Background Despite increased recognition of the urinary tract’s diverse resident microbiome (i.e., the urobiome) in postmenopausal women, the roles and functions of these microbes remain largely unknown. Further empirical research is needed to understand the physiology, interactions, and antibiotic resistance evolution of urobiome members with pathogenic potential. However, experimental work relies on viable, culturable isolates. Standard urine culturing practices are designed for identifying a narrow set of known urinary microbes, and are thus poorly suited for cultivating taxa from the resident urobiome. Here we expand the urine culturing toolkit to reliably recover diverse urobiome taxa for downstream empirical research. Methods Urine samples collected from postmenopausal women with recurrent urinary tract infections were shipped at ambient temperature to a central point for culturing. Microbial viability was maintained using boric acid preservative tubes during multi-day transport of sample aliquots. Selective media incubated under specialized conditions were used to promote recovery of diverse urobiome members, including fastidious taxa. Under 5% CO2-enriched atmospheric conditions and with longer incubation times, we leveraged a chromogenic agar (UTIC) to further differentiate isolates based on colony color and morphology. We evaluated the workflow for its ability to isolate and characterize urobiome taxa, as determined by morphological differentiation and taxonomic identification. Results Across 108 urine samples, 6.3 ± 3.2 distinct isolates were recovered, with no detectable relationship between sample shipment duration and isolate richness. On chromogenic agar, colony growth and color intensity was improved with CO2-enriched atmospheric conditions and extended incubation times. We identified diverse taxa that are typically underrepresented in standard diagnostic culture and provide novel morphological characterizations for members of the genera Actinotignum, Aerococcus , Facklamia , Lactobacillus , Latilactobacillus , Limosilactobacillus, and Streptococcus species, which have not been previously described on UTIC chromogenic agar. Discussion Using this novel workflow, we recovered a diverse collection of urobiome isolates from urine samples shipped over multiple days. We also demonstrated the utility of a chromogenic agar for the visual differentiation of key urobiome taxa. While sequencing approaches have enhanced our understanding of urobiome composition, culturing is needed to investigate microbial interactions, virulence mechanisms, and antimicrobial susceptibility. This protocol adds to the growing toolkit for the cultivation of diverse urobiome isolates needed to support downstream empirical studies and advance urinary tract infection research. ### Competing Interest Statement AJW membership scientific advisory boards Astek, Cerillo, Pathnostics, and Urobiome Therapeutics * CNA : Columbia Nalidixic Acid (agar) DNA : Deoxyribonucleic acid EQUC : Expanded Quantitative Urine Culturing GARD : Gardnerella vaginalis selective agar HCCA : α-Cyano-4-hydroxycinnamic acid MAC MacConkey (agar) MALDI-TOF : Matrix-assisted laser desorption ionization time-of-flight mass spectrometer MRS : De Man-Rogosa-Sharpe (agar) PCR : Polymerase chain reaction rRNA Ribosomal ribonucleic acid RUTI : Recurrent urinary tract infections TSA : Tryptic Soy (agar) TSB : Tryptic Soy (broth) UTI : Urinary tract infections UTIC : CondaChrome® UTIC (agar) UTIr : Urinary tract infections in postmenopausal women revisited (project) The Dutch Research Council (NWO), OCENW.XL21.XL21.088

Microbial communities are dynamic, adaptive ecosystems whose collective behavior emerges from metabolic interactions such as cross-feeding, competition, and cooperation, rather than taxonomic diversit...