Successful diagnosis of Mycobacterium marinum infection by mNGS in a patient with Human Immunodeficiency Virus: a case report.

INTRODUCTION: Mycobacterium marinum infection rarely occurs and has atypical symptoms. It is challenging to distinguish disseminated M. marinum infection from multifocal dermatosis caused by other factors clinically. CASE PRESENTATION: Herein, we reported a 68-year-old male patient with Human Immunodeficiency Virus (HIV) who presented redness and swelling in his left hand after being stabbed by marine fish for over 2 months. Mycobacterium tuberculosis infection was considered according to biochemical and pathological examinations, while empirical anti-infection treatment was ineffective. RESULTS: The metagenomic next-generation sequencing (mNGS) detected a large amount of M. marinum sequences, and the patient was finally diagnosed with M. marinum infection. After one month of combination therapy with ethambutol, rifabutin, moxifloxacin, and linezolid, the swelling disappeared significantly. In this case, the successful application of mNGS in diagnosing and treating M. marinum infection has improved the understanding of the microbe both in the laboratory and clinically, especially in patients with HIV. CONCLUSIONS: For diseases with atypical symptoms or difficulty in determining the pathogens, mNGS is suggested in clinical procedures for rapid and accurate diagnosis and treatment.

Histopathology of laboratory-reared Nothobranchius fishes: Mycobacterial infections versus neoplastic lesions.

Short-lived killifishes of the genus Nothobranchius Peters, 1868 (Cyprinodontiformes) are considered promising model organisms for biomedical research on ageing and tumorigenesis. We conducted histopathological analysis of 411 adult individuals from three Nothobranchius species to study details on spontaneous age-related neoplastic lesions. Light microscopy based on H&E and toluidine blue-stained sections revealed (a) non-proliferative liver changes with pronounced vacuolation of hepatocytes; (b) proliferation of kidney haemopoietic tissue contributing to excretory system damage; (c) proliferation of splenic mononuclear haemoblasts accompanied by reduced erythropoiesis; (d) proliferation of mononuclear cell aggregates in the liver parenchyma; and (e) rare occurrence of hepatocellular adenomas. Ziehl-Neelsen (ZN) staining revealed that the proliferative lesions are a host defence response to mycobacterial infections manifested by activation of the mononuclear phagocytic system and atypical granulomatous inflammatory reaction. 16S rRNA analysis identified three species of Mycobacterium in our samples. Our findings turn attention to lesions which mimic neoplasms by their gross appearance and question the light microscopic interpretation of lesions unless differential ZN staining is included. Beyond the limitations of our morphological approach, the intensity of mycobacterial infections is a challenging opportunity for research into the molecular-genetic background of the mononuclear phagocytic system reaction in Nothobranchius killifish.

A non-canonical type 2 immune response coordinates tuberculous granuloma formation and epithelialization.

The central pathogen-immune interface in tuberculosis is the granuloma, a complex host immune structure that dictates infection trajectory and physiology. Granuloma macrophages undergo a dramatic transition in which entire epithelial modules are induced and define granuloma architecture. In tuberculosis, relatively little is known about the host signals that trigger this transition. Using the zebrafish-Mycobacterium marinum model, we identify the basis of granuloma macrophage transformation. Single-cell RNA-sequencing analysis of zebrafish granulomas and analysis of Mycobacterium tuberculosis-infected macaques reveal that, even in the presence of robust type 1 immune responses, countervailing type 2 signals associate with macrophage epithelialization. We find that type 2 immune signaling, mediated via stat6, is absolutely required for epithelialization and granuloma formation. In mixed chimeras, stat6 acts cell autonomously within macrophages, where it is required for epithelioid transformation and incorporation into necrotic granulomas. These findings establish the signaling pathway that produces the hallmark structure of mycobacterial infection.

Mycobacterium marinum: nodular hand lesions after a fishing expedition.

