Acute Infection Model Research Articles

Antimicrobial and antibiofilm activity of human recombinant H1 histones against bacterial infections.

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.IMPORTANCEThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in biomass reduction of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

InVivo CRISPR Screening Reveals CHD7 as a Positive Regulator of Short-lived Effector Cells.

CD8+ T cells differentiate into two subpopulations in response to acute viral infection: memory precursor effector cells (MPECs) and short-lived effector cells (SLECs). MPECs and SLECs are epigenetically distinct; however, the epigenetic regulators required for formation of these subpopulations are mostly unknown. In this study, we performed an invivo CRISPR screen in murine naive CD8+ T cells to identify the epigenetic regulators required for MPEC and SLEC formation, using the acute lymphocytic choriomeningitis virus Armstrong infection model. We identified the ATP-dependent chromatin remodeler CHD7 (chromodomain-helicase DNA-binding protein 7) as a positive regulator of SLEC formation, as knockout (KO) of Chd7 reduced SLECs numerically. In contrast, KO of Chd7 increased the formation of central memory T cells following pathogen clearance yet attenuated memory cell expansion following a rechallenge. These findings establish CHD7 as a novel positive regulator of SLEC and a negative regulator of central memory T cell formation.

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.

HSV-1 infection induces phosphorylated tau propagation among neurons via extracellular vesicles.

Extracellular vesicles (EV), key players in cell-to-cell communication, may contribute to disease propagation in several neurodegenerative diseases, including Alzheimer's disease (AD), by favoring the dissemination of neurotoxic proteins within the brain. Interestingly, growing evidence supports the role of herpes simplex virus type 1 (HSV-1) infection in the pathogenesis of AD. Here, we investigated whether HSV-1 infection could promote the spread of phosphorylated tau (ptau) among neurons via EV. We analyzed the ptau species that were secreted via EV following HSV-1 infection in neuroblastoma cells and primary neurons, focusing particularly on T205, T181, and T217, the phosphorylation sites mainly associated with AD. Moreover, by overexpressing human tau tagged with GFP (htauGFP), we found that recipient tau knockout (KO) neurons uptook EV that are loaded with HSV-1-induced phtauGFP. Finally, we exploited an in vivo model of acute infection and assessed that cerebral HSV-1 infection promotes the release of ptau via EV in the brain of infected mice. Overall, our data suggest that, following HSV-1 infection, EV play a role in tau spreading within the brain, thus contributing to neurodegeneration.IMPORTANCEHerpes simplex virus type 1 (HSV-1) infection that reaches the brain has been repeatedly linked with the appearance of the pathognomonic markers of Alzheimer's disease (AD), including accumulation of amyloid beta and hyperphosphorylated tau proteins, and cognitive deficits. AD is a multifactorial neurodegenerative disease representing the most common form of dementia in the elderly, and no cure is currently available, thus prompting additional investigation on potential risk factors and pathological mechanisms. Here, we demonstrate that the virus exploits the extracellular vesicles (EV) to disseminate phosphorylated tau (ptau) among brain cells. Importantly, we provide evidence that the HSV-1-induced EV-bearing ptau can be undertaken by recipient neurons, thus likely contributing to misfolding and aggregation of native tau, as reported for other AD models. Hence, our data highlight a novel mechanism exploited by HSV-1 to propagate tau-related damage in the brain.

Oral Pyronaridine Tetraphosphate Reduces Tissue Presence of Parasites in a Mouse Model of Chagas Disease.

