Infection Model Research Articles

A cynomolgus monkey E. coli urinary tract infection model confirms efficacy of new FimH vaccine candidates.

The increase in urinary tract infections (UTI) caused by antibiotic-resistant Escherichia coli requires the development of new therapeutic agents and prophylactic vaccines. To evaluate the efficacy of new lead candidates, we implemented a cynomolgus macaque UTI challenge model that mimics human uncomplicated cystitis in response to transurethral challenge with a multidrug-resistant (MDR) E. coli serotype O25b ST131 isolate. E. coli fimbrial adhesin FimH and O-antigens are separately under clinical evaluation by others as vaccine candidates to prevent UTI and invasive urosepsis disease, respectively. Accordingly, we assessed the protective efficacy of three 50-µg intramuscular doses of a novel recombinant FimH antigen adjuvanted with liposomal QS21/MPLA compared with saline placebo in groups of nine animals. A third group was vaccinated with this FimH formulation in combination with 1 µg each of a four-valent mixture of serotype O1a, O2, O6, and O25b O-antigen CRM197 lattice glycoconjugates. Both vaccines elicited high levels of serum FimH IgG and adhesin blocking antibodies at the time of bacterial challenge and, for the combination group, O-antigen-specific antibodies. Following bacterial challenge, both vaccinated groups showed >200- and >700-fold reduction in bacteriuria at day 2 and day 7 post-infection compared with placebo, respectively. In parallel, both vaccines significantly reduced levels of inflammatory biomarkers IL-8 and myeloperoxidase in the urine at day 2 post-infection relative to placebo. Results provide preclinical proof-of-concept for the prevention of an MDR UTI infection by these new vaccine formulations.

Evaluation of Klebsiella pneumoniae pathogenicity through holistic gene content analysis

Klebsiella pneumoniae is a Gram-negative bacterium that causes both community- and healthcare-associated infections. Although various virulence factors and highly pathogenic phenotypes have been reported, the pathogenicity of K. pneumoniae is still not fully understood. In this study, we utilized whole-genome sequencing data of 168 clinical K. pneumoniae strains to assess pathogenicity. This work was based on the concept that the genetic composition of individual genomes (referred to as holistic gene content) of the strains may contribute to their pathogenicity. Holistic gene content analysis revealed two distinct groups of K. pneumoniae strains (‘major group’ and ‘minor group’). The minor group included strains with known highly pathogenic clones (ST23, ST375, ST65 and ST86). The minor group had higher rates of capsular genotype K1 and presence of nine specific virulence genes (rmpA, iucA, iutA, irp2, fyuA, ybtS, iroN, allS and clbA) compared to the major group. Pathogenicity was assessed using Galleria mellonella larvae. Infection experiments revealed lower survival rates of larvae infected with strains from the minor group, indicating higher virulence. In addition, the minor group had a higher string test positivity rate than the major group. Holistic gene content analysis predicted possession of virulence genes, string test positivity and pathogenicity as observed in the G. mellonella infection model. Moreover, the findings suggested the presence of as yet unrecognized genomic elements that are either involved in the acquisition of virulence genes or associated with pathogenicity.

Long-lived lung megakaryocytes contribute to platelet recovery in thrombocytopenia models.

Lung megakaryocytes (Mks) are largely extravascular with an immune phenotype (1). Because bone marrow (BM) Mks are short lived, it has been assumed that extravascular lung Mks are constantly "seeded" from the BM. To investigate lung Mk origins and how origin affects their functions, we developed methods to specifically label lung Mks using CFSE dye and biotin delivered via the oropharyngeal route. Labeled lung Mks were present for up to 4 months, while BM Mks had a lifespan of less than 1 week. In a parabiosis model, lung Mks were partially replaced over 1 month from a circulating source. Unlike tissue-resident macrophages, using MDS1-Cre-ERT2 TdTomato mice, we found that lung Mks arose from hematopoietic stem cells. However, studies with FlkSwitch mTmG mice showed that lung Mks were derived from a Flt3-independent lineage that did not go through a multipotent progenitor. CFSE labeling to track lung Mk-derived platelets showed that approximately 10% of circulating platelets were derived from lung-resident Mks at steady state, but in sterile thrombocytopenia this was doubled (~20%). Lung-derived platelets were similarly increased in a malaria infection model (Plasmodium yoelii) typified by thrombocytopenia. These studies indicate that lung Mks arise from a Flt3- BM source, are long-lived, and contribute more platelets during thrombocytopenia.

