The Nasal Microbiome of Predicting Bronchopulmonary Dysplasia in Preterm Infants

Abstract

Bronchopulmonary dysplasia (BPD) is chronic lung disease of prematurity and associated with substantial long-term disabilities. To characterize and compare the nasal swabs microbiome of early stage in premature infants and determine whether microbial diversity or composition in the airway associated with BPD disease. We performed a prospective observational cohort design. Preterm neonates less than 32 weeks of gestation were recruited from NICU, Children's Hospital, Zhejiang University School of Medicine from 2019 to 2020. Sterile foam swabs were collected from anterior nares at 1 and 3 weeks of postnatal age. We used PCR amplification and 16S rDNA sequencing. Neonatal demographic data including gestational age, birth weight, medication administration history were recorded. A total of 98 nasal swabs samples were collected from 54 preterm infants, 13 developed BPD infants and 41 control infants were finally involved in the study. Birth weights ranged from 700 to 2,050 g. Gestational age ranged from 25 ^2/7to 31 ^6/7. We found increased in the expression of Prevotella, Marinomonas, Enterobacteriaceae, Weissella, Selenomonas, Oribacterium, Nubsella and Antricoccus in BPD group at two time points. Prevotella was correlated with the severity of BPD (Spearman r=0.361, P=0.000). Given possible roles for noninvasive upper airway microbiota in BPD pathobiology, the nasal microbiome in BPD is a compelling area of research to continue to expand.

Introduction

Bronchopulmonary dysplasia (BPD) is a chronic lung disease caused by mechanical ventilation and oxygen therapy and is the most common complication that affects premature infants¹. The disease leads to longstanding consequences involving adverse effects on pulmonary function and neurodevelopmental outcome. BPD rates are reported 40–55% in surviving extremely low gestational age neonates over the last few decades². Despite the new advances in the field of neonatology, the incidence of BPD has largely been unchanged due to increased survival of extremely premature infants. There is still no ideal management for BPD disease and the only method to alleviate severity and improve the prognosis is early prevention. Therefore, early recognition of susceptible BPD is absolutely essential for timely intervention. Recent studies have demonstrated early airway microbiome may serve a role in modulating the infant's future susceptibility to severe BPD development³,suggesting another underlying pathway related to abnormal lung development ^4–7.

The airway microbiome can be identified early after birth and evolves over time with increasing bacterial loads and diversity⁸. Lal et al. were surprised to find that the airway microbiome of the neonates delivered by vaginal or cesarean section were similar, which indicated that the microbial DNA in the airway may be obtained through the placenta⁹. They described the composition of the airway microbiota by analyzing the airway secretions on the first day of birth: Firmicutes and Proteobacteria were dominant and Actinobacteria, Bacteroidetes, Tenericutes, Fusobacterium, Cyanobacteria, and Verrucomicrobia were observed⁹. In BPD patients, as the disease progresses, the microbial community turnover increases, the relative abundance of Proteobacteria and Firmicutes changes, and Lactobacillus decreases⁶. Some important factors affect the composition and colonization of the pulmonary microbiota including prenatal and postnatal exposure to antibiotics, sepsis, environmental microbiome, method of delivery, feeding and nutrition ^10,11. Study has also been reported the crosstalk between the lung and the intestine, which proposes a concept of the gut-lung axis, and the concomitant intestinal microbiota development also affects lung microbiota, resulting in pulmonary diseases ¹².

Pathogen detection requires sampling of lower airway secretions, which remains a challenge in non-expectorating patients. Currently, bronchoalveolar lavage (BAL) is considered as the gold standard; however, it cannot be performed and not suitable for every premature infant, so we explore a non-invasive nasal swab. The nasal cavities represent a highly accessible airway microbial community that recently was confirmed to have a pivotal role in human health and, to date, few studies focused on the microbiome of the nostrils of neonates^13,14. Nasal cavity communicates with the outside world and is exposed to a variety of exogenous and endogenous microbes. It may play important roles in protecting against nasal colonization as well as invasive disease¹⁵. Previous studies have shown that there was a large amount of overlap between the nasal microbiota and the respiratory microbiota^15,16, so the nasal microbiota could, to some extent, reflect the characteristics of the respiratory microbiota. Therefore, studying the composition and characteristic of nasal microbiota may open a window for exploring respiratory microbiota in preterm infants. The aim of this study is to: (1) characterize and compare the nasal swabs microbiome in premature infants and BPD infants (2) determine whether BPD disease is correlated with any microbial function in the airway.