The eukaryotic parasite Trypanosoma cruzi (T. cruzi) is responsible for Chagas disease, which results in heart failure in patients. The disease is more common in Latin America, and is an emerging infection with The Centers for Disease Control estimating that greater than 300,000 people are currently infected in the United States. This disease has also spread from South and Central America, where it is endemic to many other countries, including Australia, Japan, and Spain. Current therapy for Chagas disease is inadequate due to limited efficacy in the indeterminate and chronic phases of the disease, in addition to the adverse effects from nifurtimox and benznidazole, which are nitro-containing drugs used for therapy. There is a clear need for new therapies for the Chagas disease. Using a computational machine learning approach, we have previously shown that the antimalarial pyronaridine tetraphosphate is active against T. cruzi Brazil-luc in vitro against parasites infecting a myoblast cell line and is also active in vivo in an acute mouse model of Chagas disease when dosed i.p. We now further evaluated oral pyronaridine as a monotherapy to determine the minimum effective dose to treat acute and chronic models of Chagas disease. Our results for T. cruzi Brazil-luc demonstrated daily oral dosing with pyronaridine from 150 to 600 mg/kg resulted in statistically significant inhibition in the 7 day acute mouse model. Combination therapy with daily dosing of benznidazole and pyronaridine in the acute infection model demonstrated that 300 mg/kg pyronaridine could return statistically significant antiparasitic activity to a subtherapetic 10 mg/kg benznidazole. In contrast, pyronaridine as monotherapy or combined with benznidazole lacked efficacy in the chronic mouse model, whereas 100 mg/kg benznidazole alone demonstrated undetectable parasites in the heart of mice. Pyronaridine requires further assessment in other chronic models to identify if it can be used beyond the acute stage of T. cruzi infection.

Correlates of disease severity in bluetongue as a model of acute arbovirus infection.

Most viral diseases display a variable clinical outcome due to differences in virus strain virulence and/or individual host susceptibility to infection. Understanding the biological mechanisms differentiating a viral infection displaying severe clinical manifestations from its milder forms can provide the intellectual framework toward therapies and early prognostic markers. This is especially true in arbovirus infections, where most clinical cases are present as mild febrile illness. Here, we used a naturally occurring vector-borne viral disease of ruminants, bluetongue, as an experimental system to uncover the fundamental mechanisms of virus-host interactions resulting in distinct clinical outcomes. As with most viral diseases, clinical symptoms in bluetongue can vary dramatically. We reproduced experimentally distinct clinical forms of bluetongue infection in sheep using three bluetongue virus (BTV) strains (BTV-1IT2006, BTV-1IT2013 and BTV-8FRA2017). Infected animals displayed clinical signs varying from clinically unapparent, to mild and severe disease. We collected and integrated clinical, haematological, virological, and histopathological data resulting in the analyses of 332 individual parameters from each infected and uninfected control animal. We subsequently used machine learning to select the key viral and host processes associated with disease pathogenesis. We identified and experimentally validated five different fundamental processes affecting the severity of bluetongue: (i) virus load and replication in target organs, (ii) modulation of the host type-I IFN response, (iii) pro-inflammatory responses, (iv) vascular damage, and (v) immunosuppression. Overall, we showed that an agnostic machine learning approach can be used to prioritise the different pathogenetic mechanisms affecting the disease outcome of an arbovirus infection.

Multimodal mucosal and systemic immune characterization of a non-human primate trachoma model highlights the critical role of local immunity during acute phase disease.

Trachoma is a leading cause of infection-related blindness worldwide. This disease is caused by recurrent Chlamydia trachomatis (Ct) infections of the conjunctiva and develops in two phases: i) active (acute trachoma, characterized by follicular conjunctivitis), then long-term: ii) scarring (chronic trachoma, characterized by conjunctival fibrosis, corneal opacification and eyelid malposition). Scarring trachoma is driven by the number and severity of reinfections. The immune system plays a pivotal role in trachoma including exacerbation of the disease. Hence the immune system may also be key to developing a trachoma vaccine. Therefore, we characterized clinical and local immune response kinetics in a non-human primate model of acute conjunctival Ct infection and disease. The conjunctiva of non-human primate (NHP, Cynomolgus monkeys-Macaca fascicularis-) were inoculated with Ct (B/Tunis-864 strain, B serovar). Clinical ocular monitoring was performed using a standardized photographic grading system, and local immune responses were assessed using multi-parameter flow cytometry of conjunctival cells, tear fluid cytokines, immunoglobulins, and Ct quantification. Clinical findings were similar to those observed during acute trachoma in humans, with the development of typical follicular conjunctivitis from the 4th week post-exposure to the 11th week. Immunologic analysis indicated an early phase influx of T cells in the conjunctiva and elevated interleukins 4, 8, and 5, followed by a late phase monocytic influx accompanied with a decrease in other immune cells, and tear fluid cytokines returning to initial levels. Our NHP model accurately reproduces the clinical signs of acute trachoma, allowing for an accurate assessment of the local immune responses in infected eyes. A progressive immune response occurred for weeks after exposure to Ct, which subsided into a persistent innate immune response. An understanding of these local responses is the first step towards using the model to assess new vaccine and therapeutic strategies for disease prevention.

