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

In Silico Screening and In Vitro Assessment of Natural Products with Anti-Virulence Activity against Helicobacter pylori.

Helicobacter pylori is one of the most frequent human pathogens and a leading etiological agent of various gastric diseases. As stringent response, coordinated by a SpoT protein, seems to be crucial for the survivability of H. pylori, the main goal of this article was to use in silico computational studies to find phytochemical compounds capable of binding to the active site of SpoT from H. pylori and confirm the ability of the most active candidates to interfere with the virulence of this bacterium through in vitro experiments. From 791 natural substances submitted for the virtual screening procedure, 10 were chosen and followed for further in vitro examinations. Among these, dioscin showed the most interesting parameters (the lowest MIC, the highest anti-biofilm activity in static conditions, and a relatively low stimulation of morphological transition into coccoids). Therefore, in the last part, we extended the research with a number of further experiments and observed the ability of dioscin to significantly reduce the formation of H. pylori biofilm under Bioflux-generated flow conditions and its capacity for additive enhancement of the antibacterial activity of all three commonly used antibiotics (clarithromycin, metronidazole, and levofloxacin). Based on these results, we suggest that dioscin may be an interesting candidate for new therapies targeting H. pylori survivability and virulence.

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.

Transformation of Helicobacter pylori into Coccoid Forms as a Challenge for Research Determining Activity of Antimicrobial Substances.

Morphological variability is one of the phenotypic features related to adaptation of microorganisms to stressful environmental conditions and increased tolerance to antimicrobial substances. Helicobacter pylori, a gastric mucosal pathogen, is characterized by a high heterogeneity and an ability to transform from a spiral to a coccoid form. The presence of the coccoid form is associated with the capacity to avoid immune system detection and to promote therapeutic failures. For this reason, it seems that the investigation for new, alternative methods combating H. pylori should include research of coccoid forms of this pathogen. The current review aimed at collecting information about the activity of antibacterial substances against H. pylori in the context of the morphological variability of this bacterium. The collected data was discussed in terms of the type of substances used, applied research techniques, and interpretation of results. The review was extended by a polemic on the limitations in determining the viability of coccoid H. pylori forms. Finally, recommendations which can help in future research aiming to find new compounds with a potential to eradicate H. pylori have been formulated.

In Vitro Activity of Sertraline, an Antidepressant, Against Antibiotic-Susceptible and Antibiotic-Resistant Helicobacter pylori Strains.

Antibiotic resistance of Helicobacter pylori, a spiral bacterium associated with gastric diseases, is a topic that has been intensively discussed in last decades. Recent discoveries indicate promising antimicrobial and antibiotic-potentiating properties of sertraline (SER), an antidepressant substance. The aim of the study, therefore, was to determine the antibacterial activity of SER in relation to antibiotic-sensitive and antibiotic-resistant H. pylori strains. The antimicrobial tests were performed using a diffusion-disk method, microdilution method, and time-killing assay. The interaction between SER and antibiotics (amoxicillin, clarithromycin, tetracycline, and metronidazole) was determined by using a checkerboard method. In addition, the study was expanded to include observations by light, fluorescence, and scanning electron microscopy. The growth inhibition zones were in the range of 19-37 mm for discs impregnated with 2 mg of SER. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) counted for 2-8 µg/mL and 4-8 µg/mL, respectively. The time-killing assay showed the time-dependent and concentration-dependent bactericidal activity of SER. Bacteria exposed to MBCs (but not sub-MICs and MICs ≠ MBCs) underwent morphological transformation into coccoid forms. This mechanism, however, was not protective because these cells after a 24-h incubation had a several-fold reduced green/red fluorescence ratio compared to the control. Using the checkerboard assay, a synergistic/additive interaction of SER with all four antibiotics tested was demonstrated. These results indicate that SER may be a promising anti-H. pylori compound.

In Vitro Activity of 3-Bromopyruvate, an Anticancer Compound, Against Antibiotic-Susceptible and Antibiotic-Resistant Helicobacter pylori Strains.

Helicobacter pylori (H. pylori) is a bacterium capable of inducing chronic active gastritis, which in some people, develops into gastric cancers. One of the substances that may be useful in the eradication of this microorganism is 3-Bromopyruvate (3-BP), an anticancer compound with antimicrobial properties. The aim of this article was to determine the activity of 3-BP against antibiotic-susceptible and antibiotic-resistant H. pylori strains. The antimicrobial activity was determined using a disk-diffusion method, broth microdilution method, time-killing assay, and checkerboard assay. The research was extended by observations using light, fluorescence, and scanning electron microscopy. The growth inhibition zones produced by 2 mg/disk with 3-BP counted for 16⁻32.5 mm. The minimal inhibitory concentrations (MICs) ranged from 32 to 128 µg/mL, while the minimal bactericidal concentrations (MBCs) for all tested strains had values of 128 µg/mL. The time-killing assay demonstrated the concentration-dependent and time-dependent bactericidal activity of 3-BP. The decrease in culturability below the detection threshold (<100 CFU/mL) was demonstrated after 6 h, 4 h, and 2 h of incubation for MIC, 2× MIC, and 4× MIC, respectively. Bacteria treated with 3-BP had a several times reduced mean green/red fluorescence ratio compared to the control samples, suggesting bactericidal activity, which was independent from an induction of coccoid forms. The checkerboard assay showed the existence of a synergistic/additive interaction of 3-BP with amoxicillin, tetracycline, and clarithromycin. Based on the presented results, it is suggested that 3-BP may be an interesting anti-H. pylori compound.

[Role of Helicobacter pylori coccoid forms in infection and recrudescence]. / Rol de las formas cocoides de Helicobacter pylori en la infección y la recrudescencia.

Helicobacter pylori is a spiral Gram-negative bacillus, which colonizes the human stomach and plays a key role in the pathogenesis of a number of gastroduodenal diseases. However, when expose to environmental stressed conditions, such as increased oxygen tension, extended incubation and exposure to antibiotics, Helicobacter pylori is able to entering the viable but nonculturable state, in which the bacterium modifies its morphology from a spiral to coccoid form, as a manifestation of cell adaptation to these adverse conditions. In gastric tissues, viable coccoid forms may remain latent for long time and retain virulence factors, so these forms possibly contribute to the treatment failures and recurrence of Helicobacter pylori infection and gastroduodenal diseases as well. In this review, we will discuss several aspects of cellular adaptation and survival of Helicobacter pylori, antibiotic susceptibility and virulence of coccoid forms and its involvement with recrudescence.

Biofilms and Helicobacter pylori: Dissemination and persistence within the environment and host.

The presence of viable Helicobacter pylori (H. pylori) in the environment is considered to contribute to the levels of H. pylori found in the human population, which also aids to increase its genetic variability and its environmental adaptability and persistence. H. pylori form biofilms both within the in vitro and in vivo environment. This represents an important attribute that assists the survival of this bacterium within environments that are both hostile and adverse to proliferation. It is the aim of this paper to review the ability of H. pylori to form biofilms in vivo and in vitro and to address the inherent mechanisms considered to significantly enhance its persistence within the host and in external environments. Furthermore, the dissemination of H. pylori in the external environment and within the human body and its impact upon infection control will be discussed.