1. Front Microbiol. 2018 Jul 10;9:1455. doi: 10.3389/fmicb.2018.01455. eCollection

The Putative De-N-acetylase DnpA Contributes to Intracellular and
Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to

Khandekar S(1), Liebens V(2), Fauvart M(2)(3)(4), Tulkens PM(1), Michiels
J(2)(3), Van Bambeke F(1).

Author information: 
(1)Pharmacologie Cellulaire et Moléculaire, Louvain Drug Research Institute,
Université catholique de Louvain, Brussels, Belgium.
(2)Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.
(3)Center for Microbiology, Vlaams Instituut voor Biotechnologie, Leuven,
(4)imec, Leuven, Belgium.

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.

DOI: 10.3389/fmicb.2018.01455 
PMCID: PMC6048251
PMID: 30042739