Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species.

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

Identificación de la formación de cordones de Mycobacterium kansassi e implicancia en la microbiología clínica

Objetivos : Determinar si la formación de cordones ocurre en la microcolonias de M. kansasii. Material y métodos : Se sembraron en medio solido 7H11, cuatro especies de micobacterias patógenas de alta prevalencia Mycobacterium kansasii, Mycobacterium abscessus, Mycobacterium tuberculosis y Mycobacterium neonarum y se evaluaron hasta por 21 días, realizando complementariamente las coloraciones Ziehl-Neelsen para cada una de ellas. Para observar la presencia de la formación de cordones en las microcolonias, se utilizó microscopia de fase invertida. Resultados : En todas las especies se observó a nivel de las microcolonias la formación de cordones, además se identificó la formación de cordones en etapa temprana por la coloración Zhiel-Nelsen en Mycobacterium kansasii, Mycobacterium abscessus, y Mycobacterium tuberculosis. Conclusiones : Mycobacterium kansasii es capaz de desarrollar cordones a nivel microscópico, por lo que la premisa basada en la formación de cordones por M. tuberculosis como un patrón diferencial de las demás micobacterias deben ser tomadas con cautela.

SUMMARY Objectives : Determine if cord formation occurs in microcolonies of M. kansasii. Methods : 4 highly prevalent pathogenic mycobacterial species Mycobacterium kansasii, Mycobacterium abscessus, Mycobacterium tuberculosis and Mycobacterium neonarum were sown in 7H11 solid medium and observed for up to 21 days, additionally Ziehl-Neelsen staining was performed for each of them. Additionally, Ziehl-Neelsen staining was performed for each of them, observing the presence of cord formation in the microcolonies and determining their relationship with virulence and specific species Results : In all the species cultivated in solid medium 7H11, the formation of cords was observed at the level of the microcolones, in addition, the formation of cords in an early stage was identified by the Zhiel-Nelsen staining in Mycobacterium kansasii, Mycobacterium abscessus, and Mycobacterium tuberculosis. Conclusions : Mycobacterium kansasii is capable of developing beads at the microscopic level as was observed in microcoloneas, so the premise based on beads by microscopy specific for the M. tuberculosis species as a differential pattern from the other mycobacteria by forming beads in cultures must be taken with caution so as not to generate a misdiagnosis since there are other species that are capable of forming a similar pattern as has been observed in Mycobacterium kansasii.

A Murine Model of Mycobacterium kansasii Infection Reproducing Necrotic Lung Pathology Reveals Considerable Heterogeneity in Virulence of Clinical Isolates.

Among non-tuberculous mycobacteria, Mycobacterium kansasii is one of the most pathogenic, able to cause pulmonary disease indistinguishable from tuberculosis in immunocompetent susceptible adults. The lack of animal models that reproduce human-like lung disease, associated with the necrotic lung pathology, impairs studies of M. kansasii virulence and pathogenicity. In this study, we examined the ability of the C57BL/6 mice, intratracheally infected with highly virulent M. kansasii strains, to produce a chronic infection and necrotic lung pathology. As a first approach, we evaluated ten M. kansasii strains isolated from Brazilian patients with pulmonary disease and the reference strain M. kansasii ATCC 12478 for virulence-associated features in macrophages infected in vitro; five of these strains differing in virulence were selected for in vivo analysis. Highly virulent isolates induced progressive lung disease in mice, forming large encapsulated caseous granulomas in later stages (120-150 days post-infection), while the low-virulent strain was cleared from the lungs by day 40. Two strains demonstrated increased virulence, causing premature death in the infected animals. These data demonstrate that C57BL/6 mice are an excellent candidate to investigate the virulence of M. kansasii isolates. We observed considerable heterogeneity in the virulence profile of these strains, in which the presence of highly virulent strains allowed us to establish a clinically relevant animal model. Comparing public genomic data between Brazilian isolates and isolates from other geographic regions worldwide demonstrated that at least some of the highly pathogenic strains isolated in Brazil display remarkable genomic similarities with the ATCC strain 12478 isolated in the United States 70 years ago (less than 100 SNPs of difference), as well as with some recent European clinical isolates. These data suggest that few pathogenic clones have been widely spread within M. kansasii population around the world.

