Strain-resolved metagenomic analysis of the gut as a reservoir for bloodstream infection pathogens among premature infants in Singapore

Abstract

Background: Gut dysbiosis contributes to the high risk of bloodstream infection (BSI) among premature infants. Most prior studies of the premature infant gut microbiota were conducted in Western countries and prior to development of current tools for strain-resolved analysis.

Methods: We performed metagenomic sequencing of weekly fecal samples from 75 premature infants at a single hospital in Singapore. We evaluated associations between clinical factors and gut microbiota composition using PERMANOVA and mixed effects linear regression. We used inStrain to perform strain-level analyses evaluating for gut colonization by BSI-causing strains.

Results: Median (interquartile range) gestation was 27 (25, 29) weeks, and 63% of infants were born via Cesarean section. Antibiotic exposures (PERMANOVA; R² = 0.017, p = 0.001) and postnatal age (R² = 0.015, p = 0.001) accounted for the largest amount of variability in gut microbiota composition. Increasing postnatal age was associated with higher relative abundances of several common pathogens (Enterococcus faecalis: p < 0.0001; Escherichia coli: p < 0.0001; Klebsiella aerogenes: p < 0.0001; Klebsiella pneumoniae: p < 0.0001). Antibiotic exposures were generally associated with lower relative abundances of both frequently beneficial bacteria (e.g., Bifidobacterium species) and common enteric pathogens (e.g., Enterobacter, Klebsiella species). We identified strains identical to the blood culture isolate in fecal samples from 12 of 16 (75%) infants who developed BSI, including all infections caused by typical enteric bacteria.

Conclusions: Antibiotic exposures were the dominant modifiable factor affecting gut microbiota composition in a large cohort of premature infants from South-East Asia. Strain-resolved analyses indicate that the gut is an important reservoir for organisms causing BSI among premature infants.

Keywords: Early-onset sepsis; Intestinal microbiome; Late-onset sepsis; Preterm neonates; inStrain.

Fig. 1

Premature infant gut microbiome alpha diversity and composition by postnatal age. Box and whisker plots of a the Shannon diversity index and b number of observed unique microbial species are shown by week of life. The horizontal lines represent the median value; boundaries of the rectangle correspond to the 25th and 75th percentiles; whiskers extend to values at 1.5 * the interquartile range; points represent outlier values. c Relative abundances of the 20 most highly abundant microbial species and BSI-causing species by postnatal age

Fig. 2

Associations between clinical factors and gut microbiome composition. a Bubble plot depicting the amount of variation in gut microbial composition explained by given clinical variables and as measured by PERMANOVA. The size of bubbles represents the amount of variance explained by the variable; asterisks indicate statistical significance (p < 0.05). The first column depicts results from analyses of all samples adjusting for postnatal age; subsequent columns show results from analyses stratified by postnatal age. b Heatmap displaying associations between antibiotic and probiotic exposures and the relative abundances of microbial species within the gut microbiota as estimated by mixed effects linear regression. Only microbial species with a minimum mean relative abundance of 0.01% and a sample prevalence of at least 10% are shown. Boxes are shaded based on the direction and size of the effect as determined from the model beta coefficients; asterisks indicate statistical significance (p < 0.05)

Fig. 3

Time-series plots of gut microbiota composition among a subset of premature infants who developed BSI. The relative abundance of the BSI causative species is indicated by colored vertical bar for each fecal sample with sequencing data. Black vertical lines denote the timing of BSI episodes. Stars correspond to fecal samples in which the BSI strain was identified in strain-level analyses. Subject identifiers are atop each plot

Fig. 4

Putative transmission of a Streptococcus agalactiae strain between infants with an epidemiological link. Times-series plots for two infants who developed BSI from the same strain of S. agalactiae. The green horizontal line indicates the time period during which the infants resided in the same room and had shared healthcare providers. The relative abundance of the shared BSI strain is indicated by a dark blue vertical line for each fecal sample sequenced, while the light blue vertical lines represent the relative abundance of other S. agalactiae strains. Numbers below these lines correspond to the infant age in days. Black vertical lines indicate the day of the BSI episode