Gut microbial colonisation in premature neonates predicts neonatal sepsis

Abstract

Background: Neonatal sepsis due to intestinal bacterial translocation is a major cause of morbidity and mortality. Understanding microbial colonisation of the gut in prematurity may predict risk of sepsis to guide future strategies to manipulate the microbiome.

Methods: Prospective longitudinal study of premature infants. Stool samples were obtained weekly. DNA was extracted and the V6 hypervariable region of 16S rRNA was amplified followed by high throughput pyrosequencing, comparing subjects with and without sepsis.

Results: Six neonates were 24-27 weeks gestation at birth and had 18 samples analysed. Two subjects had no sepsis during the study period, two developed late-onset culture-positive sepsis and two had culture-negative systemic inflammation. 324 350 sequences were obtained. The meconium was not sterile and had predominance of Lactobacillus, Staphylococcus and Enterobacteriales. Overall, infants who developed sepsis began life with low microbial diversity, and acquired a predominance of Staphylococcus, while healthy infants had more diversity and predominance of Clostridium, Klebsiella and Veillonella.

Conclusions: In very low birth weight infants, the authors found that meconium is not sterile and is less diverse from birth in infants who will develop late-onset sepsis. Empiric, prolonged antibiotics profoundly decrease microbial diversity and promote a microbiota that is associated not only with neonatal sepsis, but the predominant pathogen previously identified in the microbiome. Our data suggest that there may be a 'healthy microbiome' present in extremely premature neonates that may ameliorate risk of sepsis. More research is needed to determine whether altered antibiotics, probiotics or other novel therapies can re-establish a healthy microbiome in neonates.

Figure 1

Subject clinical variables, sample level stacked bar plots of the most common taxonomical units and comparison of Shannon diversity indices by clinical variable. (A) Subject table of clinical variables. BW, birth weight, grams; EA, early antibiotics, days; GA, gestational age, weeks; PPROM, preterm premature rupture of membranes; SDI, Simpson diversity index at the sample level. (B) Sample level stacked bar plots. The legend shows the colour scheme for the six most abundant genera. Samples are grouped by clinical outcome and ordered sequentially for each patient. Three of the patients show samples with high abundance of Staphylococcus. Patients 2 and 3 show unusually high abundance of Veillonella and Clostridium when compared with the rest of the patients. (C) Patients comparisons based on patient subgroups. p Values are derived from pairwise t-tests, red asterisks indicate statistical significance based on 0.05 threshold. Box plots show the minimum, quartiles, median and maximum SDI values.

Figure 2

Clustered heat map demonstrating clusters of meconium (M) genera, risk for sepsis (R) genera and healthy genera (H). (A) Clustered heat map. Data are filtered based on abundance across samples, normalised and base ten log transformed. Similarity between genera based on their abundance across samples is computed with pvclust. Colours are assigned to genera in taxonomical order. Three significant and disjoint clusters appear at the 0.05 significance level, indicated with red bars. These are named eM (enriched in meconium samples), eH (enriched in healthy samples) and dH (depleted from healthy samples). Similarity between samples is computed with pvclust. SDI bar plots are shown in gray to the right of the sample names. Samples are coloured by patient type: red – sepsis, orange – systemic inflammatory response syndrome, green – healthy; with the exception of meconium samples which are blue. Three significant clusters appear at the 0.05 significance level. These are named M (meconium), R (risk), H (healthy) based on their sample composition. The R cluster shows a marked decrease in abundance for all genera and a signature overabundance of Staphylococcus. (B) SDI box plot for the three sample based clusters. Box plots show the minimum, quartiles, median and maximum SDI values.

Figure 3

Genera transition plots between the M (meconium) state and the R (risk for sepsis) and H (healthy) states. (A) Genera transition plot for the M to R state transition. Inset table shows all 12 transitions within the 18 samples. Fold-changes displayed as box plots are derived from four transitions: T1, T3, T7 and T8. p Values are based on t-tests. Asterisks highlight significance at the 0.05 level. Staphylococcus is the only significantly enriched genus when transitioning to the R cluster. (B) Genera transition plot for the M to H state transition. Fold-changes displayed as box plots are derived from three transitions: T5, T9 and T11. Kluyvera and Photobacterium are the only significantly enriched genera when transitioning to the H cluster. Data are filtered based on abundance across samples, normalised and base two log transformed.