Bacterium–host interactions monitored by time-lapse photography

Löfman, Carl; Rigo, R.; Block, Mats; Hultén, K; Enroth, Helena; Engstrand, Lars

doi:10.1038/nm0897-930

Cited by 20 publications

(21 citation statements)

References 9 publications

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“…The theory of intracellular H. pylori is still controversial but it seems that the bacteria can hide within the epithelial cells of the mucosa. This has been revealed by electron microscopy and by time-lapse photography (4,10). In the present study a fairly constant amount of intracellular bacteria was obtained in the controls for 8 h. During this period both HMR 3647 and HMR 3004 exhibited 1.8 to 2 log 10 reductions of bacteria, and the effect on intracellular H. pylori was evident.…”

supporting

confidence: 67%

“…Unexposed controls were included. To eliminate the antibiotics, both cultures were washed twice with broth by centrifugation for 10 min at 1,400 ϫ g and then diluted in fresh broth to obtain 10 4 CFU/ml. The bacterial culture in the postantibiotic phase was divided into three tubes, of which two tubes were reexposed to subinhibitory concentrations of 0.1 and 0.3 times the MIC.…”

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confidence: 99%

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In Vitro Pharmacodynamic Studies of Activities of Ketolides HMR 3647 (Telithromycin) and HMR 3004 against Extracellular or Intracellular Helicobacter pylori

Gustafsson¹,

Engstrand²,

Cars

2001

Antimicrob Agents Chemother

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The pharmacodynamic properties of the ketolides HMR 3647 (telithromycin) and HMR 3004 were studied against Helicobacter pylori. Both ketolides showed a pronounced concentration-dependent killing, a significant postantibiotic effect, a long postantibiotic sub-MIC effect, and a reduction of intracellular H. pylori.Helicobacter pylori establishes persistent infection in the gastric mucosa of humans. Antimicrobial treatment of H. pylori is now widely recommended for patients with peptic ulcer who are carrying this bacterium. H. pylori has so far been considered to colonize exclusively extracellularly. There are, however, considerable data supporting an intracellular location of the bacterium in vivo (4,8). Due to the increasing problems with macrolide resistance in H. pylori (5, 9), the development of new treatment regimens, including antibiotics with intracellular activity, is needed. The aim of our study was to evaluate the pharmacodynamic properties of the ketolides HMR 3647 (telithromycin) and HMR 3004 against extracellular and intracellular H. pylori.(This work was presented at the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, Calif., 24 to 27 September, 1998.)H. pylori NCTC 11637 was grown on chocolate agar plates (Columbia II agar base BBL; Becton Dickinson and Co., Franklin Lake, N.J.) supplemented with 10% horse serum and 8.5% horse blood. For broth cultures we used brucella broth (pH 7.3) supplemented with 10% fetal calf serum. Plates and broth cultures were incubated at 37°C in a humid atmosphere under microaerophilic conditions (7% CO 2 and 87% N 2 ). Broth cultures, incubated for 3 days, were prepared prior to the experiments. The turbidity was measured at an optical density at 530 nm of 0.5, which corresponds to 10 8 CFU/ml. HMR 3647 and HMR 3004 with known potencies were kindly provided by Hoechst Marion Roussel, Romainville, France. A stock solution was made before each experiment by dissolving 10.2 mg of the drug in 10 ml of sterile distilled water supplemented with 20 l of glacial acetic acid.MICs were determined by the macrodilution technique with an inoculum of 5 ϫ 10 5 to 1 ϫ 10 6 CFU/ml, according to NCCLS standards (11). The MIC was defined as the lowest concentration inhibiting visible growth after 3 days. To investigate whether the killing pattern was concentration dependent, bacteria at a density of 10 6 CFU/ml were exposed to 2, 5, 10, and 50 times the MIC. A growth control was also included.Samples were withdrawn after 0, 1, 2, 3, 6, 9, 12, and 24 h, and appropriate dilutions were seeded on chocolate agar in volumes of 0.1 and 0.01 ml. The plates were incubated for 5 days before the colonies were counted. The sensitivity of the viable count is estimated at 10 to 50 CFU/ml.Postantibiotic effect (PAE) and postantibiotic sub-MIC effect (PASME) determinations were performed with cultures of 10 7 CFU/ml exposed to 10 times the MIC for 2 h at 37°C. Unexposed controls were included. To eliminate the antibiotics, both cultures were washed twice with broth by ce...

