Use of zebrafish to identify host responses specific to type VI secretion system mediated interbacterial antagonism.

Interbacterial competition is known to shape the microbial communities found in the host, however the interplay between this competition and host defense are less clear. Here, we use the zebrafish hindbrain ventricle (HBV) as an in vivo platform to investigate host responses to defined bacterial communities with distinct forms of interbacterial competition. We found that antibacterial activity of the type VI secretion system (T6SS) from both Vibrio cholerae and Acinetobacter baylyi can induce host inflammation and sensitize the host to infection independent of any individual effector. Chemical suppression of inflammation could resolve T6SS-dependent differences in host survival, but the mechanism by which this occurred differed between the two bacterial species. By contrast, colicin-mediated antagonism elicited by an avirulent strain of Shigella sonnei induced a negligible host response despite being a more potent bacterial killer, resulting in no impact on A. baylyi or V. cholerae virulence. Altogether, these results provide insight into how different modes of interbacterial competition in vivo affect the host in distinct ways.

The rapidly progressing and fatal outcome of rhombencephalitis by listeriosis in a 61-year-old male.

Listeria monocytogenes is a Gram-positive facultative anaerobic bacterium that is ubiquitous in the environment and can cause severe infections in immunocompromised individuals, pregnant women, and newborns. Listeriosis can manifest as meningitis, encephalitis, or sepsis, and its diagnosis requires a high index of suspicion. The case is reported of a rare presentation of rhombencephalitis by listeriosis in a 61-year-old male who initially suffered from subacute gastric disturbances and fever. Neurological consultation showed abnormal functions of cranial nerves and meningeal signs were observed. MRI revealed a poorly demarcated focus of approximately 45 × 16 × 15mm, indicating possible inflammatory processes, necessitating a lumbar puncture. Assessment of the CSF indicated infection with the bacterium- Listeria Monocytogenes, with the final diagnosis of Listeriosis encephalitis. Despite antibiotic therapy of Ceftazidine and Ampicillin, the patient's condition deteriorated, followed by death.

Listeria monocytogenes: a rare cause of rhomboencephalitis in an immunocompetent patient.

We present an unusual case of Listeria monocytogenes rhomboencephalitis in a young, healthy patient. Although L. monocytogenes meningitis is usually associated with immunodeficiency, rhomboencephalitis is more commonly seen in immunocompetent patients. The wide differential for rhomboencephalitis can create a diagnostic challenge. Without prompt pathogen identification and appropriate antibiotic regimen, L. monocytogenes central nervous system infections can be fatal. Cerebro-Spinal Fluid (CSF) Polymerase Chain Reaction (PCR) aided a prompt diagnosis and adjustment of therapy to achieve a good patient outcome.

Epidemiology, clinical and imaging features of rhombencephalitis caused by L. monocytogenes. A retrospective observational study. / Epidemiología, clínica y resultados de imagen de rombencefalitis causada por L. monocytogenes. Un estudio observacional.

INTRODUCTION: To date, few studies have explored the specific risk factors of patients with listeriosis who develop rhombencephalitis, and there is insufficient information regarding imaging findings and clinical symptoms in patients with this disease. This work aimed to analyze the imaging findings associated with L. monocytogenes rhombencephalitis in a cohort of patients with listeriosis. MATERIALS AND METHODS: We conducted a retrospective observational study of all declared cases of listeriosis in a tertiary hospital from Granada, Spain, from 2008 to 2021. Risk factors, comorbidities, and clinical outcomes were collected for all patients. In addition, clinical symptoms and magnetic resonance imaging (MRI) findings were included for those patients who developed rhombencephalitis. Descriptive and bivariate analyses were performed using SPSS statistical software (IBM SPSS, version 21). RESULTS: Our cohort comprised 120 patients with listeriosis (41.7% women, mean age: 58.6 ± 23.8 years), of which 10 (8.3%) had rhombencephalitis. The most frequent MRI findings in patients with confirmed rhombencephalitis were T2-FLAIR hyperintensity (100%), T1 hypointensity (80%), scattered parenchymal enhancement (80%), and cranial nerve enhancement (70%), while the most frequent anatomical involvement were pons, medulla oblongata, and cerebellum. Complications occurred in 6 patients (abscess in 4, hemorrhage in 2, hydrocephalus in 1). CONCLUSIONS: Rhombencephalitis is associated with an increased in-hospital mortality in patients with listeriosis. The anatomical distribution and imaging characteristics of neurolisteriosis could be useful to suggest the diagnosis. Future studies with greater sample size should explore the association between anatomical location, imaging patterns, and associated complications (e.g., hydrocephalus, hemorrhage), and clinical outcomes.

