**5.1 Evaluation of the role of sub-MIC of ciprofloxacin and tobramycin in inducing VBNC and culturable persistent cells in starved** *P. aeruginosa in vitro* **biofilms**

The possibility of VBNC cell induction by low antibiotic doses was first explored by our group in starved *S. aureus* biofilms. Greater VBNC cell persistence and survival were found in starved cultures exposed to vancomycin and quinupristin/ dalfopristin than in those subjected to starvation alone [91].

Similar experiments were subsequently performed to test VBNC cell induction in *P. aeruginosa* biofilms exposed to starvation, alone or combined with subinhibitory (1/4 x MIC) concentrations of ciprofloxacin or tobramycin [92], which are the most widely used treatments for *P. aeruginosa* lung infection [39, 93]. Biofilms of the laboratory strain *P. aeruginosa* PAO1 and the CF isolate *P. aeruginosa* C24 were developed in rich medium in 35 mm petri dishes for 48 h at 37°C and then subcultured in non-nutrient (NN) broth, alone or supplemented with sub-MIC concentrations of ciprofloxacin or tobramycin for 170 days. The medium was refreshed once a week. Samples were evaluated at 60, 75, 90, 120, 135, 150 and 170 days. The biofilm content in VBNC cells was determined as follows:


The results are reported in **Figure 4** and are expressed as percentage of TVCs. Unlike the *S. aureus* biofilms, a culturable *P. aeruginosa* subpopulation, which can be defined as triggered persisters, was detected throughout the experiment. A VBNC subpopulation also developed and was more abundant in presence of the antibiotics. In particular, a subset of VBNC *P. aeruginosa* PAO1 cells was detected in all conditions and gradually declined in starved and ciprofloxacinexposed biofilms; in contrast, the VBNC subpopulation triggered by sub-MIC tobramycin exceeded 90% of TVCs until the end of the experiment. In *P. aeruginosa* C24, starvation alone induced a discontinuous VBNC subpopulation; starvation and ciprofloxacin triggered a persister population only between 75 and 135 days; and starvation and tobramycin induced a constant VBNC population whose abundance was similar to the one determined in the PAO1 strain at the end of the experiment.

**43**

**Figure 5.**

**Figure 4.**

*Pseudomonas aeruginosa* Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters

These data suggest that sub-MIC concentrations of tobramycin (not ciprofloxacin) play a strong role in inducing VBNC *P. aeruginosa* and are in line with previous reports of the contribution of protein synthesis inhibitors [94] and aminoglycosides [95] to VBNC cell induction. The demonstration of the role of toxin-antitoxin modules [96] and ribosome hibernation [3] in persisters development, exerted via a reduction of

*(Modified from Ref. [92]). VBNC* P. aeruginosa *cell abundance in starved biofilms exposed to sub-MIC antibiotic concentrations. The VBNC cell subpopulation was assessed at specific time points in* P. aeruginosa *PAO1 (A) and C24 (B) biofilms exposed to starvation (NN) or starvation+ ¼ x MIC ciprofloxacin (NN + CIP) or tobramycin (NN + TOB). VBNC cells were the difference between total viable cells (TVCs) and plate counts (only differences*  ≥*0.5 log were considered). Cell abundance was compared in the three stress conditions. \* = p < 0.05, \*\* = p < 0.001.*

After documenting the role of subinhibitory drug concentrations in VBNC cell induction and maintenance, we examined the effectiveness of high antibiotic concentrations on *P. aeruginosa* biofilm eradication by evaluating the abundance of persistent and VBNC cells in mature *P. aeruginosa* PAO1 biofilms exposed to 1000 x MIC/100 x MBEC (minimal biofilm eradication concentration) tobramycin for 24 h (**Figure 5**).

