Sputum Microbiota in Tuberculosis as Revealed by 16S rRNA Pyrosequencing

School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China.
PLoS ONE (Impact Factor: 3.23). 01/2013; 8(1):e54574. DOI: 10.1371/journal.pone.0054574


Background: Tuberculosis (TB) remains a global threat in the 21st century. Traditional studies of the disease are focused on the single pathogen Mycobacterium tuberculosis . Recent studies have revealed associations of some diseases with an imbalance in the microbial community. Characterization of the TB microbiota could allow a better understanding of the disease.

Methodology/Principal Findings: Here, the sputum microbiota in TB infection was examined by using 16S rRNA pyrosequencing. A total of 829,873 high-quality sequencing reads were generated from 22 TB and 14 control sputum samples. Firmicutes , Proteobacteria , Bacteroidetes , Actinobacteria , and Fusobacteria were the five major bacterial phyla
recovered, which together composed over 98% of the microbial community. Proteobacteria and Bacteroidetes were more represented in the TB samples and Firmicutes was more predominant in the controls. Sixteen major bacterial genera were recovered. Streptococcus , Neisseria and Prevotella were the most predominant genera, which were dominated by several operational taxonomic units grouped at a 97% similarity level. Actinomyces , Fusobacterium , Leptotrichia , Prevotella , Streptococcus , and Veillonella were found in all TB samples, possibly representing the core genera in TB sputum microbiota. The less represented genera Mogibacterium , Moryella and Oribacterium were enriched statistically in the TB samples, while a genus belonging to the unclassified Lactobacillales was enriched in the controls. The diversity of microbiota was similar in the TB and control samples.

Conclusions/Significance: The composition and diversity of sputum microbiota in TB infection was characterized for the first time by using high-throughput pyrosequencing. It lays the framework for examination of potential roles played by the diverse microbiota in TB pathogenesis and progression, and could ultimately facilitate advances in TB treatment.

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Available from: Man Kit Cheung
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    • "Recent studies of the respiratory tract microbiota using sputum samples and mixtures of saliva and pharyngeal secretions indicate changes and possible associations with pulmonary TB [8,9]. In this work, we examined the microbiota in three types of respiratory tract samples, nasal and oropharynx swabs and sputum, the latter taken only from patients since sputum is difficult to procure from healthy individuals, not to mention the more invasive bronchoalveolar lavage. "
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    ABSTRACT: Background Changes in respiratory tract microbiota have been associated with diseases such as tuberculosis, a global public health problem that affects millions of people each year. This pilot study was carried out using sputum, oropharynx, and nasal respiratory tract samples collected from patients with pulmonary tuberculosis and healthy control individuals, in order to compare sample types and their usefulness in assessing changes in bacterial and fungal communities. Findings Most V1-V2 16S rRNA gene sequences belonged to the phyla Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria, with differences in relative abundances and in specific taxa associated with each sample type. Most fungal ITS1 sequences were classified as Ascomycota and Basidiomycota, but abundances differed for the different samples. Bacterial and fungal community structures in oropharynx and sputum samples were similar to one another, as indicated by several beta diversity analyses, and both differed from nasal samples. The only difference between patient and control microbiota was found in oropharynx samples for both bacteria and fungi. Bacterial diversity was greater in sputum samples, while fungal diversity was greater in nasal samples. Conclusions Respiratory tract microbial communities were similar in terms of the major phyla identified, yet they varied in terms of relative abundances and diversity indexes. Oropharynx communities varied with respect to health status and resembled those in sputum samples, which are collected from tuberculosis patients only due to the difficulty in obtaining sputum from healthy individuals, suggesting that oropharynx samples can be used to analyze community structure alterations associated with tuberculosis.
    Full-text · Article · Aug 2014
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    • "Another study concluded that there was a difference between the sputum microbial composition of TB patients and healthy controls [16]. Conversely, a different paper focused on human sputum samples found no difference between TB patients and healthy controls [17]. "
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    ABSTRACT: Mycobacterium tuberculosis is an important human pathogen, and yet diagnosis remains challenging. Little research has focused on the impact of M. tuberculosis on the gut microbiota, despite the significant immunological and homeostatic functions of the gastrointestinal tract. To determine the effect of M. tuberculosis infection on the gut microbiota, we followed mice from M. tuberculosis aerosol infection until death, using 16S rRNA sequencing. We saw a rapid change in the gut microbiota in response to infection, with all mice showing a loss and then recovery of microbial community diversity, and found that pre-infection samples clustered separately from post-infection samples, using ecological beta-diversity measures. The effect on the fecal microbiota was observed as rapidly as six days following lung infection. Analysis of additional mice infected by a different M. tuberculosis strain corroborated these results, together demonstrating that the mouse gut microbiota significantly changes with M. tuberculosis infection.
    Full-text · Article · May 2014 · PLoS ONE
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    • "Another possibility is the sampling differences between the two studies, although the samples and controls in Cui’s study and ours were comparable. However, the study by Cheung et al. that recruited individuals with coughing symptoms as controls, claimed no significant differences in diversity between controls and TB sputum microbiomes [13]. It is possible that different choices of controls for comparison may have affected the bacterial species being identified from these studies. "
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    ABSTRACT: Microbiota have recently been shown to be associated with many disease conditions. However, the microbiota associated with tuberculosis (TB) infection, recurrence and treatment outcome have not been systematically characterized. Here, we used high throughput 16S RNA sequencing to analyze the sputum microbiota associated with Mycobacterium tuberculosis infection and also to identify the microorganisms associated with different outcomes of TB treatment. We recruited 25 new TB patients, 30 recurrent TB patients and 20 TB patients with treatment failure, as well as 20 healthy controls. Streptococcus, Gramulicatella and Pseudomonas were more abundant in TB patients while Prevotella, Leptotrichia, Treponema, Catonella and Coprococcus were less abundant in TB patients than in the healthy controls. We found reduced frequency and abundance of some genera such as Bulleidia and Atopobium in recurrent TB patients compared with those in new TB patients. In addition, the ratio of Pseudomonas / Mycobacterium in recurrent TB was higher than that in new TB while the ratio of Treponema / Mycobacterium in recurrent TB was lower than that in new TB, indicating that disruption of these bacteria may be a risk factor of TB recurrence. Furthermore, Pseudomonas was more abundant and more frequently present in treatment failure patients than in cured new patients, and the ratio of Pseudomonas / Mycobacterium in treatment failure was higher than that in new TB. Our data suggest that the presence of certain bacteria and the disorder of lung microbiota may be associated with not only onset of TB but also its recurrence and treatment failure. These findings indicate that lung microbiota may play a role in pathogenesis and treatment outcome of TB and may need to be taken into consideration for improved treatment and control of TB in the future.
    Full-text · Article · Dec 2013 · PLoS ONE
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