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1.
Rev Chilena Infectol ; 37(3): 252-256, 2020 Jun.
Artigo em Espanhol | MEDLINE | ID: mdl-32853316

RESUMO

BACKGROUND: Mycobacterial diseases are very important both clinically and epidemiologically. Mycobacterium tuberculosis complex (MTBc) infections confer higher morbidity and mortality rate than non-tuberculous mycobacteria (NTM) infections. Traditional species identification techniques are based on phenotypic characteristics which take a long time by laborious processes and in occasions are no conclusive. Currently, most used techniques are based on molecular methods, which are accurate but are expensive and complex. Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) is a simple, cheap and fast identification method based on comparing protein spectra with a reference database. AIM: To assess the performance of MALDI-TOF MS in the identification of MTBc and NTM, compared with molecular methods. METHODS: For that purpose, 28 isolates of 9 different species were analyzed through MALDI-TOF MS. RESULTS: 78.5% (22/28) of isolates were correctly identified, 100% (9/9) of rapidly growers NTM, 60% (9/15) of slow growing NTM and 100% (4/4) of MTBc. Every unidentified isolate (6/6) corresponded to M. avium/intracellulare complex. CONCLUSION: MALDI-TOF MS is fast, simple and cheaper than molecular methods and also has adequate accuracy.


Assuntos
Mycobacterium , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tuberculose
2.
Nat Commun ; 11(1): 4196, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826907

RESUMO

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.


Assuntos
Proteínas de Bactérias/química , DNA Primase/química , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA/química , Mycobacterium/genética , Mycobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
3.
Dermatol Online J ; 26(3)2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-32609439

RESUMO

BACKGROUND: New treatment options for warts include intralesional wart injection with agents such as vitamin D, measles, mumps, and rubella (MMR) vaccine antigen, Bacillus Calmette-Guerin (BCG) antigen, and candida antigen but there have been limited studies to compare their efficacies. OBJECTIVE: The purpose of this systematic review is to compare the efficacy and safety of injectable agents used for the treatment of warts. METHODS: A PubMed search included terms "intralesional wart therapy," "wart injection" and "verruca injection." Articles reviewed were published over 10 years. RESULTS: A total of 43 articles were reviewed; 30 covered studies with more than 10 participants and 13 were case reports, case series, and reviews. In comparison studies intralesional agents have equal or superior efficacy (66%-94.9%) compared to first-line salicylic acid or cryotherapy (65.5-76.5%). One advantage of intralesional injections is the rate of complete resolution of distant warts. LIMITATIONS: Each study varied in their agents, treatment interval, and treatment dose, making comparisons difficult. CONCLUSIONS: Intralesional wart injections are safe, affordable, and efficacious treatments for warts. Physicians should consider intralesional injections for patients with refractory warts, multiple warts, or warts in sensitive areas.


Assuntos
Injeções Intralesionais , Verrugas/tratamento farmacológico , Ácido Aminolevulínico/administração & dosagem , Antibacterianos/administração & dosagem , Antivirais/administração & dosagem , Vacina BCG/administração & dosagem , Vacinas Bacterianas/administração & dosagem , Humanos , Interferon-alfa/administração & dosagem , Mycobacterium , Tuberculina/administração & dosagem , Vitamina D/administração & dosagem
4.
Arch Microbiol ; 202(7): 1965-1976, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32474645

RESUMO

No genomic sequence of Mycobacterium isolated from orchids has been reported yet; therefore, this study intends to analyze the complete genomic sequence of a growth-promoting Mycobacterium from orchid Doritaenopsis. Mycobacterium strain Mya-zh01 was isolated from the flower stalk of Doritaenopsis Jiuhbao Red Rose. Our results show that Mya-zh01 can effectively produce and secrete the plant growth hormone indole-3-acetic acid (IAA). Inoculation of Mya-zh01 increased root number and length, plant height, leaf number, and leaf length in Doritaenopsis. Furthermore, inoculation of Mya-zh01 promotes seed germination in Doritaenopsis. We sequenced and assembled chromosome for Mya-zh01 (5,027,704 bp with 68.48% GC content), which was predicted to encode 4968 proteins with functions in oxidation reduction, growth, plasma membrane, ATP and DNA binding, carbon metabolism and biosynthesis of amino acids pathways. Mya-zh01 may trap iron from nature or host cells to facilitate the growth of the orchids by producing two siderophores (Mycobactin and Nocobactin NA). Four pathways (tryptamine, indole-3-acetamide, indole-3-pyruvate, and flavin monooxygenase) and seven enzymes [tryptophan synthase alpha chain, tryptophan synthase beta chain, amidase, monoamine oxidase, indole-3-pyruvate monooxygenase, indole-3-pyruvate decarboxylase and aldehyde dehydrogenase (NAD +)] involved in IAA biosynthesis were predicted in Mya-zh01 genome. In conclusion, this study demonstrated the significance of Mya-zh01 in facilitating plant growth and seed germination in Doritaenopsis by IAA biosynthesis, which provides a new insight into the mechanism of plant-bacteria interaction in Doritaenopsis.


