Each biofilm was scanned with CLSM at five randomly selected positions and x-z color detection, corresponding to biofilm thickness, was determined throughout the height of the biofilm. Data are representative of three independent
experiments. The results are expressed as the means ± standard deviations. SEM images of H. pylori Selleckchem CP673451 strains TK1402 (D) and SS1 (E) biofilms in Brucella broth containing 7% FCS. The 3-day biofilm of selleck screening library each strain on cover glass was investigated using SEM. The OMV-like structures are indicated by white arrows (D). Scale bars (2 μm) are shown at the bottom of each electron micrograph. *significantly different relative levels of biofilm thickness (p < 0.05; strain TK1402 versus other strains). Next we analyzed the biofilm thickness of strains TK1402, SS1, TK1029, and ATCC 49503 with CMLS observations. Strain TK1402 exhibited 2-fold or greater biofilm thickness compared to the other strains (Fig. 2C). To clarify the architectural characteristics of H. pylori biofilms, we compared TK1402 and SS1 biofilms by SEM analysis. In the biofilms of strain SS1, the bacteria attached AZD2281 clinical trial to glass surfaces in thin layers
(Fig. 2E). Interestingly, the biofilms consisted mainly of bleb-like or amorphous structures. On the other hand, the TK1402 biofilms were composed primarily of cells with bacillary morphology which were clearly outlined (Fig. 2D). In addition, these later bacteria showed layer formation with bacterial aggregates MG-132 research buy in the biofilms. The biofilm bacterial aggregates appeared to result from direct cell-cell attachment. Intriguingly,
TK1402 biofilms showed the presence of many OMV-like structures on the glass surface as well as on the bacterial cell surfaces (Fig. 2D, white arrows). These structures were not detected in the biofilms of the other strains (Fig. 2E and data not shown). A recent report indicated that OMV production from H. pylori clinical isolate MDC1 was apparent under SEM observation . We thus decided to focus our attention more on the OMV-like structures in subsequent experiments. Potential role of the OMV in TK1402 biofilm formation We observed more closely the OMV-like structures in the thin-sectioned biofilms using TEM (Fig. 3). These structures consisted primarily of bilayered proteolipids which were mainly spherical in shape (Fig. 3, black arrows). These structures also exhibited the characteristics typical of Gram negative bacterial OMV . We confirmed that the OMV-fraction did not contain flagella by observation with SM and Western blotting with anti-flagella antibody. Figure 3 TEM images of H. pylori strain TK1402 biofilms in Brucella broth supplemented with 7% FCS. The 3-day biofilm of strain TK1402 on glass slides was investigated by using TEM. We next found that the FCS concentration in the biofilm growing medium affected biofilm formation of H. pylori TK1402 (Fig. 4A). The lower concentrations of FCS (3.5%, 1.