Difference in Virulence of Mycobacterium avium Isolates Sharing Indistinguishable DNA Fingerprint Determined in Murine Model of Lung Infection

Background: Opportunistic Mycobacterium avium typically causes disease in immunocompromised patients and in some groups of apparently healthy individuals. the high virulence of some bacterial lineages increases the disease risk. High-resolution molecular genotyping studies of M. avium clinical isolates demonstrated that some genotype patterns were more prevalent than others, suggesting that close genetic relatedness of these successful isolates sharing a similar genotype could determine similar biological properties associated with high virulence.Methods and Findings: in this study, we aimed to compare the virulence and pathogenic properties of two epidemiologically unrelated M. avium isolates sharing an indistinguishable DNA fingerprint in a well-characterized model of pulmonary infection in mice, resistant or susceptible to mycobacteria. the mice, C57BL/6 wild-type or IFN-gamma gene disrupted (GKO), respectively, were intratracheally infected with two isolates, H27 (human blood isolate) and P104 (pig lymph node isolate), and the lungs were examined for bacterial loads, histopathology and cytokine gene expression. the obtained data demonstrated significant differences in the virulence properties of these strains. Although the H27 strain grew significantly faster than P104 in the early stage of infection, this bacterium induced protective immunity that started to reduce bacterial numbers in the wild-type mice, whereas the P104 strain established a chronic infection. in the GKO mice, both strains were capable of causing a chronic infection, associated with higher bacterial burdens and severe lung pathology, in a similar manner.Conclusions/Significance: the results demonstrated that the studied isolates differed in the pathogenic properties although were indistinguishable by actually widely used genotyping techniques demonstrating that the genotype similarity does not predict similarity in virulence of M. avium isolates.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Estadual Norte Fluminense, Lab Biol Recognit, Rio de Janeiro, BrazilUniv Estadual Norte Fluminense, Lab Anim Morphol & Pathol, Rio de Janeiro, BrazilInst Butantan, Lab Immunochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilFAPERJ: E-26/111.6111CNPq: 410555Web of Scienc

Microbiótica fúngica na pelagem de camundongos de linhagens isogênicas e congênicas mantidos em biotério de criação

With the development of laboratory animal housing and care technology, where animals are kept under various different schemes according to specific purposes, an appropriate evaluation of the microbiota of the environment and of the animals is important. The occurrence of fungi was studied in the fur coat of 95 mice (47 males and 48 females) aging between 37 and 55 days of the following strains: isogenic (A/Sn (10), BALB/c (16), CBA (15), CS7BL/10J (10) and congenic (B10.A (14), B10 D2/o (15) e B1 .02/n (15). The material was collected frictioning a small piece of sterilized carpet in all the extension of the mouse corporal region. Sabouraud-dextrose agar plus chloramphenicol (100 µ g/ml) and “Mycobiotic agar” were used. The first medium presented a greater number of isolations (224) than the second (61). The isolation frequencies where the following: Penicillium sp (93,68%) , Trichoderme sp (92,63%), Cladosporium sp (38,95%), Torulopsis sp (7,37%), Rhizopus sp (4,21%), Aureobasidium sp (3,16%), Absidia sp (1,05%), Aspergillus sp (1,05%), Mycellia sterilla (1,05%), Trichotecium sp (1,05%), Candida sp (1,05%) e Rhodotorula sp (1,05%). A great number of fungi genera was isolated from isogenic strains and from female mice. These results were statistically significant (p=0,05) in the signals test. The fungi isolated from the housing rooms belong to the following genera: Absidia, Aureobasidium, Cladosporium, Cephalosporium, Epicoccum, Geotrichum , Mycelia sterilia, Penicillium e Trichoderma. No pathogenic fungi were found in the mice strains or in the housing rooms studies.Como desenvolvimento da tecnologia e do manejo de animais de laboratório, mantidos em biotérios que seguem diferentes normas de trabalho, de acordo com os objetivos a que se propõem, é mister que se avalie a microbiota dos ambientes de criação, bem como dos animais que deles fazem parte. Estudou-se a frequência de fungos na pelagem de 35 camundongos (47 machos e 43 fêmeas) de linhagens isogênicas (A/Sn (10), BALB/c (16), CBA (15), CS7BL/10J (10) e congênicas (B10.A (14), B10 D2/o (15) e B1 .02/n (15), com idade que variava entre 37 e 55 dias. A coleta de material foi realizada pela fricção de um pedaço de tapete esterilizado, em toda região corpórea das camundongos, seguida da compressão do material em placas de Petri, contendo ágar Sabouraud dextrose  acrescido de cloranfenicol (100 µ g/ml) - ASD e “Mycobiotic agar” (MA), para o isolamento dos fungos. Verificou-se maior número de isolamentos no primeiro (224) do que no segundo (61). As frequências de isolamento foram, respectivamente: Penicillium sp (93,68%) , Trichoderme sp (92,63%), Cladosporium sp (38,95%), Torulopsis sp (7,37%), Rhizopus sp (4,21%), Aureobasidium sp (3,16%), Absidia sp (1,05%), Aspergillus sp (1,05%), Mycellia sterilla (1,05%), Trichotecium sp (1,05%), Candida sp (1,05%) e Rhodotorula sp (1,05%). Maior numero de gênero fúngicos foi isolado de linhagens isogênicas e de camundongos do sexo feminino. Estes resultados foram estatisticamente significativo para p=0,05 na prova dos sinais. Entre os fungos isolados nos ambientes de criacão incluíram-se os seguintes gêneros: Absidia, Aureobasidium, Cladosporium, Cephalosporium, Epicoccum, Geotrichum , Mycelia sterilia, Penicillium e Trichoderma. Não. foram encontrados fungos considerados patogênicos para as linhagens estudadas, tanto nos animais como nas diferentes salas de criação