Results

Discussion

There is no study that have analyzed the preterm infant’s nasal microbiome in BPD populations that provide mechanistic explanations for microbiome change during BPD, and its impact on host-microbiome interaction. The present study of the nasal microbiome in BPD, we found no difference in nasal microbial composition between preterm infants with BPD and controls. Our study identified Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were dominant phylums and Muribaculaceae, Escherichina, Staphylococcus, Corynebacterium were dominant genus in BPD infants relative to controls. We found increased in the expression of Prevotella in BPD group at both time points. Prevotella was correlated with the severity of BPD. Metagenomic prediction identified reduced coumarins and mannosylglycerate biosynthesis as the metabolic pathway differentially associated with BPD.

In comparison to the gut microbiome, nasal microbiome has remained under studied. The nasal cavity is a part of the mucosal system of the upper respiratory tract, and its microbial composition can reflect the entire respiratory tract composition of microorganisms ^15,18. Several studies revealed how the microbiota developed in regions of the respiratory tract in newborns and during early life^14,19. The nasal microbiota is of particular concern as the nostrils may harbor pathogens, which can cause severe respiratory diseases²⁰. Nasal microbiota compositions characterized by Moraxella, Streptococcus, or Haemophilus have been reported to be associated with upper respiratory infection²¹. Our study found that the expression of Prevotella was higher in the BPD group and it was correlated with the severity of BPD disease. Interestingly, bacterial Prevotella have been found to be prevalent commensal colonizers at mucosal sites; being the predominant genus in the respiratory system^22,23. In light of the abundant Prevotella colonization and low pathogenicity it is likely that humans have co-evolved with Prevotella. However, emerging studies have linked increased Prevotella abundance and specific strains to inflammatory disorders, suggesting that at least some strains exhibit pathobiontic properties. Increased Prevotella abundance is associated with augmented Th17 mediated mucosal inflammation²⁴. Prevotella-high profile being associated with enhanced “subclinical” lung inflammation, that is notable for enhanced expression of inflammatory cytokines and elevated Th-17 lymphocytes²⁵. Our results support the hypothesis of persistent lung inflammation after mechanical ventilation and/or lung infection, especially as Prevotella is a potential pathogen in respiratory disease in early period. Contrast to previous data from young adults born extremely preterm exhibit significant dysbiosis, which is characterized by a significant reduction in the relative abundance of genus Prevotella²⁶. However, to clearly examine this, “Prevotella” animal model should be assessed in future studies.

Coumarins are found in many bacteria, with promising pharmacological activities, including antioxidant, antimicrobial, and anti-inflammatory efficacies. The beneficial effects of coumarins include antimicrobial ²⁷²⁸. The antioxidant and anti-inflammatory activities of coumarins have been well-acknowledged in vitro and in vivo studies^29,30. In our study, we found that coumarins biosynthesis decreased in BPD infants. It may suggest BPD infants have lower anti-inflammatory activities in early period. Moreover, coumarins can effectively reduce tissue edema-associated inflammation through suppressing both lipoxygenase and cyclooxygenase enzymatic activities and prostaglandin synthesis and release³¹. Another metabolic pathway that is reduced is the mannosylglycerate biosynthesis pathway. The synthesis process is the conversion of GDP mannose and d-glycerate or d-3-phosphoglycerate to mannosylglycerate. The compatible solute mannosylglycerate has properties in terms of protein stabilization and protection under heat and freeze-drying stresses as well as against protein aggregation. Due to these characteristics, it possesses large potential for clinical applications³². The synthesis of mannosylglycerate in BPD group decreased, but the mechanism is not clear.

On the one hand, a limitation of this study is the small number of infants. Despite the high number of samples, a larger study population is needed to detect additional differences between subjects. On the other hand, although BPD is a disease of lower airways, our data are based on nasal airway samples. However, lower airway sampling is both ethically and technically challenging in prematurity. Importantly, analysis of microbiota during BPD in a higher number of infants is needed to understand the role of Prevotella. Also, the causal and mechanistic pathways between Prevotella infections and the microbiota coumarins and mannosylglycerate biosynthesis metabolic pathways remain unclear. It needs to be assessed in translational approaches or using animal models. Given possible roles for noninvasive upper airway microbiota in BPD pathobiology, monitoring and investigation of BPD infants, the nasal microbiome in BPD is a compelling area of research to continue to expand.

