**Acknowledgements**

We are grateful to Dr. Salvatore Vaiasicca for his contribution to flow cytometry assays and to Dr. Natalia Cirilli for helpful discussion on *P. aeruginosa* CF lung infections.

This work was supported by FFC grants # 13/2017 and #16/2019 of the Italian Cystic Fibrosis Research Foundation.

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

As expected, the high tobramycin concentrations chiefly affected the culturable population, which showed a reduction >4 log, whereas the TVC counts showed a

*Induction of VBNC* P. aeruginosa *cells in biofilms exposed to environmental stress factors.* P. aeruginosa *PAO1 biofilms were grown in Luria Bertani broth, with metabolite accumulation (MA T7) or in LB + 13 g/l NaCl for 7 days (NaCl T7). Culturable cells were determined by plate count (PC) and total viable cells (TVCs) were determined by qPCR/flow cytometry assays. VBNC cells were the difference between TVCs and culturable cells. These values were compared to those determined in a 24-hour-old biofilm grown in Luria Bertani (LB) broth.* 

**5.2 Evaluation of the possible involvement of additional stressors found in the** 

Finally, we examined the possible contribution of further environmental factors – especially the high salinity and metabolite accumulation that are found in the CF lung

Culturable cells and TVCs were counted as described above in *P. aeruginosa* PAO1 biofilms grown for 7 days in Luria Bertani (LB) broth, alone or added with 13 g/l NaCl. The counts were compared to those of a mature 24-hour-old biofilm (**Figure 6**). As shown in the diagram, *P. aeruginosa* biofilms tolerate and adapt to the high salinity found in the CF lung, since exposure to this stressor for 7 days failed to induce a significant difference in culturable and VBNC cell amount compared to the control condition. In contrast, the plate counts demonstrated a difference of 1 log between 7-day-old and 24-hour-old LB biofilms, whereas the number of TVCs at the two time points was not significantly different. This indicates a shift of *P. aeruginosa* cells to the VBNC state in biofilms grown for 7 days in LB medium, where bacterial metabolites accumulate. Most likely, nutrient reduction and waste accumulation induce a major shift to the persistent state, as also demonstrated for

The generation of persistent cell subpopulations is a bacterial survival strategy against adverse environmental conditions [2]. Whereas stochastic persisters are rare, external stressors can convert most bacterial population into persistent cells [45].

VBNC cells/ml after

2 log reduction, highlighting the presence of more than 1 x 107

*The results are average of three biological replicates ± standard deviation.*

[51, 58], − in the induction of persistent and VBNC *P. aeruginosa* cells.

24-hours exposure to 1000 x MIC tobramycin.

the VBNC cells in biofilms maintained in NN broth.

**CF lung**

**Figure 6.**

**44**

**6. Conclusions**

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments
