RESUMEN
Our understanding of the normal variation in the upper respiratory tract (URT) microbiota across the human lifespan and how these relate to host, environment, and health is limited. We studied the microbiota of 3,104 saliva (<10 year-olds)/oropharynx (≥10 year-olds) and 2,485 nasopharynx samples of 3,160 Dutch individuals 0-87 years of age, participating in a cross-sectional population-wide study (PIENTER-3) using 16S-rRNA sequencing. The microbiota composition was strongly related to age, especially in the nasopharynx, with maturation occurring throughout childhood and adolescence. Clear niche- and age-specific associations were found between the microbiota composition and host/environmental factors and health outcomes. Among others, social interaction, sex, and season were associated with the nasopharyngeal microbial community. By contrast, the oral microbiota was more related to antibiotics, tobacco, and alcohol use. We present an atlas of the URT microbiota across the lifespan in association with environment and health, establishing a baseline for future research.
Asunto(s)
Microbiota , Humanos , Anciano , Preescolar , Adulto , Niño , Persona de Mediana Edad , Adolescente , Anciano de 80 o más Años , Masculino , Femenino , Lactante , Adulto Joven , ARN Ribosómico 16S/genética , Estudios Transversales , Recién Nacido , Sistema Respiratorio/microbiología , Longevidad , Nasofaringe/microbiología , Saliva/microbiología , AmbienteRESUMEN
RATIONALE: Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections and hospitalizations in infants worldwide. Known risk factors, however, incompletely explain the variability of RSV disease severity, especially among healthy children. We postulate that the severity of RSV infection is influenced by modulation of the host immune response by the local bacterial ecosystem. OBJECTIVES: To assess whether specific nasopharyngeal microbiota (clusters) are associated with distinct host transcriptome profiles and disease severity in children less than 2 years of age with RSV infection. METHODS: We characterized the nasopharyngeal microbiota profiles of young children with mild and severe RSV disease and healthy children by 16S-rRNA sequencing. In parallel, using multivariable models, we analyzed whole-blood transcriptome profiles to study the relationship between microbial community composition, the RSV-induced host transcriptional response, and clinical disease severity. MEASUREMENTS AND MAIN RESULTS: We identified five nasopharyngeal microbiota clusters characterized by enrichment of either Haemophilus influenzae, Streptococcus, Corynebacterium, Moraxella, or Staphylococcus aureus. RSV infection and RSV hospitalization were positively associated with H. influenzae and Streptococcus and negatively associated with S. aureus abundance, independent of age. Children with RSV showed overexpression of IFN-related genes, independent of the microbiota cluster. In addition, transcriptome profiles of children with RSV infection and H. influenzae- and Streptococcus-dominated microbiota were characterized by greater overexpression of genes linked to Toll-like receptor and by neutrophil and macrophage activation and signaling. CONCLUSIONS: Our data suggest that interactions between RSV and nasopharyngeal microbiota might modulate the host immune response, potentially affecting clinical disease severity.
