**3. Persister development: antibiotic failure and microbiological diagnosis**

#### **3.1 Persistent and viable but non-culturable (VBNC) bacterial forms**

*P. aeruginosa* lung infections tend to be recurrent. Relapses are chiefly due to the development of persisters, bacterial forms that are unsusceptible to antibiotics and often difficult to detect by routine microbiological assays. Persistence has been defined as "the ability of a subset of the bacterial population to survive to a bactericidal antibiotic concentration" [45]. Survival is demonstrated by bacterial growth in culture once the stressor, i.e. antibiotic concentrations several times higher than the minimal inhibitory concentration (MIC), has been removed and nutrients have been restored. Accordingly, the main features distinguishing persisters from resistant cells are the inability of the former cells to grow in presence of antibiotics, though viable and metabolically active, and the lack of heritability [45].

Persisters have been considered as dormant cells that are unaffected by antibiotics [46]. However, lack of significant growth or metabolic activity does not equal

**37**

**Figure 1.**

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

Antibiotic persistence is not to be confused with antibiotic tolerance. In particular, whereas tolerance involves the whole bacterial population, persistence regards only a subset of specialized cells. Moreover, tolerant cells are killed, even if more slowly than susceptible cells, by high antibiotic doses while persisters are maintained over time (**Figure 1**). Notably, however, the two cell types share the same

Two types of persisters have been described to date: stochastic and triggered. The former cells constitute a small subpopulation that can be found in all bacterial cultures, even in exponentially growing ones, whereas the latter are induced by environmental as well as host-related stressors. Unfavorable environmental conditions, e.g. nutrient and oxygen depletion, catabolite accumulation and suboptimal pH, which can induce persistence, can occur in the lungs of CF patients, especially in the deepest layers of *P. aeruginosa* biofilms [51]. Repeated antibiotic treatment directed at eradicating chronic infection can contribute to the induction of these

VBNC cells are dormant forms described in several bacterial species, including *P. aeruginosa*. They are characterized by the inability to grow on bacteriological media despite the presence of metabolic activity [52]. VBNC cells share several features with persisters, including a number of inducing factors of which the most common are starvation, oxidative stress, suboptimal salinity and pH and low temperature [52]. Moreover, both phenotypes are highly resilient to antimicrobials. These similarities have led some researchers to conclude that "persister and VBNC cells actually represent subsequent stages of the same cycle of dormancy, adopted by non-sporulating bacteria to survive unfavorable conditions" [52]. According to this theory, stress exposure would induce the development of persisters, which in case of prolonged exposure would turn into VBNC cells, whereas stressor removal

*Behavior of susceptible, resistant, tolerant and persistent bacterial subpopulations treated with antibiotic* 

*concentrations exceeding the MIC. CFU: Colony forming unit.*

persistence, since the majority (> 99%) of dormant subpopulations are not true persisters. Persistence is a far more complex condition than dormancy [47], it shows an intense metabolic activity despite cell failure to grow or divide. Indeed, starvation-induced persisters produced more ATP per mol of carbon source consumed than nutrient supplied cells did [48]. Accordingly, persister cells seem to be able to catabolize carbon sources, which results in increased electron transport chain activity and membrane potential and increased aminoglycoside uptake [49]. However, although bacterial metabolic processes and persistence are closely related,

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

the mechanisms involved are largely unclear [50].

MIC as susceptible cells [45].

specialized bacterial forms [26].

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

persistence, since the majority (> 99%) of dormant subpopulations are not true persisters. Persistence is a far more complex condition than dormancy [47], it shows an intense metabolic activity despite cell failure to grow or divide. Indeed, starvation-induced persisters produced more ATP per mol of carbon source consumed than nutrient supplied cells did [48]. Accordingly, persister cells seem to be able to catabolize carbon sources, which results in increased electron transport chain activity and membrane potential and increased aminoglycoside uptake [49]. However, although bacterial metabolic processes and persistence are closely related, the mechanisms involved are largely unclear [50].

