Presentation Information
[P03-414]Microscopic Analysis of Antibiotic Resistance Mechanisms in Pseudomonas aeruginosa Biofilms Mediated by Hydrogen Sulfide
○Kana Higuchi1, Nobuhiko Nomura2,3,4,5, Masanori Toyofuku2,3,4, Yoshihide Tokunou2,6 (1. Graduate School of Science and Technology, Tsukuba Univ., Tsukuba, (Japan), 2. Institute of Life and Environmental Sciences, Tsukuba Univ., Tsukuba, (Japan), 3. Microbiology Research Center for Sustainability (MiCS), Tsukuba Univ., Tsukuba, (Japan), 4. Tsukuba Institute for Advanced Research (TIAR), Tsukuba Univ., Tsukuba, (Japan), 5. Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA) , Tsukuba Univ. Tsukuba, (Japan), 6. Research Center for Macromolecules and Biomaterials, National Institute for Material Science (Japan))
Keywords:
Biofilm,Pseudomonas aeruginosa,Antibiotic resistance,Hydrogen sulfide
[Purpose]
Pseudomonas aeruginosa forms aggregates at infection sites, creating biofilms that contribute to treatment refractoriness. Hydrogen sulfide (H2S) has recently been identified as a signaling molecule involved in bacterial antibiotic resistance and defense mechanisms. Since then, the physiological role of H2S in bacteria has been widely investigated; however, its effects remain controversial, with some reports suggesting that H2S does not alter antibiotic resistance. In addition, the mechanisms underlying antibiotic resistance in biofilms have not been fully elucidated. Consequently, the clinical importance of H2S has rarely been discussed. Because H2S is a gaseous molecule with extremely high cellular permeability, we hypothesized that its effects may be particularly significant in biofilms formed in stagnant environments at infection sites. The aim of this study was to clarify the relationship between H2S and antibiotic resistance in P. aeruginosa biofilms.
[Method]
A Δcse (cystathionine γ-lyase) mutant strain with reduced H2S production was constructed. Antibiotic susceptibility was evaluated in both planktonic wild-type (WT) and Δcse strains. To examine antibiotic resistance and the spatial distribution of dead cells in biofilms, microscopic observations were performed using propidium iodide (PI), a dead-cell stain. Colony-forming unit (CFU) counts were conducted to quantify viable bacteria, and Live/Dead staining with Syto9 and PI was used to determine the proportion of dead cells.
[Results]
No difference in antibiotic susceptibility was observed However, microscopic analysis of biofilms showed stronger PI fluorescence in the Δcse strain, suggesting a higher proportion of dead cells, and a possible reduction in H2S-dependent antibiotic resistance within biofilms. In contrast, CFU counts after antibiotic exposure revealed no significant difference in the number of viable cells between the two strains. Live/Dead staining of biofilms indicated a slightly higher proportion of dead cells in the Δcse strain than in the WT (p = 0.079).
[Consideration]
Deletion of cse appeared to reduce antibiotic resistance in biofilms based on microscopic observations; however, viable cell counts showed no significant difference. This discrepancy suggests that reduced H2S production may not directly decrease antibiotic resistance but instead affect PI staining, potentially reflecting altered membrane stability.
[Conclusion]
Using microscopy techniques established in our laboratory, we achieved non-destructive time-lapse imaging of biofilms. Biofilm-based observation enabled detection of phenomena that could not be fully captured by conventional in vitro assays. Elucidating the mechanisms by which H2S influences antibiotic resistance in biofilms may contribute to the development of new therapeutic strategies for infectious diseases.
Pseudomonas aeruginosa forms aggregates at infection sites, creating biofilms that contribute to treatment refractoriness. Hydrogen sulfide (H2S) has recently been identified as a signaling molecule involved in bacterial antibiotic resistance and defense mechanisms. Since then, the physiological role of H2S in bacteria has been widely investigated; however, its effects remain controversial, with some reports suggesting that H2S does not alter antibiotic resistance. In addition, the mechanisms underlying antibiotic resistance in biofilms have not been fully elucidated. Consequently, the clinical importance of H2S has rarely been discussed. Because H2S is a gaseous molecule with extremely high cellular permeability, we hypothesized that its effects may be particularly significant in biofilms formed in stagnant environments at infection sites. The aim of this study was to clarify the relationship between H2S and antibiotic resistance in P. aeruginosa biofilms.
[Method]
A Δcse (cystathionine γ-lyase) mutant strain with reduced H2S production was constructed. Antibiotic susceptibility was evaluated in both planktonic wild-type (WT) and Δcse strains. To examine antibiotic resistance and the spatial distribution of dead cells in biofilms, microscopic observations were performed using propidium iodide (PI), a dead-cell stain. Colony-forming unit (CFU) counts were conducted to quantify viable bacteria, and Live/Dead staining with Syto9 and PI was used to determine the proportion of dead cells.
[Results]
No difference in antibiotic susceptibility was observed However, microscopic analysis of biofilms showed stronger PI fluorescence in the Δcse strain, suggesting a higher proportion of dead cells, and a possible reduction in H2S-dependent antibiotic resistance within biofilms. In contrast, CFU counts after antibiotic exposure revealed no significant difference in the number of viable cells between the two strains. Live/Dead staining of biofilms indicated a slightly higher proportion of dead cells in the Δcse strain than in the WT (p = 0.079).
[Consideration]
Deletion of cse appeared to reduce antibiotic resistance in biofilms based on microscopic observations; however, viable cell counts showed no significant difference. This discrepancy suggests that reduced H2S production may not directly decrease antibiotic resistance but instead affect PI staining, potentially reflecting altered membrane stability.
[Conclusion]
Using microscopy techniques established in our laboratory, we achieved non-destructive time-lapse imaging of biofilms. Biofilm-based observation enabled detection of phenomena that could not be fully captured by conventional in vitro assays. Elucidating the mechanisms by which H2S influences antibiotic resistance in biofilms may contribute to the development of new therapeutic strategies for infectious diseases.
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