Meconium Influences Pulmonary Short-Chain Fatty Acid Concentration in Porcine Meconium Aspiration Model

Abstract

Introduction: The factors influencing meconium aspiration syndrome (MAS) severity remain poorly understood. In a piglet model of MAS, we hypothesized the respiratory microbiome would reflect the bacterial signature of meconium with short-chain fatty acid (SCFA) accumulation as a byproduct of bacterial fermentation.

Methods: Cesarean section at approximately 115-day term was performed on two sows. Male (9) and female (3) piglets were delivered, instrumented, anesthetized, and randomized into a Control (n = 6) or MAS group (n = 6). MAS received a meconium slurry (3 mL/kg) aspiration injury. Experimental animals were monitored continuously, ventilated, and resuscitated for 24 h. BALF was collected for 16S microbiome sequencing and SCFA analysis by gas chromatography. Effects of SCFAs on A549 alveolar pulmonary epithelial in vitro cell viability and inflammation were assessed.

Results: The MAS group had significantly higher fluid and vasopressor requirements than the Control group (p < 0.05) though both groups developed lung injury. The meconium microbiome demonstrated a difference in genus proportions as compared with the BALF of the Control and MAS groups. The MAS group had a relative increase in propionic acid-forming bacteria and higher BALF concentrations of propionic acid (0.6 ± 0.2 mmol/kg) than the Control group (0.2 ± 0.2 mmol/kg; p > 0.05). Propionic acid was associated with decreased pulmonary epithelial cell viability and an upregulated pro-inflammatory response.

Conclusions: Meconium may host a microbiome with byproducts that interact with the pulmonary epithelium and influence lung injury severity in MAS.

Keywords: Meconium aspiration syndrome; Microbiome; Propionic acid; Short-chain fatty acids.

Fig. 1.

Hemodynamics. Comparison of heart rate (a), mean arterial pressure (b), fluid (c), and vasopressor requirements (d) in the Control group (red) with no injury and the MAS group (blue; n = 6 per group). BL, baseline; T0, represents timepoint just after injury in the MAS group with recordings reported every 4 h until the experiment end (T24).

Fig. 2.

Lung histology with meconium aspiration syndrome. Control group at lower-power (5×; a) and higher-power (10×; b) magnification demonstrating open bronchioles and minimal atelectasis in histopathology with hematoxylin-eosin staining. MAS group at lower-power (5×; c) magnification demonstrating meconium plugs (sepia) in bronchiole with alveolar collapse in the associated lobule and higher-power (10×; d) magnification demonstrating distal alveolar duct meconium plug with associated atelectasis.

Fig. 3.

Microbiome. a Boxplot of α-diversity clusters of all samples. Bottom and top whiskers represent the minimum and maximum values. Box represents first and third quartile. Line within max is the median value. b The three eliminated samples due to contaminants (dark gray) had the lowest Shannon index as compared with meconium (medium gray) and the remaining samples including control samples and piglet BALF (light gray). c Microbial proportion by genus in the ventilator circuit (n = 5 samples) compared with meconium (n = 8 samples), Control animals mechanically ventilated for 24 h (n = 6), and animals that underwent a meconium aspiration injury (MAS; n = 6). Bacteria known to yield propionic acid fermentation byproducts are demarcated in yellow. The Naïve group did not demonstrate any microbial presence and so was not included.

Fig. 4.

SCFA quantified from bronchoalveolar lavage fluid (BALF). Acetic acid (a), isobutyric acid (b), propionic acid (c), and butyric acid (d) in BALF from Naïve animals (sacrificed immediately after birth; n = 6 samples), Control animals (ventilated for 24 h; n = 6), and animals with meconium aspiration injury (MAS; n = 6). Data reported as mean ± SEM. *p < 0.05 versus all other groups.

Fig. 5.

Inflammatory effect and viability of alveolar pulmonary epithelial cells with SCFA exposure. Pulmonary alveolar epithelial cells were cultured in a 96-well plate with acetic acid (0.1–100 mm), isobutyric acid (0.01–10 mm), and propionic acid (0.05–50 mm) for 24 h in serum-free media to determine cell viability by MTT assay (a). The culture media were collected for the determination of TNF-α (b), IL-1β (c), IL-6 (d), and HMGB-1 (e) by ELISA. Data reported as mean ± SEM, n = 4–6/group. *p < 0.05 vs. control (0 mm).