeting previously recalcitrant S. aureus infections.

Semisynthetic guanidino lipoglycopeptides with potent in vitro and in vivo antibacterial activity.

Gram-positive bacterial infections present a major clinical challenge, with methicillin- and vancomycin-resistant strains continuing to be a cause for concern. In recent years, semisynthetic vancomycin derivatives have been developed to overcome this problem as exemplified by the clinically used telavancin, which exhibits increased antibacterial potency but has also raised toxicity concerns. Thus, glycopeptide antibiotics with enhanced antibacterial activities and improved safety profiles are still necessary. We describe the development of a class of highly potent semisynthetic glycopeptide antibiotics, the guanidino lipoglycopeptides, which contain a positively charged guanidino moiety bearing a variable lipid group. These glycopeptides exhibited enhanced in vitro activity against a panel of Gram-positive bacteria including clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant strains, showed minimal toxicity toward eukaryotic cells, and had a low propensity for resistance selection. Mechanistically, guanidino lipoglycopeptides engaged with bacterial cell wall precursor lipid II with a higher binding affinity than vancomycin. Binding to both wild-type d-Ala-d-Ala lipid II and the vancomycin-resistant d-Ala-d-Lac variant was confirmed, providing insight into the enhanced activity of guanidino lipoglycopeptides against vancomycin-resistant isolates. The in vivo efficacy of guanidino lipoglycopeptide EVG7 was evaluated in a S. aureus murine thigh infection model and a 7-day sepsis survival study, both of which demonstrated superiority to vancomycin. Moreover, the minimal to mild kidney effects at supratherapeutic doses of EVG7 indicate an improved therapeutic safety profile compared with vancomycin. These findings position guanidino lipoglycopeptides as candidates for further development as antibacterial agents for the treatment of clinically relevant multidrug-resistant Gram-positive infections.

Obesity impairs ciliary function and mucociliary clearance in the murine airway epithelium.

Obesity is a risk factor for increased morbidity and mortality in viral respiratory infection. Mucociliary clearance (MCC) in the airway is the primary host defense against viral infections. However, the impact of obesity on MCC is unclear, prompting this study. Using murine tracheal tissue culture and in vitro influenza A virus (IAV) infection models, we analyzed cilia-driven flow and ciliary beat frequency (CBF) in the airway epithelium to evaluate MCC. Short-term IAV infection increased cilia-driven flow and CBF in control mice, but not in high-fat diet-induced obese mice. Basal cilia-driven flow and CBF were also lower in obese mice than in control mice. Mechanistically, the increase of extracellular adenosine triphosphate (ATP) release during IAV infection, which was observed in the control mice, was abolished in the obese mice, although the addition of ATP increased cilia-driven flow and CBF both in control and obese mice to a similar extent. Additionally, RNA sequencing and reverse transcription-polymerase chain reaction revealed the downregulation of several cilia-related genes, including Dnah1, Dnal1, Armc4, and Ttc12 (the dynein-related genes); Ulk4 (the polychaete differentiation gene); Cep164 (the ciliogenesis and intraflagellar transport gene); Rsph4a, Cfap206, and Ppil6 (the radial spoke structure and assembly gene); and Drc3(the nexin-dynein regulatory complex genes) in obese murine tracheal tissues compared to their control levels. In conclusion, our studies demonstrate that obesity attenuates MCC under basal conditions and during IAV infection by downregulating the expression of cilia-related genes and suppressing the release of extracellular ATP, thereby increasing the susceptibility and severity of IAV infection.

Unraveling the mechanism of thermotolerance by Set302 in Cryptococcus neoformans.