*Biofilm persistence to tobramycin treatment. 24-hour-old* P. aeruginosa *PAO1 biofilms were exposed for 24 h to tobramycin 1000 x MIC and assessed for their content in culturable persisters and VBNC cells before and after antibiotic treatment. Persisters were determined by plate count (PC), whereas total viable cells (TVCs) were determined by* ecfX*-qPCR and live/dead flow cytometry. The VBNC population was the difference between TVCs and PCs. The results are given as the average of three biological replicates ± standard deviation.*

protein synthesis, further supports the observed behavior of tobramycin.

*DOI: http://dx.doi.org/10.5772/intechopen.95590*

*Pseudomonas aeruginosa* Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters *DOI: http://dx.doi.org/10.5772/intechopen.95590*

#### **Figure 4.**

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

**biofilms**

particularly VBNC cells.

*in vitro* **biofilms**

and 72 h at 37°C;

**5. Evaluation of the possible role of antibiotics and other stressors in triggering VBNC cells and culturable persisters in** *P. aeruginosa*

**5.1 Evaluation of the role of sub-MIC of ciprofloxacin and tobramycin in inducing VBNC and culturable persistent cells in starved** *P. aeruginosa* 

by our group in starved *S. aureus* biofilms. Greater VBNC cell persistence and survival were found in starved cultures exposed to vancomycin and quinupristin/

dalfopristin than in those subjected to starvation alone [91].

content in VBNC cells was determined as follows:

SYBR Green 1x and propidium iodide 40 μg/ml;

culturable cells (only differences ≥0.5 log were considered).

After demonstrating VBNC *P. aeruginosa* cells in the lungs of CF patients (6), a key issue was to establish the factors that trigger their induction. We therefore examined the role of antibiotic treatments and of some environmental stressors that are found in the CF lung [51, 58], in selecting and maintaining persister cells,

The possibility of VBNC cell induction by low antibiotic doses was first explored

Similar experiments were subsequently performed to test VBNC cell induction in *P. aeruginosa* biofilms exposed to starvation, alone or combined with subinhibitory (1/4 x MIC) concentrations of ciprofloxacin or tobramycin [92], which are the most widely used treatments for *P. aeruginosa* lung infection [39, 93]. Biofilms of the laboratory strain *P. aeruginosa* PAO1 and the CF isolate *P. aeruginosa* C24 were developed in rich medium in 35 mm petri dishes for 48 h at 37°C and then subcultured in non-nutrient (NN) broth, alone or supplemented with sub-MIC concentrations of ciprofloxacin or tobramycin for 170 days. The medium was refreshed once a week. Samples were evaluated at 60, 75, 90, 120, 135, 150 and 170 days. The biofilm

• the culturable population was quantified by plate counts performed on cystine lactose electrolyte-deficient (CLED) agar after incubation for 24, 28

• total viable cells (TVCs) were expressed as the average of the counts obtained from *ecfX*-qPCR and flow cytometry assays after live/dead staining using

• the number of VBNC cells was determined as the difference between TVCs and

The results are reported in **Figure 4** and are expressed as percentage of TVCs. Unlike the *S. aureus* biofilms, a culturable *P. aeruginosa* subpopulation, which can be defined as triggered persisters, was detected throughout the experiment. A VBNC subpopulation also developed and was more abundant in presence of the antibiotics. In particular, a subset of VBNC *P. aeruginosa* PAO1 cells was detected in all conditions and gradually declined in starved and ciprofloxacinexposed biofilms; in contrast, the VBNC subpopulation triggered by sub-MIC tobramycin exceeded 90% of TVCs until the end of the experiment. In *P. aeruginosa* C24, starvation alone induced a discontinuous VBNC subpopulation; starvation and ciprofloxacin triggered a persister population only between 75 and 135 days; and starvation and tobramycin induced a constant VBNC population whose abundance was similar to the one determined in the PAO1 strain at the end

**42**

of the experiment.

