Biofilm Formation as a Complex Result of Virulence and Adaptive Responses of Helicobacter pylori

Helicobacter pylori is a bacterium that is capable of colonizing a host for many years, often for a lifetime. The survival in the gastric environment is enabled by the production of numerous virulence factors conditioning adhesion to the mucosa surface, acquisition of nutrients, and neutralization of the immune system activity. It is increasingly recognized, however, that the adaptive mechanisms of H. pylori in the stomach may also be linked to the ability of this pathogen to form biofilms. Initially, biofilms produced by H. pylori were strongly associated by scientists with water distribution systems and considered as a survival mechanism outside the host and a source of fecal-oral infections. In the course of the last 20 years, however, this trend has changed and now the most attention is focused on the biomedical aspect of this structure and its potential contribution to the therapeutic difficulties of H. pylori. Taking into account this fact, the aim of the current review is to discuss the phenomenon of H. pylori biofilm formation and present this mechanism as a resultant of the virulence and adaptive responses of H. pylori, including morphological transformation, membrane vesicles secretion, matrix production, efflux pump activity, and intermicrobial communication. These mechanisms will be considered in the context of transcriptomic and proteomic changes in H. pylori biofilms and their modulating effect on the development of this complex structure.

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Myricetin as an Antivirulence Compound Interfering with a Morphological Transformation into Coccoid Forms and Potentiating Activity of Antibiotics against Helicobacter pylori

International Journal of Molecular Sciences ◽

10.3390/ijms22052695 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2695

Author(s):

Paweł Krzyżek ◽

Paweł Migdał ◽

Emil Paluch ◽

Magdalena Karwańska ◽

Alina Wieliczko ◽

...

Keyword(s):

Helicobacter Pylori ◽

Biofilm Formation ◽

Inhibitory Effect ◽

Morphological Transformation ◽

High Tendency ◽

Minimal Inhibitory Concentrations ◽

Coccoid Form ◽

Stomach Diseases ◽

Fold Reduction ◽

H Pylori

Helicobacter pylori, a gastric pathogen associated with a broad range of stomach diseases, has a high tendency to become resistant to antibiotics. One of the most important factors related to therapeutic failures is its ability to change from a spiral to a coccoid form. Therefore, the main aim of our original article was to determine the influence of myricetin, a natural compound with an antivirulence action, on the morphological transformation of H. pylori and check the potential of myricetin to increase the activity of antibiotics against this pathogen. We observed that sub-minimal inhibitory concentrations (sub-MICs) of this compound have the ability to slow down the process of transformation into coccoid forms and reduce biofilm formation of this bacterium. Using checkerboard assays, we noticed that the exposure of H. pylori to sub-MICs of myricetin enabled a 4–16-fold reduction in MICs of all classically used antibiotics (amoxicillin, clarithromycin, tetracycline, metronidazole, and levofloxacin). Additionally, RT-qPCR studies of genes related to the H. pylori morphogenesis showed a decrease in their expression during exposure to myricetin. This inhibitory effect was more strongly seen for genes involved in the muropeptide monomers shortening (csd3, csd6, csd4, and amiA), suggesting their significant participation in the spiral-to-coccoid transition. To our knowledge, this is the first research showing the ability of any compound to synergistically interact with all five antibiotics against H. pylori and the first one showing the capacity of a natural substance to interfere with the morphological transition of H. pylori from spiral to coccoid forms.

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Bifunctional enzyme SpoT is involved in biofilm formation of Helicobacter pylori with multidrug resistance by upregulating efflux pump Hp1174 (gluP)

10.1101/321026 ◽

2018 ◽

Author(s):

Xiaoran Ge ◽

Yuying Cai ◽

Zhenghong Chen ◽

Sizhe Gao ◽

Xiwen Geng ◽

...

Keyword(s):