The underlying mechanism of thermotolerance, which is a key virulence factor essential for pathogenic fungi such as Cryptococcus neoformans, is largely unexplored. In this study, our findings suggest that Set302, a homolog of Set3 and a subunit of histone deacetylase complex Set3C, contributes to thermotolerance in C. neoformans. Specifically, the deletion of the predicted Set3C core subunit, Set302, resulted in further reduction in the growth of C. neoformans at 39°C, and survival of transient incubation at 50°C. Transcriptomics analysis revealed that the expression levels of numerous heat stress-responsive genes altered at both 30°C and 39°C due to the lack of Set302. Notably, at 39°C, the absence of Set302 led to the downregulation of gene expression related to the ubiquitin-proteasome system (UPS). Based on the GFP-α-synuclein overexpression model to characterize misfolded proteins, we observed a pronounced accumulation of misfolded GFP-α-synuclein at 39°C, consequently inhibiting C. neoformans thermotolerance. Furthermore, the loss of Set302 exacerbated the accumulation of misfolded GFP-α-synuclein during heat stress. Interestingly, the set302∆ strain exhibited a similar phenotype under proteasome stress as it did at 39°C. Moreover, the absence of Set302 led to reduced production of capsule and melanin. set302∆ strain also displayed significantly reduced pathogenicity and colonization ability compared to the wild-type strain in the murine infection model. Collectively, our findings suggest that Set302 modulates thermotolerance by affecting the degradation of misfolded proteins and multiple virulence factors to mediate the pathogenicity of C. neoformans.IMPORTANCECryptococcus neoformans is a pathogenic fungus that poses a potential and significant threat to public health. Thermotolerance plays a crucial role in the wide distribution in natural environments and host colonization of this fungus. Herein, Set302, a critical core subunit for the integrity of histone deacetylase complex Set3C and widely distributed in various fungi and mammals, governs thermotolerance and affects survival at extreme temperatures as well as the formation of capsule and melanin in C. neoformans. Additionally, Set302 participates in regulating the expression of multiple genes associated with the ubiquitin-proteasome system (UPS). By eliminating misfolded proteins under heat stress, Set302 significantly contributes to the thermotolerance of C. neoformans. Moreover, Set302 regulates the pathogenicity and colonization ability of C. neoformans in a murine model. Overall, this study provides new insight into the mechanism of thermotolerance in C. neoformans.

Depletion of essential mycobacterial gene glmM reduces pathogen survival and induces host-protective immune responses against tuberculosis

The limitations of TB treatment are the long duration and immune-dampening effects of anti-tuberculosis therapy. The Cell wall plays a crucial role in survival and virulence; hence, enzymes involved in its biosynthesis are good therapeutic targets. Here, we identify Mycobacterium tuberculosis (Mtb) GlmM, (GlmMMtb) engaged in the UDP-GlcNAc synthesis pathway as an essential enzyme. We generated a conditional knockdown strain, Rv-glmMkD using the CRISPR interference-mediated gene silencing approach. Depletion of GlmMMtb affects the morphology and thickness of the cell wall. The Rv-glmMkD strain attenuated Mtb survival in vitro, in the host macrophages (ex vivo), and in a murine mice infection model (in vivo). Results suggest that the depletion of GlmMMtb induces M1 macrophage polarization, prompting a pro-inflammatory cytokine response, apparent from the upregulation of activation markers, including IFNɣ and IL-17 that resists the growth of Mtb. These observations provide a rationale for exploring GlmMMtb as a potential therapeutic target.

Characterization of mAbs against Klebsiella pneumoniae type 3 fimbriae isolated in a target-independent phage display campaign.

