Characterization of integrated prophages within diverse species of clinical nontuberculous mycobacteria.

BACKGROUND: Nontuberculous mycobacterial (NTM) infections are increasing in prevalence, with current estimates suggesting that over 100,000 people in the United States are affected each year. It is unclear how certain species of mycobacteria transition from environmental bacteria to clinical pathogens, or what genetic elements influence the differences in virulence among strains of the same species. A potential mechanism of genetic evolution and diversity within mycobacteria is the presence of integrated viruses called prophages in the host genome. Prophages may act as carriers of bacterial genes, with the potential of altering bacterial fitness through horizontal gene transfer. In this study, we quantify the frequency and composition of prophages within mycobacteria isolated from clinical samples and compare them against the composition of PhagesDB, an environmental mycobacteriophage database. METHODS: Prophages were predicted by agreement between two discovery tools, VirSorter and Phaster, and the frequencies of integrated prophages were compared by growth rate. Prophages were assigned to PhagesDB lettered clusters. Bacterial virulence gene frequency was calculated using a combination of the Virulence Factor Database (VFDB) and the Pathosystems Resource Integration Center virulence database (Patric-VF) within the gene annotation software Prokka. CRISPR elements were discovered using CRT. ARAGORN was used to quantify tRNAs. RESULTS: Rapidly growing mycobacteria (RGM) were more likely to contain prophage than slowly growing mycobacteria (SGM). CRISPR elements were not associated with prophage abundance in mycobacteria. The abundance of tRNAs was enriched in SGM compared to RGM. We compared the abundance of bacterial virulence genes within prophage genomes from clinical isolates to mycobacteriophages from PhagesDB. Our data suggests that prophages from clinical mycobacteria are enriched for bacterial virulence genes relative to environmental mycobacteriophage from PhagesDB. CONCLUSION: Prophages are present in clinical NTM isolates. Prophages are more likely to be present in RGM compared to SGM genomes. The mechanism and selective advantage of this enrichment by growth rate remain unclear. In addition, the frequency of bacterial virulence genes in prophages from clinical NTM is enriched relative to the PhagesDB environmental proxy. This suggests prophages may act as a reservoir of genetic elements bacteria could use to thrive within a clinical environment.

Characterizing Non-Tuberculous Mycobacteria Infection in Bronchiectasis.

Chronic airway infection is a key aspect of the pathogenesis of bronchiectasis. A growing interest has been raised on non-tuberculous mycobacteria (NTM) infection. We aimed at describing the clinical characteristics, diagnostic process, therapeutic options and outcomes of bronchiectasis patients with pulmonary NTM (pNTM) disease. This was a prospective, observational study enrolling 261 adult bronchiectasis patients during the stable state at the San Gerardo Hospital, Monza, Italy, from 2012 to 2015. Three groups were identified: pNTM disease; chronic P. aeruginosa infection; chronic infection due to bacteria other than P. aeruginosa. NTM were isolated in 32 (12%) patients, and among them, a diagnosis of pNTM disease was reached in 23 cases. When compared to chronic P. aeruginosa infection, patients with pNTM were more likely to have cylindrical bronchiectasis and a "tree-in-bud" pattern, a history of weight loss, a lower disease severity and a lower number of pulmonary exacerbations. Among pNTM patients who started treatment, 68% showed a radiological improvement, and 37% achieved culture conversion without recurrence, while 21% showed NTM isolation recurrence. NTM isolation seems to be a frequent event in bronchiectasis patients, and few parameters might help to suspect NTM infection. Treatment indications and monitoring still remain an important area for future research.

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria.

Mycobacteriophage DS6A is unique among the more than 8,000 isolated mycobacteriophages due to its ability to form plaques exclusively on mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Speculation surrounding this specificity has led to unsupported assertions in published studies and patents that nontuberculous mycobacteria (NTM) are wholly resistant to DS6A infection. In this study, we identified two independent nonessential regions in the DS6A genome and replaced them with an mVenus-expressing plasmid to generate fluorescent reporter phages Φ2GFP12 and Φ2GFP13. We show that even though DS6A is able to form plaques only on MTBC bacteria, infection of various NTM results in mVenus expression in transduced cells. The efficiency of DS6A in delivering DNA varied between NTM species. Additionally, we saw a striking difference in the efficiency of DNA delivery between the closely related members of the Mycobacterium abscessus complex, M. abscessus and Mycobacterium massiliense We also demonstrated that TM4 and DS6A, two phages that do not form plaques on M. massiliense, differ in their ability to deliver DNA, suggesting that there is a phage-specific restriction between mycobacterial species. Phylogenetic analysis reveals that the DS6A genome has a characteristically mosaic structure but provided few insights into the basis for the specificity for MTBC hosts. This study demonstrates that the inability of the MTBC-specific phage DS6A to form plaques on NTM is more complex than previously thought. Moreover, the DS6A-derived fluorophages provide important new tools for the study of mycobacterial biology. IMPORTANCE: The coevolution of bacteria and their infecting phages involves a constant arms race for bacteria to prevent phage infection and phage to overcome these preventions. Although a diverse array of phage defense systems is well characterized in bacteria, very few phage restriction systems are known in mycobacteria. The DS6A mycobacteriophage is unique in the mycobacterial world in that it forms plaques only on members of the Mycobacterium tuberculosis complex. However, the novel DS6A reporter phages developed in this work demonstrate that DS6A can infect nontuberculous mycobacteria at various efficiencies. By comparing the abilities of DS6A and another phage, TM4, to infect and form plaques on various mycobacterial species, we can begin to discern new phage restriction systems employed within the genus.