Antibiotic persistence is not to be confused with antibiotic tolerance. In particular, whereas tolerance involves the whole bacterial population, persistence regards only a subset of specialized cells. Moreover, tolerant cells are killed, even if more slowly than susceptible cells, by high antibiotic doses while persisters are maintained over time (**Figure 1**). Notably, however, the two cell types share the same MIC as susceptible cells [45].

Two types of persisters have been described to date: stochastic and triggered. The former cells constitute a small subpopulation that can be found in all bacterial cultures, even in exponentially growing ones, whereas the latter are induced by environmental as well as host-related stressors. Unfavorable environmental conditions, e.g. nutrient and oxygen depletion, catabolite accumulation and suboptimal pH, which can induce persistence, can occur in the lungs of CF patients, especially in the deepest layers of *P. aeruginosa* biofilms [51]. Repeated antibiotic treatment directed at eradicating chronic infection can contribute to the induction of these specialized bacterial forms [26].

VBNC cells are dormant forms described in several bacterial species, including *P. aeruginosa*. They are characterized by the inability to grow on bacteriological media despite the presence of metabolic activity [52]. VBNC cells share several features with persisters, including a number of inducing factors of which the most common are starvation, oxidative stress, suboptimal salinity and pH and low temperature [52]. Moreover, both phenotypes are highly resilient to antimicrobials. These similarities have led some researchers to conclude that "persister and VBNC cells actually represent subsequent stages of the same cycle of dormancy, adopted by non-sporulating bacteria to survive unfavorable conditions" [52]. According to this theory, stress exposure would induce the development of persisters, which in case of prolonged exposure would turn into VBNC cells, whereas stressor removal

#### **Figure 1.**

*Behavior of susceptible, resistant, tolerant and persistent bacterial subpopulations treated with antibiotic concentrations exceeding the MIC. CFU: Colony forming unit.*

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

1 second) impair lung function hence quality of life and overall survival.

antibiotic resistance all correlate with SCVs detection in sputum [41].

**3. Persister development: antibiotic failure and microbiological** 

**3.1 Persistent and viable but non-culturable (VBNC) bacterial forms**

though viable and metabolically active, and the lack of heritability [45].

*P. aeruginosa* lung infections tend to be recurrent. Relapses are chiefly due to the development of persisters, bacterial forms that are unsusceptible to antibiotics and often difficult to detect by routine microbiological assays. Persistence has been defined as "the ability of a subset of the bacterial population to survive to a bactericidal antibiotic concentration" [45]. Survival is demonstrated by bacterial growth in culture once the stressor, i.e. antibiotic concentrations several times higher than the minimal inhibitory concentration (MIC), has been removed and nutrients have been restored. Accordingly, the main features distinguishing persisters from resistant cells are the inability of the former cells to grow in presence of antibiotics,

Persisters have been considered as dormant cells that are unaffected by antibiotics [46]. However, lack of significant growth or metabolic activity does not equal

*P. aeruginosa* adaptation to the CF lung environment ultimately results in a mucoid phenotype, a conversion first described by Lam and colleagues [42], which may take several months to years. The mucoid material has subsequently been identified as alginate. In mucoid strains, alginate may favor adhesion to lung epithelial cells, thereby inhibiting clearance. Nutrient restriction, dehydration and suboptimal antibiotic concentrations may result in mucoidity [7, 43]. Host inflammation responses are also believed to contribute to mucoid conversion, a hypothesis that is supported by the absence of mucoid variants among environmental isolates [44].

characterized by recurrent pulmonary exacerbations. Worsening of the chronic lung infection symptoms (particularly cough and sputum production), increased bacterial load and inflammation and, often, a reduction in FEV1 (forced respiratory volume in