The infectious capacity of Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus saprophyticus in a porcine model of urinary tract infection.

The purpose of this study was to establish a porcine model of urinary tract infection (UTI) with gram-positive uropathogens. Ten female domestic pigs were experimentally inoculated with human UTI isolates of Enterococcus faecalis (n = 3), Staphylococcus saprophyticus (n = 3), or Staphylococcus aureus (n = 4) and followed with regular urine samples. Bladders and kidneys were aseptically removed at termination (5-7 days post infection) and assessed by gross pathology and bacterial enumeration. Enterococcus faecalis (n = 3 of 3) and S. aureus (n = 2 of 4) successfully colonized the pig bladders. Inoculation with S. saprophyticus never resulted in detectable bacteriuria. All infected pigs had cleared the infection spontaneously before termination. Surprisingly, three (of four) pigs inoculated with S. aureus led to spontaneous infection with opportunistic pathogens. Also, one pig colonized with E. faecalis resulted in spontaneous infection with E. coli. In conlusion, the pig supports experimental UTI with E. faecalis for up to 24 h but not prolonged infection. S. aureus and S. saprophyticus fails to cause UTI in pigs and other animals should be considered for studying these pathogens.

A chronic murine model of pulmonary Acinetobacter baumannii infection enabling the investigation of late virulence factors, long-term antibiotic treatments, and polymicrobial infections.

Acinetobacter baumannii can cause prolonged infections that disproportionately affect immunocompromised populations. Our understanding of A. baumannii respiratory pathogenesis relies on an acute murine infection model with limited clinical relevance that employs an unnaturally high number of bacteria and requires the assessment of bacterial load at 24-36 hours post-infection. Here, we demonstrate that low intranasal inoculums in immunocompromised mice with a tlr4 mutation leads to reduced inflammation, allowing for persistent infections lasting at least 3 weeks. Using this "chronic infection model," we determined the adhesin InvL is an imperative virulence factor required during later stages of infection, despite being dispensable in the early phase. We also demonstrate that the chronic model enables the distinction between antibiotics that, although initially reduce bacterial burden, either lead to complete clearance or result in the formation of bacterial persisters. To illustrate how our model can be applied to study polymicrobial infections, we inoculated mice with an active A. baumannii infection with Staphylococcus aureus or Klebsiella pneumoniae. We found that S. aureus exacerbates the infection, while K. pneumoniae enhances A. baumannii clearance. In all, the chronic model overcomes some limitations of the acute pulmonary model, expanding our capabilities to study of A. baumannii pathogenesis and lays the groundwork for the development of similar models for other important opportunistic pathogens.

Vector competence of Culex quinquefasciatus for Tembusu virus and viral factors for virus transmission by mosquitoes

The ongoing epidemic of flaviviruses worldwide has underscored the importance of studying flavivirus vector competence, considering their close association with mosquito vectors. Tembusu virus is an avian-related mosquito-borne flavivirus that has been an epidemic in China and Southeast Asia since 2010. However, the reason for the outbreak of Tembusu virus in 2010 remains unclear, and it is unknown whether changes in vector transmission played an essential role in this process. To address these questions, we conducted a study using Culex quinquefasciatus as a model for Tembusu virus infection, employing both oral infection and microinjection methods. Our findings confirmed that both vertical and venereal transmission collectively contribute to the cycle of Tembusu virus within the mosquito population, with persistent infections observed. Importantly, our data revealed that the prototypical Tembusu virus MM_1775 strain exhibited significantly greater infectivity and transmission rates in mosquitoes than did the duck Tembusu virus (CQW1 strain). Furthermore, we revealed that the viral E protein and 3′ untranslated region are key elements responsible for these differences. In conclusion, our study sheds light on mosquito transmission of Tembusu virus and provides valuable insights into the factors influencing its infectivity and transmission rates. These findings contribute to a better understanding of Tembusu virus epidemiology and can potentially aid in the development of strategies to control its spread.

