Helicobacter pylori biofilm cells are metabolically distinct, express flagella, and antibiotic tolerant

Abstract

Biofilm growth protects bacteria against harsh environments, antimicrobials, and immune responses. Helicobacter pylori is a bacterium that has a robust ability to maintain colonization in a challenging environment. Over the last decade, H. pylori biofilm formation has begun to be characterized, however, there are still gaps in our understanding about how this growth mode is defined and its impact on H. pylori physiology. To provide insights into H. pylori biofilm growth properties, we characterized the antibiotic susceptibility, gene expression, and genes required for biofilm formation of a strong biofilm-producing H. pylori. H. pylori biofilms developed complex 3D structures and were recalcitrant to multiple antibiotics. Disruption of the protein-based matrix decreased this antibiotic tolerance. Using both transcriptomic and genomic approaches, we discovered that biofilm cells demonstrated lower transcripts for TCA cycle enzymes but higher ones for hydrogenase and acetone metabolism. Interestingly, several genes encoding for the natural competence Type IV secretion system 4 (tfs4) were up-regulated during biofilm formation along with several genes encoding for restriction-modification (R-M) systems, suggesting DNA exchange activities in this mode of growth. Flagella genes were also discovered through both approaches, consistent with previous reports about the importance of these filaments in H. pylori biofilm. Together, these data suggest that H. pylori is capable of adjusting its phenotype when grown as biofilm, changing its metabolism and elevating specific surface proteins including those encoding tfs4 and flagella.

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

“…The first study showing the ability of H. pylori to form biofilm took place in 1999 [ 20 ]. Since then, the knowledge on this subject has expanded significantly, and the presence of H. pylori biofilms has been demonstrated in numerous in vitro studies using plastic or glass surfaces [ 21 , 22 , 23 , 24 , 25 ], cell cultures [ 26 , 27 ], gastric biopsies from animals [ 28 ] or humans [ 29 , 30 ], and even vegetable surfaces [ 31 ] or water distribution tanks [ 32 , 33 , 34 ]. It is worth noting that initially H. pylori biofilms were strongly associated by scientists with water distribution systems and were considered as a survival mechanism outside the host and a source of fecal-oral infections [ 35 ].…”

“…It is worth noting that initially H. pylori biofilms were strongly associated by scientists with water distribution systems and were considered as a survival mechanism outside the host and a source of fecal-oral infections [ 35 ]. 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 [ 22 , 23 , 36 , 37 , 38 , 39 ].…”

“…Under laboratory conditions, H. pylori biofilm may be formed during both liquid or solid cultures. In the first case, there are several types of biofilms that can develop under such conditions, i.e., a biofilm formed on the liquid-air surface (most often as a macroscopically visible ring, possibly accompanied by a pellicle development) [ 22 , 40 , 41 , 42 ], a biofilm formed as sediment of cells adhered to a solid surface (cells are completely immersed in the medium) [ 21 , 22 , 41 ], and a biofilm present as multicellular aggregates freely drifting in the liquid phase (cells are immersed in the medium but do not sink to the bottom) [ 22 , 41 , 43 ]. In the case of culture on a solid substratum, the so called “colony biofilm” [ 38 , 39 ] or “lawn biofilm” [ 44 ] are most often indicated.…”

“…Environmental and cultivation conditions constitute parameters that may affect the process of H. pylori biofilm formation in the laboratory [ 21 , 22 , 24 , 45 , 46 ]. Adhesion of H. pylori to the surface, a first stage conditioning the biofilm formation, largely depends on the flow rate (inversely associated with the adhesion) and the surface charge (the negative surface charge is preferred), while the ambient temperature does not significantly affect the adhesion [ 24 , 45 ].…”

“…The pH of the environment is a factor that modulates the subsequent processes of biofilm formation. It was found that with decreasing pH, the ability of H. pylori to produce this structure declined, especially at very low values [ 22 ]. Exposure of the preformed biofilm to a slightly acidified environment (pH = 5.5) had no effect on the H. pylori biofilm, but still, a highly acidified medium (pH = 3) eliminated this structure.…”