Materials And Methods

Recruited infants and sample collection

The study was performed as a prospective observational cohort design, and was approved by the ethics committee of the Children’s Hospital, Zhejiang University School of Medicine(2018-IRB-090-A2). Informed consent was obtained from at least one guardian of each patient and all procedures were conducted according to the guidelines. Preterm neonates less than 32 weeks of gestation were recruited from neonatal intensive care unit (NICU), Children's Hospital, Zhejiang University School of Medicine from 2019 to 2020. Exclusion criteria were major congenital anomalies of the lung or airway, known infection, or pneumonia. Sterile foam swabs were collected from anterior nares. Swabs were collected at 1 and 3 weeks of postnatal age. The first swab was collected by 5-7 days after birth following written informed consent from parents. The second swab was collected by 15-21 days of age. Swab tips were snapped off into sterile 1.5-ml polyethylene tubes, transferred immediately to −80°C freezer for storage. All infants were followed up until 36-week postmenstrual age, when the physiological definition of BPD. We used National Institute of Child Health and Human Development (NICHD) 2019 revision to define severity of BPD ¹. All infants were stratified into the following two groups: developed BPD (BPD group) or did not develop BPD (control group). Neonatal demographic data including gestational age, birth weight, gender, delivery mode, medication administration history, infants’ diet type (human milk vs. formula) and significant events during NICU course, were extracted from the electronic medical records.

DNA extractions

DNA was extracted from swabs using the E.Z.N.A. ®Stool DNA Kit (D4015, Omega, Inc., USA) according to manufacturer’s instructions. The total DNA was eluted in 50 μL of Elution buffer and stored at -80 °C until measurement.

PCR amplification and 16S rDNA sequencing
 The V3-V4 region of the bacterial small-subunit (16S) rRNA gene was amplified with primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHVGGGTATCTAATCC-3')³³. PCR amplification was performed in a total volume of 25 μL reaction mixture containing 25 ng of template DNA, 12.5 μL PCR Premix, 2.5 μL of each primer. The PCR conditions is initial denaturation at 98 ℃ for 30 seconds; 32cycles of denaturation at 98 ℃ for 10 seconds, annealing at 54 ℃ for 30 seconds, and extension at 72 ℃ for 45 seconds; and then final extension at 72 ℃ for 10 minutes. The PCR products were purified by AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by Qubit (Invitrogen, USA). The amplicon pools were prepared for sequencing and the size and quantity of the amplicon library were assessed on Agilent 2100 Bioanalyzer (Agilent, USA) and with the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. Samples were sequenced on an Illumina NovaSeq platform according to the manufacturer's recommendations (LC-Bio Technology Co., Ltd, Hang Zhou, China).

Data analysis

Paired-end reads was assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired-end reads were merged using FLASH Quality filtering on the raw reads were performed under specific filtering conditions to obtain the high-quality clean tags according to the fqtrim(v0.94). Chimeric sequences were filtered using Vsearch software (v2.3.4). After dereplication using DADA2, we obtained feature table and feature sequence. Principal coordinate analysis (PCoA) analysis was displayed by QIIME2 and ggplot2 package. Nonmetric multidimensional scaling (NMDS) analysis was performed with the vegan package and displayed with the ggplot2 package in R software. The figures were drawn by R (v3.5.2).

Declarations

Acknowledgements We would like to thank the parents of the participants and all staff of participating NICU and assistance with patient recruitment. No compensation was received other than salary support for their contribution. We would like to thank the Wei wang for helping data analysis.
Contributors Yanping Xu, Yeqing Huang, Zhen Shen and Liping Shi had full access to all of the data in the study and take responsibility for the integrity of the data and accuracy of the data analysis. Study concept and design: Yanping Xu and Liping Shi. Acquisition, analysis or data interpretation: Yanping Xu and Yeqing Huang. Drafting of the manuscript: Yanping Xu. Critical revision of the manuscript: all authors. Statistical Analysis: Yanping Xu and Zhen Shen. Obtained funding: Yanping Xu. Study supervision: Liping Shi. All authors reviewed the manuscript.

Competing interests The author(s) declare no competing interests.

Funding This study was funded by grants from the National Natural Science Foundation of China (No.81873845). 

Data availability statement Data are available in a public, open access repository. Sequence data have been deposited to the NCBI Sequence Read Archive and are available under accession number PRJNA782204.

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Tables

Table 1. Demographics of infants enrolled in the study

BPD, bronchopulmonary dysplasia; NEC, Necrotizing enterocolitis; PDA, patent ductus arteriosus * Results significant with P<0.05