We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of Klebsiella pneumoniae. We report the discovery of monoclonal antibodies (mAbs) binding to type 3 fimbrial proteins, including MrkA. We found that anti-MrkA mAbs were cross-reactive to a diverse panel of K. pneumoniae clinical isolates, representing different O-serotypes. mAbs binding to MrkA have previously been described and have been shown to provide prophylactic protection, although only modest protection when dosed therapeutically in vivo in a murine lung infection model. Here, we used a combination of binding and opsonophagocytic killing studies using a high-content imaging platform to provide a possible explanation for the modest therapeutic efficacy in vivo reported in that model. Our work shows that expression of K. pneumoniae type 3 fimbriae in in vitro culture is not homogenous within a bacterial population. Instead, sub-populations of bacteria that do, and do not, express type 3 fimbriae exist. In a high-content opsonophagocytic killing assay, we showed that MrkA-targeting antibodies initially promote killing by macrophages; however, over time, this effect is diminished. We hypothesize the reason for this is that bacteria not expressing MrkA can evade opsonophagocytosis. Our data support the fact that MrkA is a conserved, immunodominant protein that is antibody accessible on the surface of K. pneumoniae and suggest that additional studies should evaluate the potential of using anti-MrkA antibodies in different stages of K. pneumoniae infection (different sites in the body) as well as against K. pneumoniae biofilms in the body during infection and associated with medical devices.IMPORTANCEThere is an unmet, urgent need for the development of novel antimicrobial therapies for the treatment of Klebsiella pneumoniae infections. We describe the use of phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of K. pneumoniae. We discovered monoclonal antibodies (mAbs) binding to the type 3 fimbrial protein MrkA. The anti-MrkA mAbs were found to be highly cross-reactive, binding to all K. pneumoniae strains tested from a diverse panel of clinical isolates, and were active in an opsonophagocytic killing assay at pM concentrations. MrkA is important for biofilm formation; thus, our data support further exploration of the use of anti-MrkA antibodies for preventing and/or controlling K. pneumoniae in biofilms and during infection.

Nitazoxanide synergizes polymyxin B against Escherichia coli by depleting cellular energy.

The rapid expansion of antibiotic-resistant bacterial diseases is a global burden on public health. It makes sense to repurpose and reposition already-approved medications for use as supplementary agents in synergistic combinations with existing antibiotics. Here, we demonstrate that the anthelmintic drug nitazoxanide (NTZ) synergistically enhances the effectiveness of the lipopeptide antibiotic polymyxin B in inhibiting gram-negative bacteria, including those resistant to polymyxin B. Mechanistic investigations revealed that nitazoxanide inhibited calcium influx and cell membrane depolarization, enhanced the affinity between polymyxin B and the extracellular membrane, and promoted intracellular ATP depletion and an increase in reactive oxygen species (ROS), thus enhancing the penetration and disruption of the Escherichia coli cell membrane by polymyxin B. The transcriptomic analysis revealed that the combination resulted in energy depletion by inhibiting both aerobic and anaerobic respiration patterns in bacterial cells. The increased bactericidal effect of polymyxin B on the E. coli ∆nuoC strain further indicates that NuoC could be a promising target for nitazoxanide. Furthermore, the combination of nitazoxanide and polymyxin B showed promising therapeutic effects in a mouse infection model infected with E. coli. Taken together, these results demonstrate the potential of nitazoxanide as a novel adjuvant to polymyxin B, to overcome antibiotic resistance and improve therapeutic outcomes in refractory infections.IMPORTANCEThe rapid spread of antibiotic-resistant bacteria poses a serious threat to public health. The search for potential compounds that can increase the antibacterial activity of existing antibiotics is a promising strategy for addressing this issue. Here, the synergistic activity of the FDA-approved agent nitazoxanide (NTZ) combined with polymyxin B was investigated in vitro using checkerboard assays and time-kill curves. The synergistic mechanisms of the combination of nitazoxanide and polymyxin B were explored by fluorescent dye, transmission electron microscopy (TEM), and transcriptomic analysis. The synergistic efficacy was evaluated in vivo by the Escherichia coli and mouse sepsis models. These results suggested that nitazoxanide, as a promising antibiotic adjuvant, can effectively enhance polymyxin B activity, providing a potential strategy for treating multidrug-resistant bacteria.

Improvement of a mouse infection model to capture Pseudomonas aeruginosa chronic physiology in cystic fibrosis