Assuntos
Mycobacterium/genética , Orchidaceae/microbiologia , Desenvolvimento Vegetal , Sementes/microbiologia , Flores/microbiologia , Germinação , Mycobacterium/isolamento & purificação , Mycobacterium/metabolismo
5.
An Bras Dermatol ; 95(4): 511-513, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32522448

RESUMO

The incidence of nontuberculous mycobacterial infections is increasing worldwide; by 2017, more than 190 species and subspecies have been documented. Although classically associated with immunosuppression, the recognition of these etiological agents in diseases affecting immunocompetent individuals and in healthcare-associated infections, such as after surgical and cosmetic procedures, makes the study of the epidemiology and pathogenesis of these microorganisms relevant in medical practice. Mycobacterium lentiflavum is slow-growing and rarely affects the skin. A case of cutaneous mycobacteriosis caused by M. lentiflavum is reported in an immunocompetent patient after subcutaneous injection of a lipolytic compound, treated with clarithromycin and levofloxacin.


Assuntos
Infecções por Mycobacterium não Tuberculosas , Mycobacterium , Dermatopatias Bacterianas , Antibacterianos , Humanos , Injeções Subcutâneas , Micobactérias não Tuberculosas
6.
PLoS Negl Trop Dis ; 14(5): e0008230, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32401750

RESUMO

BACKGROUND: Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB), is composed of eight subspecies. TB in West Africa, in contrast to other geographical regions, is caused by Mycobacterium africanum (MAF) in addition to M. tuberculosis (MTB), with both infections presenting similar symptoms. Nevertheless, MAF is considered to be hypovirulent in comparison with MTB and less likely to progress to active disease. In this study, we asked whether MAF and MTB infected patients possess distinct intestinal microbiomes and characterized how these microbiota communities are affected by anti-tuberculosis therapy (ATT). Additionally, we assessed if the changes in microbiota composition following infection correlate with pathogen induced alterations in host blood-gene expression. METHODS: A longitudinal, clinical study of MAF infected, MTB infected patients assessed at diagnosis and two months after start of ATT, and healthy, endemic controls was conducted to compare compositions of the fecal microbiome as determined by 16S rRNA sequencing. A blood transcriptome analysis was also performed on a subset of subjects in each group by microarray and the results cross-compared with the same individual's microbiota composition. FINDINGS: MAF participants have distinct microbiomes compared with MTB patients, displaying decreased diversity and increases in Enterobacteriaceae with respect to healthy participants not observed in the latter patient group. Interestingly, this observed elevation in Enterobacteriaceae positively correlated with enhanced inflammatory gene expression in peripheral blood and was reversed after initiation of ATT. INTERPRETATION: Our findings indicate that MAF and MTB have distinct associations with the gut microbiome that may be reflective of the differential susceptibility of West Africans to these two co-endemic infections either as biomarkers or as a contributing determinant.


Assuntos
Bactérias/isolamento & purificação , Microbioma Gastrointestinal , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium/isolamento & purificação , Tuberculose/microbiologia , Adulto , Idoso , Bactérias/classificação , Bactérias/genética , Estudos de Coortes , Fezes/microbiologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mycobacterium/classificação , Mycobacterium/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Adulto Jovem
8.
Medicine (Baltimore) ; 99(16): e19697, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32311949

