Assessment of <i>in vitro</i> biofilm formation by <i>Helicobacter pylori</i>

Yonezawa, Hideo; Osaki, Takako; Kurata, Satoru; Zaman, Cynthia; Hanawa, Tomoko; Kamiya, Shigeru

doi:10.1111/j.1440-1746.2009.06213.x

Cited by 58 publications

(50 citation statements)

References 21 publications

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“…Bacterial autoaggregation was assessed as previously described (32), with the following modifications. H. pylori cultures were prepared as described in the bacterial motility assay.…”

Section: Methodsmentioning

confidence: 99%

“…S1 and S2 in the supplemental material), we reasoned that increased aggregation in strains lacking ArsS or containing the nonphosphorylatable ArsR-D52N mutation could contribute to the enhanced biofilm formation seen with these strains. Therefore, we assessed bacterial aggregation using a quantitative aggregation assay (32). Since biofilm formation occurs as early as 24 h for some strains, 20-h cultures were used for this assessment.…”

Section: Growth Characteristics Of Fur Nikr and Arsrs Mutant Strainsmentioning

confidence: 99%

“…Biofilm formation by H. pylori has been demonstrated both in vitro (30)(31)(32) and in vivo (33)(34)(35)(36)(37). While it is unclear exactly what drives biofilm formation in H. pylori, protection from antibiotics and host defenses during infection (35,38,39) and/or protection in putative environmental reservoir (40-43) have been proposed.…”

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confidence: 99%

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Characterization of Key Helicobacter pylori Regulators Identifies a Role for ArsRS in Biofilm Formation

et al. 2016

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Helicobacter pylori must be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation, H. pylori encodes few regulatory proteins. Of the identified regulators, the ferric uptake regulator (Fur), the nickel response regulator (NikR), and the two-component acid response system (ArsRS) are each paramount to the success of this pathogen. While numerous studies have individually examined these regulatory proteins, little is known about their combined effect. Therefore, we constructed a series of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS. A growth curve analysis revealed minor variation in growth kinetics across the strains; these were most pronounced in the triple mutant and in strains lacking ArsS. Visual analysis showed that strains lacking ArsS formed large aggregates and a biofilm-like matrix at the air-liquid interface. Biofilm quantification using crystal violet assays and visualization via scanning electron microscopy (SEM) showed that all strains lacking ArsS or containing a nonphosphorylatable form of ArsR (ArsR-D52N mutant) formed significantly more biofilm than the wild-type strain. Molecular characterization of biofilm formation showed that strains containing mutations in the ArsRS pathway displayed increased levels of cell aggregation and adherence, both of which are key to biofilm development. Furthermore, SEM analysis revealed prevalent coccoid cells and extracellular matrix formation in the ArsR-D52N, ⌬nikR ⌬arsS, and ⌬fur ⌬nikR ⌬arsS mutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus, H. pylori ArsRS has a previously unrecognized role in biofilm formation. IMPORTANCEDespite a paucity of regulatory proteins, adaptation is key to the survival of H. pylori within the stomach. While prior studies have focused on individual regulatory proteins, such as Fur, NikR, and ArsRS, few studies have examined the combined effect of these factors. Analysis of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS revealed a previously unrecognized role for the acid-responsive two-component system ArsRS in biofilm formation.

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“…Bacterial autoaggregation was assessed as previously described (32), with the following modifications. H. pylori cultures were prepared as described in the bacterial motility assay.…”

Section: Methodsmentioning

confidence: 99%

Section: Growth Characteristics Of Fur Nikr and Arsrs Mutant Strainsmentioning

confidence: 99%

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Characterization of Key Helicobacter pylori Regulators Identifies a Role for ArsRS in Biofilm Formation

et al. 2016

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“…Cell aggregation was measured using the method of Yonezawa et al (33). Briefly, 1 ml of overnight culture in BHI was spun down and washed in 5 ml of phosphate-buffered saline (PBS).…”

Section: Methodsmentioning

confidence: 99%

Functional Analysis of ycfR and ycfQ in Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce

Deng

Wang

Rui

et al. 2011

Appl Environ Microbiol

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Fresh produce has been associated with multiple outbreaks of illness caused by Escherichia coli O157:H7. The mechanism of E. coli O157:H7 survival through postharvest processing of fresh produce needs to be understood to help develop more effective interventions. In our recent transcriptomic study of strain Sakai, an isolate from the 1996 sprout outbreak in Japan, and strain TW14359, an isolate from the 2006 spinach outbreak in the United States, we showed that ycfR was the most significantly upregulated gene in response to chlorine-based oxidative stress. YcfR is known to be a multiple stress resistance protein and a biofilm regulator in E. coli K-12 strains; however, its role in the pathogenic E. coli O157:H7 has not been clearly defined. In this study, ycfR was replaced with a chloramphenicol resistance cassette oriented in two different directions to construct polar and nonpolar ycfR::cat mutants of Sakai and TW14359. Chlorine resistance and survival on spinach leaf surfaces were assessed in the wild-type strains and the ycfR mutants. Both polar and nonpolar ycfR mutants of Sakai showed significantly less chlorine resistance than their parent strain. In contrast, deletion of ycfR in TW14359 did not change chlorine resistance, indicating that ycfR in these two outbreak-related E. coli O157:H7 strains may function differently. In addition, after a 24-h incubation on spinach leaves in a sublethal concentration of chlorine, the Sakai nonpolar ycfR mutant exhibited lower survival compared to the wild type. The results suggest a role for ycfR in survival of Sakai during chlorine exposure. We also found that the upstream ycfQ, which is annotated as a DNA-binding regulator, acted as a repressor of ycfR. These findings suggest that gene regulation may be a mechanism by which E. coli O157:H7 strain Sakai could survive in the postharvest processing environment.

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“…On the other hand, as with various bacteria studied to date, H. pylori can have an alternative lifestyle as a biofilm (Yonezawa et al, 2010). Biofilms are important in bacterial pathogenesis due to because allows at microbes survive and spread within the host.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of Helicobacter pylori and Its Associated Urease by Two Regional Plants of San Luis Argentina

Ibáñez¹,

Sosa²,

Ferramola³

et al. 2017

Int.J.Curr.Microbiol.App.Sci

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Assessment of in vitro biofilm formation by Helicobacter pylori

Abstract: TK1402 is a strong biofilm-forming strain of H. pylori in Brucella broth supplemented with 7% fetal calf serum. It is possible that biofilm-forming cell growth is a principal factor in biofilm development.

Cited by 58 publications

References 21 publications

Characterization of Key Helicobacter pylori Regulators Identifies a Role for ArsRS in Biofilm Formation

Characterization of Key Helicobacter pylori Regulators Identifies a Role for ArsRS in Biofilm Formation

Functional Analysis of ycfR and ycfQ in Escherichia coli O157:H7 Linked to Outbreaks of Illness Associated with Fresh Produce

Inhibition of Helicobacter pylori and Its Associated Urease by Two Regional Plants of San Luis Argentina

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