Introduction
In view of the association between intestinal dysbiosis and the development of important preterm neonatal conditions such as necrotising enterocolitis (NEC) and late-onset sepsis (LOS), efforts have been undertaken to assess the impact that different components of clinical care may have on the developing preterm intestinal microbiome. Understandably, the main focus of these works has been on assessing the impact of more microbiologically plausible interventions, such as prebiotics, probiotics and antibiotics. However, there are other interventions which may also be theorised to affect preterm intestinal microbiome development.
One such component of clinical care which is less well reported in the literature is the relationship between preterm neonatal respiratory support, and the developing microbiome. While the respiratory and gastrointestinal systems may seem only tangentially related, respiratory interventions can impact on the intestinal intraluminal environment, as evidenced by the concept of ‘CPAP-belly’,1 whereby inhaled gases enter the gastrointestinal tract via aerophagia, potentially changing the environment from its oxygen-depleted natural state.2 Swallowed gas is one of the factors that influences intraintestinal gas content, and it can be reasonably hypothesised that if aerophagia is increased (as occurs in continuous positive airway pressure (CPAP) use), then the oxygen fraction in intraintestinal gas contents will similarly increase,3 and would be compounded through the use of hyperoxic inhaled gas (as frequently occurs in preterm babies).
Where the relationship between respiratory support and microbiome development has been examined, an increased relative abundance of aerobic and facultative anaerobic bacteria is seen in association with respiratory support provision.3–5 However, these studies either do not discriminate between invasive and non-invasive respiratory modalities, with their profoundly different effects on intestinal gas delivery; or have not differentiated the purported dysbiotic effects of mechanical ventilation from the recognised and concurrent dysbiotic effects of preterm life6–9 and the medical interventions associated therewith.10 A recent clinical trial in adults examining the effect of CPAP on intestinal dysbiosis (in obstructive sleep apnoea patients) failed to recruit sufficient numbers.11
With the advent of respiratory management strategies which advocate the use of non-invasive positive pressure ventilation in more extremely preterm infants and earlier in life,12 understanding the effects of this on the developing intestinal microbiome is increasingly important, in these infants more at-risk of the serious complications of preterm life.
This study aims to assess the potential changes in microbiome development following the introduction of discrete changes in respiratory management, in isolation (as much as is feasible) from other known or potential modulators of bacterial community development, through a high-fidelity assessment of changes in the intestinal microbial community at the time of respiratory support transition.
Methodology
Subject recruitment, sample collection and handling, library preparation, sequencing and bioinformatics processing were conducted as described in our previous study,10 and are included as online supplemental file 1. The wet-lab and analysis protocols are similarly attached as a online supplemental files 2 and 3, respectively.
Assessment of the contemporaneous clinical data revealed the many ways in which respiratory support modalities could change:
Parameter changes within invasive respiratory support modes.
Changes between invasive respiratory support modes.
Extubation from invasive to non-invasive respiratory support modalities.
Parameter changes within non-invasive respiratory support modes.
Changes between non-invasive respiratory support modes.
Changing from non-invasive respiratory support modes, to self-ventilation (without pressure support, but +/− supplementary oxygen).
A pragmatic decision was made to assess changes in microbiome progression around the point of conversion from non-invasive positive pressure respiratory support (ie, bi-level positive airway pressure (BIPAP); CPAP; or heated, humidified high-flow nasal cannulae (HHFNC)) to spontaneous breathing (either with or without supplemental oxygen). This particular transition was chosen as it occurred in a significant number of subjects (maximising the power to detect any differences); and as it took place at times of clinical stability (hence the decision to progress to lesser levels of support), there was a reduced incidence of concurrent microbiome modulators (eg, antibiotics), which would have confounded the findings. This clinical event also represented the key hypothesised change in the intestinal environment: the transition from a potentially hyperoxic environment in the presence of non-invasive positive pressure respiratory support, to the increasingly anaerobic environment of the gastrointestinal tract in a spontaneously breathing subject.
Episodes were identified using the following criteria:
The transition must be from a period of non-invasive positive pressure respiratory support (eg, BIPAP/CPAP/HHFNC), to a period of self-ventilation.
Sufficient (at least three) samples must be available from the periods prior to and after the transition from non-invasive positive pressure respiratory support to self- ventilation, in order to accurately reflect the microbiome progression during these periods.
The period under analysis must occur at least 5 days clear of preceding antibiotic use, to reduce the risk of microbiome changes related to recent use and/or cessation of antibiotics being attributed to the change in respiratory support. The length of ‘washout’ period was pragmatically based on knowledge of antibiotic half-lives in this patient cohort, and our understanding of the effects of antibiotics on preterm intestinal development.10
The periods under analysis must be within 10 days either side of the respiratory support transition point.
Where antibiotics had been used within 5 days of the 10-day period prior to the respiratory support transition, the episode could still be assessed provided at least three samples were available from the pretransition period, which were taken greater than 5 days after antibiotics ceased.
Parameters of microbiome progression were studied in the pretransition and post-transition period. Longitudinal diversity progression and longitudinal progression of Enterobacteriaceae relative abundance were seen to follow roughly linear trajectories, and are quantitatively described. Using the regression coefficients of these parameters as the summary statistic for each individual’s progression prerespiratory and post respiratory support transition, a paired analysis (using t-test or a Wilcoxon signed-rank test, as appropriate) was performed to assess whether the rate of diversity progression (units/week) and rate of Enterobacteriaceae relative abundance progression (%/week) differed prerespiratory and post-respiratory support transition (see figure 1).
Ordination analyses (using adonis and permutest functions within the vegan package in R13) were undertaken to assess for changes in the microbiome composition around the time of transitioning off non-invasive positive pressure support. Representative paired samples from the pretransition and post-transition periods were identified from each subject; an NMDS (non-metric multidimensional scaling) plot constructed, and the DESeq2 R package14 used to identify taxa which differed significantly between the two periods.
Patient and public involvement
Patients or the public were not involved in the design, or conduct, or reporting or dissemination plans of our research. However, research questions which this work contributes to were identified as priorities 5 and 7 in a recent paper, which heavily incorporated patient and public involvement, addressing research priorities in neonatology.15