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

Abstract

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.

Repurposed anti-cancer drugs: the future for anti-infective therapy?

“…If viability is not compromised, in theory, the appearance of resistance is more unlikely [27]. Nevertheless, a possible drawback for anti-virulence drugs is that some clinical strains are not susceptible [28,29], and at least in vitro sporadic resistance against some of them, including brominated furanones, 5-FU and flucytosine have been found [27,28,30,31], hence its eventual utilization should include monitoring its effectivity against the specific strains before and during treatment. The aim of anti-virulence drugs is to impair processes like secretion of exotoxins, biofilm formation, adherence, evasion of the immune system, quorum sensing, etc.…”

Drugs Repurposing for Multi-Drug Resistant Bacterial Infections

“…Different agents are promising both in vitro and in vivo candidates to be repositioned as antimicrobial agents to treat infections caused by MDR Gram-negative bacilli. A variety of drugs with different mechanisms of action and targets have been selected including: DNA, RNA and proteins inhibitors [16][17][18][19][20], QS regulators [15,17,[21][22][23][24][25], biofilm formation inhibitors and disruptors [26,27], drugs that interact with cell membrane [28][29][30], drugs that interact with iron metabolism [31][32][33][34][35], and host immune system modulators [36][37][38][39]. These drugs and their mechanisms of action against critical-priority pathogens (A. baumannii, P. aeruginosa and Enterobacterales) are summarized in Figure 1 and Table 1.…”

“…Anticancer and anti-inflammatory drugs that interact with DNA, RNA and proteins have been reported. Mitomycin C, used in several types of carcinomas such Binding to DNA during DNA synthesis and causes inhibition of its synthesis and function in P. aeruginosa and E. coli [18] Cisplatin Cancer Upregulation of the recA gene of P. aeruginosa, which is known to be important for DNA repair [20] Celecoxib Inflammation Inhibition of RNA, DNA, and protein synthesis in S. aureus [19] Quorum sensing 5-fluorouracil Solid tumors Inhibition of QS formation in P. aeruginosa [23,41] Raloxifene Breast cancer Binding to PhzB2 which is involved in the production of pyocyanin, a pigment related with virulence factor and QS signalling molecule in P. aeruginosa [24] Niclosamide Helminthiasis Production of the QS signaling molecules N-3-oxododecanoyl-homeserine lactone and Nbutanoyl-homoserine lactone in P. aeruginosa [15,25] Meloxicam Inflammation Interaction with active sites of the QS of P. aeruginosa [26] Metformin Diabetes Inhibition of QS system by bind to LasR by hydrogen bonding and electrostatic interaction and to rhlR by hydrogen bonding in P. aeruginosa [21] Biofilm formation 5-Fluorouracil Solid tumors Regulation of different genes involved in the biofilm formation by P. aeruginosa [41] Meloxicam Inflammation Decrease in the extracellular Psl, Pel, and alginate production by P. aeruginosa [26,27] Glatiramer acetate as the superficial vesical carcinoma [40], has shown activity against A. baumannii, P. aeruginosa and E. coli in vitro and in vivo [16][17][18]. Mitomycin C binds to DNA during DNA synthesis and causes inhibition of its synthesis and function in P. aeruginosa and E. coli [18].…”

“…QS inhibition and regulation have been reported as antibacterial properties of anticancer, anthelminthic, anti-inflammatory and hypoglycemic drugs such as 5-fluorouracil, raloxifene, niclosamide, meloxicam and metformin. 5-fluorouracil is a potent drug indicated for the treatment of different types of solid tumors, that has shown antibacterial activity in vitro [17,22,23]. The antibacterial mechanism of this drug has been proposed as QS inhibitor [23,41].…”

“…5-fluorouracil is a potent drug indicated for the treatment of different types of solid tumors, that has shown antibacterial activity in vitro [17,22,23]. The antibacterial mechanism of this drug has been proposed as QS inhibitor [23,41]. Also, 5-fluorouracil has dual inhibition mechanisms including functioning as an alternative substrate resulting in miscoding DNA and RNA, and inhibiting thymidylate synthase [22].…”