Unraveling Imbalanced Respiratory Microbiota in Childhood Viral and "Mycoplasma pneumoniae" Pneumonia

揭示兒童病毒性和肺炎支原體肺炎中呼吸道菌群紊亂機制

Student thesis: Doctoral Thesis

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Award date29 Apr 2019

Abstract

Acute respiratory infection, especially the pneumonia, is the major risk of children morbidity and mortality in China. Emerging reports in America, Europe and Australia have demonstrated that stable respiratory microbiota could confer colonization resistance to pathogen intrusion and lower the risk of acute respiratory infection, as well as prime the immune maturation. In contrast, normal respiratory microbiota in healthy Chinese children and respiratory microbiota dysbiosis in Chinese pediatric pneumonia remain unexplored.

In this thesis, we investigated several major problems in respiratory microbiota research of Chinese children. Initially, we performed 16S rDNA gene sequencing analysis of 83 anterior nares, 60 nasopharynx and 97 oropharynx samples, which was qualified for DNA extraction from 98 healthy children in Shenzhen, China (≤12 years of age). The microbiota in the anterior nares and nasopharynx are similar at different ages and change little with age, and typical microbial species in these areas include Moraxella, Staphylococcus, Corynebacterium, Streptococcus, and Dolosigranulum. By contrast, the oropharynx is primarily colonized by Streptococcus, Prevotella, Neisseria, Veillonella, Rothia, Leptotrichia and Haemophilus. The Streptococcus and Rothia populations keep low abundance in children less than 1 year of age, whereas the Prevotella, Neisseria, Haemophilus and Leptotrichia populations accumulate significantly in individuals older than 1 year of age.

Based on normal respiratory microbiota composition at different age groups, the comparison of microbiota in upper respiratory tracts between healthy and adenovirus/Mycoplasma pneumoniae pneumonia children was conducted. Hospitalized patients harbor lower microbial diversity and simpler co-occurrence network in upper respiratory tract, as compared to healthy children. For diseased children, microbial commensals, such as Corynebacterium, reduced in nasopharynx and oropharynx microbiota. Further analysis indicated the transmission of microbial components in nasopharynx to oropharynx during acute respiratory infection. In addition, the concordance between imbalanced upper respiratory tract microbiota and that in the lung were also explored. Acute respiratory infection seemed to occur in nasopharynx firstly and then transmitted to the oropharynx as well as the lung. The lung microbiota could be stratified based on pneumonia severity in adenovirus or M. pneumoniae pneumonia. As higher severity, the lung microbiota kept less similarity with the microbiota in upper respiratory microbiota, suggesting the potential role of different immune selection between upper and lower respiratory tracts.

We also explored the association of the lung microbiota in adenovirus and M. pneumoniae pneumonia with the lung levels of inflammatory cytokines. M. pneumoniae and adenovirus are the primary pathogens causing pneumonia which is the high risk of morbidity and mortality in childhood, following increasing tracheomalacia incidence in Chinese children. We selected 52 inpatients with tracheomalacia (n=15), M. pneumoniae pneumonia (n=21) or adenovirus pneumonia (n=16). Bronchoalveolar lavage fluid was collected to compare the differences of the lung microbiota and expression of ten inflammatory cytokines between tracheomalacia, M. pneumoniae and adenovirus pneumonia. In consequence, the bacterial diversity in M. pneumoniae pneumonia is lower than that in adenovirus pneumonia and tracheomalacia, and Mycoplasma, Streptococcus and Pseudomonas predominate the lung microbiota in M. pneumoniae, adenovirus pneumonia and tracheomalacia respectively. The expression of IL-6, IL-8 and IL-10 is significantly higher in inpatients with adenovirus infection compared to children hospitalized with tracheomalacia and M. pneumoniae infection. The lung microbiota in M. pneumoniae pneumonia is remarkably correlated with IL-2, IL-4, IL-5, IL-6, TNF-α, and IL-1α, but not identified in tracheomalacia and adenovirus pneumonia. Microbiota analysis identified high load of multi-drug resistant Staphylococcus warneri, Acinetobacter baumannii or Abiotrophia defective in the lung microbiota of several inpatients, which partially explain the long length of hospitalization of these cases and intra-group differences at individual level.

Given the high proportion of novel microbial components in respiratory microbiota, we constructed a comprehensive respiratory microbial gene catalogue which is crucial for functional metagenomic analysis by metagenomic sequencing data of 334 respiratory samples of 171 healthy children and 76 pneumonia children in China. Established gene catalogue contains 2.24 million non-redundant genes, 50.92% and 27.52% of which can be assigned to a known microbial genus and species, respectively. This microbial gene catalogue will improve the understanding of host-microbiota interaction in Mycoplasma pneumoniae and adenoviral pneumonia. Microbial profiling by aligning metagenomic sequencing data of oropharyngeal samples to the established catalogue shows similar results to that which base on 16S rDNA analysis and clinical detection. Microbial functions in oropharynx of healthy children mainly belong to Prevotella, while Streptococcus represents the pivotal player in the microbial functions of patients. In addition, the metabolism of various monosaccharides, membrane transport and signal transduction-associated functional orthologues are found to accumulate significantly in the oropharyngeal microbiome of diseased children. A total of 17 microbial strains are recovered in oropharyngeal microbiota, among which 99.82% of M. pneumoniae genome is assembled and enriched in diseased children. Genomic analysis M. pneumoniae indicates the components of antibiotics resistance and virulence genes, which could be explained clinically.

This study will fill gaps in understanding the baseline microbiota structures in microbiota of upper respiratory tracts in healthy Chinese children, which will improve the interpretation of imbalanced upper respiratory tracts microbiota imbalance under acute respiratory infection. Exploration of the concordance between microbiota in upper and lower respiratory tracts indicated the microbiota transmission trajectory during adenovirus and M. pneumoniae infection. Establishment of respiratory microbial gene catalogue will also promote the exploration of unidentified and keystone microbial components, as well as microbial functions in microbiota resilience in upper respiratory tracts of Chinese children.