Acute Immune Response to Mycobacterium massiliense in C57BL/6 and BALB/c Mice ▿

Mycobacterium massiliense is an environmental opportunistic pathogen that has been associated with soft tissue infection after minor surgery. We studied the acute immune response of C57BL/6 and BALB/c mice infected intravenously with 106 CFU of an M. massiliense strain isolated from a nosocomial infection in Brazil. The results presented here show that M. massiliense is virulent and pathogenic to both C57BL/6 and BALB/c mice, inducing a granulomatous inflammatory reaction that involves the activation of macrophages, dendritic cells, and natural killer cells induced by gamma interferon and interleukin-17 (IL-17) in C57BL/6 mice and by IL-12 in BALB/c mice

Production of pro-inflammatory mediators in culture of cells obtained from lungs of C57BL/6 WT mice infected with <i>M. avium</i> H27 or P104 strains.

<p>Lung cells were isolated on day 30 after i/t infection of mice and cultured in 96 well-plates, 5×10⁴ cells/well. Culture supernatants were collected after 48 h incubation and concentrations of IFN-γ and TNF-α were measured by sandwich ELISA. Nitric oxide production was measured by Griess reaction. The data were obtained in two independent experiments. Asterisks represent statistical significance (***p<0.001).</p

Expression of cytokine genes in lung cells of mice infected with H27 strain.

<p>Experiments were performed and results of cytokine gene expression determination were presented exactly as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021673#pone-0021673-g005" target="_blank">Fig. 5</a>.</p

Comparison of lung, liver and spleen bacillary loads in C57BL/6 WT and GKO mice infected with <i>M. avium</i> strains.

<p>Mice were infected as indicated in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021673#pone-0021673-g002" target="_blank">Fig. 2</a>. At the indicated time-points, six mice were sacrificed, and bacterial burdens (CFU) in the lungs, livers and spleens were measured. The data were obtained in two independent experiments. Asterisks represent statistical significance (**p<0.01, ***p<0.001).</p

Expression of cytokine genes in lung cells of mice infected with P104 strain.

<p>C57BL/6 and GKO mice were infected as indicated in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021673#pone-0021673-g002" target="_blank">Fig.2</a>. mRNA was extracted from lungs of these mice on days 5, 15, 30 and 60 post-infection. Levels of mRNA were quantified by real-time PCR using specific primers for inflammatory cytokine genes and normalized to the level of β-actin expression. Cytokine gene expression determination was performed with four animals per time-point. The data were obtained in two independent experiments. Results are expressed as fold increase ± SEM compared to non-infected animals. Asterisks represent statistical significance (**p<0.01, ***p<0.001) when compared WT and GKO mice.</p