Chemical composition and in vitro antibacterial activity of essential oils from Murraya paniculata (L. ) Jack (Rutaceae) ripe and unripe fruits against bacterial genera Mycobacterium and Streptococcus

This study aims to investigate chemical composition of essential oils from Murraya paniculata (L.) Jack (Rutaceae) ripe and unripe fruits and determine their in vitro antibacterial activity. Essential oils were extracted by hydrodistillation from Murraya paniculata (L.) Jack ripe and unripe fruits collected in the Cerrado, in Rio Verde, southwestern Goiás, Brazil. They were analyzed by gas chromatography with flame ionization detector (GC-FID) and by gas chromatography-mass spectrometry (GC-MS). Sesquiterpenes, which represent the most abundant class of compounds in oils, predominated in both ripe and unripe fruits. Major constituents of essential oils extracted from ripe fruits (RF-EO) were (-caryophyllene (21.3%), (-ylangene (13.3%), germacrene-D (10.9%) and (-zingiberene (9.7%) whereas the ones of unripe fruits (UF-EO) were sesquithujene (25.0%), (-zingiberene (18.2%), germacrene-D (13.1%) and (-copaene (12.7%). In vitro antibacterial activity of essential oils was evaluated in terms of its minimum inhibitory concentration (MIC) values by the broth microdilution method in 96-well microplates. Both essential oils under investigation showed moderate anti-streptococcal activity against the following bacteria: Streptococcus mutans, S. mitis, S. sanguinis, S. sobrinus and S. salivarius. MIC values ranged between 100 and 400 µg/mL. Regarding the antimycobacterial activity, essential oils from M. paniculata (L.) Jack unripe and ripe fruits were active against Mycobacterium kansasii (MIC = 250 µg/mL), moderately active against M. tuberculosis (MIC = 500 µg/mL) and inactive against M. avium (MIC = 2000 µg/mL). This study was pioneer in revealing similar chemical profiles of both essential oils extracted from Murraya paniculata (L.) Jack unripe and ripe fruits, besides describing their in vitro anti-streptococcal and antimycobacterial activities.

Características clínicas e epidemiológicas de casos de infecção pulmonar por Mycobacterium kansasii no Rio de Janeiro, no período de 2006 a 2016 / Clinical and epidemiological characteristics of M. kansasii pulmonary infections from Rio de Janeiro, Brazil, between 2006 and 2016

RESUMO Objetivo Avaliar características clínicas, tomográficas e microbiológicas dos pacientes com doença pulmonar causada pela M. kansasii (DPMK) atendidos em unidade ambulatorial no período 2006-2016. Métodos Estudo descritivo, em que foram analisados 38 pacientes. Foram analisadas as características demográficas, clínico-radiológicas, laboratoriais e terapêuticas. Resultados A média de idade foi 64 anos (DP=10,6; IIQ=57-72; mediana=65,0) e 22 (57,9%) eram pacientes do sexo masculino. Comorbidade pulmonar estava presente em 89,5%. A comorbidade mais frequente foi a bronquiectasia (78,9%). Tratamento anterior para tuberculose pulmonar (TBP) foi relatado em 65,9%. O esquema terapêutico mais utilizado foi rifampicina, isoniazida e etambutol (44,7%). A tomografia de tórax (TCT) mostrou bronquiectasia (94,1%), distorção arquitetural (76,5%), espessamento de septo (67,6%) e cavidades (64,7%). A doença foi bilateral em 85,2%. Houve 10,7% de resistência à rifampicina, 67,9% resistentes ao etambutol e sensibilidade à claritromicina. Conclusão Em pacientes com doença pulmonar estrutural, é importante a busca de DPMNT, principal diagnóstico diferencial com TBP. TC de tórax demonstra diferentes padrões que se sobrepõem ao de doença estrutural causada por TBP ou outras enfermidades pulmonares. Destaca-se a resistência ao etambutol, fármaco componente do esquema preconizado.

ABSTRACT Objective To evaluate clinical, tomographic, and microbiological characteristics of pulmonary disease caused by M. kansasii (MKPD) in patients treated at an outpatient unit from 2006-2016. Methods We studied thirty eight patients, and analyzed socio-demographic, clinical-radiological, laboratory, and therapeutic characteristics. Results The mean age was 64 years (SD = 10.6; IIQ = 57-72; median = 65.0), and 22 (57.9%) male patients. Pulmonary comorbidity was present in 89.5% of the patients. The most frequent comorbidity was bronchiectasis (78.9%). Previous treatment for pulmonary tuberculosis (PTB) was found in 65.9%. The most used therapeutic regimen was rifampicin, isoniazid and ethambutol (44.7%). Chest tomography (CT) showed bronchiectasis (94.1%), architectural distortion (76.5%), septum thickening (67.6%), and cavities (64.7%). Disease was bilateral in 85.2%. We observed 10.7% resistance to rifampicin, 67.9% resistance to ethambutol, and sensitivity to clarithromycin. Conclusion In patients with structural lung disease, it is important to search for NTM, the main differential diagnosis with PTB. Chest CT showed different patterns that overlapped with structural disease caused by PTB or other lung diseases. We observed resistance to ethambutol, a drug component of the recommended regimen.