Development of a natural product optimization strategy for inhibitors against MraY, a promising antibacterial target

MraY (phospho-N-acetylmuramoyl-pentapeptide-transferase) inhibitory natural products are attractive molecules as candidates for a new class of antibacterial agents to combat antimicrobial-resistant bacteria. Structural optimization of these natural products is required to improve their drug-like properties for therapeutic use. However, chemical modifications of these natural products are painstaking tasks due to complex synthetic processes, which is a bottleneck in advancing natural products to the clinic. Here, we develop a strategy for a comprehensive in situ evaluation of the build-up library, which enables us to streamline the preparation of the analogue library and directly assess its biological activities. We apply this approach to a series of MraY inhibitory natural products. Through construction and evaluation of the 686-compound library, we identify promising analogues that exhibit potent and broad-spectrum antibacterial activity against highly drug-resistant strains in vitro as well as in vivo in an acute thigh infection model. Structures of the MraY-analogue complexes reveal distinct interaction patterns, suggesting that these analogues represent MraY inhibitors with unique binding modes. We further demonstrate the generality of our strategy by applying it to tubulin-binding natural products to modulate their tubulin polymerization activities.

Tafenoquine for Relapsing Babesiosis: A Case Series.

Relapsing babesiosis often occurs in highly immunocompromised patients and has been attributed to the acquisition of resistance against drugs commonly used for treatment such as atovaquone, azithromycin, and clindamycin. Tafenoquine, which is approved for malaria prophylaxis and presumptive antirelapse treatment of Plasmodium vivax malaria, has shown activity against Babesia microti in several animal models of acute infection and in a single human case of relapsing babesiosis. Here, we report 5 cases of relapsing babesiosis treated with tafenoquine, including the previous case, and begin to define the conditions for optimal use of tafenoquine in relapsing babesiosis. A definitive diagnosis of babesiosis was made by microscopic examination of Giemsa-stained thin blood smears or a real-time polymerase chain reaction (PCR) that targets the parasite 18S rRNA gene. Clearance of B. microti infection was ascertained by use of blood smear and real-time PCR. Tafenoquine was initiated with a loading dose of 600 mg. A weekly maintenance dose consisted of 200 mg or 300 mg; the lower dose was associated with a delayed clearance of B. microti. In 2 cases, all antimicrobial agents but tafenoquine were discontinued prior to clearance of infection. In 2 other cases, clearance was achieved while tafenoquine was administered along with other antimicrobial agents. In 3 of these 4 cases, tafenoquine was used in combination with atovaquone-proguanil. Other agents included atovaquone, azithromycin, and/or clindamycin. In 1 case, tafenoquine was administered alone and failed to prevent relapse. Tafenoquine can be a useful adjunct for the treatment of highly immunocompromised patients experiencing relapsing babesiosis caused by B. microti.

A Gram-negative-selective antibiotic that spares the gut microbiome.

