Studies that require bacterial growth in urine, such as transcriptomic studies, utilize pools of urines which pose an issue when trying to replicate the data presented. Comparing results from different pools of urines can be problematic when gender, age, and hormonal status are factors. Here we show that pooling urine prevents identification of factors that are important for understanding bacterial growth in urine.

Following IRB approval on urine collection, 3 strains of Escherichia coli were grown in five urines from 3 groups of post-menopausal women: (1) with an infection (average age 69), (2) with a history of infections (average age 72), and (3) never had an infection (average age 71). The urines were filtered with a 0.2-micron filter to remove bacterial and viral contaminants. To create the pooled urine, 400 microliters of each individual urine was pooled to create a 2mL stock. Single colonies of the uropathogenic E. coli (UPEC) strain UTI89, recurrent UPEC strain LRPF007, and the nonpathogenic E. coli W3110, were grown in 1mL of Luria-Bertani (LB) broth for 2 hours. Cells were collected from the pre-culture, centrifuged, washed twice with minimal media to remove nutrients from the LB, and each strain was resuspended to an equal density. 2 microliters of cells were added to 198 microliters of either individual or pooled urine, and grown for 24 hours in a 96-well dish at 37°C. After the growth period, the lag times, doubling times, CFU per mL (using OD600), final OD600, and change in OD600 were determined in triplicates.

The pooled urine supported growth that was not an average of the individual urines but tended to support near optimal growth. In other words, individual urines had inhibitory or limiting factors that were not apparent when the urines were pooled. The same results were obtained regardless of the bacterial strain that was tested. The urines from individuals who have never had a urinary tract infection tended to be less inhibitory.

The pooled urine showed no averaging effect in terms of final cell count or growth rate, which shows that pooling urine obscures the factors that limit growth. The observation that urine from individuals who have had or have a urinary tract infection are often more inhibitory suggests that an infection induces a defense mechanism.

The confounding factors created by pooling urine shields researchers from valuable individualized result findings. Pooled urine should not be used as a means to study infections in urine. This being said, reproducibility of results using individual urine creates similar issues. Instead, further development of synthetic urine may be the future of bacterial growth analysis in urine.