RESUMO

This study aims to elucidate the strains and drug resistance of mycobacterium isolated from osteoarticular tuberculosis (OATB) patients and provide a reference for the diagnosis and treatment of OATB.Sixty-nine clinically diagnosed and surgically treated OATB patients were collected in time period of January 2017 to December 2018 at the First Affiliated Hospital of Xinjiang Medical University. The BACTEC MGIT 960 system was used for mycobacteria culturing, strain identification, and drug susceptibility testing, and the mycobacteria culture positive rate, species distribution, and drug resistance were analyzed.Within 4 weeks, 24 (34.78%) isolates of mycobacteria culture were positive; 40 (57.97%) isolates were positive, when culturing time was expanded to 8 weeks, and the difference was statistically significant (P < .05). Among the 40 isolates, 24 (60%) were identified as mycobacterium tuberculosis (MTB), 10 (25%) were Mycobacterium bovis, and 6 (15%) were non-tuberculous mycobacteria (NTM). Among total 69 isolates, 40 were enrolled in drug sensitivity test, and 15 (37.5%) isolates were confirmed drug resistant strains, in which 5 isolates were MTB, 4 isolates were M. bovis, and 6 isolates of NTM.The pathogen of clinically diagnosed OATB was mainly MTB. However, M. bovis and NTM also accounted for a considerable proportion, and their drug resistance rate was higher. Extending the culturing time appropriately could improve the culture positive rate. NTM was a drug resistant strain, and mycobacteria culturing, strain identification, and drug resistance analysis should be carried out to serve as a guide for individual treatment.


Assuntos
Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana , Mycobacterium/efeitos dos fármacos , Tuberculose Osteoarticular/tratamento farmacológico , Tuberculose Osteoarticular/microbiologia , Adolescente , Adulto , Idoso , Criança , China , Feminino , Humanos , Masculino , Técnicas Microbiológicas , Pessoa de Meia-Idade , Mycobacterium/isolamento & purificação , Adulto Jovem
9.
PLoS One ; 15(4): e0231372, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324750

RESUMO

BACKGROUND: Rwanda conducted a national tuberculosis (TB) prevalence survey to determine the magnitude of TB in the country and determine to what extent the national surveillance system captures all TB cases. In addition we measured the patient diagnostic rate, comparing the measured TB burden data with the routine surveillance data to gain insight into how well key population groups are being detected. METHODS: A national representative nationwide cross-sectional survey was conducted in 73 clusters in 2012 whereby all enrolled participants (residents aged 15 years and above) were systematically screened for TB by symptoms and chest X-ray (CXR). Those with either clinical symptoms (cough of any duration) and/or CXR abnormalities suggestive of TB disease were requested to provide two sputum samples (one spot and one morning) for smear examination and solid culture. RESULTS: Of the 45,058 eligible participants, 43,779 were enrolled in the survey. Participation rate was high at 95.7% with 99.8% of participants undergoing both screening procedures and 99.0% of those eligible for sputum examination submitting at least one sputum sample. Forty cases of prevalent mycobacterium tuberculosis (MTB) and 16 mycobacteria other than tuberculosis (MOTT) cases were detected during the survey. Chest x-ray as screening tool had 3 and 5 times greater predictive odds for smear positive and bacteriological confirmed TB than symptom screening alone respectively. A TB prevalence of 74.1 (95% CI 48.3-99.3) per 100,000 adult population for smear positive TB and 119.3 (95% CI 78.8-159.9) per 100,000 adult population for bacteriological confirmed MTB was estimated for Rwanda. CONCLUSIONS: The survey findings indicated a lower TB prevalence than previously estimated by WHO providing key lessons for national TB control, calling for more sensitive screening and diagnostic tools and a focus on key populations. Use of chest x-ray as screening tool was introduced to improve the diagnostic yield of TB.


Assuntos
Tuberculose/diagnóstico , Adolescente , Adulto , Estudos Transversais , Feminino , Inquéritos Epidemiológicos , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Prevalência , Ruanda/epidemiologia , Escarro/microbiologia , Tuberculose/diagnóstico por imagem , Tuberculose/epidemiologia , Adulto Jovem
10.
PLoS Negl Trop Dis ; 14(4): e0008276, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339201