Infections caused by Gram-negative pathogens are increasingly prevalent and are typically treated with broad-spectrum antibiotics, resulting in disruption of the gut microbiome and susceptibility to secondary infections1-3. There is a critical need for antibiotics that are selective both for Gram-negative bacteria over Gram-positive bacteria, as well as for pathogenic bacteria over commensal bacteria. Here we report the design and discovery of lolamicin, a Gram-negative-specific antibiotic targeting the lipoprotein transport system. Lolamicin has activity against a panel of more than 130 multidrug-resistant clinical isolates, shows efficacy in multiple mouse models of acute pneumonia and septicaemia infection, and spares the gut microbiome in mice, preventing secondary infection with Clostridioides difficile. The selective killing of pathogenic Gram-negative bacteria by lolamicin is a consequence of low sequence homology for the target in pathogenic bacteria versus commensals; this doubly selective strategy can be a blueprint for the development of other microbiome-sparing antibiotics.

Biomimetic protein structural transitions regulate activation and inhibition of the broad-spectrum bactericidal activity of cationic nanoparticles

The development of cationic polymers as alternative materials to antibiotics necessitates addressing the challenge of balancing their antimicrobial activity and toxicity. Here we propose a precise switching strategy inspired by biomimetic voltage-gated ion channels, enabling controlled activation and inhibition of cationic antimicrobial functions through protein conformational transitions in diverse physiological environments. Following thermodynamic studies on the specific recognition between mannose end groups on polycations and concanavalin A (ConA), we synthesized a type of ConA-polycation nanoparticle. The nanoparticle was inhibited under neutral conditions, with cationic moieties shielded by ConA's β-sheet. This shielding suppresses their antimicrobial activity, thereby ensuring satisfactory biocompatibility. In mildly acidic environments, however, the transition of a portion of ConA to an α-helix conformation exposed cations at the particle periphery, activating antibacterial functionality. Compared to inhibited nanoparticles, those in the activated state exhibited a 32–256 times reduction in the minimum bactericidal concentration against bacteria and fungi (2–16 µg/mL). In a murine acute pulmonary infection model, intravenous administration of inhibited nanoparticles effectively reduced bacterial counts by 4-log within 12 h. The biomimetic design, regulating cationic antimicrobial functionality through the alteration in protein secondary structure, significantly retards bacterial resistance development, holding great promise for intelligent antimicrobial materials. Statement of significanceCationic antimicrobial polymers exhibit advantages distinct from antibiotics due to their lower propensity for resistance development. However, the presence of cationic moieties also poses a threat to healthy cells and tissues, significantly constraining their potential for clinical applications. To address this challenge, we propose a biomimetic strategy that mimics voltage-gated ion channels to activate the antimicrobial functionality of cations selectively in bacterial environments through the conformational transitions of proteins between β–sheets and α–helices. In healthy tissues, the antimicrobial functionality is inhibited, ensuring satisfactory biocompatibility. Antimicrobial cationic materials capable of intelligent switching between an activated state and an inhibited state in response to environmental changes offer an effective strategy to prevent the development of resistance and mitigate potential side effects.

Universal antibacterial and anti-inflammatory treatment using chitosan-prussian blue nanozyme

Bacterial infections are a growing global public health problem, exacerbated by the widespread and often inappropriate use of antibiotics, leading to the emergence of non-antibiotic pathogens. Herein, we synthesized a chitosan-Prussian blue nanozyme (CS@PB), a non-antibiotic agent, for universal antibacterial and anti-inflammatory treatment of bacterial infections. Confocal microscopy images showed that CS@PB significantly enhanced the physical interaction between chitosan and bacteria, thereby increasing the antibacterial ability. Moreover, these nanozymes exhibited potent antioxidant and anti-inflammatory properties, promoting macrophage polarization toward the M2-like phenotype, reducing oxidative stress, and alleviating inflammation. This dual-action approach effectively accelerates the healing of bacteria-infected inflammatory wounds. The synergistic bactericidal and anti-inflammatory properties of CS@PBs inhibited wound infection and promoted the healing of skin infections in a mouse model. In addition, CS@PB displayed remarkable lung retention and potent bactericidal effects, resulting in significantly improved survival rates in mouse models of acute pulmonary bacterial infections. In conclusion, CS@PBs exhibited exceptional bactericidal capabilities, anti-inflammatory properties, and minimal toxicity, suggesting that they are promising candidates for a new generation of non-antibiotic antimicrobial agents for the treatment of bacterial infections.