Asunto(s)
Perfilación de la Expresión Génica , Microbiota , Cavidad Nasal/microbiología , Faringe/microbiología , Infecciones por Virus Sincitial Respiratorio/microbiología , Estudios de Casos y Controles , Corynebacterium , Femenino , Haemophilus influenzae , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Microbiota/genética , Moraxella , Estudios Prospectivos , ARN Ribosómico 16S/genética , Índice de Severidad de la Enfermedad , Staphylococcus aureus , StreptococcusRESUMEN
RATIONALE: Cystic fibrosis (CF) is characterized by early structural lung disease caused by pulmonary infections. The nasopharynx of infants is a major ecological reservoir of potential respiratory pathogens. OBJECTIVES: To investigate the development of nasopharyngeal microbiota profiles in infants with CF compared with those of healthy control subjects during the first 6 months of life. METHODS: We conducted a prospective cohort study, from the time of diagnosis onward, in which we collected questionnaires and 324 nasopharynx samples from 20 infants with CF and 45 age-matched healthy control subjects. Microbiota profiles were characterized by 16S ribosomal RNA-based sequencing. MEASUREMENTS AND MAIN RESULTS: We observed significant differences in microbial community composition (P < 0.0002 by permutational multivariate analysis of variance) and development between groups. In infants with CF, early Staphylococcus aureus and, to a lesser extent, Corynebacterium spp. and Moraxella spp. dominance were followed by a switch to Streptococcus mitis predominance after 3 months of age. In control subjects, Moraxella spp. enrichment occurred throughout the first 6 months of life. In a multivariate analysis, S. aureus, S. mitis, Corynebacterium accolens, and bacilli were significantly more abundant in infants with CF, whereas Moraxella spp., Corynebacterium pseudodiphtericum and Corynebacterium propinquum and Haemophilus influenzae were significantly more abundant in control subjects, after correction for age, antibiotic use, and respiratory symptoms. Antibiotic use was independently associated with increased colonization of gram-negative bacteria such as Burkholderia spp. and members of the Enterobacteriaceae bacteria family and reduced colonization of potential beneficial commensals. CONCLUSIONS: From diagnosis onward, we observed distinct patterns of nasopharyngeal microbiota development in infants with CF under 6 months of age compared with control subjects and a marked effect of antibiotic therapy leading toward a gram-negative microbial composition.
Asunto(s)
Portador Sano/microbiología , Fibrosis Quística/microbiología , ADN Bacteriano/genética , Microbiota/genética , Nasofaringe/microbiología , ARN Ribosómico 16S/genética , Antibacterianos/uso terapéutico , Burkholderia/genética , Infecciones por Burkholderia/tratamiento farmacológico , Infecciones por Burkholderia/epidemiología , Infecciones por Burkholderia/microbiología , Portador Sano/epidemiología , Estudios de Casos y Controles , Estudios de Cohortes , Corynebacterium/genética , Infecciones por Corynebacterium/tratamiento farmacológico , Infecciones por Corynebacterium/epidemiología , Infecciones por Corynebacterium/microbiología , Fibrosis Quística/epidemiología , Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Infecciones por Enterobacteriaceae/epidemiología , Infecciones por Enterobacteriaceae/microbiología , Femenino , Infecciones por Haemophilus/tratamiento farmacológico , Infecciones por Haemophilus/epidemiología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/genética , Humanos , Lactante , Recién Nacido , Masculino , Moraxella/genética , Infecciones por Moraxellaceae/tratamiento farmacológico , Infecciones por Moraxellaceae/epidemiología , Infecciones por Moraxellaceae/microbiología , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/epidemiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Infecciones Estreptocócicas/tratamiento farmacológico , Infecciones Estreptocócicas/epidemiología , Infecciones Estreptocócicas/microbiología , Streptococcus mitis/genéticaRESUMEN
Early-life microbiota seeding and subsequent development is crucial to future health. Cesarean-section (CS) birth, as opposed to vaginal delivery, affects early mother-to-infant transmission of microbes. Here, we assess mother-to-infant microbiota seeding and early-life microbiota development across six maternal and four infant niches over the first 30 days of life in 120 mother-infant pairs. Across all infants, we estimate that on average 58.5% of the infant microbiota composition can be attributed to any of the maternal source communities. All maternal source communities seed multiple infant niches. We identify shared and niche-specific host/environmental factors shaping the infant microbiota. In CS-born infants, we report reduced seeding of infant fecal microbiota by maternal fecal microbes, whereas colonization with breastmilk microbiota is increased when compared with vaginally born infants. Therefore, our data suggest auxiliary routes of mother-to-infant microbial seeding, which may compensate for one another, ensuring that essential microbes/microbial functions are transferred irrespective of disrupted transmission routes.
