Targetting E.coli growth in vivo in intervention against urinary tract infections

Abstract

UTI is an everyday encounter in the primary health sector, and in the US, UTI accounts for 8.1% of all hospital admissions 1. In Denmark, with 5.6 mill citizens, over 500,000 prescriptions of antibiotics for UTIs are issued per year 2and even in the primary sector, one finds a high incidence of antibiotic resistance in bacteria that cause UTI 3. This stresses the need for new approaches to supplement standard antibiotic treatment to control UTIs. UTIs are most frequently caused by E. coli that produce a variety of virulence factors 4– 8, of which α‐haemolysin (HlyA) is the most consistent. Our previous in vitro studies demonstrate that the biological effects of HlyA are to a large extent are secondary to extracellular ATP signalling. Interestingly, insertion of HlyA into a cells membrane inflicts non‐lytic ATP release from cells directly through the HlyA‐pore. In our murine model of ascending UTI, it is easily confirmed that HlyA is a virulence factor that promotes development of pyelonephritis and we have shown that HlyA is responsible for the majority of the septic symptoms in a murine model of urosepsis. Therefore, we hypothesise that release of host ATP may be essential for the virulence promoted by HlyA.We test the hypothesis by a combination of bacterial growth studies (flow cytometry and OD660) and in vivo murine model of pyelonephritis. Our data show that E.coli grow exceedingly well in urine from healthy volunteers, despite its low iron content17. Within the first couple of hours, the growth rate of E.coli is significantly higher in urine than in Lysogeny Broth (LB). In contrast, the E.coli practically does not grow in physiological saline (HEPES buffered salt solution, HBS) containing 5.6 mM glucose. Moreover, we found that E.coli grows faster in hyperosmotic compared to dilute urine and addition of as little as 25μl human urine per ml HBS was sufficient to stimulate E.coli growth maximally. This supports that the initial accelerated growth in urine compared to LB medium must be a substance found explicitly in the urine. By screening, we exclude urea, iron, creatinine, proteins/polypeptides and taurin to have any effect on bacterial growth. We did, however, find that NH4+ was necessary as a nitrogen donor. Moreover, it was observed that ATP degradation products ADP and AMP, which are present in the urine in micromolar concentrations, markedly supported and accelerated E. coli growth in HBS. Our in vitro data signifies that dilution of the urine potentially could protect against ascending UTI and our initial in vivo studies of ascending UTI in mice support that the urine has to be diluted to values below 300 mosmol/l to be able to detect this effect. Therefore, simple dilution would not provide sufficient protection against ascending UTIs. Consequently, we are currently specifically targeting the ADP/AMP and NH4+ pathways in our strive to develop new treatment strategies for prevention of UTI.Support or Funding InformationThis project was funden byThe Independent Research fund ‐DenmarkThe Augustinus Foundation