The identification of effective treatments requires a greater understanding of the factors underpinning the exacerbations. Notably, the lung of CF patients is initially colonized by *Haemophilus influenzae* and *Staphylococcus aureus*; then, patients gradually become susceptible to infection with a variety of environmental Gramnegative bacteria carrying a broad range of constitutive and acquired antibiotic resistance determinants [39]. *P. aeruginosa* is the main pathogen triggering airway inflammation and the leading cause of CF morbidity and mortality [40]. Most CF patients are susceptible to *P. aeruginosa* respiratory infections from infancy. The 30% of them acquire a strain from the environment resulting in acute infections in the first year of life, this rate increases to about 50% before turning 3 years, while mucoid phenotype and chronic infection usually raise from 3 to 16 years [10]. Lung colonization generally involves alternate asymptomatic periods and relapses with progressive tissue deterioration that eventually lead to lung failure and to premature death. Over the years *P. aeruginosa* develops multiple phenotypic variants such as SCVs, mucoid and persistent forms. In particular, SCVs are typically isolated from the lungs of chronic CF patients. They are small (1–3 mm in diameter) usually non-motile and resistant to several classes of antibiotics; produce high amounts of exopolysaccharide and form biofilms that strongly adhere to surfaces [41]. *In vitro* and *in vivo* tests have demonstrated that exposure to sublethal concentrations of antibiotics, such as aminoglycosides, selects for SCVs. In CF patients, prolonged persistent infection, deterioration of pulmonary function and increased

**36**

**diagnosis**

and nutrient restoration would involve recovery of the full metabolic state typical of exponential growth [52, 53]. Unlike culturable persisters, VBNC cells can regain culturability only through the action of specific activators (**Figure 2**), a phenomenon known as resuscitation [54]. The activators can be specific for the bacterial species and even for a single strain; while not completely understood they seem easily found *in vivo* [54].

#### **3.2 VBNC** *P. aeruginosa* **and issues related to the diagnosis of CF lung infection**

In CF patients, the microbiological diagnosis of *P. aeruginosa* lung infection is still performed by culture-based assays, which cannot detect VBNC cells [6]. Such assays involve microorganism isolation using rich (Columbia blood or chocolate) and selective (MacConkey or Pseudomonas) agar followed by isolate identification by biochemical or mass spectrometry analysis [55, 56]. Though effective in diagnosing primary colonization and pulmonary exacerbations, these methods suffer from considerable limitations, first and foremost poor sensitivity, due to the multiple phenotypic variants found in *P. aeruginosa* isolated from chronic CF biofilm-related infections [57].

A variety of stressors, principally nutrient depletion, oxidative and osmotic stress, an acid pH, a strong immune response and the presence of subinhibitory antibiotic concentrations [51, 58], make the CF lung an unfavorable environment for *P. aeruginosa*. The bacterial response involves the development of different phenotypes. The best known is the mucoid phenotype [59], alongside the loss of motility and pigmentation [60], the formation of auxotrophic variants and SCVs [61]. All these phenotypes are characterized by slow growth, which hampers culture-based diagnosis. However, the main problem is detecting VBNC cells. These cells – albeit not necessarily virulent – given suitable conditions can revert to full metabolically active forms capable of quick duplication and full virulence [54], which trigger a new infection. Developing a diagnostic technique that detects these forms is therefore critical to forecast symptom relapse and start early treatment.

#### **3.3 The multifaceted role of antibiotics**

Antibiotic treatment can play two different roles as regards persistent cells: it can either select a pre-existing persistent subpopulation or induce the persistent phenotype [45]. The hypothesis has also been advanced that antibiotics exert a biphasic dose-dependent action, i.e. inhibition of bacterial growth at high (≥ the MIC) doses

#### **Figure 2.**

*Differences between culturable persistent and VBNC cells after stressor removal and nutrient restoration. Whereas persistent cells quickly begin to grow and divide again, VBNC cells require exposure to a growth activator (the resuscitation-promoting factor) before regaining full metabolic activity and doubling ability. The progeny of both cell types will contain a mixed population as the starting culture.*

**39**

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

and stimulation of a specific bacterial response by acting as a molecular signal at

