Airway microbiome and lung injury

Introduction

It has been determined that only 1% of all bacteria can be cultured and many of the microbial species that normally (or abnormally) inhabit the human body are not identified by culture. Culture-independent molecular techniques have demonstrated that body sites, including the lung, are not sterile and are host to communities of microorganisms. The term microbiota refers specifically to the community of microorganisms living in a particular environment while the term microbiome refers to the communities of microorganisms and their encoded genes. Microbial diversity measures how much variety exists in a microbial community and is characterized by richness (the number of different bacterial species present) and evenness (the relative abundance of the various species within the microbial community), while the term dysbiosis describes a microbial pattern associated with disease states.

Culture-independent molecular methods show that the microbiota of humans is far greater than previously recognized. The Human Microbiome Project focused particularly on the normal microbiome of the skin, mouth, nose, digestive tract, and vagina and found that even healthy individuals differ remarkably in the diversity and abundance of the microbiome at the different sites. The relative abundance of members of a microbiome is most often determined by sequencing of the variables regions (V1–3 or V4–6) of the bacterial 16S ribosomal RNA (rRNA), but more extensive metagenomic and metatranscripomic sequencing provides more in-depth data on microbial gene function and possible host interactions.

Due to the relative inaccessibility of the lower airways and the lungs, there has been less research on the airway and pulmonary microbiome as compared to the gastrointestinal or oral microbiome. Hilty et al. found that lower airways of adults are not sterile, with approximately 2000 bacterial genomes per cm ² surface sampled. They also found that the tracheobronchial tree contained a characteristic microbial flora that differs from the nares and oropharynx and between health and disease. ^, The major colonists in normal people are anaerobes such as Bacteroidetes (e.g., Prevotella spp.) grown with difficulty in culture while Proteobacteria (e.g., Haemophilus , Moraxella , Neisseria spp.) are strongly associated with airway disease in chronic obstructive pulmonary disease (COPD) and asthma. It is possible that microbial immigration from the oral cavity contributes to the lung microbiome during health, although the lungs selectively eliminate Prevotella bacteria derived from the upper airways. In healthy adult lungs, spatial variation in microbiota within an individual is significantly less than variation across individuals, and bronchoalveolar lavage (BAL) of a single lung segment is probably acceptable for sampling the healthy lung microbiome. A limitation of bronchoscopic sampling of the airways is the possible contamination from the oral or nasal flora. However, direct sampling of lung tissue derived from nonmalignant lung tissue samples from cancer patients determined that Proteobacteria is the dominant phylum, and other common phyla include Firmicutes , Bacteroidetes , and Actinobacteria. Microbiota taxonomic alpha diversity increased with environmental exposures to air particulates, residency in high-density population areas, and smoking pack-years.

Newborn lung microbiome

The newborn lung microbiome is even more technologically challenging to sample, since available sampling is limited to intubated infants with upper airways sampled by tracheal aspirates of the endotracheal tube and distal airways sampled by tracheal lavage. Despite this significant limitation, there have been some recent studies evaluating the airway microbiome in preterm infants, specifically in relation to development of bronchopulmonary dysplasia (BPD). Mourani et al. evaluated serial tracheal aspirates (<72 hours, 7 days, 14 days, and 21 days) from 10 preterm infants who required mechanical ventilation for at least 21 days. Samples were analyzed by quantitative real-time polymerase chain reaction (PCR) assays for total bacterial load and by pyrosequencing for bacterial identification. Seventy-two organisms were observed in total. Seven organisms represented the dominant organism (>50% of total sequences) in 31 of the 32 samples with positive sequences. Staphylococcus and the genital mycoplasmas Ureaplasma parvum and Ureaplasma urealyticum were the most frequently identified dominant organisms, but Pseudomonas , Enterococcus , and Escherichia were also identified. Most infants in this series established either Staphylococcus spp. ( Firmicutes ) or Ureaplasma spp. ( Tenericutes ) as the predominant organism by 7 days of age. Lohmann et al. evaluated tracheal aspirates of 25 preterm infants obtained at birth and on days 3, 7, and 28. Bacterial DNA was extracted, and 16S rRNA genes were amplified and sequenced. It was found that Acinetobacter was the predominant genus in the airways of all infants at birth. Infants who developed BPD had reduced bacterial diversity at birth.