GATA2 mutation in long stand Mycobacterium kansasii infection, myelodysplasia and MonoMAC syndrome: a case-report.

BACKGROUND: GATA2 is a transcription factor that is a critical regulator of gene expression in hematopoietic cells. GATA2 deficiency presents with multi-lineage cytopenia, mycobacterial, fungal and viral infections. Patients with GATA2 mutation have a high risk of developing myelodysplastic syndrome or acute myeloid leukemia. CASE PRESENTATION: We described a 43 years-old white male with 20-year follow-up of autoimmune and thrombotic phenomena, hypothyroidism, disseminated refractory Mycobacterium kansasii infection and MonoMAC syndrome. GATA2 c.1061 C > T; p.T354 M mutation was identified after he progressed from myelodysplastic pancytopenia to refractory anemia with excess blasts type II. His relatives were also investigated and he underwent unsuccessful haematopoietic stem cell transplantation. We discuss the clinical features, genetic diagnosis and treatment of this immunodeficiency disorder. CONCLUSIONS: This case illustrates the challenge how a multidisciplinary disease should be handle. Once usual causes of immunodeficiency were excluded, clinicians should considerGATA2 deficiency in patients with myelodysplasia and long-standing Mycobacterium kansasii infection.

Drug susceptibility profile of Mycobacterium kansasii clinical isolates from Brazil.

OBJECTIVES: Mycobacterium kansasii (M. kansasii) pulmonary infection can cause disease with clinical and radiological features similar to tuberculosis. Failure to treat M. kansasii infection is usually associated with resistance; to increase the chance of successful treatment it is important to identify the species and know the susceptibility profile. This study aimed to evaluate the antimycobacterial susceptibility profiles of M. kansasii isolates from Brazil. METHODS: Sixty-nine M. kansasii isolates from 69 patients were identified by partial sequencing of the hsp65 gene, and their susceptibility profiles were analysed by minimal inhibitory concentration (MIC) assays. RESULTS: From 69 isolates, 68 showed susceptibility to clarithromycin, amikacin, and moxifloxacin. Most strains showed high rates of resistance to trimethoprim-sulfamethoxazole and ciprofloxacin. Resistance to rifampicin and ethambutol was found in 12% and 25% of isolates, respectively. CONCLUSIONS: Worrying results were found regarding susceptibility to some drugs used as first-line agents in the treatment of diseases caused by M. kansasii.

Cutaneous Mycobacterial Infections.

