Aerococcus urinae Isolated from Women with Lower Urinary Tract Symptoms: In Vitro Aggregation and Genome Analysis

Abstract

Aerococcus urinae is increasingly recognized as a potentially significant urinary tract bacterium. A. urinae has been isolated from urine collected from both males and females with a wide range of clinical conditions, including urinary tract infection (UTI), urgency urinary incontinence (UUI), and overactive bladder (OAB). A. urinae is of particular clinical concern because it is highly resistant to many antibiotics and, when undiagnosed, can cause invasive and life-threatening bacteremia, sepsis, or soft tissue infections. Previous genomic characterization studies have examined A. urinae strains isolated from patients experiencing UTI episodes. Here, we analyzed the genomes of A. urinae strains isolated as part of the urinary microbiome from patients with UUI or OAB. Furthermore, we report that certain A. urinae strains exhibit aggregative in vitro phenotypes, including flocking, which can be modified by various growth medium conditions. Finally, we performed in-depth genomic comparisons to identify pathways that distinguish flocking and nonflocking strains.IMPORTANCEAerococcus urinae is a urinary bacterium of emerging clinical interest. Here, we explored the ability of 24 strains of A. urinae isolated from women with lower urinary tract symptoms to display aggregation phenotypes in vitro We sequenced and analyzed the genomes of these A. urinae strains. We performed functional genomic analyses to determine whether the in vitro hyperflocking aggregation phenotype displayed by certain A. urinae strains was related to the presence or absence of certain pathways. Our findings demonstrate that A. urinae strains have different propensities to display aggregative properties in vitro and suggest a potential association between phylogeny and flocking.

Keywords: Gram positive; aggregation; bladder; lower urinary tract symptoms; urinary microbiome.

FIG 1

A. urinae urinary strains display in vitro aggregation phenotypes. (A) The flocking phenotype. A. urinae forms aggregates in a liquid culture when grown statically in TSB or BHI for 48 h under 5% CO₂ atmospheric conditions. A. urinae cells gather at the bottom of the tube, but once the tube is disturbed by flicking, the ability of A. urinae cells to form aggregates becomes apparent. (B) Hockey puck phenotype. When A. urinae is grown on BHI+Glu (or TSA+Glu [not shown]) agar plates for 48 h under 5% CO₂ atmospheric conditions (strain UMB0080), the resultant colonies maintain their shape when pushed across the agar surface (indicated by the arrow and inset of top image). When A. urinae does not have the hockey puck phenotype (UMB0722), the colonies are unable to maintain their shape (indicated by the arrow and inset of bottom image). Experiments to observe the in vitro phenotypes were performed in triplicates more than 20 times.

FIG 2

Core genome phylogenetic comparison of A. urinae urinary strains. A total of 29 A. urinae genomes were compared using 40 universal single-copy marker genes. Three other species of Aerococcus were used to root the tree. Each A. urinae strain from our collection was color coded based on its ability to form either the flocking or hockey puck phenotype. The tree on the left is color coded based on the flocking phenotype and the tree on the right is color coded for the hockey puck phenotype.

FIG 3

Percent viability of urothelial cells with various treatments at 3 h. Three different strains of A. urinae were chosen for testing based on their ability to flock in two independent experiments with three intra-assay replicates in each experiment: UMB0080 is a +/− strain, UMB0722 is a −/− strain, and UMB3669 is a +/+ strain. For positive controls, HUCS were treated with two different strains of UPEC (NU14 and UTI89) and two strains of GBS from our UMB strain collection (UMB1026 and UMB1047). For negative controls, the HUCs were treated with various TLR agonists (LPS, Pam2, and Pam3) as well as Lactobacillus crispatus (LC40). *, P < 0.05, **, P < 0.001. Statistics are a comparison to untreated control.

FIG 4

Functional annotation comparison of hyperflocking strains versus nonflocking strains. (A) Coding sequence difference. Average coding sequence difference between the hyperflocking and nonflocking strains. The graph is pared down to the seven specific subsystems that depict either CDS being higher in the hyperflocking strains than in nonflocking strains or completely absent from the hyperflocking strains. The colored dot under each pair of bars corresponds to the name of the subsystem in panel B. (B) Phylogenetic tree with specific subsystem comparison. This phylogenetic tree is a modified version of the trees shown in Fig. 2. This tree focuses on only the hyperflocking and nonflocking strains. Next to each strain is an array of colored dots that correspond to the specific subsystems that are present in the genome of that strain.