, 2008) with Alexa Fluor 546-labeled phalloidin (Invitrogen) (1 : 200 dilution in BSP) for 1 h, washed twice with BSP and then mounted on a glass slide using mounting solution with 4′,6-diamidino-2-phenylindole. Once the slides were dry, coverslips were sealed using clear nail polish, and images were captured using green and red filters on an Olympus IX70 inverted fluorescence microscope equipped with a DP70 digital camera. The images were merged using imagej software (National Institute of Health). In our previous study (Puttamreddy et al., 2010), a mini-Tn5 transposon insertion library was constructed in E. coli O157:H7 strain EDL933
and 51 distinct genes/intergenic regions were identified to be involved either directly or indirectly Pembrolizumab in vivo in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation in a quantitative biofilm assay at P<0.05. To test whether biofilms on different surfaces may require different gene products, we subjected all 51 Bnp mutants to a quantitative biofilm test on polystyrene, polypropylene, polyvinyl chloride and glass. There was no statistical difference in the quantity of biofilms produced on any of the surfaces tested (Supporting Information, Fig. S1). To analyze the Enzalutamide cell line role of biofilm formation of E.
coli O157:H7 in adherence to T84 (human colonic epithelial) and HEp2 (human laryngeal epithelial) cells, wild type and Bnp mutants were tested for adherence in an in vitro adherence assay by both microscopic and quantitative analysis. We also constructed eae and esp deletion mutations as controls for potential adhesin–biofilm interactions.
Additionally, we tested a biofilm-positive transposon mutant (M1) as a control for nonspecific transposon effects on cellular adherence. The quantitative adherence assay (significance P<0.05) showed that all 51 Bnp mutants lost their adherence to both T84 and HEp2 cells after a 90-min infection when compared with wild type (Fig. 1, Table S1). This was also true for the eae and espAB deletion mutants as expected (Fig. 1, Table S1) because both of these genes have been shown previously to be important to cellular adherence (Yu & Kaper, 1992; Ebel et al., 1998). For microscopic analysis, all of the bacterial strains were transformed with a green fluorescent protein-expressing plasmid, pISM31. pheromone Escherichia coli O157:H7 EDL933 wild type adhered to both cell types while all of the 51 Bnp mutants failed to adhere. A biofilm-positive mini-Tn5Km2 mutant of E. coli O157:H7 EDL933 (M1) showed no significant loss in either biofilm formation or adherence when compared with the wild type (Figs 2 and 3, also see Fig. S2 for a higher magnification of wild-type adherence to the two cell types). This eliminates the possibility of a nonspecific effect between mini-Tn5Km2 insertions and loss of adherence. To identify whether loss of adherence leads to a loss of biofilm phenotype, deletions in eae and espAB, both well-known adhesins (Jerse et al.