Introduction: A decade ago, the ”healthy” bladder was presumed to be a sterile organ. The advent of expanded quantitative urine culture (EQUC) and incorporation of 16S rRNA sequencing in studying the urinary microbiome have now disproven this long-held dogma. To date, the pediatric urinary microbiome of healthy infants has not been characterized using EQUC and 16S rRNA. The aim of this study is to identify the cultivatable urobiome of male infants and characterize a representation of the core microbiome using 16S rRNA sequencing. Methods: IRB approval was obtained to collect catheterized urine samples from healthy infants less than 12 months of age undergoing an elective circumcision. Exclusion criteria included prior history of UTI, genitourinary anomaly, or urethral catheterization. Urine aliquots were plated in 5% Sheep’s Blood and Brucella agar plates and incubated anaerobically and aerobically for up to 5 days. Speciation of bacterial isolates were verified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Environmental control samples were collected from the operating room also analyzed. Urine samples were shipped to University of California at San Diego Microbiome Center for DNA extraction and sequencing. Following DNA extraction, the V4 hypervariable 16S rRNA region was amplified using standardized primers from the Earth Microbiome Project and reads were processed using R programming. Results: Fifty patients were enrolled. Sixty-four percent of patients grew organisms using EQUC, ranging from 1 to 5 unique species. Infants born full term were more likely to grow an organism using EQUC (p=0.03), but there was no difference in microbiome diversity with 16S rRNA sequencing based on method of infant delivery, birth term status, or prior antibiotic exposure. Forty different cultivatable species were identified, with the highest representation in the Actinomycetaceae (18%), Enterobacteriaceae and Staphylococcaceae families (13%). The most common species was Actinotignum schaalii seen in 18% of infants. 16S sequencing demonstrated that the core microbiome of infants is composed of bacteria from the phyla Actinobacteria, Bacteroides, Firmicutes, and Proteobacteria. Conclusions: Using EQUC we identified uncommon bacterial isolates in 64% of healthy infant samples, with 40 distinct organisms. A core microbiome representation of the infant bladder was identified with 16S rRNA sequencing. Our results demonstrate that a urinary microbiome can be detected in healthy infants using culturing and sequencing methods. SOURCE OF Funding: none