Mycobacterium marinum is a slow-growing, acid-fast bacillus in the category of non-tuberculous mycobacteria which most commonly cause skin and soft tissue infections in patients, particularly those with aquatic exposure. Classically, M. marinum skin and soft tissue infections clinically manifest with formation of nodular or sporotrichoid extremity lesions, or deeper space infections such as tenosynovitis and osteomyelitis. Disseminated disease may occur in immunocompromised hosts. M. marinum is a slow-growing organism that is challenging to culture, as it typically requires 5-14 days (yet may take up to several weeks) with low temperatures of approximately 30°C to yield growth. In terms of treatment, further data are needed to elucidate the optimal regimen and duration for M. marinum infections. Combination therapy with clarithromycin and ethambutol is recommended for treatment of skin and soft tissue infections, with addition of rifampicin for deeper space infections. Surgery may be needed in addition to medical management.

Mycobacterium marinum infection simulating chromomycosis: a case report.

Skins infections caused by Mycobacterium marinum occur only rarely. We report one case of chronic and extensive M. marinum cutaneous infection simulating chromoblastomycosis and review the pertinent literature. A 52-year-old farmer reported a 32-year chronic skin problem on his right lower limb, resulting from contact with cacti. It consisted of skin lesion presenting with dyschromic atrophic center plate and verrucous borders with hematic crusts, extending from the knee anteriorly to the inferior third of the right leg. Mycobacterium marinum infection was detected by histopathological examination of a skin fragment, culture for mycobacteria and genetic mapping of the culture material. The patient was successfully treated with Ethambutol, Rifampicin and Trimethoprim-Sulfamethoxazole. The clinical and histopathological findings of M. marinum infection is nonspecific showing clinical polymorphism and bacilli are rarely evident on histopathological examination. Given these difficulties, it is essential to perform tissue culture in a suspicious case and it is important keep this infection in mind in patients with long-lasting indolent verrucous lesions and a history of exposure to sea water, freshwater, aquaria or fish.

Solid lipid nanoparticles containing anti-tubercular drugs attenuate the Mycobacterium marinum infection.

The present study aimed to formulate anti-tubercular drugs (Rifampicin, Isoniazid and Pyrazinamide) loaded solid lipid nanoparticles (ATDs-SLNs) using microemulsion technique for oral administration. Central composite designed (CCD) was applied to study the effect of stearic acid (X1), Compritol® 888 ATO (X2) and equal ratio of poloxamer 188: sodium taurocholate (% w/w) (X3) on particle size, zeta potential and entrapment efficiency. The optimised formulation (SLN8) was found to be spherical in shape with mean particle size 187.9 ± 10.73 nm and zeta potential -47.4 mV. The maximum percentage entrapment of RIF, INH and PYZ in the optimised formulation was found to be 86.40 ± 0.274, 83.84 ± 0.269 and 81.43 ± 0.576, respectively. The in-vitro drug release study demonstrated that the release of drug from SLNs was slow in comparison to marketed formulation and pure ATDs. Cytotoxicity of the ATDs-SLNs was studied on murine macrophage cell line (RAW 264.7) using modified MTT assay demonstrated two folds growth inhibition of M. marinum as compared to standard antitubercular drugs. Overall, the developed SLNs may be considered as a promising anti-mycobacterial nano-drug, providing a new direction to the tuberculosis clinics.

Infección por Mycobacterium marinum en una paciente en tratamiento con adalimumab / Mycobacterium marinum Infection in a Woman Taking Adalimumab

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First Etiologically Confirmed Cases of Mycobacterium Marinum Infection in Bulgaria.

This study aimed to describe the first two microbiologically confirmed cases of cutaneous and soft tissue Mycobacterium marinum infection in Bulgaria. The isolation of the Nontuberculous Mycobacteria (NTM) strains and their species identification was performed at NRL TB, NCIPD using specific media and cultivation conditions, and PCR based Line Probe Assay (LPA) from the positive cultures. The two patients had closely related jobs to fishes and water reservoirs and both of them had a similar clinical manifestation of M. mari-num infection known as "swimming pool" or "fish tank" granuloma. The prolonged specific treatment with at least two-drug combina-tion of rifampicin plus ethambutol and some complications were a big challenge for clinicians as well as the patients.

Quantification of Natural Growth of Two Strains of Mycobacterium Marinum for Translational Antituberculosis Drug Development.

