Patterned progression of bacterial populations in the premature infant gut

Patricio S. La Rosa; Barbara B. Warner; Yanjiao Zhou; George M. Weinstock; Erica Sodergren; Carla M. Hall-Moore; Harold J. Stevens; William E. Bennett; Nurmohammad Shaikh; Laura A. Linneman; Julie A. Hoffmann; Aaron Hamvas; Elena Deych; Berkley A. Shands; William D. Shannon; Phillip I. Tarr

doi:10.1073/pnas.1409497111

New Research In

Edited by E. Peter Greenberg, University of Washington, Seattle, WA, and approved July 15, 2014 (received for review May 22, 2014)

This article has a Correction. Please see:

Correction for La Rosa et al., Patterned progression of bacterial populations in the premature infant gut

Article Figures & SI

Figures

Tables

Download figure

Open in new tab

Download powerpoint

Fig. 1.
Bacterial taxa composition at class level as function of day of life of a single subject. These samples are from subject 32 who was born after 29 wk gestation. Each bar represents the bacterial proportions within single samples. From left to right, bars correspond to stools obtained on days of life 3, 4, 6, 8, 9, 10, 12, 14, 14, 16, 17, 19, 25, and 39, respectively. The predominant classes are Bacilli (dark blue), Gammaproteobacteria (red), and Clostridia (green). Eleven additional classes (light blue) were also present, mostly in stools obtained on days of life 3 and 4.
Download figure

Open in new tab

Download powerpoint

Fig. 2.
Percent bacterial class abundance. Proportion of each bacterial class is represented in y axes in each graph. The x axes represent samples obtained at various postconceptional ages (weeks). The columns represent class proportions, as indicated. (A–C) All infants in cohort (n = 58 subjects). (D–F) Infants in <26 wk gestational age subcohort (n = 15 subjects). (G–I) Infants in 26–28 wk gestational age subcohort (n = 20 subjects). (J–L) Infants in >28 wk gestational age subcohort (n = 23 subjects). To reduce the effect of random fluctuations in the data (noise), the lines were produced by fitting a cubic smooth spline (thick black line) to the data with 95% confidence bands (thinner lines) generated using a nonparametric bootstrap method.

Tables

Figures

View popup

Table 1.

Details of subject groups

Gestational ages at birth (n)	All (58)	<26 wk (15)	26–28 wk (20)	>28 wk (23)
Perinatal
Vaginal birth, n (%)	13 (22)	4 (27)	6 (30)	3 (13)
Birth weight g, median (IQR)	960 (800, 1220)	750 (690, 900)	895 (770, 980)	1,250 (1,110, 1,445)
Gestational age at birth weeks, median (IQR)	27.1 (25.6, 29.0)	25 (24.9, 25.3)	26.7 (26.3, 27.1)	29.1 (28.9, 31.6)
Male/Female	24/34	4/11	10/10	10/13
Caucasian/African American	30/28	5/10	10/10	15/8
APGAR 5 min, median (IQR)	7.0 (6.0, 8.0)	6.0 (4.0, 7.0)	7.0 (5.5, 8.0)	8.0 (6.0, 9.0)
Postnatal
First antibiotic free day of life, median (IQR)	5 (4, 9)	5 (4, 9)	8.5 (4, 9.5)	4 (2, 5)
Percent of days of life on antibiotics,^* median (IQR)	14 (5.8, 25.6)	22.2 (11.4, 30.6)	17.3 (8.6, 29.0)	6.4 (5.4, 14.3)
Day of life on which enteral feedings initiated, median (IQR)	5.5 (4.0, 8.0)	7.0 (6.0, 9.0)	5 (4.0, 7.0)	4.0 (3.0, 7.0)
Human milk use
None^†, n (%)	2 (3)	0	1 (5)	1 (4)
<10%, n (%)	16 (28)	4 (27)	8 (40)	4 (17)
10–50%, n (%)	18 (31)	3 (20)	4 (20)	11 (48)
>50%, n (%)	22 (38)	8 (53)	7 (35)	7 (31)
Outcome
Discharged to home from NICU, n (%)	57 (98)	15 (100)	19 (95)	23 (100)
Day of life discharged home (IQR)	61 (49, 73)	78 (75, 81)	65 (60, 71)	47 (37, 51)
Days to death (n)	66 (1)	N/A	66 (1)	N/A

APGAR score, a measure of physical condition at birth scored from 0 to 10, 10 being highest; N/A, not applicable.
↵* Percent of days of life antibiotics were administered before each specimen.
↵^† Percentage of enteral volume provided by human milk.