Asunto(s)
Microbiota , Madres , Femenino , Embarazo , Humanos , Lactante , Parto Obstétrico , Cesárea , HecesRESUMEN
BACKGROUND: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia in school-aged children and can be preceded by asymptomatic carriage. However, its role in recurrent respiratory tract infections is unclear. We studied the prevalence of M.pneumoniae carriage in children with recurrent respiratory infections and identified associated factors. METHODS: We tested M.pneumoniae carriage by qPCR in children with recurrent infections and their healthy family members in a cross-sectional study. Serum and mucosal total and M.pneumoniae-specific antibody levels were measured by ELISA and nasopharyngeal microbiota composition was characterized by 16S-rRNA sequencing. FINDINGS: Prevalence of M.pneumoniae carriage was higher in children with recurrent infections (68%) than their family members without infections (47% in siblings and 27% in parents). M.pneumoniae carriage among family members appeared to be associated with transmission within the household, likely originating from the affected child. In logistic regression corrected for age and multiple comparisons, IgA (OR 0.16 [0.06-0.37]) and total IgG deficiency (OR 0.15 [0.02-0.74]) were less prevalent in M.pneumoniae carriers (n = 78) compared to non-carriers (n = 36). In multivariable analysis, the nasopharyngeal microbiota of M.pneumoniae carriers had lower alpha diversity (OR 0.27 [0.09-0.67]) and a higher abundance of Haemophilus influenzae (OR 45.01 [2.74-1608.11]) compared to non-carriers. INTERPRETATION: M.pneumoniae carriage is highly prevalent in children with recurrent infections and carriers have a less diverse microbiota with an overrepresentation of disease-associated microbiota members compared to non-carriers. Given the high prevalence of M.pneumoniae carriage and the strong association with H. influenzae, we recommend appropriate antibiotic coverage of M.pneumoniae and H. influenzae in case of suspected pneumonia in children with recurrent respiratory tract infections or their family members. FUNDING: Wilhelmina Children's Hospital Research Fund, 'Christine Bader Stichting Irene KinderZiekenhuis', Sophia Scientific Research Foundation, ESPID Fellowship funded by Seqirus, Hypatia Fellowship funded by Radboudumc and The Netherlands Organisation for Health Research and Development (ZonMW VENI grant to LM Verhagen).
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Microbiota , Infecciones Neumocócicas , Neumonía , Infecciones del Sistema Respiratorio , Niño , Humanos , Lactante , Streptococcus pneumoniae/genética , Mycoplasma pneumoniae/genética , Infecciones Neumocócicas/epidemiología , Estudios Transversales , Reinfección , Nasofaringe , Haemophilus influenzae , Portador Sano/epidemiologíaRESUMEN
One of the most widely used techniques in microbiota research is 16S-rRNA-sequencing. Several laboratory processes have been shown to impact sequencing results, especially in low biomass samples. Low biomass samples are prone to off-target amplification, where instead of bacterial DNA, host DNA is erroneously amplified. Knowledge on the laboratory processes influencing off-target amplification and detection is however scarce. We here expand on previous findings by demonstrating that off-target amplification is not limited to invasive biopsy samples, but is also an issue in low bacterial biomass respiratory (mucosal) samples, especially when below 0.3 pg/µL. We show that off-target amplification can partly be mitigated by using gel-based library purification methods. Importantly, we report a higher off-target amplicon detection rate when using MiSeq reagent kit v3 compared to v2 (mean 13.3% vs 0.1% off-target reads/sample, respectively), possibly as a result of differences in reagents or sequencing recipes. However, since after bioinformatic removal of off-target reads, MiSeq reagent kit v3 still results in a twofold higher number of reads when compared to v2, v3 is still preferred over v2. Together, these results add to the growing knowledge base on off-target amplification and detection, allowing researchers to anticipate this problem in 16S-rRNA-based microbiome studies involving low biomass samples.