TITLE: Epidemiología, clínica y resultados de imagen de rombencefalitis causada por L. monocytogenes. Un estudio observacional.Introducción. Hasta la fecha, pocos estudios han explorado los factores de riesgo específicos de los pacientes con listeriosis que desarrollan rombencefalitis, y no hay suficiente información sobre los hallazgos de imagen y los síntomas clínicos en pacientes con esta enfermedad. El objetivo de este trabajo fue analizar los hallazgos de imagen asociados a la rombencefalitis por L. monocytogenes en una cohorte de pacientes con listeriosis. Materiales y métodos. Se realizó un estudio observacional retrospectivo de todos los casos declarados de listeriosis en un hospital terciario de Granada, España, desde 2008 hasta 2021. Se recogieron los factores de riesgo, las comorbilidades y los resultados clínicos de todos los pacientes. Además, se incluyeron los síntomas clínicos y los hallazgos de resonancia magnética (RM) de los pacientes que desarrollaron rombencefalitis. Se realizaron análisis descriptivos y bivariados utilizando el software estadístico SPSS (IBM SPSS, versión 21). Resultados. Nuestra cohorte incluyó a 120 pacientes con listeriosis (41,7%, mujeres; edad media: 58,6 ± 23,8 años), de los cuales 10 (8,3%) tenían rombencefalitis. Los hallazgos más frecuentes en la RM de los pacientes con rombencefalitis confirmada fueron hiperintensidad en T2-FLAIR (100%), hipointensidad en T1 (80%), realce parenquimatoso disperso (80%) y realce de los nervios craneales (70%), mientras que la afectación anatómica más frecuente fue en la protuberancia, la médula oblongada y el cerebelo. Se produjeron complicaciones en seis pacientes (absceso en cuatro, hemorragia en dos e hidrocefalia en uno). Conclusiones. La rombencefalitis se asocia a un aumento de la mortalidad intrahospitalaria en pacientes con listeriosis. La distribución anatómica y las características de imagen de la neurolisteriosis podrían ser útiles para sugerir el diagnóstico. Futuros estudios con mayor tamaño muestral deberían explorar la asociación entre la localización anatómica, los patrones de imagen y las complicaciones asociadas (por ejemplo, hidrocefalia y hemorragia), y los resultados clínicos.

The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with macrophages.

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity.This article has an associated First Person interview with the first author of the paper.

Analysis tools to quantify dissemination of pathology in zebrafish larvae.

We describe new open source software called QuantiFish for rapid quantitation of fluorescent foci in zebrafish larvae, to support infection research in this animal model. QuantiFish extends the conventional measurements of bacterial load and number of bacterial foci to include measures for dissemination of infection. These are represented by the proportions of bacteria between foci and their spatial distribution. We showcase these measures by comparison of intravenous and hindbrain routes of Mycobacterium marinum infection, which are indistinguishable by measurement of bacterial load and not consistently differentiated by the number of bacterial foci. The intravenous route showed dose dependent dissemination of infection, reflected by increased spatial dispersion of bacteria and lower proportions of bacteria distributed across many foci. In contrast, hindbrain infection resulted in localised disease, limited to a smaller area and higher proportions of bacteria distributed across fewer foci. The application of QuantiFish may extend beyond models of infection, to study other pathologies such as metastatic cancer.

Rapid detection of Listeria monocytogenes rhombencephalitis in an immunocompetent patient by multiplexed PCR.

A 46-year-old previously healthy man presented with 1 week of headache, nausea, vomiting and dizziness. He was found to have cranial nerve deficits, his cerebrospinal fluid (CSF) demonstrated a lymphocytic pleocytosis and brain MRI suggested rhombencephalitis. Although Gram stains and cultures of his CSF did not identify a pathogen, Listeria monocytogenes DNA was detected by the FilmArray Meningitis/Encephalitis panel within 2 hours of performing a lumbar puncture. He was treated with ampicillin and gentamicin and had a near-complete recovery. This case highlights the importance of recognising L. monocytogenes infection as a cause of acute cranial nerve impairment with MRI findings suggestive of brainstem encephalitis. It also highlights the frequently atypical CSF profile and low yield of culture in L. monocytogenes rhombencephalitis and the value of multiplex PCR testing of CSF to rapidly identify this pathogen and permit targeted therapy.

Early trigeminal nerve involvement in Listeria monocytogenes rhombencephalitis: case series and systematic review.

