Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa

ORIGINAL RESEARCH published: 11 March 2019 doi: 10.3389/fcimb.2019.00049 Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa Francesco Imperi 1,2*, Ersilia V. Fiscarelli 3 , Daniela Visaggio 1 , Livia Leoni 1 and Paolo Visca 1 1 Department of Science, Roma Tre University, Rome, Italy, 2 Laboratory affiliated to Istituto Pasteur Italia–Fondazione Cenci Bolognetti, Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy, 3 Laboratory of Cystic Fibrosis Microbiology, Bambino Gesú Hospital, Rome, Italy Edited by: Natalia V. Kirienko, Rice University, United States Reviewed by: Carlos Juan, Instituto de Investigación Sanitaria de Palma (IdISPa), Spain Pierre Cornelis, Vrije University Brussel, Belgium Michael L. Vasil, University of Colorado School of Medicine, United States *Correspondence: Francesco Imperi Specialty section: This article was submitted to Clinical Microbiology, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 10 December 2018 Accepted: 15 February 2019 Published: 11 March 2019 Citation: Imperi F, Fiscarelli EV, Visaggio D, Leoni L and Visca P (2019) Activity and Impact on Resistance Development of Two Antivirulence Fluoropyrimidine Drugs in Pseudomonas aeruginosa. Front. Cell. Infect. Microbiol. 9:49. doi: 10.3389/fcimb.2019.00049 The rise in antibiotic resistance among bacterial pathogens has prompted the exploitation of alternative antibacterial strategies, such as antivirulence therapy. By inhibiting virulence traits, antivirulence drugs are expected to lessen pathogenicity without affecting bacterial growth, therefore avoiding the spread of resistance. However, some studies argued against this assumption, and the lack of antivirulence drugs in clinical use hampers the empirical assessment of this concept. Here we compared the mode of action and range of activity of two drugs which have been proposed for repurposing as quorum sensing and pyoverdine inhibitors in the human pathogen Pseudomonas aeruginosa: the anticancer drug 5-fluorouracil (5-FU) and the antimycotic drug 5-fluorocytosine (5-FC), respectively. The effect on bacterial growth, emergence and spread of resistance, and activity against clinical isolates were assessed. Our results confirm that 5-FU has growth inhibitory activity on reference strains and can rapidly select for spontaneous resistant mutants with loss-of-function mutations in the upp gene, responsible for uracil conversion into UMP. These mutants were also insensitive to the anti-pyoverdine effect of 5-FC. Conversely, 5-FC did not cause relevant growth inhibition, likely because of poor enzymatic conversion into 5-FU by cytosine deaminase. However, coculturing experiments showed that 5-FU resistant mutants can outcompete sensitive cells in mixed populations, in the presence of not only 5-FU but also 5-FC. Moreover, we observed that serial passages of wild-type cells in 5-FC-containing medium leads to the appearance and spread of 5-FC insensitive sub-populations of 5-FU resistant cells. The different effect on growth of 5-FU and 5-FC was overall conserved in a large collection of cystic fibrosis (CF) isolates, corresponding to different infection stages and antibiotic resistance profiles, although high variability was observed among strains. Notably, this analysis also revealed a significant number of pyoverdine-deficient isolates, whose proportion apparently increases over the course of the CF infection. This study demonstrates that the efficacy of an antivirulence drug with no apparent effect on growth can be significantly influenced by the emergence of insensitive mutants, and highlights the importance of the assessment of resistance-associated fitness cost and activity on clinical isolates for the development of “resistance-proof” antivirulence drugs. Keywords: acquired resistance, antimetabolite, antivirulence drug, cystic fibrosis, Pseudomonas aeruginosa, siderophore, virulence Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 March 2019 | Volume 9 | Article 49 Imperi et al. Fluorinated Pyrimidines Against Pseudomonas aeruginosa INTRODUCTION biofilm formation (Ueda et al., 2009). The antivirulence potential of fluorinated pyrimidines was later expanded by a drug repurposing screening campaign, which identified 5-FC as a potent inhibitor of pyoverdine siderophore production, and showed that this antimycotic drug can also suppress P. aeruginosa lethality in a mouse model of acute lung infection (Imperi et al., 2013), in line with the crucial role of pyoverdine-mediated iron uptake and virulence in this infection model (Minandri et al., 2016). The anti-P. aeruginosa efficacy of fluorinated pyrimidines was also supported by an in vivo screening in the Caenorhabditis elegans infection model, that revealed antipathogenic and anti-pyoverdine activities in 5-FU, 5-FC, and 5fluorouridine (Kirienko et al., 2016). Notably, 5-FU had a broad inhibitory effect on several virulence phenotypes (Ueda et al., 2009), while 5-FC appeared to exert its antivirulence activity by targeting mainly the production of the pyoverdine siderophore and of pyoverdine-regulated virulence factors (Imperi et al., 2013; Kirienko et al., 2016). These works also reported that, while 5-FC does not affect P. aeruginosa growth even at high concentrations, 5-FU has a strong bacteriostatic effect on P. aeruginosa, in agreement with an older report describing the growth inhibitory activity of 5-FU, but not of 5-FC, against this bacterium (West, 1986). Here we provide evidence that 5-FC and 5-FU likely share the same mechanism of action, and that the modest growth inhibitory activity of 5-FC is due to poor uptake and/or limited conversion into 5-FU by P. aeruginosa cytosine deaminase. By using co-culturing approaches and in vitro evolution experiments, we also demonstrated that 5-FC/5-FU insensitive spontaneous mutants with a defective pyrimidine salvage pathway readily emerge and spread in 5-FU treated populations and that, unexpectedly, these resistant mutants are also selected by 5-FC treatment, though at lower frequency. Finally, we found that the growth inhibitory and/or antipyoverdine activities of these two drugs are overall conserved in a large collection of cystic fibrosis (CF) isolates, although some inter-strain variability was observed. Antibiotic resistance is a serious public health concern at the global level, as an alarmingly high level of drug resistance has been reported in most common bacterial pathogens (Tommasi et al., 2015), calling for the investigation of alternative therapeutic options. In the last decades, researchers started looking at virulence factors as targets for the development of novel anti-infective drugs aimed at inhibiting pathogen-dependent host damage rather than bacterial growth (Finlay and Falkow, 1997). Such molecules are referred to as antivirulence drug (...truncated)