View popup

Table 2.
Distributions of sequences by class and intervals of sampling
Colored rows correspond to age postconception [<30 (blue), 30–33 (red), and > 33 (green) wk] and day of life [<28 (blue), 28–56 (red), and >56 (green) d] groupings.
* Sum of all reads across all classes for each interval. Values in these columns include the sums of the Bacilli, Clostridia, and Gammaproteobacteria reads in this table, as well as the 572,463 reads that are assigned to 38 other classes (including those designated “unclassified”) as provided in the extended dataset.
^† Percentage of reads for each class within each designated interval divided by the 6,927,236 reads in study.
^‡ Comparison between adjacent age postconception and day of life values differ by more than twofold.
^§ Percentage of reads within each class within each designated interval divided by the sum of all reads for the respective intervals (Sum columns values for age postconception and day of life are denominators).
^¶ Percentage of reads within each class within each designated interval divided by the total number of reads for that class (Sum row values are denominators).
^‖ Sum of all reads, each class. Pie charts represent proportions of classes, by age postconception or day of life intervals, as designated at top of columns. Segments are earliest (blue), middle (red), and latest (green) age postconception or day of life intervals in which samples obtained.

View popup

Table 3.

Relationship of clinical factors to predominant taxa (class level)

Variable	GA at birth (wk)	Bacilli	Clostridia	Gammaproteobacteria
Day of life	<26	−0.007 (−0.011, −0.004)↓	0.007 (0.004, 0.011)↑	0.000 (−0.004, 0.004)
	26–28	−0.007 (−0.010, −0.003)↓	0.009 (0.005, 0.012)↑	0.000 (−0.005, 0.003)
	>28	−0.010 (−0.014, −0.006)↓	0.010 (0.005, 0.014)↑	0.000 (−0.004, 0.005)
Days on antibiotics^*	<26	0.004 (−0.005, 0.013)	−0.012 (−0.021, −0.003)↓	0.009 (0.000, 0.0182)
	26–28	−0.001 (−0.011, 0.010)	−0.016 (−0.026, −0.005)↓	0.017 (0.006, 0.027)↑
	>28	−0.012 (−0.031, 0.007)	−0.008 (−0.027, 0.012)	0.021 (0.002, 0.040)↑
Breast milk^†	<26	−0.045 (−0.116, 0.026)	−0.031 (−0.102, 0.040)	0.073 (0.001, 0.143)↑
	26–28	−0.080 (−0.185, 0.025)	−0.048 (−0.153, 0.0577)	0.115 (0.010, 0.221)↑
	>28	−0.016 (−0.101, 0.070)	−0.074 (−0.159, 0.012)	0.079 (−0.007, 0.164)
C-section	<26	−0.187 (−0.346, −0.028)↓	−0.011 (−0.171, 0.148)	0.201 (0.042, 0.360)↑
	26–28	0.226 (0.027, 0.425)↑	0.010 (−0.189, 0.209)	−0.089 (−0.289, 0.110)
	>28	0.212 (−0.017, 0.441)	0.021 (−0.208, 0.249)	−0.083 (−0.312, 0.146)
Sampled after^‡ 01/01/2011	<26	0.266 (0.131, 0.401)↑	−0.151 (−0.286, −0.016)↓	−0.140 (−0.275, −0.005)↓
	26–28	0.037 (−0.178, 0.252)	−0.159 (−0.373, 0.056)	−0.129 (−0.344, 0.086)
	>28	0.063 (−0.129, 0.254)	−0.049 (−0.240, 0.142)	−0.039 (−0.231, 0.152)

A mixed model regression analysis investigated the relationship between clinical variables and taxa fractional abundance in repeated samples of subjects, by gestational age group. Statistically significant linear trends are indicated by arrows. Values are beta coefficients with 95% confidence intervals. Negative values indicate taxa decrements and positive values indicate taxa increments per unit increase in predictor variable. ↓ Significant decrease in taxa, P < 0.05; ↑ Significant increase in taxa, P < 0.05. GA, gestational age at birth.
↵* Percent of days of life antibiotics were administered before each specimen.
↵^† Breast milk (BM) categorized by volume (0,0%; 1, <10%; 2,10–50%; 3, >50% of enteral volume).
↵^‡ Samples collected in first half of study or second half; positive values represent greater representation in second half.

Data supplements

Supporting Information

Files in this Data Supplement:

Download Supporting Information (PDF)
Download Dataset_S01 (XLSX)
Download Movie_S01 (MP4) - Movie S1. Day-by-day progression of microbial content for three different subjects. These three cases exemplify the general trend in the progression from Bacilli to Gammaproteobacteria, to Clostridia, in an animation format used in prior studies of microbial populations over time (1, 2). Other general characteristics portrayed by these subjects are frequent abruptions in microbial content, and the slower rate of accumulation of Clostridia in the most premature subject, and the most rapid rate of accumulation of this bacterial class in the least premature subject. Arrows change at one second intervals, but their pace of appearance is not proportional to the days between samples. Numbers at bottom of each graph provide the days of life of each sample. Dots are color-coded, with unique shading for each subject. Examples A, B, and C represent subjects 53, 47, and 39, born after 25, 28, and 31 wk gestation, respectively.