Laboratory models are central to microbiology research, advancing the understanding of bacterial physiology by mimicking natural environments, from soil to the human microbiome. When studying host-bacteria interactions, animal models enable investigators to examine bacterial dynamics associated with a host, and in the case of human infections, animal models are necessary to translate basic research into clinical treatments. Efforts toward improving animal infection models are typically based on reproducing host genotypes/phenotypes and disease manifestations, leaving a gap in how well the physiology of microbes reflects their behavior in a human host. Understanding bacterial physiology is vital because it dictates host response and bacterial interactions with antimicrobials. Thus, our goal was to develop an animal model that accurately recapitulates bacterial physiology in human infection. The system we chose to model was a chronic Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF). To accomplish this goal, we leveraged a framework that we recently developed to evaluate model accuracy by calculating the percentage of bacterial genes that are expressed similarly in a model to how they are expressed in their infection environment. We combined two complementary models of P. aeruginosa infection-an in vitro synthetic CF sputum model (SCFM2) and a mouse acute pneumonia model. This combined model captured the chronic physiology of P. aeruginosa in CF better than the standard mouse infection model, showing the power of a data-driven approach to refining animal models. In addition, the results of this work challenge the assumption that a chronic infection model requires long-term colonization.

In vivo multiscale analyses of spring viremia of carp virus (SVCV) infection: From model organism to target species.

Spring viremia of carp virus (SVCV) has a broad fish host spectrum and is responsible for a disease that generally affects juvenile fishes with a mortality rate of up to 90%. In the absence of treatments or vaccines against SVCV, the search for prophylactic or therapeutic solutions is thus relevant, particularly to identify solutions compatible with mass vaccination. In addition to being a threat to aquaculture and ecosystems, SVCV is a unique pathogen to study virus-host interactions in the zebrafish model. Establishing the first reverse genetics system for SVCV and the design of recombinant SVCV (rSVCV) expressing fluorescent or bioluminescent proteins adds a new dimension for the study of these interactions using innovative imaging techniques. The infection by bath immersion of zebrafish larvae with rSVCV expressing mCherry allows us to define the first SVCV replication sites and the host innate immune responses using different transgenic lines of zebrafish. The fins were found as the main initial sites of infection in both zebrafish and carp, its natural host. Hence, new insights into the physiopathology of SVCV infection have been described. We report that neutrophils are recruited at the sites of infection and persist up to the death of the animal leading to an uncontrolled inflammation correlated with the expression of the pro-inflammatory cytokine IL1β. Tissue damage was observed at the site of initial replication, a likely consequence of virus-induced injury or the pro-inflammatory response. Interestingly, SVCV infection by bath immersion triggers a persistent pro-inflammatory response rather than activation of the antiviral IFN signaling pathway as observed following intravenous injection, highlighting the importance of the route of infection on the progression of pathogenicity. Thus, this model of zebrafish larva infection by rSVCV offers new perspectives to study in detail virus-host interactions and to discover new prophylactic or therapeutic solutions.

Tunicate cellulose nanocrystal reinforced multifunctional hydrogel with super flexible, fatigue resistant, antifouling and self-adhesive capability for effective wound healing

Hydrogels as skin wound dressings have been extensively studied owing to their good flexibility and biocompatibility. Nevertheless, the mechanical performance, adhesive capability, antifouling and antibacterial properties of conventional hydrogels are still unsatisfactory, which hinder the application of hydrogel for cutaneous healing. Here, we developed a novel biocompatible multifunctional hydrogel with super flexible, fatigue resistant, antifouling and self-adhesive capability for effective wound healing, where naturally rigid polymers including quaternized chitosan (QCS) and Tunicate cellulose nanocrystals (TCNCs) are used as bioactive cross-linkers and reinforcers to endow the hydrogel with excellent mechanical and antibacterial property, and the synergistic contributions from the poly(acrylic acid/methacrylate anhydride dopamine/sulfobetaine methacrylate) (poly(AA/DMA/SBMA)) chains and QCS endow the hydrogel with excellent adhesive property, antioxidant, antifouling and pH-responsive sustained drug release capabilities. The optimized hydrogel exhibited high tensile strength (77.69 KPa), large tensile strain (889.9 %), large toughness (307.51KJ.m−3), high adhesive strength (35.57 KPa) and ideal compressive property. The in vivo infected full-thickness skin model demonstrated that the hydrogel with vanvomycin sustained release ability efficiently improved the granulation tissue formation, facilitating collagen deposition and reducing inflammatory expression, thus effectively accelerating wound healing. This superiorly skin-adhesive antibacterial biocompatible hydrogel appears to be a promising candidate for wound therapy.