Identifiability investigation of within-host models of acute virus infection.

Uncertainty in parameter estimates from fitting within-host models to empirical data limits the model's ability to uncover mechanisms of infection, disease progression, and to guide pharmaceutical interventions. Understanding the effect of model structure and data availability on model predictions is important for informing model development and experimental design. To address sources of uncertainty in parameter estimation, we use four mathematical models of influenza A infection with increased degrees of biological realism. We test the ability of each model to reveal its parameters in the presence of unlimited data by performing structural identifiability analyses. We then refine the results by predicting practical identifiability of parameters under daily influenza A virus titers alone or together with daily adaptive immune cell data. Using these approaches, we present insight into the sources of uncertainty in parameter estimation and provide guidelines for the types of model assumptions, optimal experimental design, and biological information needed for improved predictions.

Effects of Vancomycin/Tobramycin-Doped Ceramic Composite (Polyvinyl Alcohol Composite–Vancomycin/Tobramycin–Polymeric Dicalcium Phosphate Dihydrate) in a Rat Femur Model Implanted With Contaminated Porous Titanium Cylinders

BackgroundPeriprosthetic joint infection (PJI) remains common and problematic. We hypothesized that using a bioceramic that provided rapid release of the antibiotics (vancomycin [VAN] or VAN and tobramycin [VAN and TOB]) from a polyvinyl-alcohol-composite (PVA) combined with a delayed and sustained antibiotic release from polymeric-dicalcium-phosphate-dihydrate (PDCPD) ceramic would inhibit S. aureus-associated implant infections. MethodsA total of 50 male Sprague Dawley rats were randomly divided into 5 groups—I: negative control; II: bacteria only; III: bacteria + saline wash; IV: bacteria + PVA-VAN-PDCPD, and V: bacteria + PVA-VAN-TOB-PDCPD. A porous titanium (Ti) implant was press-fit into the rat knee. S. aureus-containing broth was added into the joint space creating a PJI. After 1 week, the joints from groups III to V were washed with saline and the fluid collected for bacterial quantification. This was followed by saline irrigation treatment (groups III to V) and application of the antibiotic-loaded PVA-PDCPD bioceramic (groups IV and V). On day 21, joint fluid was collected, and the implants harvested for bacterial quantification. ResultsNo bacteria were isolated from the negative control (group I). The positive control (group II) was positive on both days 7 and 21. Bacteria were still present on day 21 in the fluid and implant in group III. Groups (IV and V) showed a decrease in the bacterial burden in the fluid and implant on day 21. There were significant differences in bacteria levels in the collected wash fluid and on the implant at day 21 between the saline wash (group III) and treatment groups (IV and V). ConclusionsIn this animal model of acute periprosthetic infection, treatment with PVA-VAN-PDCPD and PVA-VAN/TOB-PDCPD reduced bacterial load in the infected joint and the infected Ti implant. Application of PVA-VAN-PDCPD and/or PVA-VAN/TOB-PDCPD after saline irrigation could be used as an addition to the treatment of PJI.