The zebrafish infected with Mycobacterium marinum (M. marinum) is an attractive tuberculosis disease model, showing similar pathogenesis to Mycobacterium tuberculosis (M. tuberculosis) infections in humans. To translate pharmacological findings from this disease model to higher vertebrates, a quantitative understanding of the natural growth of M. marinum in comparison to the natural growth of M. tuberculosis is essential. Here, the natural growth of two strains of M. marinum, E11 and MUSA , is studied over an extended period using an established model-based approach, the multistate tuberculosis pharmacometric (MTP) model, for comparison to that of M. tuberculosis. Poikilotherm-derived strain E11 and human-derived strain MUSA were grown undisturbed up to 221 days and viability of cultures (colony forming unit (CFU)/mL) was determined by plating at different time points. Nonlinear mixed effects modeling using the MTP model quantified the bacterial growth, the transfer among fast, slow, and non-multiplying states, and the inoculi. Both strains showed initial logistic growth, reaching a maximum after 20-25 days for E11 and MUSA , respectively, followed by a decrease to a new plateau. Natural growth of both E11 and MUSA was best described with Gompertz growth functions. For E11, the inoculum was best described in the slow-multiplying state, for MUSA in the fast-multiplying state. Natural growth of E11 was most similar to that of M. tuberculosis, whereas MUSA showed more aggressive growth behavior. Characterization of natural growth of M. marinum and quantitative comparison with M. tuberculosis brings the zebrafish tuberculosis disease model closer to the quantitative translational pipeline of antituberculosis drug development.

Fish tank granuloma: An emerging skin disease in Iran mimicking Cutaneous Leishmaniasis.

OBJECTIVE: Mycobacterium marinum causes a rare cutaneous disease known as fish tank granuloma (FTG). The disease manifestations resemble those associated with Cutaneous Leishmaniasis (CL). The aim of this study was to determine whether FTG was the cause of cutaneous lesions in patients who were referred to the Parasitology laboratory of Imam Reza Hospital in Mashhad to be investigated for CL. MATERIALS/METHODS: One hundered patients, clinically diagnosed with CL between April 2014 and March 2015, were included in this study. Ziehl-Neelsen staining was performed to identify acid-fast Mycobacterium in addition to bacterial cultures using Löwenstein-Jensen medium. Skin lesion samples were also collected and kept on DNA banking cards for PCR testing. RESULTS: Twenty-nine of the 100 individuals with skin lesions, and therefore suspected of suffering from CL, tested positive for Mycobacterium marinum by PCR. Of these, 21 (72.4%) were male and 8(27.6%) were female. In 97% of these cases the lesions were located on hands and fingers. These patients had a history of manipulating fish and had been in contact with aquarium water. A sporotrichoid appearance was observed in 58.6% of the patients with mycobacterial lesions; 67% of patients had multiple head appearance. CONCLUSION: Patients suspected to have CL and who test negative for CL could be affected by FTG. Therefore, after obtaining an accurate case history, molecular diagnosis is recommended for cases that give a negative result by conventional methods.

Deep infection with Mycobacterium marinum: successful treatment of a frequently misdiagnosed disease.

We report a rare yet typical presentation of a severe infection with Mycobacterium marinum that affected the deep structure of the hand and wrist of a 43-old fish breeder. A combination therapy of surgical debridement and antibiotic treatment with clarithromycin and ethambutol for 6 months led to a total resolution of the symptoms. Intensive rehabilitation completely restored the function of the hand.

Establishment of loop-mediated isothermal amplification for rapid and convenient detection of Mycobacterium marinum complex.

Mycobacterium marinum is a zoonotic pathogen that can cause dermatological infection mainly from contaminated water or fish. Some well-known genetically similar species and subspecies are M. lifrandii and M. pseudoshottsii from amphibians and fish in aquaculture, and M. ulcerans, a causative agent of a neglected tropical disease (NTD), Buruli ulcer. They are believed to survive in water as their major niche, which might be related to their source of infection, but detailed ecological surveillance of the species complex remains to be done. Herein, we present a new detection system for M. marinum complex based on isothermal DNA amplification that can be conducted conveniently with high sensitivity and specificity. The target was a chromosomal gene, mrsA, including a restriction polymorphism between M. ulcerans (except for the most ancestral subspecies, M. ulcerans subsp. shinshuense) and the other species. The system was able to detect less than 500 fg (approximately 70 copies) of genomic DNA of M. marinum, within 60 min, and caused no amplification from mycobacterial species other than M. marinum complex species. It was also verified that restriction of the amplified DNA fragments was able to discriminate M. ulcerans as expected. This easy, quick, and convenient system is expected to facilitate detection of M. marinum complex from various resources.

