OMV fraction and could affect biofilm formation, a control fraction from Brucella broth supplemented with 7% FCS without the microorganism was used. The levels of biofilm formation in the 0.2% β-cyclodextrin medium supplemented with the control OMV fraction was similar to that of the 0.2% β-cyclodextrin medium alone (Fig. 5B, lane β-cyclodextrin-control). On the other hand, the addition of the 0.1 mg OMV fraction from TK1402 showed significantly higher levels of biofilm formation than those in 0.2% β-cyclodextrin medium with the control fraction (Fig. 4B, β-cyclodextrin-FCS OMV 0.1). The levels of biofilm formation with OMV addition were similar to that in Brucella broth supplemented with 7% FCS (Fig. 4B. β-cyclodextrin-FCS OMV 0.2). We further determined that the 0.1 mg OMV fraction from H. pylori cultured in Brucella broth containing 0.2% β-cyclodextrin could also enhance biofilm formation Batimastat but at levels lower than 0.2 mg of this fraction. The OMV fraction induced more biofilm formation than 0.1 mg of the OMV fraction from 7% FCS medium (Fig. 5B, β-cyclodextrin-β-cyclo OMV 0.1). Evaluation of biofilm formation by other
isolated H. pylori strains In order to detect other strains having similar biofilm forming ability to strain TK1402, we assessed the biofilm forming ability of ten additional clinical isolates of H. pylori. Only strain TK1049 showed similar levels of biofilm formation to that of strain TK1402 (Fig. 6A). The other strains showed lower levels of biofilm formation than strain TK1402 (the biofilm OD595 values ranged from 0.1 to Aspartate 0.3). The structure of TK1049 biofilms was then observed by using SEM (Fig. 6C). Cellular aggregation was observed to be similar to that of TK1402 biofilms and many vesicle-like structures were also detected with TK1409. Moreover, 3-day biofilm formation with strain TK1049 in Brucella broth supplemented with 0.2% β-cyclodextrin was weaker than that in Brucella broth supplemented with 7% FCS. However, the addition of the OMV fraction from TK1402 in Brucella broth
supplemented with 0.2% β-cyclodextrin restored biofilm formation similar to that in Brucella broth supplemented with 7% FCS (Fig. 6B). Figure 6 (A) Biofilm formation by strain TK1049. Graph shows quantification of biofilms formed after 3-day (Day 3) and 5-days (Day 5) in Brucella broth supplemented with 7% FCS. (B) Biofilm formation by strain TK1049 in Brucella broth supplemented with 0.2% β-cyclodextrin and addition of the OMV-fraction from TK1402 grown in 0.2% β-cyclodextrin medium. (C) SEM observation of TK1049 biofilms. *significantly different (p < 0.05). Discussion In this study, we characterized biofilm formation in H. pylori strains and demonstrated differential abilities to form biofilms in reference and clinical isolates.