Drug Resistance ◽

Helicobacter Pylori ◽

Multidrug Resistance ◽

Biofilm Formation ◽

Efflux Pump ◽

Efflux Pumps ◽

Multi Drug Resistance ◽

Bifunctional Enzyme ◽

Average Expression Level ◽

H Pylori

ABSTRACTThe drug resistance of Helicobacter pylori (H. pylori) is gradually becoming a serious problem. Biofilm formation is an important factor that leads to multidrug resistance in bacteria. The ability of H. pylori to form biofilms on the gastric mucosa has been known. However, there are few studies on the regulation mechanisms of H. pylori biofilm formation and multidrug resistance. Guanosine 3’-diphosphate 5’-triphosphate and guanosine 3’,5’-bispyrophosphate [(p)ppGpp] are global regulatory factors and are synthesized in H. pylori by the bifunctional enzyme SpoT. It has been reported that (p)ppGpp is involved in the biofilm formation and multidrug resistance of various bacteria. In this study, we found that SpoT also plays an important role in H. pylori biofilm formation and multidrug resistance. Therefore, it is necessary to carry out some further studies regarding its regulatory mechanism. Considering that efflux pumps are of great importance in the biofilm formation and multidrug resistance of bacteria, we tried to find if efflux pumps controlled by SpoT participate in these activities. Then, we found that Hp1174 (glucose/galactose transporter, gluP), an efflux pump of the MFS family, is highly expressed in biofilm-forming and multi-drug resistance (MDR) H. pylori and is upregulated by SpoT. Through further research, we determined that gluP involved in H. pylori biofilm formation and multidrug resistance. Furthermore, the average expression level of gluP in clinical MDR strains was considerably higher than that in clinical drug-sensitive strains. Taken together, our results revealed a novel molecular mechanism of H. pylori tolerance to multidrug.

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Biofilm Formation and Antibiotic Resistance Phenotype of Helicobacter pylori Clinical Isolates

Toxins ◽

10.3390/toxins12080473 ◽

2020 ◽

Vol 12 (8) ◽

pp. 473 ◽

Cited By ~ 2

Author(s):

Kartika Afrida Fauzia ◽

Muhammad Miftahussurur ◽

Ari Fahrial Syam ◽

Langgeng Agung Waskito ◽

Dalla Doohan ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Crystal Violet ◽

Antibiotic Susceptibility ◽

Biofilm Formation ◽

Clinical Isolates ◽

Resistance Phenotype ◽

Resistance To Antibiotics ◽

Inhibition Concentration ◽

H Pylori

We evaluated biofilm formation of clinical Helicobacter pylori isolates from Indonesia and its relation to antibiotic resistance. We determined the minimum inhibition concentration (MIC) of amoxicillin, clarithromycin, levofloxacin, metronidazole and tetracycline by the Etest to measure the planktonic susceptibility of 101 H. pylori strains. Biofilms were quantified by the crystal violet method. The minimum biofilm eradication concentration (MBEC) was obtained by measuring the survival of bacteria in a biofilm after exposure to antibiotics. The majority of the strains formed a biofilm (93.1% (94/101)), including weak (75.5%) and strong (24.5%) biofilm-formers. Planktonic resistant and sensitive strains produced relatively equal amounts of biofilms. The resistance proportion, shown by the MBEC measurement, was higher in the strong biofilm group for all antibiotics compared to the weak biofilm group, especially for clarithromycin (p = 0.002). Several cases showed sensitivity by the MIC measurement, but resistance according to the MBEC measurements (amoxicillin, 47.6%; tetracycline, 57.1%; clarithromycin, 19.0%; levofloxacin, 38.1%; and metronidazole 38.1%). Thus, biofilm formation may increase the survival of H. pylori and its resistance to antibiotics. Biofilm-related antibiotic resistance should be evaluated with antibiotic susceptibility.

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Genetic Determinants of Biofilm Formation and Antibiotic Resistance of Helicobacter Pylori using Whole Genome Sequencing

10.21203/rs.3.rs-800655/v1 ◽

2021 ◽

Author(s):

Kartika Afrida Fauzia ◽

Hafeza Aftab ◽

Muhammad Miftahussurur ◽

Langgeng Agung Waskito ◽

Vo Phuoc Tuan ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibiotic Resistance ◽

Biofilm Formation ◽

Cross Sectional Study ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Genetic Determinants ◽

Genome Sequences ◽

Cross Sectional ◽

H Pylori

Abstract The nucleotide polymorphisms (SNPs) associated with the biofilm formation phenotype of Helicobacter pylori were investigated. Fifty-six H. pylori isolates from Bangladeshi patients were included in this cross-sectional study. Crystal violet was used to classify the phenotypes into high- and low-biofilm formers. Whole genome sequences were analyzed using the “Antimicrobial Resistance Identification By Assembly” (ARIBA) pipeline. The results indicated 19.6% high- and 81.4% low-biofilm formers. These phenotypes were not related to specific clades in the phylogenetic analysis. Biofilm formation was significantly associated with SNPs of alpA, alpB, cagE, cgt, csd4, csd5, futB, gluP, homD, and murF (P < 0.05). Among the SNPs reported in alpB, strains encoding the N156K, G160S, and A223V mutations were high-biofilm formers. Mutations associated with antibiotic resistance can be detected. This study revealed the potential role of SNPs to biofilm formation, and propose a method to detect mutation in antibiotic resistance and biofilm from whole genome sequences.

