E selection of nosocomial infections, including pneumonia, bacteremia and urinary tract infections [41,42]. K. pneumoniae also can be located within the intestine, exactly where it might colonize the regional microbiota. Conditions similar to those utilised with EAEC strain 55989a resulted in mixed biofilm composed of 75 MG1655 F9 and 25 KpLM21. We then monitored expression of six genes absent from the K. pneumoniae genome and expressed in MG1655 F9 in response to E. coli 55989a colonization. yiaF, yliE and stfE were selected for their contribution to MG1655 F9 colonization resistance against 55989a, even though yceP and yliH (bssR and bssS, respectively, for “regulator of biofilm through signal secretion”, see Discussion) were chosen since they are overexpressed in mixed CP biofilm when compared with uninfected and/or selfinfected biofilm (Table three) [43,44]. RTPCR on RNA extracted from mixed 24 h CP (E. coli K. pneumoniae) biofilms showed that, even though colonization of commensal MG1655 F9 biofilm by KpLM21 had no influence on stfE expression and lowered yliE expression, it led to induction of yiaF, yceP and yliH (Table four and Fig. S1) These observations indicated the existence of frequent genetic responses upon colonization of E. coli commensal biofilm by two different exogenous bacterial pathogens.Correlation amongst in vitro and in vivo reduction of pathogen colonizationTo test the in vivo part of colonization resistance genes identified by way of our method, we employed a mouse model of intragastric infection to evaluate the extent of intestinal colonization by 55989a and KpLM21 pathogens in streptomycintreated mice, in which enterobacteria which include E. coli and Klebsiella had been shown to colonize exactly the same niches [45,46].94-75-7 custom synthesis Streptomycintreated mice had been precolonized with wildtype or mutant commensal E. coli MG1655 F9 and all 3 strains efficiently colonized the mouse intestine [4749] (Fig. 4A). We chose to test the function of: i) yiaF, which affects in vitro colonization resistance to 55989a and was also induced upon Table four. Gene expression level in mixed MG1655F9 K. pneumoniae biofilms.Fold inductiona 1.6260.13 0.7260.04 0.8360.09 1.4160.11 1.6960.KpLM21 colonization (Fig. 1B, and Table four); ii) yliE, which was similarly involved in in vitro colonization resistance to 55989a, but was not induced upon KpLM21 colonization (Fig. 1B and Table 4 and Fig. S1); and iii) yceP, a gene induced in response to each pathogens, even though without having detectable in vitro effects on 55989a colonization (Fig.7-Bromo-4-chloroisoindolin-1-one manufacturer 1B and Table four and Fig.PMID:23912708 S1). To carry out colonization experiments in streptomycintreated mice, we initially made E. coli 55989a and KpLM21 streptomycinresistant derivatives (respectively, 55989as and KpLM21s). We also introduced yiaF, yliE and yceP mutants into the similar streptomycinresistant derivative of MG1655 F9, (MG1655s F9) and checked that these strains have been not considerably affected with regards to growth and in vitro biofilm capacity against this background (data not shown). We then determined bacterial counts in feces recovered from individually inoculated mice (n = at the least 8 for each and every strain) and observed that wildtype and MG1655s F9 yiaF, yliE and yceP mutants reached equivalent intestinal colonization capacity at day ten (in between 107 to 108 cfu/g of feces) prior to pathogen inoculation (Fig. 4B and 5B). At day 11 postinoculation, streptomycintreated mice colonized with wildtype MG1655s F9 or corresponding yiaF, yliE and yceP mutants had been inoculated intragastrically with either 55989as (Fig. 4) or KpLM21s (F.