BackgroundHelicobacter 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.ResultsStrain 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% β-cyclodextrin. However, the addition of the OMV-fraction collected from TK1402 could enhance biofilm formation.ConclusionThe results suggested that OMV produced from TK1402 play an important role in biofilm formation in strain TK1402.

The enteric pathogen Salmonella enterica serovar Typhimurium, similar to other facultative intracellular pathogens, has been shown to respond to the hostile conditions inside macrophages of the host organism by producing a set of stress proteins that are also induced by various environmental stresses. The stress-induced ClpXP protease is a member of the ATP-dependent proteases, which are known to be responsible for more than 90% of all proteolysis in Escherichia coli. To investigate the contribution of the ClpXP protease to the virulence of serovar Typhimurium we initially cloned the clpP and clpX operon from the pathogenic strain serovar Typhimurium 3306 and then created insertional mutations in the clpP and/or clpX gene. The ⌬clpP and ⌬clpX mutants were used to inoculate BALB/c mice by either the intraperitoneal or the oral route and found to be limited in their ability to colonize organs of the lymphatic system and to cause systemic disease in the host. A variety of experiments were performed to determine the possible reasons for the loss of virulence. An oxygendependent killing assay using hydrogen peroxide and paraquat (a superoxide anion generator) and a serum killing assay using murine serum demonstrated that all of the serovar Typhimurium ⌬clpP and ⌬clpX mutants were as resistant to these killing mechanisms as the wild-type strain. On the other hand, the macrophage survival assay revealed that all these mutants were more sensitive to the intracellular environment than the wild-type strain and were unable to grow or survive within peritoneal macrophages of BALB/c mice. In addition, it was revealed that the serovar Typhimurium ClpXP-depleted mutant was not completely cleared but found to persist at low levels within spleens and livers of mice. Interferon gamma-deficient mice and tumor necrosis factor alpha-deficient mice failed to survive the attenuated serovar Typhimurium infections, suggesting that both endogenous cytokines are essential for regulation of persistent infection with serovar Typhimurium.

This study investigated the influence of urease-positive non-Helicobacter pylori bacteria on the results of a urea breath test (UBT) to evaluate the diagnostic utility of a UBT using film-coated [ 13 C]urea tablets. The UBT was performed in 102 patients treated with a proton pump inhibitor and antibiotics for the eradication of H. pylori. Urease-producing bacteria other than H. pylori were isolated and identified from the oral cavity and stomach. In 4/102 patients, the UBT gave false-positive results. These false-positive results were found to be caused by the presence of urease-positive bacteria in the oral cavity and stomach. Five bacterial species with urease activity (Proteus mirabilis, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter cloacae and Staphylococcus aureus) were subsequently isolated from the oral cavity and/or stomach. As there was no correlation between the in vitro urease activity of urease-positive non-H. pylori bacteria and the UBT value, and all of the patients with a false-positive UBT result were suffering from atrophic gastritis, it is possible that the false-positive results in the UBT were a result of colonization of urease-positive bacteria and gastric hypochlorhydric conditions. Thus, for the diagnosis of H. pylori infection using a UBT, the influence of stomach bacteria must be considered when interpreting the results.

The human gastric pathogen Helicobacter pylori forms biofilms in vitro and in vivo. The purpose of this study was to evaluate the effects of H. pylori biofilm formation in vitro on clarithromycin (CLR) susceptibility. CLR susceptibility of H. pylori intermediate (2-day) and mature (3-day) biofilms on glass coverslips was determined at concentrations from 0.03 to 0.5 µg/ml. H. pylori biofilm biomass was increased after treatment with CLR at minimum inhibitory concentration levels by up to 4-fold (2-day biofilm) and 16-fold (3-day biofilm). Minimum bactericidal concentrations of CLR against cells in a biofilm were higher (1.0 µg/ml) than that for planktonic cells (0.25 µg/ml). It was shown that the expression of efflux pump genes was significantly increased in biofilm cells. In addition, exposure of biofilms to CLR resulted in high level resistance generation compared to planktonic cells with increased resistance associated with the presence of a point mutation at either position 2142 or 2143 in the domain V loop of the 23S rRNA gene. These results demonstrate that H. pylori biofilm formation decreases the susceptibility to CLR and that H. pylori CLR resistance mutations are more frequently generated in biofilms than in planktonic cells.

No abstract