Growth of Gram-negative uropathogens in human urine under hypoxic conditions produces clinically relevant metabolomic profiles

SUMMARY Background Much work on uropathogens is done in rich laboratory media that do not reflect the nutrient availability of urine. Also, the environment of the bladder is microaerobic (∼5 % O 2 ) but routine laboratory work with uropathogens is done under aerobic conditions. Aims To understand the influence of oxygen conditions (aerobic, microaerobic) and physiologically relevant growth substrates (artificial urine, human urine) on the ability of bacteria isolated from catheter-associated urinary tract infections (CAUTIs) to form biofilms, and to begin to define the CAUTI bacterial metabolome. Methods Biofilm assays were conducted in rich lab medium (tryptone soy broth supplemented with glucose), and commercially available artificial and pooled human urine for 29 well-characterized uropathogens representing five different genera of Gram-negative bacteria. Spent media were collected and analysed using 400 MHz 1 H-NMR, to quantify major metabolites produced by uropathogens in artificial urine and human urine. Findings There was a significant decrease in biofilm formation for all uropathogens when grown in physiologically relevant growth substrates. Untargeted metabolomic analyses of artificial and human urine showed the artificial urine used in this study did not recapitulate the complexity of major metabolites present in human urine. Further analyses of spent human urine highlighted significant increases in acetate production by the uropathogens when they were grown under microaerobic compared with aerobic conditions. Conclusions Growth of uropathogenic Enterobacteriaceae under physiologically relevant conditions (i.e. human urine, 5 % O 2 ) generates data more relevant to clinical disease and is an important consideration for future work on bacteria causing urinary tract infections.