Cell-intrinsic Tim-3 is Required for an Optimal Acute CD8+ T Cell Response

Abstract Tim-3, or transmembrane immunoglobulin and mucin domain-3, is a type I membrane protein expressed by various immune cells, best known as a negative regulator of anti-tumor immunity. However, Tim-3 also has a co-stimulatory role in T cells under some conditions, via enhancement of PI3K signaling. We hypothesize that Tim-3 signaling enhances CD8+ T cell activation during acute infection and contributes to enhanced CD8+ effector function. To test this hypothesis, we used LCMV-specific TCR transgenic (P14) mice expressing a truncated, non-signaling, allele of Tim-3 or CD8-specific deletion of Tim-3 and used LCMV Armstrong as an acute infection model. At the effector stage, endogenous Tim-3+ T cells had increased expression of effector markers, and cytokine production compared with Tim-3- cells. Tim-3 knockout mice also showed a significantly lower number of LCMV-specific T cells. Bulk RNA sequencing and TCR sequencing revealed that Tim-3-expressing effector CD8+ T cells had a distinct pattern of effector gene upregulation, with increased expansion of a subset of TCR clones. Mechanistically, Tim-3 signals enhances phosphorylation of Foxo1, inhibiting its entry into the nucleus, conferring enhanced effector function. Thus, Tim-3 signaling contributes positively to an effective CD8+ T cell response during acute infection. These findings may lead to a better understanding of CD8+ T cell function in different settings, including in tumors.

A Role for NOD2 in Intestinal Memory T cell Formation and Function

Abstract Aberrant resident memory T cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Genetic mutations in NOD2 are associated with the highest risk of CD development. NOD2 has been shown to be critical for initiating adaptive immune responses at systemic sites. However, the role of NOD2 in establishing memory T cell responses in the intestine remains unclear. Using an acute model of LCMV infection, we interrogated the role of NOD2 in intestinal memory T cell generation and function. Littermate wildtype or NOD2-deficient mice were infected intraperitoneally with LCMV-Armstrong, and the intestinal T cell priming, effector, and memory T cell responses were profiled in the mesenteric lymph nodes (mLN) and small intestinal lamina propria (SILP). Augmented NOD2 signalling at homeostasis and during infection increased endogenous and LCMV-specific CD4+ T cell numbers in the mLN, but not in the spleen. This effect was due to a NOD2-dependent increase in T cell homing to the mLN. Furthermore, NOD2-deficiency resulted in decreased effector and memory T cell numbers in the SILP. This effect was associated with decreased memory T cell functions in NOD2-deficient mice during ex vivo and in vivo recall experiments. Taken together, our experiments suggest a role for NOD2 in mediating optimal T cell priming in the mesenteric lymph nodes, which later affects effector and memory T cell functions in the intestine.

Elimination of Decapod iridovirus 1 (DIV1) infection at high water temperature: a new environmental control strategy

AbstractDecapod iridovirus 1 (DIV1) poses a major challenge to sustainable shrimp farming and poses a serious hazard to aquaculture industry. This study investigated the complex interaction between DIV1 infection and water temperature, focusing on the effect of high temperature on DIV1 infection due to Penaeus monodon. Using models of latent and acute infection, the study revealed the response of P. monodon to DIV1 under different conditions. In the experimental set-up, the effect of high water temperature (34 ± 1 °C) compared with room temperature (26 ± 1 °C) was investigated. DIV1 replication was significantly inhibited in the high-temperature group (H), resulting in complete viral elimination within 15 days. DIV1 did not resurface even after return to room temperature (26 ± 1 °C), indicating sustained antiviral effects. Compared with the room temperature (26 ± 1 °C) group (N), the H group showed a 100% reduction in the incidence of latent and acute infection. Exposure to high water temperature directly impaired the viability of DIV1, enhancing the immune system of P. monodon, and expediting metabolic processes for efficient DIV1 clearance. The study highlights the significant inhibitory effects of high water temperature (34 ± 1 °C) on DIV1 infection in P. monodon, resulting in viral eradication. This discovery offers a potential strategy for mitigating DIV1 infections in shrimp aquaculture, prompting further investigation into underlying mechanisms. Optimising parameters and protocols for high-temperature treatment is crucial for viral control. Exploring the broader implications of the findings on other viral infections in crustacean aquaculture could provide valuable insights for comprehensive disease prevention and control.

Contribution of intramacrophage stages to Pseudomonas aeruginosa infection outcome in zebrafish embryos: insights from mgtC and oprF mutants.