Placa granulomatosa en la rodilla derecha de un niño de 10 años de edad / Granulomatous plaque of the right knee in a 10-year-old boy

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Investigation of a community cluster of cutaneous Mycobacterium marinum infection, an emerging zoonotic pathogen in aquaculture industry, Haliburton, Kawartha, Pine Ridge District Health Unit, Ontario, Canada, July-August 2015.

In July 2015, a cluster of five suspect cases of clinically diagnosed Mycobacterium marinum (M. marinum) skin infections were reported to the Haliburton, Kawartha, Pine Ridge District Health Unit (HKPRDHU), Ontario, Canada, with two additional cases subsequently identified through case finding. All seven cases presented with cutaneous lesions located on the finger, hand and/or elbow regions typical of M. marinum infection. Specimens were collected by skin biopsy for two of the seven cases; both cases tested positive for M. marinum by molecular detection (hsp65 gene amplification and sequencing), and one was confirmed positive for M. marinum by culture. All seven cases reported handling raw shrimp from an aquaculture facility in the Health Unit's jurisdiction. M. marinum is not a reportable disease in Ontario, and there are no known previous reports of a cluster of M. marinum reported in Ontario, Canada. A cluster investigation working group was struck that included representation from various agencies including Public Health Ontario (PHO), Public Health Ontario Laboratories (PHOL), Ontario Ministry of Agriculture and Rural Affairs (OMAFRA) and the two health units involved in case investigations. Several public health and aquaculture farming recommendations were made to mitigate further risks associated with handling of raw shrimp from the facility. Several challenges were faced during the investigation process. The paper discusses these challenges and public health recommendations made in order to mitigate occupational and public health risks related to the hazard identified.

Extensive genomic diversity among Mycobacterium marinum strains revealed by whole genome sequencing.

Mycobacterium marinum is the causative agent for the tuberculosis-like disease mycobacteriosis in fish and skin lesions in humans. Ubiquitous in its geographical distribution, M. marinum is known to occupy diverse fish as hosts. However, information about its genomic diversity is limited. Here, we provide the genome sequences for 15 M. marinum strains isolated from infected humans and fish. Comparative genomic analysis of these and four available genomes of the M. marinum strains M, E11, MB2 and Europe reveal high genomic diversity among the strains, leading to the conclusion that M. marinum should be divided into two different clusters, the "M"- and the "Aronson"-type. We suggest that these two clusters should be considered to represent two M. marinum subspecies. Our data also show that the M. marinum pan-genome for both groups is open and expanding and we provide data showing high number of mutational hotspots in M. marinum relative to other mycobacteria such as Mycobacterium tuberculosis. This high genomic diversity might be related to the ability of M. marinum to occupy different ecological niches.

Quantitative N-Terminal Footprinting of Pathogenic Mycobacteria Reveals Differential Protein Acetylation.

N-terminal acetylation (NTA) is a post-transcriptional modification of proteins that is conserved from bacteria to humans. In bacteria, the enzymes that mediate protein NTA also promote antimicrobial resistance. In pathogenic mycobacteria, which cause human tuberculosis and other chronic infections, NTA has been linked to pathogenesis and stress response, yet the fundamental biology underlying NTA of mycobacterial proteins remains unclear. We enriched, defined, and quantified the NT-acetylated populations of both cell-associated and secreted proteins from both the human pathogen, Mycobacterium tuberculosis, and the nontuberculous opportunistic pathogen, Mycobacterium marinum. We used a parallel N-terminal enrichment strategy from proteolytic digests coupled to charge-based selection and stable isotope ratio mass spectrometry. We show that NTA of the mycobacterial proteome is abundant, diverse, and primarily on Thr residues, which is unique compared with other bacteria. We isolated both the acetylated and unacetylated forms of 256 proteins, indicating that NTA of mycobacterial proteins is homeostatic. We identified 16 mycobacterial proteins with differential levels of NTA on the cytoplasmic and secreted forms, linking protein modification and localization. Our findings reveal novel biology underlying the NTA of mycobacterial proteins, which may provide a basis to understand NTA in mycobacterial physiology, pathogenesis, and antimicrobial resistance.