PROBLEMS OF MANAGING THE IMMUNE RESPONSE UNDER CONDITIONS OF COMPETITIVE ADSORPTION, DIFFUSION PERTURBATIONS AND TEMPERATURE RESPONSE OF THE ORGANISM

Analysis of the problems associated with the rapid spread of infectious diseases indicates the need for new approaches to the diagnosis and develop- ment of effective personalized treatment programs. One of the important aspects of solving this problem is the creation of a mathematical modeling toolkit for predicting the dynamics of infectious diseases, taking into account spatial effects, concentrated effects, various mechanisms of body protec- tion in the conditions of the application of various types of therapeutic methods of treatment. An important component of complex methods of treat- ment of a wide range of infectious diseases is the use of adsorption drugs, which, along with detoxification of the body, are able to provide an addi- tional mechanism of neutralization of viral elements. In the process of using such means, not only pathogenic elements and toxins will be adsorbed, but also various types of immune agents, which will affect the dynamics of the immune response. In the paper the infectious disease model is generalized to take into account the features of competitive adsorption of antigens and immune agents in the conditions of diffusion perturbations, concentrated influences and temperature reaction of the body. By synthesizing the ideas of step-by-step procedures, asymptotic and numerical methods, an effective computational technology of step-by-step approximation of the solution of the original model singularly perturbed problem was built. The results of the computer experiments presented in the paper illustrate the features of reducing the predicted concentration of viral elements during their competi- tive adsorption together with immune agents, in particular, the effect of reducing the efficiency of the use of non-specific adsorbents. It is emphasized that taking into account the features of the action of competitive adsorption is important for making decisions regarding the formation of rational treatment programs with the complex use of adsorbing substances and immunological drugs.

Fast Computer Model Calibration using Annealed and Transformed Variational Inference

Computer models play a crucial role in numerous scientific and engineering domains. To ensure the accuracy of simulations, it is essential to properly calibrate the input parameters of these models through statistical inference. While Bayesian inference is the standard approach for this task, employing Markov chain Monte Carlo methods often encounters computational hurdles due to the costly evaluation of likelihood functions and slow mixing rates. Although variational inference (VI) can be a fast alternative to traditional Bayesian approaches, VI has limited applicability due to boundary issues and local optima problems. To address these challenges, we propose flexible VI methods based on deep generative models that do not require parametric assumptions on the variational distribution. We embed a surjective transformation in our framework to avoid posterior truncation at the boundary. Additionally, we provide theoretical conditions that guarantee the success of the algorithm. Furthermore, our temperature annealing scheme and fine-tuning can prevent being trapped in local optima through a series of intermediate posteriors and weight adjustment. We apply our method to infectious disease models and a geophysical model, illustrating that the proposed method can provide fast and accurate inference compared to its competitors.

Long-time behavior and quasi-density function for a stochastic epidemic model with relapse and reinfection

A stochastic susceptible–infected–recovered–infected (SIRI) epidemic model with relapse and reinfection is established in this paper. First, we prove that the solution to the epidemic model is unique and globally positive. Next, we determine some sufficient conditions for the extinction of the disease when [Formula: see text] and for the persistence in mean in the case of [Formula: see text]. Furthermore, we prove the existence of at least one ergodic stationary distribution of the stochastic model if [Formula: see text]. Additionally, by solving the corresponding three-dimensional Fokker–Planck equation, it is theoretically shown that the epidemic model has a log-normal probability density function when [Formula: see text], then we obtain the exact expression of density function of the stationary distribution. Finally, we give some numerical simulations to support our theoretical results.

