Respiratory tract clinical sample selection for microbiota analysis in patients with pulmonary tuberculosis
- Luz Elena Botero†1, 2, 3,
- Luisa Delgado-Serrano†1,
- Martha Lucía Cepeda1,
- Jose Ricardo Bustos1,
- Juan Manuel Anzola1,
- Patricia Del Portillo1,
- Jaime Robledo2, 3 and
- María Mercedes Zambrano1Email author
© Botero et al.; licensee BioMed Central Ltd. 2014
Received: 26 March 2014
Accepted: 7 July 2014
Published: 25 August 2014
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.
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.
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.
KeywordsMicrobiota Respiratory tract Pulmonary tuberculosis ITS1 16S rRNA Microbial diversity Mycobacterium tuberculosis
Recent studies suggest that microbial communities inhabiting the human body can influence the host's health status and contribute to disease . The human upper respiratory tract represents the major portal of entry for numerous airborne microorganisms, such as bacteria, fungi, or viruses . High-throughput sequencing methods have provided great insight regarding the composition of the respiratory tract-associated microbiota, which has been recently related with the development of diseases such as asthma , nosocomial pneumonia, pulmonary cystic fibrosis , and chronic obstructive pulmonary disease .
Tuberculosis (TB), a respiratory disease caused by Mycobacterium tuberculosis (Mtb), is a major global public health problem that affects millions of people each year and ranks as the second leading cause of death from an infectious disease worldwide, with 8.6 million new cases and 1.3 million deaths in 2012 (25% of them were HIV-associated) . The Mtb pathogen typically affects the lungs (pulmonary TB) but can affect other sites as well (extrapulmonary TB). Individuals with pulmonary TB can expel bacteria by talking, coughing, or sneezing, spreading the pathogen through airborne particles that are inhaled by others. The complex Mtb-human host interaction and the resulting infectious process indicate that TB disease development may be a multifactorial process . Microorganism characteristics coupled to local host immune response determine whether bacilli are cleared or will lead to either acute or latent disease .
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. Previous studies have shown that oropharyngeal swabs can be a reasonable proxy for lung samples , and an analysis in healthy individuals indicated that lung and upper airway bacterial populations, which include the oropharynx, were largely indistinguishable from one another . Given that the resemblance between oropharyngeal and sputum communities is still unclear and the difficulty of getting sputum samples from healthy individuals, the aim of this work was to use different sample types and determine which one could be used to evaluate the composition of the respiratory tract microbiota associated with TB patients and healthy controls.
Population and sampling
Age; median (range) in years
Body mass index (BMI; kg/m2) median (max/min)*
Sequence data and diversity indexes
Phyla that differ significantly between sample types
Mean relative abundance
O vs N
O vs S
N vs S
Samples were also analyzed in order to see changes in respiratory tract bacterial communities associated to health status. The only difference between patient and control groups, using either nasal and oropharynx samples separately or both sample types (nasal and oropharynx) together, was found in oropharynx samples, where unclassified sequences belonging to the Streptococcaceae family were more abundant in TB patients (p = 0.00878, not shown). Taken together, these observations indicate alterations in these communities and raise the possibility that such imbalances could affect, or result from, infection and/or colonization.
Differences in community diversity indexes and in abundance of particular taxa, specifically in oropharynx communities, between TB patients and healthy controls suggest disturbance of respiratory tract microbial communities, despite the overall similarity in terms of the major phyla identified. These altered communities could either result from or influence infection and/or colonization by M. tuberculosis, a possibility that can be further examined by studying changes in particular taxa or in functionality via metagenomic sequencing using samples collected at various time points. More importantly, there was a resemblance between communities from sputum in TB patients and those present in the oropharynx, both of which were distinct from the nasal microbiota. This study therefore indicates that oropharynx samples can be valuable for probing respiratory tract microbiota and sets the groundwork for more extensive comparison and analysis of possible microbial community imbalances associated with a diseased state such as TB.
The research complied with the standards and recommendations for biomedical research involving human subjects adopted by the 18th World Medical Assembly, Helsinki, Finland, June 1964 and the 59th Meeting, Seoul, 2008. Ethical standards also complied with resolution N°008430 (1993) established by the Colombian Ministry of Health for work with humans. Informed written consent was obtained from all participants prior to enrollment with approval by the Ethics Committee of Corporación Corpogen (Bogotá), Corporación para Investigaciones Biológicas-CIB (Medellín) and with the approval of the Research Committee METROSALUD, ESE (Medellín).
- BMI :
Body mass index
- HIV :
Human immunodeficiency virus
- ITS1 :
Internal transcribed spacer region 1
- Mtb :
- OTU :
Operational taxonomic unit
- PCoA :
Principal coordinate analyses
- QIIME :
Quantitative Insights Into Microbial Ecology
- TB :
- UPGMA :
Unweighted pair group method with arithmetic mean.
This work was funded by Colciencias (Grant No. 657049326148). We would like to thank the program ‘Habitante de Calle,’ Secretaría de Salud, Medellín, Colombia, and Dr. Lucas Arias, for facilitating access to the institution and to patients with pulmonary TB diagnosis. We would also like to thank Alejandro Reyes and Silvia Restrepo (Universidad de Los Andes, Bogotá, Colombia) for their input and experimental assistance during the development of this research.
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