Humans encounter mycobacterial species due to their ubiquity in different environmental niches. In many individuals, pathogenic mycobacterial species may breach our first-line barrier defenses of the innate immune system and modulate the activation of phagocytes to cause disease of the respiratory tract or the skin and soft tissues, sometimes resulting in disseminated infection. Cutaneous mycobacterial infections may cause a wide range of clinical manifestations, which are divided into four main disease categories: (i) cutaneous manifestations of Mycobacterium tuberculosis infection, (ii) Buruli ulcer caused by Mycobacterium ulcerans and other related slowly growing mycobacteria, (iii) leprosy caused by Mycobacterium leprae and Mycobacterium lepromatosis, and (iv) cutaneous infections caused by rapidly growing mycobacteria. Clinically, cutaneous mycobacterial infections present with widely different clinical presentations, including cellulitis, nonhealing ulcers, subacute or chronic nodular lesions, abscesses, superficial lymphadenitis, verrucous lesions, and other types of findings. Mycobacterial infections of the skin and subcutaneous tissue are associated with important stigma, deformity, and disability. Geography-based environmental exposures influence the epidemiology of cutaneous mycobacterial infections. Cutaneous tuberculosis exhibits different clinical phenotypes acquired through different routes, including via extrinsic inoculation of the tuberculous bacilli and dissemination to the skin from other sites, or represents hypersensitivity reactions to M. tuberculosis infection. In many settings, leprosy remains an important cause of neurological impairment, deformity, limb loss, and stigma. Mycobacterium lepromatosis, a mycobacterial species related to M. leprae, is linked to diffuse lepromatous leprosy of Lucio and Latapí. Mycobacterium ulcerans produces a mycolactone toxin that leads to subcutaneous tissue destruction and immunosuppression, resulting in deep ulcerations that often produce substantial disfigurement and disability. Mycobacterium marinum, a close relative of M. ulcerans, is an important cause of cutaneous sporotrichoid nodular lymphangitic lesions. Among patients with advanced immunosuppression, Mycobacterium kansasii, the Mycobacterium avium-intracellulare complex, and Mycobacterium haemophilum may cause cutaneous or disseminated disease. Rapidly growing mycobacteria, including the Mycobacterium abscessus group, Mycobacterium chelonei, and Mycobacterium fortuitum, are increasingly recognized pathogens in cutaneous infections associated particularly with plastic surgery and cosmetic procedures. Skin biopsies of cutaneous lesions to identify acid-fast staining bacilli and cultures represent the cornerstone of diagnosis. Additionally, histopathological evaluation of skin biopsy specimens may be useful in identifying leprosy, Buruli ulcer, and cutaneous tuberculosis. Molecular assays are useful in some cases. The treatment for cutaneous mycobacterial infections depends on the specific pathogen and therefore requires a careful consideration of antimicrobial choices based on official treatment guidelines.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

Micobacteriosis pulmonar por Mycobacterium kansasii / Micobacteriosis pulmonar por Mycobacterium kansasii

Micobacterias no tuberculosas (MNT) es una designación utilizada para referirse a un gran número de especies de micobacterias ambientales potencialmente patógenas y no patógenas, distintas de la Mycobacterium tuberculosis y Mycobacterium leprae. Mycobacterium kansasii (M. Kansasii) es una MNT oportunista causante de infecciones pulmonares, cutáneas, entre otras, cuya tasa de incidencia ha ido incrementando en los últimos años a nivel mundial. A través de presentar el siguiente caso se pretende aportar al conocimiento con respecto al abordaje de pacientes con infección por MNT a nivel pulmonar, dirigido a médicos que trabajan en atención primaria de salud (APS). Se trata del caso de una paciente de 46 años de edad que acude al Hospital Provincial General Docente de Riobamba (HPGDR) con infección por MNT a nivel pulmonar. En el examen microscópico se detectaron Bacilos­Ácido­Alcohol­Resistentes (BAAR) mientras en el cultivo de esputo más antibiograma se aisló M. kansasii resistente a los antibióticos utilizados para la terapia convencional de tuberculosis. Se trata de un caso raro en la práctica clínica. Es crucial saber cómo manejar una infección con M. kansasii debido a su implicación para la salud del paciente y el sistema de salud nacional. El médico de APS debe reconocer su papel fundamental y la importancia que tiene un diagnóstico oportuno y tratamiento adecuado.

Non­tuberculous mycobacteria (NTM) is a designation for a large number of mycobacterial species potentially pathogenous and non­pathogenous, different than Mycobacterium tuberculosis and Mycobacterium leprae. Mycobacterium kansasii (M. kansasii) is an opportunistic NTM that causes among other things, pulmonary and cutaneous infections, whose incidence is increasing worldwide. Trough the following case report we seek to provide a guide to physicians working on primary health care (PHC) on the management of patients with pulmonary infection caused by NTM. We report the case of a 46­year­old female patient who came to the Hospital Provincial General Docente de Riobamba (HPGDR) with a pulmonary infection caused by NTM. In the microscopic examination it was identified Acid­Fast Bacilli (AFB), meanwhile the microbiological culture and antibiogram it was isolated M. kansasii resistant to common antibiotics used for conventional tuberculosis therapy. It is a rare case in the clinical practice. It is crucial to know how to manage an infection with M. Kansasii due to its implication on the health of affected subjects and the national health system. A physician working on PHC has to know his/her fundamental role and the importance of an early diagnosis and adequate treatment.