3D-printed porous titanium rods equipped with vancomycin-loaded hydrogels and polycaprolactone membranes for intelligent antibacterial drug release

Implant-related infections pose significant challenges to orthopedic surgeries due to the high risk of severe complications. The widespread use of bioactive prostheses in joint replacements, featuring roughened surfaces and tight integration with the bone marrow cavity, has facilitated bacterial proliferation and complicated treatment. Developing antibacterial coatings for orthopedic implants has been a key research focus in recent years to address this critical issue. Researchers have designed coatings using various materials and antibacterial strategies. In this study, we fabricated 3D-printed porous titanium rods, incorporated vancomycin-loaded mPEG750-b-PCL2500 gel, and coated them with a PCL layer. We then evaluated the antibacterial efficacy through both in vitro and in vivo experiments. Our coating passively inhibits bacterial biofilm formation and actively controls antibiotic release in response to bacterial growth, providing a practical solution for proactive and sustained infection control. This study utilized 3D printing technology to produce porous titanium rod implants simulating bioactive joint prostheses. The porous structure of the titanium rods was used to load a thermoresponsive gel, mPEG750-b-PCL2500 (PEG: polyethylene glycol; PCL: polycaprolactone), serving as a novel drug delivery system carrying vancomycin for controlled antibiotic release. The assembly was then covered with a PCL membrane that inhibits bacterial biofilm formation early in infection and degrades when exposed to lipase solutions, mimicking enzymatic activity during bacterial infections. This setup provides infection-responsive protection and promotes drug release. We investigated the coating’s controlled release, antibacterial capability, and biocompatibility through in vitro experiments. We established a Staphylococcus aureus infection model in rabbits, implanting titanium rods in the femoral medullary cavity. We evaluated the efficacy and safety of the composite coating in preventing implant-related infections using imaging, hematology, and pathology. In vitro experiments demonstrated that the PCL membrane stably protects encapsulated vancomycin during PBS immersion. The PCL membrane rapidly degraded at a lipase concentration of 0.2 mg/mL. The mPEG750-b-PCL2500 gel ensured stable and sustained vancomycin release, inhibiting bacterial growth. We investigated the antibacterial effect of the 3D-printed titanium material, coated with PCL and loaded with mPEG750-b-PCL2500 hydrogel, using a rabbit Staphylococcus aureus infection model. Imaging, hematology, and histopathology confirmed that our composite antibacterial coating exhibited excellent antibacterial effects and infection prevention, with good safety in trials. Our results indicate that the composite antibacterial coating effectively protects vancomycin in the hydrogel from premature release in the absence of bacterial infection. The outer PCL membrane inhibits bacterial growth and prevents biofilm formation. Upon contact with bacterial lipase, the PCL membrane rapidly degrades, releasing vancomycin for antibacterial action. The mPEG750-b-PCL2500 gel provides stable and sustained vancomycin release, prolonging its antibacterial effects. Our composite antibacterial coating demonstrates promising potential for clinical application.

The effects of a high-flavonoid corn cultivar on the gastrointestinal tract microbiota in chickens undergoing necrotic enteritis.

