Average nucleotide identity analysis, based on whole genome sequences of 115 strains previously identified as Aerococcus urinae , an emerging uropathogen, discriminates at least six unique genomic taxa. The whole genome analysis affords clearer species boundaries over 16S rRNA gene sequencing and traditional phenotypic approaches for the identification and phylogenetic organization of Aerococcus species. The newly described species can be differentiated by matrix-assisted laser desorption ionization time-of-flight analysis of protein signatures. We propose the emendation of the description of A. urinae (type strain ATCC 51268^T = CCUG 34223^T=NCFB 2893) and the names of Aerococcus tenax sp. nov. (ATCC TSD-302^T = DSM 115700^T = CCUG 76531^T=NR-58630^T), Aerococcus mictus sp. nov. (ATCC TSD-301^T = DSM 115699^T = CCUG 76532^T=NR-58629^T), and Aerococcus loyolae sp. nov. (ATCC TSD-300^T = DSM 115698^T = CCUG 76533^T=NR-58628^T) for three of the newly identified genomic taxa.

Bacterial isolates from the human urinary microbiome have been extensively studied for their antibiotic resistance; however, little work has been done on those isolates that are difficult to grow in vitro . This study was designed to qualify a serum-based medium, New York City Broth III (NYCIII), and a broth microdilution method to determine the antibiotic susceptibility of previously underreported or undescribed microbes that have a difficult time growing in standard Mueller-Hinton broth. Here, we demonstrate that NYCIII microbroth dilution can be an effective method for the determination of antibiotic susceptibility of species found in the human urinary microbiome. We show that this method serves well to characterize fastidious and anaerobic urinary microbes that have no Clinical and Laboratory Standards Institute (CLSI) guidelines, including several in the families Aerococcaceae , Lactobacillaceae , or Actinomycetaceae . Previous studies using expanded quantitative urine culture reveal that urine samples from clinical patients are commonly polymicrobial in composition. Thus, we test whether NYCIII can serve as a viable harmonized medium, capable of supporting antibiotic susceptibility testing in a range of fastidious, non-fastidious, and anaerobic urinary microbes. We propose this methodology to be standardized comparable to CLSI standards to allow for resistance testing in uncharacterized urinary bacteria. IMPORTANCE: Antibiotic susceptibilities of fastidious and anaerobic bacteria of the human urinary microbiome are largely underreported due to difficulty in growing them in the lab environment. The current standard medium, Muller-Hinton broth, has difficulty supporting the growth of many of these species, leaving microbiologists without a standardized method. To address this need, this study offers a methodology to survey susceptibilities in a high-throughput manner of these understudied microbes with a proposed harmonized medium, NYCIII, which is capable of supporting the growth of both fastidious and non-fastidious urinary microbes. Broader standardization of this method can allow for the development of antibiotic-resistant breakpoints of the many uncharacterized urinary microbes.

Urinary tract infections represent one of the most prevalent bacterial diseases, yet current diagnostic and research methodologies are hampered by inadequate culture media that fail to replicate the bladder biochemical environment. Conventional artificial urine formulations contain undefined components, lack essential nutrients, or inadequately support urinary microbiome (urobiome) growth. To address these limitations, we developed SimUrine, a fully defined synthetic urine medium that aims to replicate human bladder chemistry while supporting diverse microbial growth requirements. SimUrine was systematically developed through iterative optimization of multi-purpose artificial urine, incorporating defined concentrations of carbon sources, vitamins, trace elements, and amino acids within physiologically relevant ranges. The modular design enables component substitution without complete reformulation, facilitating customization for culturomics, antimicrobial susceptibility testing, and microbial ecology studies, while reducing batch-to-batch variability associated with authentic urine. Performance evaluation demonstrated SimUrine's capability to support the growth of fastidious urobiome members, including Lactobacillus species, Aerococcus urinae , and Corynebacterium riegelii , which fail to proliferate in conventional minimal media. Physicochemical characterization confirmed that SimUrine formulation exhibits properties within normal human urine ranges for density, conductivity, osmolarity, and viscosity, ensuring physiological relevance. Clinical applications revealed reduced antibiotic susceptibility compared to standard media, suggesting a more accurate representation of in vivo conditions. Co-culture experiments using Escherichia coli and Enterococcus faecalis demonstrated previously unobserved microbial interactions, highlighting SimUrine's utility for investigating urobiome dynamics. SimUrine represents a significant advancement in urobiome research methodology, providing a standardized, reproducible platform for investigating the urobiome under physiologically relevant conditions, potentially improving fundamental understanding and clinical diagnostic approaches. IMPORTANCE: Urinary tract infections (UTIs) affect millions globally, yet current research and diagnostic methods rely on inadequate culture media that fail to replicate the bladder's unique biochemical environment. This fundamental limitation has hindered accurate UTI research and potentially compromised clinical treatment decisions. SimUrine addresses this critical gap as the first fully defined synthetic urine medium that mimics human bladder chemistry while supporting the growth of diverse urinary microbes. The breakthrough enables the cultivation of urobiome organisms in a minimal medium that resembles natural conditions, revealing novel microbial interactions that influence urinary health. Crucially, SimUrine demonstrates different antimicrobial susceptibility patterns compared to standard clinical media, suggesting current testing protocols may inaccurately predict treatment outcomes. This standardized, reproducible platform eliminates the variability of authentic urine samples while maintaining physiological relevance, potentially transforming urobiome research methodology and providing a new tool for the study of UTIs worldwide.

Previously identified under the single designation of Aerococcus urinae , three distinct taxonomic species have been distinguished as Aerococcus loyolae, Aerococcus mictus, and Aerococcus tenax . Here, we present the complete genome sequences of the type strains of these species assembled via a combination of short-read and long-read sequencing techniques. Registered at ClinicalTrials.gov ( NCT01166438 ).

The recognition of the Aerococcus urinae complex (AUC) as an emerging uropathogen has led to growing concerns due to a limited understanding of its disease spectrum and antibiotic resistance profiles. Here, we investigated the prevalence of macrolide resistance within urinary AUC isolates, shedding light on potential genetic mechanisms. Phenotypic testing revealed a high rate of macrolide resistance: 45%, among a total of 189 urinary AUC isolates. Genomic analysis identified integrative and conjugative elements (ICEs) as carriers of the macrolide resistance gene ermA, suggesting horizontal gene transfer as a mechanism of resistance. Furthermore, comparison with publicly available genomes of related pathogens revealed high ICE sequence homogeneity, highlighting the potential for cross-species dissemination of resistance determinants. Understanding mechanisms of resistance is crucial for developing effective surveillance strategies and improving antibiotic use. Furthermore, the findings underscore the importance of considering the broader ecological context of resistance dissemination, emphasizing the need for community-level surveillance to combat the spread of antibiotic resistance within the urinary microbiome.

Trueperella bernardiae is infrequently isolated, usually in polymicrobial communities, from human hosts with a wide variety of symptoms and diseases. Here, we report a complete genome sequence of Trueperella bernardiae (UMB8254), isolated from the bladder of a human female with metabolic syndrome and nephrolithiasis.