Distortions in development of intestinal microbiota associated with late onset sepsis in preterm infants

Abstract

Late onset sepsis (LOS) is a major contributor to neonatal morbidity and mortality, especially in premature infants. Distortions in the establishment of normal gut microbiota, commensal microbes that colonize the digestive tract, might increase the risk of LOS via disruption of the mucosal barrier with resultant translocation of luminal contents. Correlation of distortions of the intestinal microbiota with LOS is a necessary first step to design novel microbiota-based screening approaches that might lead to early interventions to prevent LOS in high risk infants. Using a case/control design nested in a cohort study of preterm infants, we analyzed stool samples that had been prospectively collected from ten preterm infants with LOS and from 18 matched controls. A 16S rRNA based approach was utilized to compare microbiota diversity and identify specific bacterial signatures that differed in their prevalence between cases and controls. Overall α-diversity (Chao1) was lower in cases two weeks before (p<0.05) but not one week before or at the time of diagnosis of LOS. Overall microbiota structure (Unifrac) appeared distinct in cases 2 weeks and 1 week before but not at diagnosis (p<0.05). Although we detected few operational taxonomic units (OTUs) unique or enriched in cases, we found many OTUs common in controls that were lacking in cases (p<0.01). Bifidobacteria counts were lower in cases at all time points. Our results support the hypothesis that a distortion in normal microbiota composition, and not an enrichment of potential pathogens, is associated with LOS in preterm infants.

Figure 1. Heatmap showing the distribution of the most prevalent OTUs at 98% similarity level.

List of full names for closest matches shown by abbreviation in Figure 1 are listed below: ArActinomyces_radingae AsNAchromobacter_sp._N2 bCbacterium_C20 BdBacteroides_dorei BdBacteroides_dorei bDbacterium_DR304 BsCBacteroides_sp._CB53 BsTBosea_sp._TPR12 bTbacterium_Te14R BuBacteroides_uniformis BvBacteroides_vulgatus CdClostridium_disporicum CfCitrobacter_freundii CkCitrobacter_koseri ClClostridium_lavalense CpClostridium_perfringens Cs1Clostridium_sporogenes Cs2Clostridium_sordellii Cs3Clostridium_sp._75064 CsCClostridium_sp._CS4 CsPClostridium_sp._PN6–15 EaEnterococcus_avium EcEnterobacter_cancerogenus EcEscherichia_coli Ef1Enterococcus_faecalis Ef2Enterococcus_faecium EmEnterococcus_malodoratus EsmEnterobacter_sp._mcp11b FmFinegoldia_magna FnFusobacterium_nucleatum KoKlebsiella_oxytoca KpKlebsiella_pneumoniae LaLactobacillus_animalis LcLeuconostoc_citreum LlLactococcus_lactis LpLactococcus_plantarum LrLactococcus_raffinolactis Pa1Propionibacterium_acnes Pa2Pantoea_agglomerans PgPropionibacterium_granulosum PsEPhyllobacterium_sp._EBBLQ01 PsgPeptoniphilus_sp._gpac121 PsHPropionibacterium_sp._H456 RsPRhodococcus_sp._PN8 Sa1Staphylococcus_aureus Sa2Streptococcus_agalactiae SeStaphylococcus_epidermidis Sg1Serratia_grimesii Sg2Streptococcus_gallolyticus ShStaphylococcus_hominis SmSerratia_marcescens SmStenotrophomonas_maltophilia SpStaphylococcus_pasteuri SsStreptococcus_salivarius Ss1Streptococcus_sp._10aVMg3 SsCStaphylococcus_sp._C-12 SsCSerratia_sp._CJB2 SsGStreptococcus_sp._G3 SvStreptococcus_vestibularis VaVeillonella_atypica VbAVeillonellaceae_bacterium_ADV_07/08/06-B-1388 VdVeillonella_dispar WcWeissella_confusa.

Figure 2. Chao rarefaction diversity.

Chao diversity was calculated from sequence distribution A) 2 weeks before; B) 1 week before; and C) within 72 hours of LOS diagnosis. Error bars indicate SE.

Figure 3. Unifrac diversity measures.

Principal component analysis (PCA) of overall diversity based on UniFrac (unweighted) metric A) 2 weeks before; B) 1 week before; and C) within 72 hours of LOS diagnosis Squares represent controls and triangles represent cases. P1 is component 1 and P2 component 2.

Figure 4. Changes in proportion of bacterial phyla.

Proportions of the four major phyla 2 weeks before and within 72 hours of LOS diagnosis.

Figure 5. Differences in OTU abundance.

Heat map of selected OTUs at 98% similarity by subject A) 2 weeks before and B) within 72 hours of LOS diagnosis. Sepsis cases are shown on the top (dark blue shaded area) and controls in the bottom (light blue shaded area). Cell Colors indicate the ratio for a particular OTU in a sample to the average ratio of this OTU in all samples. Dark red: >10, light red: >5 and < = 10, light green: >1.5 and < = 5 and light blue: < = 1.5. Numbers indicate how often the OTU was detected. List of full names for closest matches shown by abbreviation in Figure 5 are listed below: AsN Achromobacter_sp._N2 Bb Bifidobacterium_breve Cp Clostridium_perfringens Cs Cronobacter_sakazakii CsC Clostridium_sp._CS4 Ct Corynebacterium_tuberculostearicum Ea Enterococcus_avium Ec Escherichia_coli Ec Enterobacter_cloacae Ef Enterococcus_faecalis Ef Enterococcus_faecium Ef Escherichia_fergusonii EsC Enterobacter_sp._CTSP23 Fm Finegoldia_magna Ko Klebsiella_oxytoca Kp Klebsiella_pneumoniae Lc Leuconostoc_citreum Pb Pseudomonas_brenneri Pm Phyllobacterium_myrsinacearum PsE Phyllobacterium_sp._EBBLQ01 Sa Streptococcus_agalactiae Se Staphylococcus_epidermidis Sg Streptococcus_gallolyticus Sh Staphylococcus_hominis Sm Stenotrophomonas_maltophilia Sp Staphylococcus_pasteuri Ss1 Streptococcus_sp._10aMclG2 Sv Streptococcus_vestibularis Va Veillonella_atypica VbA0 Veillonellaceae_bacterium_ADV_07/08/06-B-1388 VbA1 Veillonellaceae_bacterium_ADV_12/01/04-B-1195 Vd Veillonella_dispar Vp Veillonella_parvula.

Figure 6. qPCR for fecal counts of Bifidobacteria.

* p-value 2 weeks before LOS - 0.05; **p-value week of sepsis –0.03.