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ADN , Secuenciación de Nucleótidos de Alto Rendimiento , ADN/genética , ADN Bacteriano/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Indicadores y Reactivos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/métodosRESUMEN
The respiratory tract is populated by a specialized microbial ecosystem, which is seeded during and directly following birth. Perturbed development of the respiratory microbial community in early-life has been associated with higher susceptibility to respiratory tract infections (RTIs). Given a consistent gap in time between first signs of aberrant microbial maturation and the observation of the first RTIs, we hypothesized that early-life host-microbe cross-talk plays a role in this process. We therefore investigated viral presence, gene expression profiles and nasopharyngeal microbiota from birth until 12 months of age in 114 healthy infants. We show that the strongest dynamics in gene expression profiles occurred within the first days of life, mostly involving Toll-like receptor (TLR) and inflammasome signalling. These gene expression dynamics coincided with rapid microbial niche differentiation. Early asymptomatic viral infection co-occurred with stronger interferon activity, which was related to specific microbiota dynamics following, including early enrichment of Moraxella and Haemophilus spp. These microbial trajectories were in turn related to a higher number of subsequent (viral) RTIs over the first year of life. Using a multi-omic approach, we found evidence for species-specific host-microbe interactions related to consecutive susceptibility to RTIs. Although further work will be needed to confirm causality of our findings, together these data indicate that early-life viral encounters could impact subsequent host-microbe cross-talk, which is linked to later-life infections.
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Interacciones Microbiota-Huesped , Microbiota/genética , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/virología , Virosis/inmunología , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Haemophilus/inmunología , Humanos , Lactante , Recién Nacido , Inflamasomas , Masculino , Microbiota/inmunología , Moraxella/inmunología , Nasofaringe/virología , Recurrencia , Infecciones del Sistema Respiratorio/fisiopatología , Especificidad de la EspecieRESUMEN
The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray-Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.
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Benchmarking/métodos , Microbiota , Juego de Reactivos para Diagnóstico/normas , Mucosa Respiratoria/microbiología , Biomasa , Humanos , Metagenómica/métodos , Metagenómica/normas , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , ARN Ribosómico 16S/genética , Saliva/microbiologíaRESUMEN
Respiratory tract infections are a major cause of morbidity and mortality worldwide in young children. Concepts such as the gut-lung axis have highlighted the impact of microbial communities at distal sites in mediating disease locally. However, little is known about the extent to which microbial communities from multiple body sites are linked, and how this relates to disease susceptibility. Here, we combine 16S-based rRNA sequencing data from 112 healthy, term born infants, spanning three body sites (oral cavity, nasopharynx, gut) and the first six months of life. Using a cross-niche microbial network approach, we show that, already from the first week of life on, there is a strong association between both network structure and species essential to these structures (hub species), and consecutive susceptibility to respiratory tract infections in this cohort. Our findings underline the crucial role of cross-niche microbial connections in respiratory health.
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Bacterias/aislamiento & purificación , Tracto Gastrointestinal/microbiología , Microbiota , Boca/microbiología , Nasofaringe/microbiología , Infecciones del Sistema Respiratorio/microbiología , Estudios de Cohortes , Microbioma Gastrointestinal , Humanos , Lactante , Recién Nacido , Países BajosRESUMEN
OBJECTIVES: Patients with Cystic Fibrosis (CF) suffer from pancreatic insufficiency, lipid malabsorption and gastrointestinal complaints, next to progressive pulmonary disease. Altered mucosal homoeostasis due to malfunctioning chloride channels results in an adapted microbial composition of the gastrointestinal and the respiratory tract. Additionally, antibiotic treatment has the potential to distort resident microbial communities dramatically. This study aims to investigate early life development of the gut microbial community composition of children with CF compared to healthy infants and to study the independent effects of antibiotics taking into account other clinical and lifestyle factors. STUDY DESIGN: Faecal samples from 20 infants with CF and 45 healthy infants were collected regularly during the first 18 months of life and microbial composition was determined using 16S rRNA based sequencing. RESULTS: We observed significant differences in the overall microbiota composition between infants with CF and healthy infants (p<0.001). Akkermansia and Anaerostipes were significantly more abundant in control infants, whereas Streptococci and E. coli were significantly more abundant in infants with CF, also after correction for several clinical factors (p<0.05). Antibiotic use in infants with CF was associated with a lower alpha diversity, a reduced abundance of Bifidobacterium and Bacteroides, and a higher abundance of Enterococcus. CONCLUSION: Microbial development of the gut is different in infants with CF compared to healthy infants from the first months of life on, and further deviates over time, in part as a result of antibiotic treatment. The resulting dysbiosis may have significant functional consequences for the microbial ecosystem in CF patients.