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A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral-shape to coccoids

10.1101/585380 ◽

2019 ◽

Author(s):

Lamya El Mortaji ◽

Alejandro Tejada-Arranz ◽

Aline Rifflet ◽

Ivo G Boneca ◽

Gérard Pehau-Arnaudet ◽

...

Keyword(s):

Oxidative Stress ◽

Helicobacter Pylori ◽

Membrane Integrity ◽

Type I ◽

Morphological Transformation ◽

Bacterial Chromosomes ◽

Concentration Result ◽

H Pylori ◽

Plasmid Stabilization

SummaryToxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which in type I systems is an antisense-RNA. While the regulatory mechanisms of these systems are mostly well-defined, the toxins’ biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori. We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by Cryo-EM. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or ATP concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression.Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment.Significance StatementHelicobacter pylori, a gastric pathogen causing 800,000 deaths in the world annually, is encountered both in vitro and in patients as spiral-shaped bacteria and as round cells named coccoids. We discovered that the toxin from a chromosomal type I toxin-antitoxin system is targeting H. pylori membrane and acting as an effector of H. pylori morphological conversion to coccoids. We showed that these round cells maintain their membrane integrity and metabolism, strongly suggesting that they are viable dormant bacteria. Oxidative stress was identified as a signal inducing toxin expression and coccoid formation. Our findings reveal new insights into a form of dormancy of this bacterium that might be associated with H. pylori infections refractory to treatment.

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Biological characteristics and virulence of Helicobacter pylori

Clinical Microbiology and Antimicrobial Chemotherapy ◽

10.36488/cmac.2018.1.14-23 ◽

2018 ◽

Vol 20 (1) ◽

pp. 14-23 ◽

Cited By ~ 2

Author(s):

G.Sh. Isaeva ◽

R.I. Valieva

Keyword(s):

Helicobacter Pylori ◽

Clinical Outcomes ◽

Genetic Heterogeneity ◽

Biofilm Formation ◽

Clinical Manifestations ◽

Epidemiological Studies ◽

Biological Characteristics ◽

Pathogenicity Factors ◽

H Pylori

This review summarizes the most recent data on the biological characteristics of Helicobacter pylori (morphological, cultural, biochemical). H. pylori pathogenicity factors promoting colonization, adhesion, biofilm formation, aggression, and cytotoxicity, their contribution to the pathogenesis of diseases as well as the possible relationships with various clinical outcomes are described in detail. The genetic heterogeneity of H. pylori strains which can determine different clinical manifestations and have significance for conducting epidemiological studies is also considered.

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Genetic requirements and transcriptomics of Helicobacter pylori biofilm formation on abiotic and biotic surfaces

npj Biofilms and Microbiomes ◽

10.1038/s41522-020-00167-3 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Skander Hathroubi ◽

Shuai Hu ◽

Karen M. Ottemann

Keyword(s):

Helicobacter Pylori ◽

Biofilm Formation ◽

Tca Cycle ◽

Harsh Environments ◽

Laboratory Model ◽

Secretion Systems ◽

Biofilm Growth ◽

Tca Cycle Enzymes ◽

Growth Properties ◽

H Pylori

AbstractBiofilm growth is a widespread mechanism that protects bacteria against harsh environments, antimicrobials, and immune responses. These types of conditions challenge chronic colonizers such as Helicobacter pylori but it is not fully understood how H. pylori biofilm growth is defined and its impact on H. pylori survival. To provide insights into H. pylori biofilm growth properties, we characterized biofilm formation on abiotic and biotic surfaces, identified genes required for biofilm formation, and defined the biofilm-associated gene expression of the laboratory model H. pylori strain G27. We report that H. pylori G27 forms biofilms with a high biomass and complex flagella-filled 3D structures on both plastic and gastric epithelial cells. Using a screen for biofilm-defective mutants and transcriptomics, we discovered that biofilm cells demonstrated lower transcripts for TCA cycle enzymes but higher ones for flagellar formation, two type four secretion systems, hydrogenase, and acetone metabolism. We confirmed that biofilm formation requires flagella, hydrogenase, and acetone metabolism on both abiotic and biotic surfaces. Altogether, these data suggest that H. pylori is capable of adjusting its phenotype when grown as biofilm, changing its metabolism, and re-shaping flagella, typically locomotion organelles, into adhesive structures.