The search for alternative therapies to antimicrobial growth promoters (AGP) in poultry production has gained momentum in the past years because of consumer preference and government restrictions on the use of AGP in animal production. Flavonoids are plant-derived metabolites that have been studied for their health-promoting properties that could potentially be used as an alternative to AGP in poultry. In a previous study, we showed that the inclusion of a flavonoid-rich corn cultivar (PennHFD1) in the diet improved the health of broilers undergoing necrotic enteritis. However, the mechanisms of action by which the PennHFD1-based diet ameliorated necrotic enteritis are unknown. This study describes the microbial diversity and composition of the jejunum and ileum of chickens co-infected with Eimeria maxima and Clostridium perfringens and treated with a high-flavonoid corn-based diet. Luminal content and mucosal samples from the jejunum and ileum were collected for DNA extraction, 16S rRNA amplicon sequencing and data analyses. The infection model and the dietary treatments significantly changed the alfa diversity indices (Mucosal samples: ASVs, P = 0.04; Luminal content samples: ASVs, P = 0.03), and beta diversities (Mucosal samples: P < 0.01, Luminal content: P < 0.01) of the ileal samples but not those of the jejunal samples. The microbial composition revealed that birds fed the high-flavonoid corn diet had a lower relative abundance of C. perfringens compared to birds fed the commercial corn diet. The treatments also changed the relative abundance of other bacteria that are related to gut health, such as Lactobacillus. We concluded that both the infection model and the dietary high-flavonoid corn changed the broilers' gut microbial diversity and composition. In addition, the decrease in the relative abundance of C. perfringens corroborates with a decrease in mortality and intestinal lesions due to necrotic enteritis. Collecting different segments and sample types provided a broader understanding of the changes in the gut microbiota among treatments.

Infiltrating monocytes drive cardiac dysfunction in a cardiomyocyte-restricted mouse model of SARS-CoV-2 infection.

Cardiovascular manifestations of coronavirus disease 2019 (COVID-19) include myocardial injury, heart failure, and myocarditis and are associated with long-term disability and mortality. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and antigens are found in the myocardium of COVID-19 patients, and human cardiomyocytes are susceptible to infection in cell or organoid cultures. While these observations raise the possibility that cardiomyocyte infection may contribute to the cardiac sequelae of COVID-19, a causal relationship between cardiomyocyte infection and myocardial dysfunction and pathology has not been established. Here, we generated a mouse model of cardiomyocyte-restricted infection by selectively expressing human angiotensin-converting enzyme 2 (hACE2), the SARS-CoV-2 receptor, in cardiomyocytes. Inoculation of Myh6-Cre Rosa26loxP-STOP-loxP-hACE2 mice with an ancestral, non-mouse-adapted strain of SARS-CoV-2 resulted in viral replication within the heart, accumulation of macrophages, and moderate left ventricular (LV) systolic dysfunction. Cardiac pathology in this model was transient and resolved with viral clearance. Blockade of monocyte trafficking reduced macrophage accumulation, suppressed the development of LV systolic dysfunction, and promoted viral clearance in the heart. These findings establish a mouse model of SARS-CoV-2 cardiomyocyte infection that recapitulates features of cardiac dysfunctions of COVID-19 and suggests that both viral replication and resultant innate immune responses contribute to cardiac pathology.IMPORTANCEHeart involvement after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection occurs in multiple ways and is associated with worse outcomes in coronavirus disease 2019 (COVID-19) patients. It remains unclear if cardiac disease is driven by primary infection of the heart or immune response to the virus. SARS-CoV-2 is capable of entering contractile cells of the heart in a culture dish. However, it remains unclear how such infection affects the function of the heart in the body. Here, we designed a mouse in which only heart muscle cells can be infected with a SARS-CoV-2 strain to study cardiac infection in isolation from other organ systems. In our model, infected mice show viral infection, worse function, and accumulation of immune cells in the heart. A subset of immune cells facilitates such worsening heart function. As this model shows features similar to those observed in patients, it may be useful for understanding the heart disease that occurs as a part of COVID-19.

VSV infection and LPS treatment alter serum bile acid profiles, bile acid biosynthesis, and bile acid receptors in mice.

This study focuses on the crosstalk between bile acid (BA) metabolism and immune response in VSV infection and LPS treatment models and depicts the effect of infection on circulating BA profiles, the biosynthesis-related enzymes, and their receptors. These findings provide insights into the effect of infection on BA metabolism and signaling, adding a more comprehensive understanding to the relationship between infection, BA metabolism and immune responses.

RfaH contributes to maximal colonization and full virulence of hypervirulent Klebsiella pneumoniae.

