The recurrence of Pseudomonas aeruginosa (PA) biofilm infections is a major issue in cystic fibrosis (CF) patients. A pivotal role is played by the presence of antibiotic-unresponsive persisters and/or viable but non-culturable (VBNC) forms, whose development might be favored by subinhibitory antibiotic concentrations. The involvement of tobramycin and ciprofloxacin, widely used to treat CF PA lung infections, in the abundance of VBNC cells was investigated in PA biofilms models. In vitro biofilms of the laboratory strain PAO1-N and the clinical strain C24 were developed and starved by subculture for 170 days in a non-nutrient (NN) broth, unsupplemented or supplemented with one-quarter minimal inhibitory concentration (MIC) of tobramycin or ciprofloxacin. VBNC cells abundance, estimated as the difference between total live (detected by qPCR and flow cytometry) and colony forming unit (CFU) counts, showed a strain- and drug-specific pattern. A greater and earlier abundance of VBNC PAO1-N cells was detected in all conditions. Exposure of the C24 strain to NN and NN + ciprofloxacin induced only a transient VBNC subpopulation, which was more abundant and stable until the end of the experiment in tobramycin-exposed biofilms. The same response to tobramycin was observed in the PAO1-N strain. These findings suggest that low tobramycin concentrations might contribute to PA infection recurrence by favoring the development of VBNC forms.

Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones.

The occurrence of Pseudomonas aeruginosa (PA) persisters, including viable but non-culturable (VBNC) forms, subpopulations of tolerant cells that can survive high antibiotic doses, is the main reason for PA lung infections failed eradication and recurrence in Cystic Fibrosis (CF) patients, subjected to life-long, cyclic antibiotic treatments. In this paper, we investigated the role of subinhibitory concentrations of different anti-pseudomonas antibiotics in the maintenance of persistent (including VBNC) PA cells in in vitro biofilms. Persisters were firstly selected by exposure to high doses of antibiotics and their abundance over time evaluated, using a combination of cultural, qPCR and flow cytometry assays. Two engineered GFP-producing PA strains were used. The obtained results demonstrated a major involvement of tobramycin and bacterial cell wall-targeting antibiotics in the resilience to starvation of VBNC forms, while the presence of ciprofloxacin and ceftazidime/avibactam lead to their complete loss. Moreover, a positive correlation between tobramycin exposure, biofilm production and c-di-GMP levels was observed. The presented data could allow a deeper understanding of bacterial population dynamics during the treatment of recurrent PA infections and provide a reliable evaluation of the real efficacy of the antibiotic treatments against the bacterial population within the CF lung.

Pseudomonas aeruginosa lung infection is difficult to eradicate due to the multiple (intrinsic and acquired) antibiotic resistance of bacteria and to their ability to produce a thick biofilm. Antibiotic treatment is hampered by poor antibiotic diffusion, efflux pump overexpression and the development of a persistent subpopulation with low metabolic activity. This is a cause for special concern in Cystic Fibrosis (CF) patients, where P. aeruginosa lung infection is the chief cause of morbidity and mortality. Combined tobramycin-ciprofloxacin treatment is routinely adopted due to the low frequency of resistant strains and its ostensible ability to control the infection. Nevertheless, symptoms usually recur, mainly due to the antibiotic persisters, which are difficult to detect in routine cultural microbiological assays. This chapter describes the issues involved in the microbiological diagnosis of P. aeruginosa lung infection in CF patients and the possible role of subinhibitory antibiotic concentrations in persister development and infection recurrence.