Failed infection eradication even after prolonged antibiotic treatment is a major clinical problem in patients with microbial biofilms. Antibiotic unresponsiveness has been explained by poor drug penetration in the biofilm matrix and by the development of dormant/persistent cells in the deepest biofilm layers [22]. Since low antibiotic concentrations are probably found for extended periods in the lung of CF patients with intermittent/chronic infection, who undergo repeated antibiotic treatment, the development of persistent forms is likely to be stimulated by the drugs themselves. Evidence to this effect has been reported for different classes of antibiotics, including quinolones and aminoglycosides, although more comprehen-

**4.** *P. aeruginosa* **VBNC cell detection and quantification in CF respiratory** 

Given the wide phenotypic variability of *P. aeruginosa*, encompassing difficult-to grow phenotypes, several culture-independent approaches have been devised to provide reliable infection diagnosis. DNA-based techniques are useful because they are able to detect the whole bacterial population. Most protocols are based on PCR

To find a suitable target gene on which to base *P. aeruginosa* detection, most protocols have been tested on a variety of bacterial isolates of different origins. The *oprL* gene, encoding a peptidoglycan-associated protein, has long been considered as one of the best targets [64–67]. However, its specificity was questioned when Anuj and colleagues [68] obtained cross-reactions with other species. Notably, the selection of multiple targets is considered as the best approach to *P. aeruginosa* detection, since it excludes false-negative results due to mutations in the amplified gene sequences. The *gyrB* and *ecfX* genes are two other widely used targets. The former gene encodes the DNA gyrase subunit B. Tests against several CF *P. aeruginosa* isolates have identified a species-specific internal sequence [69, 70]. The *ecfX* gene – found in 19 copies/genome – encodes a σ factor belonging to the ECF subfamily, which is involved in the synthesis of proteins with an extracytoplasmic function and seems to play a role in *P. aeruginosa* haem uptake and virulence [71]. The gene has been reported to be specific of *P. aeruginosa* and has been used to achieve its detection in environmental as well as clinical samples [6, 71]. Further proposed targets are the *algD* gene [72]

After reliable detection, a key issue is direct pathogen quantification in sputum samples. Most of the work in this field has been performed after 2010 using specific

The main drawback of DNA-based approaches is that they do not detect only live bacterial cells and may be affected by the presence of dead cells as well as by eDNA [77]. An efficient and widely used approach to this problem is to treat samples with propidium monoazide before DNA extraction [66]. The dye penetrates the cells via damaged walls/membranes and binds their DNA; after photo-activation, binding to the nucleic acids prevents DNA polymerase binding, hence DNA amplification in PCR assays. Since live cells commonly have an intact wall, they are not affected by the dye and only their DNA is detected. The same objective can be achieved with

low concentrations (< the MIC), a phenomenon known as hormesis [62].

sive investigations are required to draw firm conclusions.

and some 16 s [73] and 23 s rDNA sequences [74].

extraction kits and lysis protocols [66, 67, 74–76].

other treatments such as ethidium monoazide and DNase [6, 77].

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

**4.1 Total live cell detection strategies**

**samples**

or qPCR assays [63].

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

and stimulation of a specific bacterial response by acting as a molecular signal at low concentrations (< the MIC), a phenomenon known as hormesis [62].

Failed infection eradication even after prolonged antibiotic treatment is a major clinical problem in patients with microbial biofilms. Antibiotic unresponsiveness has been explained by poor drug penetration in the biofilm matrix and by the development of dormant/persistent cells in the deepest biofilm layers [22]. Since low antibiotic concentrations are probably found for extended periods in the lung of CF patients with intermittent/chronic infection, who undergo repeated antibiotic treatment, the development of persistent forms is likely to be stimulated by the drugs themselves. Evidence to this effect has been reported for different classes of antibiotics, including quinolones and aminoglycosides, although more comprehensive investigations are required to draw firm conclusions.

## **4.** *P. aeruginosa* **VBNC cell detection and quantification in CF respiratory samples**

#### **4.1 Total live cell detection strategies**

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

easily found *in vivo* [54].