Micobacterias atípicas en cinco pacientes adultos sin evidencias de inmunosupresión: Construyendo una experiencia / Atypical mycobacterial infections in five adult patients without evidence of immunosuppression: Making an experience

We aim to communicate the experience gathered during the management of infections by atypical mycobacteria in immunocompetent patients in a general practice. Between 2008 and 2013, 5 patients with non-tuberculous mycobacterial infections were identified: 2 with cutaneous involvement and 3 with lung infection. None of them had evidence of immunosuppression. A patient with elbow bursitis by M. chelonae presented with a high mononuclear count in fluid analysis with mycobacterial growth at the fifth day of culture. He evolved satisfactorily with clarithromycin. A case with M. fortuitum skin infection had a delayed initial diagnosis with progression to local draining lymph nodes; the culture when requested was positive after 13 days of incubation. Patients with pulmonary infection presented with prolonged cough and sputum and had in common to be postmenopausal women displaying small nodules and bronchiectases at lung images, a classical pattern. Time elapsed between respiratory sampling and a definitive inform ranged from 40 to 89 days. Non-tuberculous mycobacterial infections in non-immunosuppresed patients can generate diagnostic and therapeutic challenges. Delay in identification contributes to this problem.

El objetivo de este trabajo es reportar la experiencia acumulada sobre infecciones por micobacterias atípicas en pacientes sin inmunosupresión. Entre el año 2008 y 2013 se observaron cinco pacientes con infección por micobacterias atípicas: dos con infección cutánea y tres con infección pulmonar. Ninguno de estos pacientes tenía evidencias de inmunosupresión. Un paciente con bursitis de codo por M. chelonae tuvo un estudio citoquímico con aumento de celularidad de predominio mononuclear y desarrollo de bacterias al quinto día; respondió favorablemente a claritromicina. Un caso con infección cutánea por M. fortuitum evolucionó en forma prolongada con supuración ganglionar antes del diagnóstico y el cultivo solicitado a los 13 días fue positivo. Los tres pacientes con aislados pulmonares presentaron tos y expectoración y tenían en común ser mujeres en edad post-menopáusica y presentar pequeños infiltrados nodulares asociados a bronquiectasias en el estudio de imágenes pulmonares, un patrón descrito en la literatura científica. En estos tres casos, la latencia entre la toma de muestra y el informe definitivo tuvo un rango de 40 a 89 días. El aislamiento de micobacterias atípicas en muestras de expectoración en pacientes sin inmunosupresión se da en un contexto típico pero plantea dificultades diagnósticas y terapéuticas. El lento crecimiento de estos microorganismos en el laboratorio contribuye a este problema.

Abscess resulting from Mycobacterium kansasii in the left thigh of AIDS patient

A case of abscess resulting from Mycobacterium kansasii, in the left thigh of a 53-year-old woman infected with the Human Immunodeficiency virus, is reported. Curiously, there was no pulmonary or systemic involvement as is usual with these Mycobacterium infections. The patient had CD4 T lymphocyte count of 257 cells/µL and a viral load of 60,154 copies. Despite presenting a relatively preserved immunity, the patient also presented Criptococcic meningoencephalitis and Esophageal candidiasis. The patient responded satisfactorily to treatment for infections and after 51 days was discharged.

Investigation of the antimycobacterial activity of 36 plant extracts from the Brazilian Atlantic Forest / Análise da atividade antimicobacteriana de 36 extratos vegetais da Mata Atlântica brasileira

Thirty-six plant extracts from the brazilian Atlantic Forest were tested for their antimycobacterial activity against Mycobacterium tuberculosis H37Rv and M. kansasii, using the method REMA in seriate concentrations of 100 to 0.20 µg/mL. Among the thirty six extracts tested, five were active against M. tuberculosis, and three of these extracts also showed activity against M. kansasii. Cytotoxicity test with VERO cells was performed with the five extracts active against M. tuberculosis. Only the extract of Peschiera affinis was identified as non-toxic in the concentration of 100µg/mL.

Trinta e seis extratos vegetais originários da Mata Atlântica foram testados quanto à sua atividade antimicobacteriana frente ao M. tuberculosis H37Rv e M. kansasii, utilizando o método REMA em concentrações seriadas de 100 a 0,20 µg/mL. Dentre os trinta e seis extratos testados, cinco mostraram atividade frente ao M. tuberculosis, e destes apenas, três mostraram atividade ao M. kansasii, que apresentou susceptibilidade a outros dez. O teste de citotoxicidade com células VERO foi realizado com os cinco extratos ativos frente ao M. tuberculosis em que identificou-se a não toxicidade em apenas um extrato (Peschiera affinis) na concentração de 100 µg/mL.