This project (which follows up on project FFC#13/2017) aims to provide information on the role of antibiotics in the development of viable but non-culturable (VBNC) P. aeruginosa (Pa) forms and to develop a flow cytometry (FC) protocol to support qPCR in detecting VBNC cells (i.e., the difference between total viable and culturable cells), which are responsible for lung infection recurrence.
In vitro GFP-expressing Pa biofilms were exposed to ceftazidime, ciprofloxacin, colistin (COL), fosfomycin, meropenem (MER), tobramycin (TOB), ceftolozane/tazobactam (C/T) and ceftazidime/avibactam (CZA). Viable Pa cells were detected by culture, qPCR and FC. TOB and all cell wall-targeting drugs, except CZA, favoured the development and persistence of VBNC forms.
A species-specific FC protocol was then developed to detect VBNC Pa cells in clinical samples. The protocol was first validated using in vitro Pa biofilms. Its specificity was confirmed using samples containing different cystic fibrosis (CF) pathogens; sensitivity was hampered by the sample matrix.  The protocol thus proved reliable for PA infection diagnosis, but not for pathogen quantification, which was better achieved by qPCR. To correlate the presence of VBNC Pa cells with infection status and antibiotic treatment, we analysed 50 sputum samples from 22 intermittent or chronic patients. VBNC forms were found in 28/50 from15 patients (12 chronic). The results showed a persistence in VBNC cells after treatment with TOB, β-lactams and their combinations (including TOB+ C/T or CZA), their absence after treatment with COL, C/T or CZA and their presence (in the absence of culturable cells) after treatment with COL + TOB/MER. 

In conclusion, FC and qPCR provided similar results on VBNC Pa cell detection in CF (including culture-negative) samples. The persistence of a VBNC Pa subpopulation seems to be a feature of chronic patients, related to treatment with aminoglycosides and β-lactams, in line with in vitro findings.

• Mangiaterra, Gianmarco et al. Contribution of Drugs Interfering with Protein and Cell Wall Synthesis to the Persistence of Pseudomonas aeruginosa Biofilms: An In Vitro Model. International journal of molecular sciences vol. 22,4 1628. 5 Feb. 2021
• King, John et al. Pseudomonas aeruginosa in the Cystic Fibrosis Lung. Advances in experimental medicine and biology vol. 1386 (2022): 347-369
• Mangiaterra, Gianmarco et al. Role of Tobramycin in the Induction and Maintenance of Viable but Non-Culturable Pseudomonas aeruginosa in an In Vitro Biofilm Model. Antibiotics (Basel, Switzerland) vol. 9,7 399. 10 Jul. 2020