Abstract
Persisters are the fraction of antibiotic-exposed bacteria transiently refractory to killing and are recognized as a cause of antibiotic treatment failure. The putative de-N-acetylase DnpA increases persister levels in Pseudomonas aeruginosa upon exposure to fluoroquinolones in broth. In this study the wild-type PAO1 and its dnpA insertion mutant (dnpA::Tn) were used in parallel and compared for their capacity to generate persisters in broth (surviving fraction after exposure to high antibiotic concentrations) and their susceptibility to antibiotics in models of intracellular infection of THP-1 monocytes and of biofilms grown in microtiter plates. Multiplication in monocytes was evaluated by fluorescence dilution of GFP (expressed under the control of an inducible promoter) using flow cytometry. Gene expression was measured by quantitative RT-PCR. When exposed to fluoroquinolones (ciprofloxacin or levofloxacin) but not to meropenem or amikacin, the dnpA::Tn mutant showed a 3- to 10-fold lower persister fraction in broth. In infected monocytes, fluoroquinolones (but not the other antibiotics) were more effective (difference in Emax: 1.5 log cfu) against the dnpA::Tn mutant than against the wild-type PAO1. Dividing intracellular bacteria were more frequently seen (1.5 to 1.9-fold) with the fluoroquinolone-exposed dnpA::Tn mutant than with its parental strain. Fluoroquinolones (but not the other antibiotics) were also 3-fold more potent to prevent biofilm formation by the dnpA::Tn mutant than by PAO1 as well as to act upon biofilms (1–3 days of maturity) formed by the mutant than by the parental strain. Fluoroquinolones induced the expression of gyrA (4.5–7 fold) and mexX (3.6–5.4 fold) in the parental strain but to a lower extent (3–4-fold for gyrA and 1.8–2.8-fold for mexX, respectively) in the dnpA::Tn mutant. Thus, our data show that a dnpA insertion mutant of P. aeruginosa is more receptive to fluoroquinolone antibacterial effects than its parental strain in infected monocytes or in biofilms. The mechanism of this higher responsiveness could involve a reduced overexpression of the fluoroquinolone target.
Highlights
Besides plain resistance most often associated with genomic changes, persistence is increasingly recognized as a cause of therapeutic failures
Previous work has shown that the inactivation of dnpA::Tn insertion mutant and PAO1 (dnpA) significantly lowers the persister fraction in PAO1 when exposed to low concentrations of ofloxacin, a fluoroquinolone antibiotic (De Groote et al, 2009)
We first compared the activity of both fluoroquinolones and of meropenem and amikacin against the dnpA::Tn and PAO1 strains in infected human THP-1 monocytes after 24 h of incubation and using a wide range of extracellular concentrations to obtain full concentration-response curves
Summary
Besides plain resistance most often associated with genomic changes, persistence is increasingly recognized as a cause of therapeutic failures. Persisters represent the fraction of antibiotic-treated bacteria that are transiently refractory to antibiotic killing. This phenotype is not genetically inherited and is reversible upon antibiotic removal (Van den Bergh et al, 2017). Few reports have investigated the mechanism(s) of persister formation in Pseudomonas aeruginosa. These have highlighted a role for the quorum-sensing determinants lasI and lasR (Kayama et al, 2009; Moker et al, 2010), the alternative sigma factor RpoN (Viducic et al, 2007), and the stationary-phase regulators RpoS, DskA, RelA, and SpoT (Murakami et al, 2005; Viducic et al, 2006). Preliminary data suggest that LPS synthesis is unaffected in the deletion mutant
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