RESUMO

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae) and the more recently discovered Mycobacterium lepromatosis (M. lepromatosis). The two leprosy bacilli cause similar pathologic conditions. They primarily target the skin and the peripheral nervous system. Currently it is considered a Neglected Tropical Disease, being endemic in specific locations within countries of the Americas, Asia, and Africa, while in Europe it is only rarely reported. The reason for a spatial inequality in the prevalence of leprosy in so-called endemic pockets within a country is still largely unexplained. A systematic review was conducted targeting leprosy transmission research data, using PubMed and Scopus as sources. Publications between January 1, 1945 and July 1, 2019 were included. The transmission pathways of M. leprae are not fully understood. Solid evidence exists of an increased risk for individuals living in close contact with leprosy patients, most likely through infectious aerosols, created by coughing and sneezing, but possibly also through direct contact. However, this systematic review underscores that human-to-human transmission is not the only way leprosy can be acquired. The transmission of this disease is probably much more complicated than was thought before. In the Americas, the nine-banded armadillo (Dasypus novemcinctus) has been established as another natural host and reservoir of M. leprae. Anthroponotic and zoonotic transmission have both been proposed as modes of contracting the disease, based on data showing identical M. leprae strains shared between humans and armadillos. More recently, in red squirrels (Sciurus vulgaris) with leprosy-like lesions in the British Isles M. leprae and M. lepromatosis DNA was detected. This finding was unexpected, because leprosy is considered a disease of humans (with the exception of the armadillo), and because it was thought that leprosy (and M. leprae) had disappeared from the United Kingdom. Furthermore, animals can be affected by other leprosy-like diseases, caused by pathogens phylogenetically closely related to M. leprae. These mycobacteria have been proposed to be grouped as a M. leprae-complex. We argue that insights from the transmission and reservoirs of members of the M. leprae-complex might be relevant for leprosy research. A better understanding of possible animal or environmental reservoirs is needed, because transmission from such reservoirs may partly explain the steady global incidence of leprosy despite effective and widespread multidrug therapy. A reduction in transmission cannot be expected to be accomplished by actions or interventions from the human healthcare domain alone, as the mechanisms involved are complex. Therefore, to increase our understanding of the intricate picture of leprosy transmission, we propose a One Health transdisciplinary research approach.


Assuntos
Reservatórios de Doenças , Transmissão de Doença Infecciosa , Hanseníase/transmissão , Hanseníase/veterinária , Animais , Tatus/microbiologia , Saúde Global , Humanos , Incidência , Hanseníase/epidemiologia , Mycobacterium/isolamento & purificação , Mycobacterium leprae/isolamento & purificação , Prevalência , Sciuridae/microbiologia
12.
BMC Infect Dis ; 20(1): 258, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32234012

RESUMO

BACKGROUND: Mycobacterial species other than Mycobacterium tuberculosis and Mycobacterium leprae are generally free-living organisms and Mycobacterium simiae is one of the slowest growing Non-tuberculous mycobacteria. This is the first case report of Mycobacterium simiae infection in Sri Lanka and only very few cases with extrapulmonary manifestation reported in the literature. CASE PRESENTATION: A 24-year-old, previously healthy Sri Lankan male presented with generalized lymphadenopathy with discharging sinuses, evening pyrexia, weight loss, poor appetite and splenomegaly. Lymph node biopsies showed sheets of macrophages packed with organisms in the absence of granulomata. Ziehl Neelsen, Wade Fite and Giemsa stains revealed numerous red coloured acid-fast bacilli within foamy histiocytes. Slit skin smear for leprosy was negative and tuberculosis, fungal and bacterial cultures of the lymph node and bone marrow did not reveal any growth. Later he developed watery diarrhea and colonoscopy revealed multiple small polyps and ulcers throughout the colon extending up to the ileum, Which was confirmed to be due to cytomegalovirus confirmed by PCR and successfully treated with ganciclovir. Positron emission tomography scan guided biopsies of the gut and lymph nodes confirmed presence of mycobacterial spindle cell pseudo-tumours and PCR assays revealed positive HSP65. The culture grew Mycobacterium Simiae. Flow cytometry analysis on patient's blood showed extremely low T and B cell counts and immunofixation revealed low immunoglobulin levels. His condition was later diagnosed as adult onset immunodeficiency due to anti- interferon - gamma autoantibodies. He was initially commenced on empirical anti-TB treatment with atypical mycobacterial coverage. He is currently on a combination of daily clarithromycin, ciprofloxacin, linezolid with monthly 2 g/kg/intravenous immunoglobulin to which, he had a remarkable clinical response with complete resolution of lymphadenopathy and healing of sinuses. CONCLUSIONS: This infection is considered to be restricted to certain geographic areas such as mainly Iran, Cuba, Israel and Arizona and this is the first case report from Sri lanka. Even though the infection is mostly seen in the elderly patients, our patient was only 24 years old. In the literature pulmonary involvement was common presentation, but in this case the patient had generalized lymphadenopathy and colonic involvement without pulmonary involvement.