*(Modified from Ref. [92]). VBNC* P. aeruginosa *cell abundance in starved biofilms exposed to sub-MIC antibiotic concentrations. The VBNC cell subpopulation was assessed at specific time points in* P. aeruginosa *PAO1 (A) and C24 (B) biofilms exposed to starvation (NN) or starvation+ ¼ x MIC ciprofloxacin (NN + CIP) or tobramycin (NN + TOB). VBNC cells were the difference between total viable cells (TVCs) and plate counts (only differences*  ≥*0.5 log were considered). Cell abundance was compared in the three stress conditions. \* = p < 0.05, \*\* = p < 0.001.*

These data suggest that sub-MIC concentrations of tobramycin (not ciprofloxacin) play a strong role in inducing VBNC *P. aeruginosa* and are in line with previous reports of the contribution of protein synthesis inhibitors [94] and aminoglycosides [95] to VBNC cell induction. The demonstration of the role of toxin-antitoxin modules [96] and ribosome hibernation [3] in persisters development, exerted via a reduction of protein synthesis, further supports the observed behavior of tobramycin.

After documenting the role of subinhibitory drug concentrations in VBNC cell induction and maintenance, we examined the effectiveness of high antibiotic concentrations on *P. aeruginosa* biofilm eradication by evaluating the abundance of persistent and VBNC cells in mature *P. aeruginosa* PAO1 biofilms exposed to 1000 x MIC/100 x MBEC (minimal biofilm eradication concentration) tobramycin for 24 h (**Figure 5**).

#### **Figure 5.**

*Biofilm persistence to tobramycin treatment. 24-hour-old* P. aeruginosa *PAO1 biofilms were exposed for 24 h to tobramycin 1000 x MIC and assessed for their content in culturable persisters and VBNC cells before and after antibiotic treatment. Persisters were determined by plate count (PC), whereas total viable cells (TVCs) were determined by* ecfX*-qPCR and live/dead flow cytometry. The VBNC population was the difference between TVCs and PCs. The results are given as the average of three biological replicates ± standard deviation.*

#### **Figure 6.**

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

As expected, the high tobramycin concentrations chiefly affected the culturable population, which showed a reduction >4 log, whereas the TVC counts showed a 2 log reduction, highlighting the presence of more than 1 x 107 VBNC cells/ml after 24-hours exposure to 1000 x MIC tobramycin.

#### **5.2 Evaluation of the possible involvement of additional stressors found in the CF lung**

Finally, we examined the possible contribution of further environmental factors – especially the high salinity and metabolite accumulation that are found in the CF lung [51, 58], − in the induction of persistent and VBNC *P. aeruginosa* cells.

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 VBNC cells in biofilms maintained in NN broth.

#### **6. Conclusions**

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].

**45**

of CF lung infection.

**Acknowledgements**

Cystic Fibrosis Research Foundation.

lung infections.

*Pseudomonas aeruginosa* Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters

In infectious biofilms, a combination of stress factors can induce the development of persistent forms which can trigger infection recurrence. This is a cause of special concern in *P. aeruginosa* CF lung infection, where VBNC cells undermine treatment and hamper microbiological diagnosis, which is still routinely performed by culture-based assays. A routine diagnostic workup including culture-independent

The evidence described in this chapter demonstrates that VBNC *P. aeruginosa* cells are found in sputum samples from CF patients and that several months after a culture-negative and qPCR-positive assay some patients experience infection recurrence and have culture-positive sputum samples. These data also highlight the reliability of qPCR in detecting the whole bacterial population, including the phenotypic variants that are missed by culture-based assays. Notably, flow cytometry has demonstrated the accuracy of the *ecfX*-targeting qPCR protocol in detecting all viable *P. aeruginosa* cells and can provide a sound alternative for routine monitoring of the infection. Together, qPCR and flow cytometry supply a clear picture of *P. aeruginosa* population dynamics in the lungs of CF patients with intermittent and chronic infection and can be harnessed to monitor the effectiveness of the antibiotic