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Bacterias , Fibrosis Quística , Disbiosis , Microbioma Gastrointestinal , Antibacterianos/efectos adversos , Antibacterianos/uso terapéutico , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/epidemiología , Fibrosis Quística/microbiología , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Disbiosis/diagnóstico , Disbiosis/etiología , Disbiosis/microbiología , Heces/microbiología , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/fisiología , Humanos , Lactante , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/fisiopatología , Masculino , Países Bajos/epidemiología , ARN Ribosómico 16S/análisis , Análisis de Secuencia de ARNRESUMEN
Birth by Caesarian section is associated with short- and long-term respiratory morbidity. We hypothesized that mode of delivery affects the development of the respiratory microbiota, thereby altering its capacity to provide colonization resistance and consecutive pathobiont overgrowth and infections. Therefore, we longitudinally studied the impact of mode of delivery on the nasopharyngeal microbiota development from birth until six months of age in a healthy, unselected birth cohort of 102 children (n=761 samples). Here, we show that the respiratory microbiota develops within one day from a variable mixed bacterial community towards a Streptococcus viridans-predominated profile, regardless of mode of delivery. Within the first week, rapid niche differentiation had occurred; initially with in most infants Staphylococcus aureus predominance, followed by differentiation towards Corynebacterium pseudodiphteriticum/propinquum, Dolosigranulum pigrum, Moraxella catarrhalis/nonliquefaciens, Streptococcus pneumoniae, and/or Haemophilus influenzae dominated communities. Infants born by Caesarian section showed a delay in overall development of respiratory microbiota profiles with specifically reduced colonization with health-associated commensals like Corynebacterium and Dolosigranulum, thereby possibly influencing respiratory health later in life.
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Bacterias/aislamiento & purificación , Microbiota , Nasofaringe/microbiología , Infecciones del Sistema Respiratorio/microbiología , Bacterias/clasificación , Bacterias/genética , Cesárea , Estudios de Cohortes , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Femenino , Edad Gestacional , Humanos , Recién Nacido , Filogenia , Embarazo , Staphylococcus aureus/clasificación , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Short-term in vitro expansion of antigen-specific T cells is an appreciated assay for the analysis of small memory T-cell populations. However, how well short-term expanded T cells represent the direct ex vivo situation remains to be elucidated. In this study we compared the clonality of Epstein-Barr virus (EBV) and cytomegalovirus (CMV)-specific CD8(+) T cells directly ex vivo and after in vitro stimulation with antigen. Our data show that the antigen-specific T cell repertoire significantly alters after in vitro culture. Clear shifts in clonotype hierarchy were observed, with the most dominant ex vivo clonotype decreasing after stimulation at the expense of several previously subdominant clonotypes. Notably, these alterations were more pronounced in polyclonal T-cell populations compared to mono- or oligoclonal repertoires. Furthermore, TCR diversity significantly increased after culture with antigen. These results suggest that the T-cell repertoire is highly subjective to variation after in vitro stimulation with antigen. Hence, although short-term expansion of T cells provides a simple and efficient tool to examine antigen-specific immune responses, caution is required if T-cell populations are expanded prior to detailed, clonotypic analyses or other repertoire-based investigations.