Assuntos
Síndromes de Imunodeficiência/imunologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium/patogenicidade , Autoanticorpos/sangue , Ciprofloxacino/uso terapêutico , Claritromicina/uso terapêutico , Humanos , Biópsia Guiada por Imagem , Interferon gama/sangue , Linfonodos/microbiologia , Linfadenopatia/etiologia , Masculino , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/patologia , Tomografia por Emissão de Pósitrons , Sri Lanka , Adulto Jovem
14.
Bioresour Technol ; 309: 123307, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32315913

RESUMO

The biotransformation of phytosterol to androstenedione (AD) by mycobacteria is a unique process accompanied by energy-producing. However, high intracellular ATP content can severely inhibit the efficient production of AD. In this study, a novel citrate-based ATP futile cycle (AFC) and pyruvate-based AFC were constructed for the first time. Application of AFCs reduced intracellular ATP and propionyl-CoA levels and increased NAD+/NADH ratios and cell viability. The forced consumption of ATP promotes the transcription of critical genes in propionyl-CoA metabolism. The synergistic effect of enhanced propionyl-CoA metabolism and AFC increased AD conversion yield from 60.6% to 97.3%. The AD productivity was further improved by repeated batch fermentation using untreated cane molasses. The maximum productivity was 181% higher than that of the original strain. Therefore, the strategy of combining AFC and repeated batch fermentation is a valuable tool for the efficient and low-cost production of AD and other steroidal pharmaceutical precursors.


Assuntos
Melaço , Mycobacterium , Trifosfato de Adenosina , Androstenodiona , Bengala , Fermentação , Ciclização de Substratos
15.
BMC Infect Dis ; 20(1): 215, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164551

RESUMO

BACKGROUND: The poorly known mycobacterial species Mycobacterium monacense is a rapidly growing non-tuberculous mycobacterium that was first described in 2006 (Reischl et al., Int J Syst Evol Microbiol 56:2575-8, 2006); it has been reported that its isolation is usually associated with skin and lung infections, especially in immunosuppressed patients (Hogardt et al., Jpn J Infect Dis 61:77-8, 2008; Taieb et al., J Hand Surg Am 33:94-6, 2008; Therese et al., Lung India 28:124-6, 2011; Shojaei et al., Ann Lab Med 32:87-90, 2012; Romero et al., New Microbes New Infect 10:112-5, 2016 ). The clinical significance of Mycobacterium monacense is not yet fully understood. Here, we report the first isolation of Mycobacterium monacense from the blood culture of a patient in China with severe pneumonia. CASE PRESENTATION: On June 26, 2018, a 38-year-old man was admitted to the intensive care unit with breathing difficulty. One day prior, he was discovered with his face immersed in a small pond (non-chlorinated water) and with limb convulsions. He had undergone craniocerebral surgery after trauma 5 years earlier, which left him with epilepsy as a sequela. Bilateral diffuse ground-glass opacity was found in the lungs on chest X ray and chest CT image at admission. The result of the HIV serology test of the patient was negative. The patient was diagnosed with severe pneumonia. Drug-susceptible Klebsiella pneumoniae and Candida glabrata were isolated in the BALF, and yellow-pigmented colonies were isolated from blood cultures of the patient. The strain isolated from blood was identified by 16S rDNA sequencing as Mycobacteria monacense, which is a rapidly growing mycobacterium (RGM). The patient was treated with a combination of cefoperazone sulbactam, linezolid and voriconazole for 10 days, and the symptoms improved. During the one-year follow-up time, the patient did not relapse. CONCLUSIONS: We report the first case of M. monacense isolated from blood cultures in a patient with severe pneumonia, which provided evidence that the environmental microorganism possessed pathogenic characteristics.


