Biofilms, communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances, pose a substantial health risk and are key contributors to many chronic and recurrent infections. Chronicity and recalcitrant infections are also common features associated with the ulcer-causing human pathogen H. pylori. However, relatively little is known about the role of biofilms in H. pylori pathogenesis, as well as the biofilm structure itself and the genes associated with this mode of growth. In the present study, we found that H. pylori biofilm cells highly expressed genes related to cell envelope and stress response, as well as those encoding the flagellar apparatus. Flagellar filaments were seen in high abundance in the biofilm. Flagella are known to play a role in initial biofilm formation, but typically are downregulated after that state. H. pylori instead appears to have coopted these structures for nonmotility roles, including a role building a robust biofilm.

Helicobacter pylori, a WHO class I carcinogen, is one of the most successful human pathogens colonizing the stomach of over 4.4 billion of the world’s population. Antibiotic therapy represents the best solution but poor response rates have hampered the elimination of H. pylori. A growing body of evidence suggests that H. pylori forms biofilms, but the role of this growth mode in infection remains elusive. Here, we demonstrate that H. pylori cells within a biofilm are tolerant to multiple antibiotics in a manner that depends partially on extracellular proteins. Biofilm-forming cells were tolerant to multiple antibiotics that target distinct pathways, including amoxicillin, clarithromycin, and tetracycline. Furthermore, this tolerance was significantly dampened following proteinase K treatment. These data suggest that H. pylori adapts its phenotype during biofilm growth resulting in decreased antibiotic susceptibility but this tolerance can be partially ameliorated by extracellular protease treatment.

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.

Helicobacter pylori has an impressive ability to persist chronically in the human stomach. Similar characteristics are associated with biofilm formation in other bacteria. The H. pylori biofilm process, however, is poorly understood. To gain insight into this mode of growth, we carried out comparative transcriptomic analysis between H. pylori biofilm and planktonic cells, using the mouse colonizing strain SS1. Optimal biofilm formation was obtained with low serum and three-day growth, conditions which caused both biofilm and planktonic cells to be ∼80% coccoid. RNA-seq analysis found that 8.18% of genes were differentially expressed between biofilm and planktonic cell transcriptomes. Biofilm-downregulated genes included those involved in metabolism and translation, suggesting these cells have low metabolic activity. Biofilm-upregulated genes included those whose products were predicted to be at the cell envelope, involved in regulating a stress response, and surprisingly, genes related to formation of the flagellar apparatus. Scanning electron microscopy visualized flagella that appeared to be a component of the biofilm matrix, supported by the observation that an aflagellated mutant displayed a less robust biofilm with no apparent filaments. We observed flagella in the biofilm matrix of additional H. pylori strains, supporting that flagellar use is widespread. Our data thus supports a model in which H. pylori biofilm involves a multi-gene stress-biased response, and that flagella play an important role in H. pylori biofilm formation.IMPORTANCEBiofilms, communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances, pose a substantial health risk and are key contributors to many chronic and recurrent infections. Chronicity and recalcitrant infections are also common features associated with the ulcer-causing human pathogen H. pylori. However, relatively little is known about the role of biofilms in H. pylori pathogenesis as well as the biofilm structure itself and the genes associated with this mode of growth. In the present study, we found that H. pylori biofilm cells highly expressed genes related to cell envelope, stress response and those encoding the flagellar apparatus. Flagellar filaments were seen in high abundance in the biofilm. Flagella are known to play a role in initial biofilm formation, but typically are downregulated after that state. H. pylori instead appears to have co-opted these structures for non-motility roles, including a role building a robust biofilm.