Recently, we evaluated the airway microbiome of extremely preterm and term infants soon after birth and in preterm infants with established BPD. Tracheal aspirates were collected from a discovery cohort of 23 extremely low-birth-weight (ELBW) infants and 10 full-term (FT) infants (with no respiratory disease) at birth or within 6 hours of birth at the time of intubation, as well as from 18 infants with established BPD in whom samples were obtained at 36 weeks postmenstrual age (PMA) at the time of endotracheal tube change. A validation cohort was used consisting of tracheal aspirates from extremely preterm infants at a different institution. We were able to detect and characterize bacterial DNA by 16S rRNA sequencing in tracheal aspirates of all ELBW and FT infants soon after birth. The lung microbiome was similar at birth in ELBW and FT infants irrespective of gestational age. Both ELBW and FT infants had a predominance of Firmicutes and Proteobacteria on the first day of life, in addition to Actinobacteria , Bacteroidetes , Tenericutes , Fusobacterium , Cyanobacteria , and Verrucomicrobia ( Fig. 4.1 ). The relative abundance of bacterial phyla and Shannon alpha diversity did not differ between ELBW and FT infants. Compared to newborn FT infants matched for PMA, the airway microbiome of infants after the diagnosis of BPD was characterized by increased phylum Proteobacteria and decreased phyla Firmicutes and Fusobacteria ( Fig. 4.2 ). At the genus level, the most abundant Proteobacteria in BPD patients were Enterobacteriaceae . To confirm the presence of Proteobacteria in the BPD patient samples, we also performed specific endotoxin assays. Endotoxin concentrations in the airway were similar between term and preterm infants at birth, but endotoxin levels were increased in infants with established BPD compared to concentrations at birth. Serial samples in five ELBW infants who went on to develop BPD demonstrated a distinct temporal dysbiotic change with decreases in Firmicutes and increases in Proteobacteria over time. It was observed both in the discovery cohort and the validation cohort that genus Lactobacillus was less abundant even as early as birth in infants who later developed BPD, compared to the infants who did not develop BPD ( Fig. 4.2 ). Interestingly, preterm birth was associated with alterations in the vaginal microbial community with decreased relative abundance of Lactobacillus.

Analyzing airway microbial community turnover in sequentially sampled tracheal aspirates from ventilated preterm infants, Wagner et al. found that infants who eventually developed severe BPD had greater bacterial community turnover with age, acquired less Staphylococcus soon after birth, and had higher initial relative abundance of Ureaplasma .

As both extremely preterm and term infants had a similar diverse microbiome at birth, it is probable that the airway and lung microbiomes are established before birth, potentially through transplacental passage of bacterial products. This contention is supported by culture-independent studies of the uterine microbiome demonstrating the presence of bacteria in the placenta, fetal membranes, and amniotic fluid of healthy pregnancies that have challenged the notion that the fetus develops in a sterile environment and have increased our understanding of infection-related preterm birth as a polymicrobial diease. Using next-generation sequencing and metagenomic analyses, Aagaard et al. identified a low abundance microbiota in the placenta of term and preterm placentas including Escherichia coli , Prevotella tannerae , Bacteroides species, and Fusobacterium species. Using 16S rDNA pyrosequencing to identify bacteria in placental membranes from term and preterm deliveries, Doyle et al. found six genera ( Fusobacterium , Streptococcus , Mycoplasma , Aerococcus , Gardnerella , and Ureaplasma ) and one family ( Enterobacteriaceae ) that were more abundant in preterm membranes or absent in term membranes. There was reduced abundance of genus Lactobacillus and increased abundance of genera Streptococcus , Aerococcus , and Ureaplasma in membranes from preterm infants delivered by the vaginal route. There is some evidence for a low biomass microbiome in both fetal lung and placenta even very early in gestation in the first trimester, which shows maturational changes with advancing gestational age.