Pseudomonas aeruginosa often colonizes immunocompromised patients, causing acute and chronic infections. This bacterium can reside transiently inside cultured macrophages, but the contribution of the intramacrophic stage during infection remains unclear. MgtC and OprF have been identified as important bacterial factors when P. aeruginosa resides inside culturedmacrophages. In this study, we showed that P. aeruginosa mgtC and oprF mutants, particular the latter one, had attenuated virulence in both mouse and zebrafish animal models of acute infection. To further investigate P. aeruginosa pathogenesis in zebrafish at a stage different from acute infection, we monitored bacterial load and visualized fluorescent bacteria in live larvae up to 4days after infection. Whereas the attenuated phenotype of the oprF mutant was associated with a rapid elimination of bacteria, the mgtC mutant was able to persist at low level, a feature also observed with the wild-type strain in surviving larvae. Interestingly, these persistent bacteria can be visualized in macrophages of zebrafish. In a short-time infection model using a macrophage cell line, electron microscopy revealed that internalized P. aeruginosa wild-type bacteria were either released after macrophage lysis or remained intracellularly, where they were localized in vacuoles or in the cytoplasm. The mgtC mutant could also be detected inside macrophages, but without causing cell damage, whereas the oprF mutant was almost completely eliminated after phagocytosis, or localized in phagolysosomes. Taken together, our results show that the main role of OprF for intramacrophage survival impacts both acute and persistent infection by this bacterium. On the other hand, MgtC plays a clear role in acute infection but is not essential for bacterial persistence, in relation with the finding that the mgtC mutant is not completely eliminated by macrophages.

Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection.

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([111In]-WBC SPECT, [18F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[11C]-methionine, d-[3-11C]-alanine, and d-[3-11C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [18F]3,3,3-trifluoro-d-alanine (d-[18F]-CF3-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[18F]-CF3-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[19F]-CF3-ala was stable in human and mouse serum. d-[19F]-CF3-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[19F]-CF3-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[18F]-CF3-ala via displacement of a bromo-precursor with [18F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[18F]-CF3-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[18F]-CF3-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [18F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.

Possibilities of local effect the healing of small bowel sutures in a rat model of acute intra-abdominal infection

Aims: Investigate the regeneration of the small bowel suture line regeneration after suturing with a material with antioxidant properties. Methodology: 7 intact rats, 63 with simulated intra-abdominal infection (IAI). A laparotomy was performed in 12 h after simulating. The small bowel was cut and sutured in 56 rats (2 groups of 28) after an abdominal lavage. Standard Kapron was used in the control group (CG). Kapron impregnated with a 5% solution of ethylmethylhydroxypyridine succinate was used in the research group (RG). Sections of the small bowel were collected from a total of 21 rats (7 intact rats, 7 rats with intra-abdominal infection (IAI) models, and 7 rats at different time points (1, 3, 5, and 7 days) post suturing) for subsequent histological examination. The preparations were stained with hematoxylin-eosin and by Mikel Calvo. The R/B coefficient (R/BC) was determined. The experiment's time duration was 7.5 days in December 2022. Results: R/BC increased (p<0.05) in the connective tissue fibers of the small bowel submucosal base in 12 h after the simulating of IAI. R/BC increased (p<0.05) in both groups in 1 day after suturing. R/BC increased in CG, decreased (p<0.05) in RG in 3 days. Fibrin was found between the ligature’s fibers in RG. R/BC decreased (p<0.05) in both groups in 5 days, indicators were smaller (p<0.01) in RG. Fibrin was found between the ligature’s fibers in CG, granulation tissue was in RG. R/BC decreased in both groups in 7 days, but indicators were smaller in RG (p<0.01). Unfilled areas in the granulation tissue between the ligature fibers were in CG. Conclusion: 1. The use of ligatures impregnated with the 5% ethylmethylhydroxypyridine succinate solution for suturing the small bowel in rats with intra-abdominal infection models is associated with the significant decrease in the activity of local proteins oxidation and the significant acceleration of local regeneration processes. 2. The creation of a suture material with antioxidant properties may be a promising direction for the prevention of suture leakage