Primeiro isolamento de Mycobacterium kyorinense em espécime clínico no Brasil / First isolation of Mycobacterium kyorinense in a clinical specimen in Brazil

Os testes bioquímicos realizados, o seqüenciamento de diferentes alvos genéticos e a construção de uma árvore concatenada, construída a través do método Neighbor-Joining, permitiram a identificação das cepas brasileiras como M. kyorinense.

Primeiro isolamento de Mycobacterium kyorinense em espécime clínico no Brasil / First isolation of Mycobacterium kyorinense in a clinical specimen in Brazil

Os testes bioquímicos realizados, o seq³enciamento de diferentes alvos genéticos e a construþÒo de uma árvore concatenada, construída a través do método Neighbor-Joining, permitiram a identificaþÒo das cepas brasileiras como M. kyorinense. (AU)

Human immunodeficiency virus-associated atypical mycobacterial skeletal infections.

The clinical and laboratory features of six human immunodeficiency virus (HIV)-positive patients with atypical mycobacterial skeletal infections, seen at a county outpatient HIV facility or university outpatient clinic, are reviewed and compared with other reported cases. Atypical mycobacterial skeletal infections are a manifestation of advanced HIV disease, with most cases having CD4 counts < 100/mm3 at the time these infections become clinically apparent. Multiple sites are frequently involved, and concomitant skin infection with the same organism is common, especially with Mycobacterium haemophilum. The incidence of atypical mycobacterial skeletal infection in HIV-infected individuals was significantly higher than in the general county hospital district patient population, whereas the frequency of Myobacterium tuberculosis skeletal infection did not differ significantly between the two populations. The clinician therefore should maintain a high index of suspicion for atypical mycobacteria in a patient presenting with skeletal infection in the setting of a markedly depressed CD4 count.

Micobacterioses em pacientes portadores da síndrome da imunodeficiência adquirida: importância de uma investigação sistemática / Mycobacteriosis in patients with acquired immunodeficiency syndrome: the importance of systematic research

Com o objetivo de demonstrar a importância da investigação sistemática de micobactérias em pacientes com SIDA, independente dos sintomas apresentados, a autora pesquisou a presença de Mycobacterium tuberculosis e micobactéria atípica em materiais obtidos em três sitios: pulmão (escarro espontâneo ou induzido), urina e medula óssea. Para tal, realizou um estudo transversal em 24 pacientes com SIDA, internados em um hospital público de Salvador. 12 pacientes (50%) tiveram diagnóstico de micobacteriose: através da cultura foi identificado um paciente (4%) com uma associação de micobactérias atípicas e seis (25%) com Mycobacterium tuberculosis. Cinco pacientes (21%) apresentaram positividade apenas à baciloscopia, que correspondiam provavelmente ao Mycobacterium tuberculosis. Para a obtenção de secreção das vias aéreas inferiores nos pacientes que não expectoraram espontaneamente para três amostras, foi utilizada a técnica da indução do escarro através da nebulização com solução hipertônica, o que levou a um acréscimo de 8% no diagnóstico de micobacteriose. No entanto, a obtenção de secreção com esta técnica só ocorreu nos pacientes com anormalidade clinica do aparelho respiratório, sendo improdutiva a sua utilização naqueles pacientes sem alteração deste aparelho. A pesquisa em sitios extra-pulmonares de fácil acesso, através da urocultura e do aspirado de medula óssea, acrescentou mais 17%, cada exame, ao diagnóstico de micobacteriose. Quando analisados em associação ao escarro induzido, acrescentaram 25% ao número de casos diagnosticados. A presença de poucos bacilos foi uma constante em todas as amostras dos pacientes com exames positivos, o que evidenciou a importância da obtenção de amostras seriadas do escarro para aumentar a chance de diagnóstico de micobactéria pulmonar. Neste estudo, foram ainda observados aspectos clinicos, concluindo-se que micobactérias devem ser pesquisadas em todos os pacientes que expectorem espontaneamente ou...

Ocorrencia de micobacterias em aguas poluidas com residuos industriais e domesticos / Occurrence of mycobacteria in waters pollution with industrial and domestic residues

Foram isoladas 158 cepas de micobacterias a partir de amostras de agua poluida notadamente com residuos de petroleo apos a depuração com hidroxido de sodio 4 por cento (111 cepas) ou cloreto de benzalconio de 0,34 por cento (47 cepas) A maioria das micobacterias isoladas comportou se como pertencente ao grupo III de Runyon (78 cepas), seguindo-se as de crescimento rapido 61 cepas e as escotocromogenicas, de crescimento lento 19 cepas. Aproiximadamente 32 por cento das cepas foi caracterizada como micobacterias potencialmente patogenicas incluindo M avium intracelulare 3,8 por cento. M scrofulaceum 6,3 por cento e m fortutum 22,2 por cento. Conclui se que a agua do Rio de Jacare esta contaminada com micobacterias potencialmente patogenicas podendo servir como fonte de doença humana.