Hypervirulent K. pneumoniae (hvKp) have emerged as clinically important pathogens, posing a serious threat to human health. RfaH, a transcriptional elongation factor, has been regarded as implicated in facilitating the transcription of long virulence operons in certain bacterial species. In K. pneumoniae, RfaH plays a vital role in promoting CPS synthesis and hypermucoviscosity, as well as mediating bacterial fitness during lung infection. In this study, we aim to conduct a systematic investigation of the roles of rfaH in the survival, dissemination, and colonization of hvKp through in vitro and in vivo assays. We found that bacterial cells and colonies displayed capsule -deficient phenotypes subsequent to the deletion of rfaH in K. pneumoniae NTUH-K2044. We confirmed that rfaH is required for the synthesis of capsule and lipopolysaccharide (LPS) by positively regulating the expression of CPS and LPS gene clusters. We found that the ΔrfaH mutant led to a significantly decreased mortality of K. pneumoniae in a mouse intraperitoneal infection model. We further demonstrated that the absence of rfaH was associated with slower bacterial growth under conditions of low nutrition or iron limitation. ΔrfaH displayed reduced survival rates in the presence of human serum. Besides, the engulfment of the ΔrfaH mutant was significantly higher than that of NTUH-K2044 by macrophages in vivo, indicating an indispensable role of RfaH in the phagocytosis resistance of hvKp in mice. Both mouse intranasal and intraperitoneal infection models revealed a higher bacterial clearance rate of ΔrfaH in lungs, livers, and spleens of mice compared to its wild type, suggesting an important role of RfaH in the bacterial survival, dissemination, and colonization of hvKp in vivo. Histopathological results supported that RfaH contributes to the pathogenicity of hvKp in mice. In conclusion, our study demonstrates crucial roles of RfaH in the survival, colonization and full virulence of hvKp, which provides several implications for the development of RfaH as an antibacterial target.

Conformational ensembles in Klebsiella pneumoniae FimH impact uropathogenesis

Klebsiella pneumoniae is an important pathogen causing difficult-to-treat urinary tract infections (UTIs). Over 1.5 million women per year suffer from recurrent UTI, reducing quality of life and causing substantial morbidity and mortality, especially in the hospital setting. Uropathogenic E. coli (UPEC) is the most prevalent cause of UTI. Like UPEC, K. pneumoniae relies on type 1 pili, tipped with the mannose-binding adhesin FimH, to cause cystitis. However, K. pneumoniae FimH is a poor binder of mannose, despite a mannose-binding pocket identical to UPEC FimH. FimH is composed of two domains that are in an equilibrium between tense (low-affinity) and relaxed (high-affinity) conformations. Substantial interdomain interactions in the tense conformation yield a low-affinity, deformed mannose-binding pocket, while domain-domain interactions are broken in the relaxed state, resulting in a high-affinity binding pocket. Using crystallography, we identified the structural basis by which domain-domain interactions direct the conformational equilibrium of K. pneumoniae FimH, which is strongly shifted toward the low-affinity tense state. Removal of the pilin domain restores mannose binding to the lectin domain, thus showing that poor mannose binding by K. pneumoniae FimH is not an inherent feature of the mannose-binding pocket. Phylogenetic analyses of K. pneumoniae genomes found that FimH sequences are highly conserved. However, we surveyed a collection of K. pneumoniae isolates from patients with long-term indwelling catheters and identified isolates that possessed relaxed higher-binding FimH variants, which increased K. pneumoniae fitness in bladder infection models, suggesting that long-term residence within the urinary tract may select for higher-binding FimH variants.