Assuntos
Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium/isolamento & purificação , Pneumonia Bacteriana/microbiologia , Adulto , Hemocultura , Cefoperazona/uso terapêutico , China , DNA Ribossômico/genética , Humanos , Linezolida/uso terapêutico , Masculino , Mycobacterium/genética , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Pneumonia Bacteriana/tratamento farmacológico , Sulbactam/uso terapêutico , Voriconazol/uso terapêutico
16.
PLoS One ; 15(3): e0230282, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160243

RESUMO

Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several different expression vectors are available for gene expression in mycobacteria, they lack the required versatility of expression and the inclusion of reporter tags. We here present the construction of a set of nine E. coli-Mycobacterium shuttle plasmids, which offer a combination of three mycobacterial promoter systems (heat shock inducible-hsp60, tetracycline-, and acetamide-inducible) along with three polypeptide tags (Green Fluorescent Protein (GFP), Glutathione S-transferase (GST) and hexa-histidine tag). These vectors offer the cloning of a target gene in all the nine given vectors in parallel, thus allowing the generation of recombinant plasmids that will express the target gene from different promoters with different tags. Here, while the hexa-histidine and GST tags can be used for protein purification and pull-down experiments, the GFP-tag can be used for protein localization within the cell. Additionally, the vectors also offer the choice of positioning of the reporter tag either at the N-terminus or at the C-terminus of the expressed protein, which is achieved by cloning of the gene at any of the two blunt-end restriction enzyme sites available in the vector. We believe that these plasmids will be extremely useful in the gene expression studies in mycobacteria by offering the choices of promoters and reporters. Our work also paves the way to developing more such plasmids with other tags and promoters that may find use in mycobacterial biology.


Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Mycobacterium/genética , Escherichia coli/genética , Genes Reporter , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/genética , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional
17.
J Zoo Wildl Med ; 51(1): 177-187, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32212561

RESUMO

This case series includes a single case of disseminated tuberculous disease due to Mycobacterium pinnipedii in a New Zealand fur seal (Arctocephalus forsteri), which was being cared for by a zoo in New Zealand. The remaining five pinnipeds in the colony underwent extensive mycobacterial disease surveillance over the following 4 yr, involving a total of 26 anesthetic procedures and numerous diagnostic tests that included comparative intradermal tuberculin skin tests, mycobacterial antibody serology, respiratory and gastric lavages, and computed tomography (CT) scans. An additional case of chronic sinusitis due to Mycobacterium marinum and Pseudomonas aeruginosa was identified in a California sea lion (Zalophus californianus). Results from CT and the respiratory lavages were the most helpful antemortem diagnostic tests for active mycobacterial disease in this case series. Of the remaining four animals, two were euthanatized and two remain alive, and none of them had evidence of active mycobacterial disease. Further mycobacterial disease surveillance in staff and animals was performed, and no other case was identified. There are no validated mycobacterial surveillance tests available for pinnipeds and so it remains unknown whether the two surviving pinnipeds are truly negative or whether they have latent mycobacterial infection that could develop into active mycobacterial disease in the future. For this reason, increased levels of biosecurity and quarantine remain permanently in place for the pinniped colony.


Assuntos
Otárias , Mycobacterium/isolamento & purificação , Leões-Marinhos , Tuberculose/veterinária , Animais , Animais de Zoológico , Evolução Fatal , Feminino , Masculino , Nova Zelândia , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
18.
Adv Exp Med Biol ; 1204: 31-56, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32152942

RESUMO

Mincle (macrophage inducible C-type lectin, Clec4e, Clecsf9) was originally identified as a member of the C-type lectin receptor family in 1999. Then, the function of Mincle to control antifungal immunity by binding to Candida albicans was reported in 2008. Around the same time, it was reported that Mincle recognized damaged cells and induced sterile inflammation by coupling with the ITAM-adaptor molecule FcRγ. In the following year, a breakthrough discovery reported that Mincle was an essential receptor for mycobacterial cord factor (trehalose-6,6'-dimycolate, TDM). Mincle gained increasing attention immediately after this critical finding. Although our understanding of the recognition of Mycobacteria has been advanced significantly, it was also revealed that Mincle interacts with pathogens other than Mycobacteria. In addition, endogenous ligands of Mincle were identified recently. Therefore, Mincle is now considered a danger receptor both for self and non-self ligands, so-called damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). This chapter will give an overview of the accumulated knowledge of the multi-task danger receptor Mincle from its discovery to the latest findings.