The two techniques have enabled us to gain insight into the role of antibiotics in VBNC cell development and infection persistence. Notably, whereas high antibiotic doses can select persistent subpopulations, subinhibitory concentrations – which are found in the CF lung between treatment cycles and in the deepest biofilm layers [51, 58] – can stimulate the development of persistent phenotypic variants, including VBNC cells [53]. Besides the fact that starvation proved to be a necessary condition for VBNC cell induction in our *in vitro* biofilms, our findings highlight a different behavior of tobramycin and ciprofloxacin. Although large amounts of VBNC cells were induced in all test conditions over the first 120 days, their number was maintained more consistently in presence of tobramycin, whereas ciprofloxacin exerted a discontinuous effect similar to the one of starvation alone. These findings can partly be explained by the ability of tobramycin to act as a signal molecule that interferes with QS signals, thus modulating gene expression in biofilm-growing *P. aeruginosa* [58], and by its adverse effect on protein synthesis via ribosome binding [3].

In conclusion, more detailed information on the main gene pathways and persistence regulators and on the effects of different antibiotics is essential to meet the challenge of antibiotic-resilient *P. aeruginosa* infectious biofilms and the eradication

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

This work was supported by FFC grants # 13/2017 and #16/2019 of the Italian

therapy and to foster the development of new eradication treatments.

*DOI: http://dx.doi.org/10.5772/intechopen.95590*

approaches should thus be urgently adopted.

#### *Pseudomonas aeruginosa* Biofilm Lung Infection in Cystic Fibrosis: The Challenge of Persisters *DOI: http://dx.doi.org/10.5772/intechopen.95590*

In infectious biofilms, a combination of stress factors can induce the development of persistent forms which can trigger infection recurrence. This is a cause of special concern in *P. aeruginosa* CF lung infection, where VBNC cells undermine treatment and hamper microbiological diagnosis, which is still routinely performed by culture-based assays. A routine diagnostic workup including culture-independent approaches should thus be urgently adopted.

The evidence described in this chapter demonstrates that VBNC *P. aeruginosa* cells are found in sputum samples from CF patients and that several months after a culture-negative and qPCR-positive assay some patients experience infection recurrence and have culture-positive sputum samples. These data also highlight the reliability of qPCR in detecting the whole bacterial population, including the phenotypic variants that are missed by culture-based assays. Notably, flow cytometry has demonstrated the accuracy of the *ecfX*-targeting qPCR protocol in detecting all viable *P. aeruginosa* cells and can provide a sound alternative for routine monitoring of the infection. Together, qPCR and flow cytometry supply a clear picture of *P. aeruginosa* population dynamics in the lungs of CF patients with intermittent and chronic infection and can be harnessed to monitor the effectiveness of the antibiotic therapy and to foster the development of new eradication treatments.

The two techniques have enabled us to gain insight into the role of antibiotics in VBNC cell development and infection persistence. Notably, whereas high antibiotic doses can select persistent subpopulations, subinhibitory concentrations – which are found in the CF lung between treatment cycles and in the deepest biofilm layers [51, 58] – can stimulate the development of persistent phenotypic variants, including VBNC cells [53]. Besides the fact that starvation proved to be a necessary condition for VBNC cell induction in our *in vitro* biofilms, our findings highlight a different behavior of tobramycin and ciprofloxacin. Although large amounts of VBNC cells were induced in all test conditions over the first 120 days, their number was maintained more consistently in presence of tobramycin, whereas ciprofloxacin exerted a discontinuous effect similar to the one of starvation alone. These findings can partly be explained by the ability of tobramycin to act as a signal molecule that interferes with QS signals, thus modulating gene expression in biofilm-growing *P. aeruginosa* [58], and by its adverse effect on protein synthesis via ribosome binding [3].

In conclusion, more detailed information on the main gene pathways and persistence regulators and on the effects of different antibiotics is essential to meet the challenge of antibiotic-resilient *P. aeruginosa* infectious biofilms and the eradication of CF lung infection.
