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Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation.

Yonezawa H, Osaki T, Kurata S, Fukuda M, Kawakami H, Ochiai K, Hanawa T, Kamiya S - BMC Microbiol. (2009)

Bottom Line: The strong biofilm forming ability of TK1402 is reflected the relative thickness of the biofilms.In addition, outer membrane vesicles (OMV) were detected within the matrix of only the TK1402 biofilms.However, the addition of the OMV-fraction collected from TK1402 could enhance biofilm formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Disease, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo, 181-8611, Japan. yonezawa@ks.kyorin-u.ac.jp

ABSTRACT

Background: Helicobacter pylori forms biofilms on glass surfaces at the air-liquid interface in in vitro batch cultures; however, biofilms of H. pylori have not been well characterized. In the present study, we analyzed the ability of H. pylori strains to form biofilms and characterized the underlying mechanisms of H. pylori biofilm formation.

Results: Strain TK1402 showed strong biofilm forming ability relative to the other strains in Brucella broth supplemented with 7% FCS. The strong biofilm forming ability of TK1402 is reflected the relative thickness of the biofilms. In addition, outer membrane vesicles (OMV) were detected within the matrix of only the TK1402 biofilms. Biofilm formation was strongly correlated with the production of OMV in this strain. We further observed that strain TK1402 did not form thick biofilms in Brucella broth supplemented with 0.2% beta-cyclodextrin. However, the addition of the OMV-fraction collected from TK1402 could enhance biofilm formation.

Conclusion: The results suggested that OMV produced from TK1402 play an important role in biofilm formation in strain TK1402.

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Related in: MedlinePlus

CLSM images of H. pylori strains TK1402 (A) and SS1 (B) biofilms in Brucella broth containing 7% FCS. Each image represents the layer in the Z-stack that has the maximum bacterial coverage. The 3-day biofilms of each strain were stained with BacLight LIVE/DEAD stain. Viable cells are colored green and nonviable cells are colored red. x-z and y-z reconstructions of each biofilm are shown on the right and upper sides of each x-y image. The scale bars equal 50 μm in both panels. (C) Biofilm thickness of each strain. 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 strains TK1402 (D) and SS1 (E) biofilms in Brucella broth containing 7% FCS. The 3-day biofilm of 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).
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Figure 2: CLSM images of H. pylori strains TK1402 (A) and SS1 (B) biofilms in Brucella broth containing 7% FCS. Each image represents the layer in the Z-stack that has the maximum bacterial coverage. The 3-day biofilms of each strain were stained with BacLight LIVE/DEAD stain. Viable cells are colored green and nonviable cells are colored red. x-z and y-z reconstructions of each biofilm are shown on the right and upper sides of each x-y image. The scale bars equal 50 μm in both panels. (C) Biofilm thickness of each strain. 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 strains TK1402 (D) and SS1 (E) biofilms in Brucella broth containing 7% FCS. The 3-day biofilm of 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).

Mentions: The biofilms were stained with a BacLight LIVE/DEAD bacterial viability kit solution which could differentiate between live cells (green) and dead cells (red). Strain TK1402 formed strong biofilms covering the entire visible area (Fig. 2A) but the other strain SS1 formed relatively poor biofilms (Fig. 2B). In the biofilms of both strains, the majority of the biofilm cells were stained green (Fig. 2A, and 2B). In order to confirm that the TK1402 biofilm cells were viable, the 2-day and 3-day biofilms cells were scrapped into PBS and the optical densities and the CFU values of the mixtures were evaluated (Table 1). The 2-day and 3-day cultures of this strain in Brucella broth supplemented with 7% FCS were also measured as controls. The optical densities and CFU of the 3-day biofilm cells showed increases compared to those of 2-day biofilm cells. Further, the CFU values were normalized to optical densities. The values for the 2-day biofilm cells were similar compared to the controls (broth culture). However, the normalized values for 3-day biofilm cells tended to be decreased, although there was no significant difference in the normalized values, suggesting that 3-day biofilm cells might contain some dead cells or morphologically altered cells.


Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation.

Yonezawa H, Osaki T, Kurata S, Fukuda M, Kawakami H, Ochiai K, Hanawa T, Kamiya S - BMC Microbiol. (2009)

CLSM images of H. pylori strains TK1402 (A) and SS1 (B) biofilms in Brucella broth containing 7% FCS. Each image represents the layer in the Z-stack that has the maximum bacterial coverage. The 3-day biofilms of each strain were stained with BacLight LIVE/DEAD stain. Viable cells are colored green and nonviable cells are colored red. x-z and y-z reconstructions of each biofilm are shown on the right and upper sides of each x-y image. The scale bars equal 50 μm in both panels. (C) Biofilm thickness of each strain. 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 strains TK1402 (D) and SS1 (E) biofilms in Brucella broth containing 7% FCS. The 3-day biofilm of 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: CLSM images of H. pylori strains TK1402 (A) and SS1 (B) biofilms in Brucella broth containing 7% FCS. Each image represents the layer in the Z-stack that has the maximum bacterial coverage. The 3-day biofilms of each strain were stained with BacLight LIVE/DEAD stain. Viable cells are colored green and nonviable cells are colored red. x-z and y-z reconstructions of each biofilm are shown on the right and upper sides of each x-y image. The scale bars equal 50 μm in both panels. (C) Biofilm thickness of each strain. 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 strains TK1402 (D) and SS1 (E) biofilms in Brucella broth containing 7% FCS. The 3-day biofilm of 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).
Mentions: The biofilms were stained with a BacLight LIVE/DEAD bacterial viability kit solution which could differentiate between live cells (green) and dead cells (red). Strain TK1402 formed strong biofilms covering the entire visible area (Fig. 2A) but the other strain SS1 formed relatively poor biofilms (Fig. 2B). In the biofilms of both strains, the majority of the biofilm cells were stained green (Fig. 2A, and 2B). In order to confirm that the TK1402 biofilm cells were viable, the 2-day and 3-day biofilms cells were scrapped into PBS and the optical densities and the CFU values of the mixtures were evaluated (Table 1). The 2-day and 3-day cultures of this strain in Brucella broth supplemented with 7% FCS were also measured as controls. The optical densities and CFU of the 3-day biofilm cells showed increases compared to those of 2-day biofilm cells. Further, the CFU values were normalized to optical densities. The values for the 2-day biofilm cells were similar compared to the controls (broth culture). However, the normalized values for 3-day biofilm cells tended to be decreased, although there was no significant difference in the normalized values, suggesting that 3-day biofilm cells might contain some dead cells or morphologically altered cells.

Bottom Line: The strong biofilm forming ability of TK1402 is reflected the relative thickness of the biofilms.In addition, outer membrane vesicles (OMV) were detected within the matrix of only the TK1402 biofilms.However, the addition of the OMV-fraction collected from TK1402 could enhance biofilm formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious Disease, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo, 181-8611, Japan. yonezawa@ks.kyorin-u.ac.jp

ABSTRACT

Background: Helicobacter pylori forms biofilms on glass surfaces at the air-liquid interface in in vitro batch cultures; however, biofilms of H. pylori have not been well characterized. In the present study, we analyzed the ability of H. pylori strains to form biofilms and characterized the underlying mechanisms of H. pylori biofilm formation.

Results: Strain TK1402 showed strong biofilm forming ability relative to the other strains in Brucella broth supplemented with 7% FCS. The strong biofilm forming ability of TK1402 is reflected the relative thickness of the biofilms. In addition, outer membrane vesicles (OMV) were detected within the matrix of only the TK1402 biofilms. Biofilm formation was strongly correlated with the production of OMV in this strain. We further observed that strain TK1402 did not form thick biofilms in Brucella broth supplemented with 0.2% beta-cyclodextrin. However, the addition of the OMV-fraction collected from TK1402 could enhance biofilm formation.

Conclusion: The results suggested that OMV produced from TK1402 play an important role in biofilm formation in strain TK1402.

Show MeSH
Related in: MedlinePlus