Assuntos
Fatores Corda/imunologia , Lectinas Tipo C/imunologia , Mycobacterium/química , Mycobacterium/imunologia , Receptores Imunológicos/imunologia , Animais , Humanos
19.
Epidemiol Infect ; 148: e49, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32054545

RESUMO

A new fast-growing mycobacterium, designated strain QGD101T, was isolated from the sputum of an 84-year-old man suspected of tuberculosis in Wuhan Medical Treatment Center, Hubei, China. This strain was a gram-staining-negative, aerobic, non-spore-forming and catalase-positive bacterium, which was further identified as the NTM by PNB and TCH tests. The moxifloxacin and levofloxacin exhibited strong suppressing function against QGD101T with MIC values of 0.06 and 0.125 µg/ml after drug susceptibility testing of six main antimicrobial agents on mycobacteria. Based on the sequence analysis of 16S rRNA, rpoB, hsp65 and 16S-23S rRNA internal transcribed spacer, the strain QGD101T could not be identified to a species level. Mycobacterium moriokaense ATCC43059T that shared the highest 16S rRNA gene sequence similarity (98%) with strain QGD101T was actually different in genomes average nucleotide identity (78.74%). In addition, the major cellular fatty acids of QGD101T were determined as C18:1ω9c, C16:0 and C18:2ω6c. The DNA G + C content was 64.9% measured by high performance liquid chromatography. Therefore, the phenotypic and genotypic characterisation of this strain led us to the conclusion that it represents a novel species of mycobacteria, for which the name Mycobacterium hubeiense sp. nov. (type strain QGD101T = CCTCCAA 2017003T = KCTC39927T) was proposed. Thus, the results of this study are very significant for the clinical diagnosis of tuberculosis and future personalised medicine.


Assuntos
Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Escarro/microbiologia , Idoso de 80 Anos ou mais , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Composição de Bases , Chaperonina 60/genética , China , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , RNA Polimerases Dirigidas por DNA/genética , Ácidos Graxos/análise , Humanos , Levofloxacino/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Mycobacterium/genética , Mycobacterium/fisiologia , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
20.
Int J Nanomedicine ; 15: 1073-1094, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32103956

RESUMO

Purpose: This study demonstrated improved transdermal delivery of rifampicin-loaded cationic nanoemulsion gel to treat systemic and cutaneous tuberculosis using capmul, labrasol, and acconon, which exert anti-Mycobacterium activities. This approach enhanced drug permeation across the skin, increased therapeutic efficacy, and reduced dose-related side effects. Methods: Design Expert® was used to optimize formulations (Smix ratio and capmul as independent factors), which were prepared using a slow spontaneous titration method. The optimized nanoemulsion was incorporated into carbopol gel to allow for topical application and comparative assessments. Nanoemulsions and gels were evaluated for size, size distribution, shape, zeta potential, percent spread, viscosity, in vitro hemolysis, in vitro release, and ex vivo skin permeation and deposition. A mechanistic evaluation was performed using scanning electron microscopy. Furthermore, in vivo pharmacokinetic and irritation studies were performed. Results: The optimized cationic nanoemulsion (OCNE-1) was characterized by small particle size (≤100 nm), had optimal viscosity, percent spread, zeta potential, and percent drug release, and was hemocompatible. The OCNE-1T gel exhibited higher permeation flux (51.32 ± 0.5 µg/cm2 hr), permeation coefficient (2.566 ± 0.08 cm/hr), drug deposition (994.404 µg/cm2), and enhancement ratio (7.16) than those of the OCNE-1 nanoemulsion or drug solution. Scanning electron microscopy was used to characterize the mechanism of enhanced permeation. An In vivo study showed that the Cmax and area under the curve following transdermal application were 4.34- and 4.74-fold higher than those following oral administration. Conclusion: Transdermal delivery of rifampicin could be a promising alternative to conventional approaches to treat systemic and local tuberculosis, and other bacterial infections.


Assuntos
Antituberculosos/administração & dosagem , Emulsões/administração & dosagem , Rifampina/administração & dosagem , Tuberculose/tratamento farmacológico , Resinas Acrílicas/química , Administração Cutânea , Animais , Antituberculosos/farmacocinética , Antituberculosos/farmacologia , Disponibilidade Biológica , Caprilatos/química , Cátions/química , Emulsões/farmacologia , Excipientes/química , Géis/química , Géis/farmacologia , Glicerídeos/química , Mycobacterium/efeitos dos fármacos , Nanoestruturas/administração & dosagem , Nanoestruturas/química , Tamanho da Partícula , Ratos Sprague-Dawley , Rifampina/farmacocinética , Rifampina/farmacologia , Pele/efeitos dos fármacos , Tuberculose Cutânea/tratamento farmacológico
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