Bacterial diversity in meconium of preterm neonates and evolution of their fecal microbiota during the first month of life

Abstract

The establishment and succession of bacterial communities in infants may have a profound impact in their health, but information about the composition of meconium microbiota and its evolution in hospitalized preterm infants is scarce. In this context, the objective of this work was to characterize the microbiota of meconium and fecal samples obtained during the first 3 weeks of life from 14 donors using culture and molecular techniques, including DGGE and the Human Intestinal Tract Chip (HITChip) analysis of 16S rRNA amplicons. Culture techniques offer a quantification of cultivable bacteria and allow further study of the isolate, while molecular techniques provide deeper information on bacterial diversity. Culture and HITChip results were very similar but the former showed lower sensitivity. Inter-individual differences were detected in the microbiota profiles although the meconium microbiota was peculiar and distinct from that of fecal samples. Bacilli and other Firmicutes were the main bacteria groups detected in meconium while Proteobacteria dominated in the fecal samples. Culture technique showed that Staphylococcus predominated in meconium and that Enterococcus, together with Gram-negative bacteria such as Escherichia coli, Escherichia fergusonii, Klebsiella pneumoniae and Serratia marcescens, was more abundant in fecal samples. In addition, HITChip results showed the prevalence of bacteria related to Lactobacillus plantarum and Streptococcus mitis in meconium samples whereas those related to Enterococcus, Escherichia coli, Klebsiella pneumoniae and Yersinia predominated in the 3(rd) week feces. This study highlights that spontaneously-released meconium of preterm neonates contains a specific microbiota that differs from that of feces obtained after the first week of life. Our findings indicate that the presence of Serratia was strongly associated with a higher degree of immaturity and other hospital-related parameters, including antibiotherapy and mechanical ventilation.

Figure 1. Counts of microbial species isolated and identified by species-specific PCR or 16S rRNA sequencing PCR.

Forty-four different species were isolated and identified in some or all the samples analyzed (meconium (·), 1^st week (··), 2^nd week (···) and 3^rd week (····) fecal samples). Concentrations of the identified species are represented in a grey scale from non detected (nd) in white to 8–10 log₁₀ CFU/mL in black.

Figure 2. Cultivable bacteria in meconium and fecal samples analyzed in this study.

Frequency of occurrence is the percentage of samples in which a particular bacterial genus was detected at each sampling time (meconium, 1^st week, 2^nd week and 3^rd week feces). The total number of weekly samples analyzed is indicated under each sampling time. Results of the Fisher exact probability test (Freeman-Halton extension for a 2×4 contingency table) are shown by asterisks. * = P<0.05; ** = P<0.01; *** = P<0.001.

Figure 3. Principal component analysis (PCA) of microbiological profiles of cultivable bacteria.

PCA was performed based on the bacterial counts of cultivable bacteria detected at each sampling time: meconium, 1^st, 2^nd and 3^rd week feces.

Figure 4. Cluster analysis of DGGE profiles.

The analysis was performed using the Neighbor Joining method based on the Dice similarity coefficient in meconium and 3^rd week feces from infants 2, 3, 4, 5, and 10.

Figure 5. Phylogenetic fingerprints of the meconium and 3^rd week fecal microbiota of 14 preterm infants.

The highest phylogenetic level of specificity of probes is described on the right side of the figure. Darkness of the spot corresponds to the bacterial abundance in the sample. Pearson correlation and Ward’s clustering method were used.

Figure 6. Phyla in the microbiota of meconium and feces analyzed by HITChip and culture-based techniques.

The relative contribution of the phyla to the microbiota of meconium and 3^rd week fecal samples of fourteen infants detected by culture techniques is shown. In the figure, data for these phyla obtained by HITChip is compared.

Figure 7. Relative contribution of the phylum/order-like phylogroups to the microbiota of studied infants assessed by HITChip.

Phylum/order-like phylogroups detected in meconium and 3^rd week fecal samples by HITChip are presented. Only phylum/order-like phylogroups that contributed for at least 0.5% to a given profile are shown.

Figure 8. Shannon-Weaver diversity indices from culture and HITChip results.

Boxes in blue represent Shannon-Weaver diversity index obtained with culture techniques in meconium (0 week), 1^st, 2^nd and 3^rd week feces. Boxes in green represent Shannon-Weaver diversity index obtained with HITChip in meconium (0 week) and 3^rd week feces.