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confidence: 67%

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confidence: 99%

In Vitro Pharmacodynamic Studies of Activities of Ketolides HMR 3647 (Telithromycin) and HMR 3004 against Extracellular or Intracellular Helicobacter pylori

Gustafsson¹,

Engstrand²,

Cars

2001

Antimicrob Agents Chemother

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“…For a very long time, H. pylori was considered an extracellular pathogen (52,53,56), but an increasing number of re- ports based on biospy examination and in vitro infection experiments have indicated that at least some host-associated H. pylori bacteria are located within host epithelial cells (19,28,31,50,62,64,73,79,85). In the present study, we used AGS cells as an infection model in order to demonstrate that the penetration of gastric epithelial cells is a multifactorial process resulting from an intricate interplay between H. pylori and host cells.…”

Section: Discussionmentioning

confidence: 99%

“…Early reports indicated that H. pylori was not present in the gastric mucosa but was present in the mucus layer overlying the gastric tissue (52,53,56). However, in recent years, a number of biospy studies (28,30,62) and cell culture infection models (19,31,50,64,73,79,85) have provided increasing evidence for the intracellular localization of H. pylori. Clearance of intracellular H. pylori has been demonstrated in vitro by using antibiotics with known intracellular activity (40).…”

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confidence: 99%

Specific Entry ofHelicobacter pyloriinto Cultured Gastric Epithelial Cells via a Zipper-Like Mechanism

et al. 2002

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Although Helicobacter pylori has generally been considered an extracellular pathogen, a number of in vitro infection experiments and biopsy examinations have shown that it is capable of occasionally entering mammalian host cells. Here, we characterized this entry process by using AGS cells as a host cell model. In gentamicin protection-invasion assays, the number of H. pylori colonies recovered was lower than that for Salmonella enterica serovar Typhimurium X22, Escherichia coli expressing InvA, and Yersinia enterocolitica YO:9 grown at 25°C but higher than that for Neisseria gonorrhoeae VP1 and Y. enterocolitica YO:9 grown at 37°C. At the ultrastructural level, the entry process was observed to occur via a zipper-like mechanism. Internalized H. pylori was bound in tight LAMP-1-containing vacuoles in close association with condensed filamentous actin and tyrosine phosphorylation signals. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, and calphostin C, an inhibitor of protein kinase C, both inhibited the entry of H. pylori in a sensitive and dosedependent manner; however, the level of entry was enhanced by sodium vanadate, an inhibitor of tyrosine phosphatases and ATPases. Furthermore, the cytokine tumor necrosis factor alpha antagonized the entry of H. pylori into AGS cells. Collectively, these results demonstrate that the entry of H. pylori into AGS cells occurs via a zipper-like mechanism which involves various host signal transduction events.

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“…However, this notion has been challenged by in vitro studies on cell lines and primary cultures of gastric epithelial cells, as well as observations of intracellular H. pylori in gastric tissue sections from infected individuals. These studies have indicated that a diminutive fraction of the colonizing population might enter epithelial cells (Lofman et al, 1997;Su et al, 1999;Bjorkholm et al, 2000Bjorkholm et al, , 2003Papadogiannakis et al, 2000). In addition Dunn et al (1997) have demonstrated a fourth mechanism of surface localization of cytoplasmic bacterial proteins.…”

Section: Discussionmentioning

confidence: 99%

Helicobacter pylori heat shock protein B (HspB) localizes in vivo in the gastric mucosa and MALT lymphoma

Luca

Falco

Manente

et al. 2008

Journal Cellular Physiology

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Heat shock protein B (HspB) is one of the dominant proteins recognized by most Helicobacter pylori-infected persons and is being considered as potential candidates for subunit vaccines. In the present study we describe the generation of an antibody against HspB and its use in immunohistochemical assays on gastric biopsies. We have demonstrated that our rabbit polyclonal antibody against HspB did not recognize any protein in lysates from a lung human epithelial cell (H1299) line and did not cross-react with the other members of human heat shock proteins. Secondly, we have observed that in gastric biopsies, HspB immunostaining was present inside the cytoplasm of human epithelial cells with a particular localization in the apical portion of gastric epithelial cells other than in the extracellular spaces among gastric cells of human stomach. Finally, we have demonstrated a cytoplasmic HspB immunostaining in groups of neoplastic cells of MALT lymphoma. In conclusion, our observations suggest a possible involvement of HspB in the pathogenesis of H. pylori-related pathologies such as gastritis, ulcer and gastric cancer.

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Bacterium–host interactions monitored by time-lapse photography

Cited by 20 publications

References 9 publications

In Vitro Pharmacodynamic Studies of Activities of Ketolides HMR 3647 (Telithromycin) and HMR 3004 against Extracellular or Intracellular Helicobacter pylori

In Vitro Pharmacodynamic Studies of Activities of Ketolides HMR 3647 (Telithromycin) and HMR 3004 against Extracellular or Intracellular Helicobacter pylori

Specific Entry ofHelicobacter pyloriinto Cultured Gastric Epithelial Cells via a Zipper-Like Mechanism

Helicobacter pylori heat shock protein B (HspB) localizes in vivo in the gastric mucosa and MALT lymphoma

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