Role of <i>psl</i> Genes in Antibiotic Tolerance of Adherent Pseudomonas aeruginosa

<jats:title>ABSTRACT</jats:title> <jats:p> Bacteria attached to a surface are generally more tolerant to antibiotics than their planktonic counterparts, even without the formation of a biofilm. The mechanism of antibiotic tolerance in biofilm communities is multifactorial, and the genetic background underlying this antibiotic tolerance has not yet been fully elucidated. Using transposon mutagenesis, we isolated a mutant with reduced tolerance to biapenem (relative to that of the wild type) from adherent cells. Sequencing analysis revealed a mutation in the <jats:italic>pslL</jats:italic> gene, which is part of the polysaccharide biosynthesis operon. The <jats:named-content content-type="genus-species">Pseudomonas aeruginosa</jats:named-content> PAO1Δ <jats:italic>pslBCD</jats:italic> mutant demonstrated a 100-fold-lower survival rate during the exposure of planktonic and biofilm cells to biapenem; a similar phenotype was observed in a mouse infection model and in clinical strains. Transcriptional analysis of adherent cells revealed increased expression of both <jats:italic>pslA</jats:italic> and <jats:italic>pelA</jats:italic> , which are directly regulated by bis-(3′,5′)-cyclic dimeric GMP (c-di-GMP). Inactivation of <jats:italic>wspF</jats:italic> resulted in significantly increased tolerance to biapenem due to increased production of c-di-GMP. The loss of <jats:italic>pslBCD</jats:italic> in the Δ <jats:italic>wspF</jats:italic> mutant background abolished the biapenem-tolerant phenotype of the Δ <jats:italic>wspF</jats:italic> mutant, underscoring the importance of <jats:italic>psl</jats:italic> in biapenem tolerance. Overexpression of PA2133, which can catalyze the degradation of c-di-GMP, led to a significant reduction in biapenem tolerance in adherent cells, indicating that c-di-GMP is essential in mediating the tolerance effect. The effect of <jats:italic>pslBCD</jats:italic> on antibiotic tolerance was evident, with 50- and 200-fold-lower survival in the presence of ofloxacin and tobramycin, respectively. We speculate that the <jats:italic>psl</jats:italic> genes, which are activated by surface adherence through elevated intracellular c-di-GMP levels, confer tolerance to antimicrobials. </jats:p>