It is not currently known what proportion of the transferred bacterial DNA from the placenta are from live bacteria and what proportion is from “processed” bacterial products (DNA fragments, cell wall fragments, etc.). Our recent study indicated the presence of both bacterial DNA and bacterial lipopolysaccharide in neonatal airways at the time of birth. Microbiome analysis evaluates bacterial DNA but does not indicate if the DNA is from live bacteria. It has been suggested that evaluation of susceptibility of the bacterial DNA to DNAse I may indicate the proportion of DNA from live bacteria, as live bacterial DNA is DNAse I resistant, but bacterial DNA from dead bacteria is DNAse I sensitive—63% of DNA in porcine BALF is DNAse I sensitive, suggesting the majority of airway bacterial DNA is from dead bacteria.

It may be speculated that the establishment of the lung microbiome during fetal life enables the priming of the immune system in the fetus and later recognition of and response to bacterial flora encountered after birth. Alterations in the airway microbiome are associated with childhood pulmonary disorders such as asthma. ^, It is also likely that the lung microbiome contributes to normal alveolar development. Yun et al. studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non–specific pathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF, or germ-free (GF) facilities. Metabolically active murine lung microbiota were found in all but GF mice. Bacteria were detectable by fluorescent in situ hybridization (FISH) on alveolar epithelia in the absence of inflammation. A higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae (consistent with better alveolarization), which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. There is increasing evidence that lung and gut microbiota are altered by hyperoxia, with selective relative growth advantage of oxygen-tolerant microbes (e.g., Staphylococcus aureus ), which may contribute to oxygen-induced lung injury in mice. Willis et al. have shown that perinatal maternal antibiotic exposure augments lung injury in newborn mice exposed to hyperoxia, while Dolma and colleagues have shown that GF mice without a microbiome were protected from hyperoxia injury, showing improved lung structure and mechanics, and decreased inflammation compared to hyperoxia-exposed, non-GF mice. These studies indicate that dysbiosis is a contributor to lung injury and abnormal lung development in the BPD model.

There are many potential mechanisms by which the microbiome may modulate lung injury and repair. We have recently demonstrated that bacterial lipopolysaccharide (LPS) exposure alters exosomal microRNA in tracheal aspirates and reduces specific microRNA such as miR-876-3p that contributes to a BPD phenotype. We have also shown that the early airway microbiome alters the metabolome, and that the airway metabolome of BPD-predisposed infants was enriched for metabolites involved in fatty acid activation and androgen and estrogen biosynthesis compared with BPD-resistant infants. It is known that manipulation of the gut microbiota may influence lung pathology via the gut-lung axis, but it is not clear if the manipulation of the gut microbiome also simultaneously alters the lung microbiome or if the effects in the lung can be due solely to alterations of the microbiome in the gut. It is possible that bacteria or bacterial products from the gut may be translocated to the systemic circulation and filtered from the pulmonary circulation into the lungs. It is known that the lung microbiome is enriched with gut bacteria in sepsis and the acute respiratory distress syndrome.

In the next section, we will review the human and experimental evidence that the low-virulence pathogens U. parvum and U. urealyticum that are common members of the vaginal, amniotic fluid, placental, and preterm lung microbiota contribute to preterm birth and lung injury due to an augmented dysregulated inflammatory response that contributes to the development of BPD. Recent studies provide new insights into how these organisms evade the host immune response to establish colonization in the intrauterine cavity and fetal/newborn lung and identify these mechanisms as potential therapeutic targets.