Survey of the infant male urobiome and genomic analysis of Actinotignum spp.

Abstract

The urinary bladder harbors a community of microbes termed the urobiome, which remains understudied. In this study, we present the urobiome of healthy infant males from samples collected by transurethral catheterization. Using a combination of enhanced culture and amplicon sequencing, we identify several common bacterial genera that can be further investigated for their effects on urinary health across the lifespan. Many genera were shared between all samples suggesting a consistent urobiome composition among this cohort. We note that, for this cohort, early life exposures including mode of birth (vaginal vs. Cesarean section), or prior antibiotic exposure did not influence urobiome composition. In addition, we report the isolation of culturable bacteria from the bladders of these infant males, including Actinotignum spp., a bacterial genus that has been associated with urinary tract infections in older male adults. Herein, we isolate and sequence 9 distinct strains of Actinotignum spp. enhancing the genomic knowledge surrounding this genus and opening avenues for delineating the microbiology of this urobiome constituent. Furthermore, we present a framework for using the combination of culture-dependent and sequencing methodologies for uncovering mechanisms in the urobiome.

Synthesis of current pediatric urinary microbiome research

“…However, EQUC and culture-independent 16S sequencing do not always reconcile. For example, the families detected most frequently by EQUC by Reasoner et al ( 21 ) were not detected in culture-independent sequencing results at all, and several families detected most frequently by 16S sequencing were not detected in EQUC isolates. 16S sequencing methods are limited by available mapping to known genetic sequences in available databases, and EQUC still is not a fully comprehensive method of culturing all species.…”

“…Swabs of adjacent niches had similar results (>90%). Reasoner et al ( 21 ) modified the EQUC protocol and with its use, identified growth in 65% of urine samples in at least one growth condition. They were also able to detect additional families and species in their samples using MALDI-TOF.…”

“…The latest of the two studies, by Reasoner et al ( 21 ), also examined a tighter age range of 50 prepubertal males—infants aged 6–12 months—and was the first to examine a cohort that did not contain any individuals possessing any structural or functional urinary tract abnormalities nor had experienced any prior UTI. Nocardiopsis and Acinetobacter were detected in all TUC samples, and Staphylococcus, Escherichia-Shigella , and Pseudomonas were detected in over 90% of samples.…”

“…This method cannot provide functional genomic information needed for analysis of traits such as virulence and fitness factors and required for understanding urobiome development. While there are now several compositional studies of the pediatric urobiome, with a couple including EQUC isolation of constituents, only one study proceeded with complete genome sequencing and analyses of identified constituents: Actinotignum schaali and Actinotignum sanguinis in Reasoner et al ( 21 ). In their enhanced culture Actinotignum spp.…”

“…Additionally, they reported two potential virulence factors: ermX , which confers macrolide resistance, and Esx-1 Type VII secretion/ esxA , which is an anti-eukaryotic membrane-permeabilizing toxin found in Mycobacterium tuberculosis pathogen and gram-positive bacteria. This is of special interest because A. schaalii is now being recognized as an opportunistic causative agent of UTI with increasing incidence ( 21 ).…”