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Helicobacter pylori Biofilm and New Strategies to Combat it

Current Molecular Medicine ◽

10.2174/1566524020666201203165649 ◽

2020 ◽

Vol 20 ◽

Author(s):

Majid Taati Moghadam ◽

Zahra Chegini ◽

Amin Khoshbayan ◽

Iman Farahani ◽

Aref Shariati

Keyword(s):

Helicobacter Pylori ◽

Biofilm Formation ◽

Extracellular Polymeric Substances ◽

Combination Therapies ◽

Chronic Infections ◽

Chemical Substances ◽

And Performance ◽

H Pylori ◽

Initial Binding ◽

New Strategies

: Helicobacter pylori, the most frequent pathogens worldwide that colonize around 50% of the world’s population, cause important diseases such as gastric adenocarcinoma, chronic gastritis, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. In recent years, various studies have reported that H. pylori biofilm may be one of the critical barriers to the eradication of this bacterial infection. Biofilms inhibit the penetration of antibiotics, increase the expression of efflux pumps and mutations, multiple therapeutic failures, and chronic infections. Nanoparticles and natural products can demolish H. pylori biofilm by destroying the outer layers and inhibiting the initial binding of bacteria. Also, the use of combination therapies destroying extracellular polymeric substances decreases coccoid forms of bacteria and degrading polysaccharides in the outer matrix that lead to an increase in the permeability and performance of antibiotics. Different probiotics, antimicrobial peptides, chemical substances, and polysaccharides by inhibiting adhesion and colonization of H. pylori can prevent biofilm formation by this bacterium. Of note, many of the above are applicable to acidic pH and can be used to treat gastritis. Therefore, H. pylori biofilm may be one of the major causes of failure to the eradication of infections caused by this bacterium, and antibiotics are not capable of destroying the biofilm. Thus, it is necessary to use new strategies to prevent recurrent and chronic infections by inhibiting biofilm formation.

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A Helicobacter pylori TolC Efflux Pump Confers Resistance to Metronidazole

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.4.1477-1482.2005 ◽

2005 ◽

Vol 49 (4) ◽

pp. 1477-1482 ◽

Cited By ~ 56

Author(s):

Karin van Amsterdam ◽

Aldert Bart ◽

Arie van der Ende

Keyword(s):

Helicobacter Pylori ◽

Efflux Pump ◽

Sodium Deoxycholate ◽

Loss Of Function ◽

Knockout Mutant ◽

Double Knockout ◽

Active Efflux ◽

Knockout Mutants ◽

H Pylori ◽

Susceptibility Profiles

ABSTRACT In Helicobacter pylori, the contribution of efflux proteins to antibiotic resistance is not well established. As translocases that act in parallel may have overlapping substrate specificities, the loss of function of one such translocase may be compensated for by that of another translocase with no effect on susceptibilities to antibiotics. The genome of H. pylori 26695 was assessed for the presence of putative translocases and outer membrane efflux or TolC-like proteins which could interact to form efflux systems involved in drug resistance. Twenty-seven translocases were identified, of which HP1184 was the sole representative of the multidrug and toxic compound extrusion family of translocases and which could thus have a unique substrate specificity. In addition, four TolC-like proteins (HP0605, HP0971, HP1327, and HP1489) were identified. Thus, it is feasible that inactivation of a TolC-like protein would affect the functions of multiple translocases. We aimed to determine whether efflux systems contribute to antimicrobial susceptibility by evaluation of the susceptibility profiles of an HP1184-knockout mutant, four mutants in which one of the four TolC homologs was inactivated, as well as a mutant in which both HP0605 and HP0971 were inactivated. The HP1184- and HP1489-knockout mutants both showed increased susceptibilities to ethidium bromide, while the HP0605-knockout mutant exhibited increased susceptibilities to novobiocin and sodium deoxycholate. The HP0605 and HP0971 double-knockout mutant was also more susceptible to metronidazole, in addition to being susceptible to novobiocin and sodium deoxycholate. Thus, active efflux is an eminent means of resistance to antimicrobials in H. pylori and resembles the situation in other bacteria.

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

Journal of Bacteriology ◽

10.1128/jb.00324-16 ◽

2016 ◽

Vol 198 (18) ◽

pp. 2536-2548 ◽

Cited By ~ 18

Author(s):

Stephanie L. Servetas ◽

Beth M. Carpenter ◽

Kathryn P. Haley ◽

Jeremy J. Gilbreath ◽

Jennifer A. Gaddy ◽

...

Keyword(s):

Helicobacter Pylori ◽

Biofilm Formation ◽

Regulatory Proteins ◽

Content Type ◽

Isogenic Mutant ◽

Two Component ◽

Mutant Strains ◽

Regulatory Mutations ◽

H Pylori

ABSTRACTHelicobacter pylorimust be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation,H. pyloriencodes 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, ΔnikRΔarsS, and ΔfurΔnikRΔarsSmutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus,H. pyloriArsRS has a previously unrecognized role in biofilm formation.IMPORTANCEDespite a paucity of regulatory proteins, adaptation is key to the survival ofH. pyloriwithin 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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