Listeria monocytogenes is associated with rhombencephalitis. However, the exact mechanisms of brainstem invasion remains poorly understood. Here, we demonstrate clinical and radiological data suggesting that Listeria may invade the brainstem via the trigeminal nerve. Three females (41, 64 and 70 years) with culture proven L. monocytogenes bacteremia and rhombencephalitis were investigated in the period of 2014-16. T2-weighted and contrast-enhanced T1-weighted MRI revealed a cerebellopontine abscess in all three patients, including the involvement of the trigeminal nerve root. In two patients, MRI also revealed selective contrast enhancement of the sensory trigeminal tract in the pons and medulla oblongata. Prior to any other neurological symptoms, two patients complained of hypoesthesia and a tingling sensation in the ipsilateral half of the face, consistent with sensory trigeminal nerve dysfunction on that side. In addition, we identified another 120 cases of Listeria rhombencephalitis following a systematic review. Cranial nerves VII, V, IX, and X, respectively, medulla oblongata, cerebellum and pons, were the most frequently involved brain structures. The present clinical and radiological findings corroborate earlier data from animal experiments, indicating that L. monocytogenes may be capable of retrograde intra-axonal migration along the cranial nerves. We suggest that in a subset of patients with rhombencephalitis L. monocytogenes enters the cerebellopontine angle through the trigeminal nerve, invading the brainstem via the sensory trigeminal nuclei.

Listeria monocytogenes sequence type 1 is predominant in ruminant rhombencephalitis.

Listeria (L.) monocytogenes is an opportunistic pathogen causing life-threatening infections in diverse mammalian species including humans and ruminants. As little is known on the link between strains and clinicopathological phenotypes, we studied potential strain-associated virulence and organ tropism in L. monocytogenes isolates from well-defined ruminant cases of clinical infections and the farm environment. The phylogeny of isolates and their virulence-associated genes were analyzed by multilocus sequence typing (MLST) and sequence analysis of virulence-associated genes. Additionally, a panel of representative isolates was subjected to in vitro infection assays. Our data suggest the environmental exposure of ruminants to a broad range of strains and yet the strong association of sequence type (ST) 1 from clonal complex (CC) 1 with rhombencephalitis, suggesting increased neurotropism of ST1 in ruminants, which is possibly related to its hypervirulence. This study emphasizes the importance of considering clonal background of L. monocytogenes isolates in surveillance, epidemiological investigation and disease control.

Brote de enfermedad neurológica aguda asociada a enterovirus en Cataluña: aspectos epidemiológicos / Outbreak of acute neurological disease associated to enterovirus in Catalonia: epidemiological aspects

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Rhombencephalitis due to Listeria monocytogenes: A Clinicopathologic Study of a Case.

INTRODUCTION: Central nervous system infections caused by Listeria monocytogenes usually manifest in the form of meningitis or meningoencephalitis, and are more common among immunosuppressed patients. Brainstem encephalitis (rhombencephalitis) is less common and fatal if not recognized and treated early. CASE REPORT: We describe the case of a 40-year-old, immunocompetent male patient, who presented with initial symptoms of high fever and productive cough. Signs of brainstem involvement appeared later. A magnetic resonance imaging of the brain revealed a lesion of inflammatory appearance in the right medulla oblongata, and the cerebrospinal fluid test showed mononuclear pleocytosis. Blood and cerebrospinal fluid cultures were negative. He presented with a significant improvement with the start of ceftriaxone and subsequent association of corticosteroids, until he developed respiratory failure and died. The third blood cultures grew after his death and they were positive for L. monocytogenes. An autopsy was carried out, which showed necrotizing inflammation, with gram-positive bacilli in the brainstem and the cerebellum. CONCLUSIONS: A fatal delay in the diagnosis occurred, mainly because of the favorable clinical response to ceftriaxone and corticosteroids. This case reminds us that a febrile clinical presentation with brainstem involvement must generate the suspicion of a Listeria infection, and therefore ampicillin must be a part of the empirical treatment.

A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides.

Mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. Individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. These individuals often present with defects in phagocytic effector cell function. Research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence against mucormycosis. However, these model systems have not been satisfactory for direct analysis of the interaction between innate immune effector cells and infectious sporangiospores in vivo. Here, we report the first real-time in vivo analysis of the early innate immune response to mucormycete infection using a whole-animal zebrafish larval model system. We identified differential host susceptibility, dependent on the site of infection (hindbrain ventricle and swim bladder), as well as differential functions of the two major phagocyte effector cell types in response to viable and non-viable spores. Larval susceptibility to mucormycete spore infection was increased upon immunosuppressant treatment. We showed for the first time that macrophages and neutrophils were readily recruited in vivo to the site of infection in an intact host and that spore phagocytosis can be observed in real-time in vivo. While exploring innate immune effector recruitment dynamics, we discovered the formation of phagocyte clusters in response to fungal spores that potentially play a role in fungal spore dissemination. Spores failed to activate pro-inflammatory gene expression by 6 h post-infection in both infection models. After 24 h, induction of a pro-inflammatory response was observed only in hindbrain ventricle infections. Only a weak pro-inflammatory response was initiated after spore injection into the swim bladder during the same time frame. In the future, the zebrafish larva as a live whole-animal model system will contribute greatly to the study of molecular mechanisms involved in the interaction of the host innate immune system with fungal spores during mucormycosis.

Rhombencephalitis by Listeria Monocytogens in Two Diabetic Patients.

Brainstem involvement is a characteristic feature and accounts for the high mortality associated with listeriosis especially in immunosuppressed patients. We report two cases of rhombencephalitis infection by Listeria monocytogenes in a 65 and 63-year-old men with diabetes. They were referred to a neurologist due to fever and drowsiness after 3 weeks. The 65-year-old man had vertigo, diplopia, ataxia, bidirectional nystagmus and the 63-year-old man complained of perioral numbness, dysphagia and dysartheria. Treatment with ampicillin (12 g/day) was started empirically and modified when the culture results were available. The CSF cultures were positive to Listeria monocytogenes and brain MRI findings were suggestive of rhomboencephalitis. Despite delays in treatment, they had a complete clinical recovery with resolution of MRI abnormalities. In contrast to our results, in most reports, a bi-phasic illness has been described and late treatment was associated with unfavorable courses or long lasting sequelae.

Visualizing and quantifying Pseudomonas aeruginosa infection in the hindbrain ventricle of zebrafish using confocal laser scanning microscopy.

Pseudomonas aeruginosa colonizes surfaces using a stepwise process that involves several phases, including attachment, production of exopolysaccharides, formation of microcolonies and the eventual development of biofilms. This process has been extensively characterized in vitro using both light and electron microscopic techniques. However, our ability to visualize this process in situ at the site of infection has been limited by the nature of the vertebrate models available. The optically clear zebrafish (Danio rerio) is an emerging model well suited for imaging bacterial infections. In this study, we infected the hindbrain ventricle of 54 h post-fertilization zebrafish with P. aeruginosa PAO1 and visualized and quantified microcolony formation using confocal laser scanning microscopy and image analyses. In comparison to wildtype PAO1, infection with a P. aeruginosa mutant deficient in the ability to produce the exopolysaccharide Psl caused less zebrafish mortality and fewer, smaller microcolonies per zebrafish at both 18 h and 29 h post-infection. The work presented here demonstrates reproducible in situ visualization and quantification methods for determining the extent of P. aeruginosa infection in a vertebrate model. We demonstrate how this model system can be manipulated to understand the effect of virulence factors on pathogenicity. Furthermore, this model can be adapted to study biofilm formation in situ, thereby extending our understanding of how bacterial persistence leads to chronic infections.

Listeria rhomboencephalitis in a patient on a tumor necrosis factor α inhibitor (etanercept).

We describe a case of an 87-year-old woman with rheumatoid arthritis on etanercept who developed subacute encephalopathy. Magnetic resonance imaging and blood cultures led to the diagnosis of Listeria monocytogenes rhomboencephalitis, which proved to be fatal. Postmortem examination of the brain revealed abscesses with extensive necrosis. Gram stain showed small gram-positive rods in the necrotic tissue and the wall lining the abscesses. While rhomboencephalitis is a rare entity, and clinical recognition may be hampered by immunosuppression such as in this case, early blood cultures, lumbar puncture, and empiric treatment with ampicillin should be initiated in all patients with suspected rhomboencephalitis if suggested by imaging and by a clinical concern for infection.

Listeria monocytogenes brainstem infection (rhombencephalitis) mimicking ischemic stroke.

The frequency of Listeria monocytogenes (Lm) infection of the central nervous system is increasing. We report a patient recently treated with chemotherapeutic drugs for pulmonary adenocarcinoma who suddenly developed hemiparesis, was initially diagnosed with stroke, and was then found to be affected by Lm rhombencephalitis accompanied by a brain abscess. Lm meningoencephalitis mimicking ischemic stroke is rare but must be considered, especially in specific patients.