Structured multicellular intestinal spheroids (SMIS) as a standardized model for infection biology

Background3D cell culture models have recently garnered increasing attention for replicating organ microarchitecture and eliciting in vivo-like responses, holding significant promise across various biological disciplines. Broadly, 3D cell culture encompasses organoids as well as single- and multicellular spheroids. While the latter have found successful applications in tumor research, there is a notable scarcity of standardized intestinal models for infection biology that mimic the microarchitecture of the intestine. Hence, this study aimed to develop structured multicellular intestinal spheroids (SMIS) specifically tailored for studying molecular basis of infection by intestinal pathogens.ResultsWe have successfully engineered human SMIS comprising four relevant cell types, featuring a fibroblast core enveloped by an outer monolayer of enterocytes and goblet cells along with monocytic cells. These SMIS effectively emulate the in vivo architecture of the intestinal mucosal surface and manifest differentiated morphological characteristics, including the presence of microvilli, within a mere two days of culture. Through analysis of various differentiation factors, we have illustrated that these spheroids attain heightened levels of differentiation compared to 2D monolayers. Moreover, SMIS serve as an optimized intestinal infection model, surpassing the capabilities of traditional 2D cultures, and exhibit a regulatory pattern of immunological markers similar to in vivo infections after Campylobacter jejuni infection. Notably, our protocol extends beyond human spheroids, demonstrating adaptability to other species such as mice and pigs.ConclusionBased on the rapid attainment of enhanced differentiation states, coupled with the emergence of functional brush border features, increased cellular complexity, and replication of the intestinal mucosal microarchitecture, which allows for exposure studies via the medium, we are confident that our innovative SMIS model surpasses conventional cell culture methods as a superior model. Moreover, it offers advantages over stem cell-derived organoids due to scalability and standardization capabilities of the protocol. By showcasing differentiated morphological attributes, our model provides an optimal platform for diverse applications. Furthermore, the investigated differences of several immunological factors compared to monotypic monolayers after Campylobacter jejuni infection underline the refinement of our spheroid model, which closely mimics important features of in vivo infections.

Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus.

The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.

Screening of the Pandemic Response Box library identified promising compound candidate drug combinations against extensively drug-resistant Acinetobacter baumannii

Infections caused by antimicrobial-resistant Acinetobacter baumannii pose a significant threat to human health, particularly in the context of hospital-acquired infections. As existing antibiotics lose efficacy against Acinetobacter isolates, there is an urgent need for the development of novel antimicrobial agents. In this study, we assessed 400 structurally diverse compounds from the Medicines for Malaria Pandemic Response Box for their activity against two clinical isolates of A. baumannii: A. baumannii 5075, known for its extensive drug resistance, and A. baumannii QS17-1084, obtained from an infected wound in a Thai patient. Among the compounds tested, seven from the Pathogen box exhibited inhibitory effects on the in vitro growth of A. baumannii isolates, with IC50s ≤ 48 µM for A. baumannii QS17-1084 and IC50s ≤ 17 µM for A. baumannii 5075. Notably, two of these compounds, MUT056399 and MMV1580854, shared chemical scaffolds resembling triclosan. Further investigations involving drug combinations identified five synergistic drug combinations, suggesting potential avenues for therapeutic development. The combination of MUT056399 and brilacidin against A. baumannii QS17-1084 and that of MUT056399 and eravacycline against A. baumannii 5075 showed bactericidal activity. These combinations significantly inhibited biofilm formation produced by both A. baumannii strains. Our findings highlight the drug combinations as promising candidates for further evaluation in murine wound infection models against multidrug-resistant A. baumannii. These compounds hold potential for addressing the critical need for effective antibiotics in the face of rising antimicrobial resistance.

Multifunctional EGCG@ZIF-8 Nanoplatform with Photodynamic Therapy/Chemodynamic Therapy Antibacterial Properties Promotes Infected Wound Healing.

