Background: Early onset sepsis (EOS) causes morbidity and mortality in preterm infants, yet diagnosis is inadequate. 16S rRNA gene amplicon sequencing of cord blood (CB) is a culture-independent method of pathogen detection that may improve EOS diagnosis. Ureaplasma and Mycoplasma spp. have been isolated from CB with uncertain clinical significance, and PCR is more sensitive. Is cord blood normally sterile? Our objective was to compare Sanger (SS) and next generation (NGS) 16S rRNA sequencing results of CB from preterm infants with confirmed sepsis (cEOS), presumed sepsis (PS) and no sepsis (control).
Methods: This is a nested case-control study within a prospective cohort with archived CB collected by sterile venipuncture at birth. Infants (31±3 wks gestation) were selected using the following criteria: 1) cEOS [+ blood culture <72 hours of life, antibiotics], n=8; 2) PS [ culture, ≥2 abnormal labs, antibiotics], n=8; 3) control [ culture, no antibiotic course, did well], n=8. After DNA extraction, 16S rRNA gene sequencing using SS and NGS was performed. BLAST and QIIME were used for annotation of sequences and microbe identification.
Results: Postnatal cEOS organisms were E. coli and S. agalactiae. SS identified 4 of 8 cEOS species in CB; NGS identified 7 of 8. One E. coli cEOS case had G. vaginalis identified by SS and Gardnerella, Lactobacillus and Ureaplasma by NGS. Three PS had a predominant taxon: Enterobacteriaceae, Sneathia and Actinomyces. Other PS and all controls had a variety of bacteria identified: Enterobacteriaceae, Streptococcus, Lactobacillus, Ureaplasma, Mycoplasma, Prevotella, Bacteroides and Clostridia spp., among others.
Conclusion: CB 16S rRNA gene amplicon sequencing correctly identified pathogens in most cEOS cases. A predominant bacterial taxon was identified in some cEOS and PS, while others had negative SS and multiple organisms by NGS. NGS detected bacteria associated with vaginal, oral and gastrointestinal flora in controls. According to this preliminary data, CB does not appear to be sterile. The significance of bacterial DNA in neonatal disease is unclear. Further investigation of CB molecular diagnostics for EOS and possible maternal-fetal origins of the microbiome is warranted. Deep sequencing may be a tool to delineate the presence and role of rare or novel organisms in CB.
L. B. Mithal,
Thrasher Research Fund:
S. J. Green, None
C. Qi, None
R. Yogev, None
K. Mestan, Viacord/Perkin-Elmer: Investigator , Research support
National Institutes of Health- K23: Grant Investigator , Research grant
Northwestern Memorial Foundation: Grant Investigator , Research grant