Damaged skin is susceptible to invasion by harmful microorganisms, especially Staphylococcus aureus and Escherichia coli, which can delay healing. Epigallocatechin-3-gallate (EGCG) is a natural compound known for effectively promoting wound healing and its potent anti-inflammatory effects. However, its application is limited due to its susceptibility to oxidation and isomerization, which alter its structure. The use of zeolitic imidazolate framework-8 (ZIF-8) can effectively tackle these issues. This study introduces an oxygen (O2) and hydrogen peroxide (H2O2) self-supplying ZIF-8 nanoplatform designed to enhance the bioavailability of EGCG, combining photodynamic therapy (PDT) and chemodynamic therapy (CDT) to improve antibacterial properties and ultimately accelerate wound healing. For this purpose, EGCG and indocyanine green (ICG), a photosensitizer, were successively integrated into a ZIF-8, and coated with bovine serum albumin (BSA) to enhance biocompatibility. The outer layer of this construct was further modified with manganese dioxide (MnO2) to promote CDT and calcium peroxide (CaO2) to supply H2O2 and O2, resulting in the final nanoplatform EGCG-ICG@ZIF-8/BSA-MnO2/CaO2 (EIZBMC). In in vitro experiments under 808 nm laser, EIZBMC exhibited synergistic antibacterial effects through PDT and CDT. This combination effectively released reactive oxygen species (ROS), which mediated oxidative stress to inhibit the bacteria. Subsequently, in a murine model of wound infection, EIZBMC not only exerted antibacterial effects through PDT and CDT but also alleviated the inflammatory condition and promoted the regeneration of collagen fibers, which led to accelerated wound healing. Overall, this research presents a promising approach to enhancing the therapeutic efficacy of EGCG by leveraging the synergistic antibacterial effects of PDT and CDT. This multifunctional nanoplatform maximizes EGCG's anti-inflammatory properties, offering a potent solution for promoting infected wound healing.

Berberine alleviates ETEC-induced intestinal inflammation and oxidative stress damage by optimizing intestinal microbial composition in a weaned piglet model.

Enterotoxigenic Escherichia coli (ETEC) is the main diarrhea-causing pathogen in children and young animals and has become a global health concern. Berberine is a type of "medicine and food homology" and has a long history of use in China, particularly in treating gastrointestinal disorders and bacterial diarrhea. In this study, we explored the effects of berberine on growth performance, intestinal inflammation, oxidative damage, and intestinal microbiota in a weaned piglet model of ETEC infection. Twenty-four piglets were randomly divided into four groups-a control group (fed a basal diet [BD] and infused with saline), a BD+ETEC group (fed a basal diet and infused with ETEC), a LB+ETEC group (fed a basal diet with 0.05% berberine and infused with ETEC infection), and a HB+ETEC group (fed a basal diet with 0.1% berberine and infused with ETEC). Berberine significantly improved the final body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) (P<0.05) of piglets, and effectively decreased the incidence of diarrhea among the animals (P<0.05). Additionally, berberine significantly downregulated the expression levels of the genes encoding TNF-α, IL-1β, IL-6, IL-8, TLR4, MyD88, NF-κB, IKKα, and IKKβ in the small intestine of piglets (P<0.05). ETEC infection significantly upregulated the expression of genes coding for Nrf2, CAT, SOD1, GPX1, GST, NQO1, HO-1, GCLC, and GCLM in the small intestine of the animals (P<0.05). Berberine significantly upregulated 12 functional COG categories and 7 KEGG signaling pathways. A correlation analysis showed that berberine significantly increased the relative abundance of beneficial bacteria (Gemmiger, Pediococcus, Levilactobacillus, Clostridium, Lactiplantibacillus, Weissella, Enterococcus, Blautia, and Butyricicoccus) and decreased that of pathogenic bacteria (Prevotella, Streptococcus, Parabacteroides, Flavonifractor, Alloprevotella) known to be closely related to intestinal inflammation and oxidative stress in piglets. In conclusion, ETEC infection disrupted the intestinal microbiota in weaned piglets, upregulating the TLR4/MyD88/NF-κB and Nrf2 signaling pathways, and consequently leading to intestinal inflammation and oxidative stress-induced damage. Our data indicated that berberine can optimize intestinal microbiota balance and modulate the TLR4/MyD88/NF-κB and Nrf2 signaling pathways, thus helping to alleviate intestinal inflammation and oxidative damage caused by ETEC infection in weaned piglets.