**3.3 The multifaceted role of antibiotics**

and nutrient restoration would involve recovery of the full metabolic state typical of exponential growth [52, 53]. Unlike culturable persisters, VBNC cells can regain culturability only through the action of specific activators (**Figure 2**), a phenomenon known as resuscitation [54]. The activators can be specific for the bacterial species and even for a single strain; while not completely understood they seem

**3.2 VBNC** *P. aeruginosa* **and issues related to the diagnosis of CF lung infection**

found in *P. aeruginosa* isolated from chronic CF biofilm-related infections [57]. A variety of stressors, principally nutrient depletion, oxidative and osmotic stress, an acid pH, a strong immune response and the presence of subinhibitory antibiotic concentrations [51, 58], make the CF lung an unfavorable environment for *P. aeruginosa*. The bacterial response involves the development of different phenotypes. The best known is the mucoid phenotype [59], alongside the loss of motility and pigmentation [60], the formation of auxotrophic variants and SCVs [61]. All these phenotypes are characterized by slow growth, which hampers culture-based diagnosis. However, the main problem is detecting VBNC cells. These cells – albeit not necessarily virulent – given suitable conditions can revert to full metabolically active forms capable of quick duplication and full virulence [54], which trigger a new infection. Developing a diagnostic technique that detects these forms is therefore critical to forecast symptom relapse and start early treatment.

In CF patients, the microbiological diagnosis of *P. aeruginosa* lung infection is still performed by culture-based assays, which cannot detect VBNC cells [6]. Such assays involve microorganism isolation using rich (Columbia blood or chocolate) and selective (MacConkey or Pseudomonas) agar followed by isolate identification by biochemical or mass spectrometry analysis [55, 56]. Though effective in diagnosing primary colonization and pulmonary exacerbations, these methods suffer from considerable limitations, first and foremost poor sensitivity, due to the multiple phenotypic variants

Antibiotic treatment can play two different roles as regards persistent cells: it can either select a pre-existing persistent subpopulation or induce the persistent phenotype [45]. The hypothesis has also been advanced that antibiotics exert a biphasic dose-dependent action, i.e. inhibition of bacterial growth at high (≥ the MIC) doses

*Differences between culturable persistent and VBNC cells after stressor removal and nutrient restoration. Whereas persistent cells quickly begin to grow and divide again, VBNC cells require exposure to a growth activator (the resuscitation-promoting factor) before regaining full metabolic activity and doubling ability. The* 

*progeny of both cell types will contain a mixed population as the starting culture.*

**38**

**Figure 2.**

Given the wide phenotypic variability of *P. aeruginosa*, encompassing difficult-to grow phenotypes, several culture-independent approaches have been devised to provide reliable infection diagnosis. DNA-based techniques are useful because they are able to detect the whole bacterial population. Most protocols are based on PCR or qPCR assays [63].

To find a suitable target gene on which to base *P. aeruginosa* detection, most protocols have been tested on a variety of bacterial isolates of different origins. The *oprL* gene, encoding a peptidoglycan-associated protein, has long been considered as one of the best targets [64–67]. However, its specificity was questioned when Anuj and colleagues [68] obtained cross-reactions with other species. Notably, the selection of multiple targets is considered as the best approach to *P. aeruginosa* detection, since it excludes false-negative results due to mutations in the amplified gene sequences. The *gyrB* and *ecfX* genes are two other widely used targets. The former gene encodes the DNA gyrase subunit B. Tests against several CF *P. aeruginosa* isolates have identified a species-specific internal sequence [69, 70]. The *ecfX* gene – found in 19 copies/genome – encodes a σ factor belonging to the ECF subfamily, which is involved in the synthesis of proteins with an extracytoplasmic function and seems to play a role in *P. aeruginosa* haem uptake and virulence [71]. The gene has been reported to be specific of *P. aeruginosa* and has been used to achieve its detection in environmental as well as clinical samples [6, 71]. Further proposed targets are the *algD* gene [72] and some 16 s [73] and 23 s rDNA sequences [74].

After reliable detection, a key issue is direct pathogen quantification in sputum samples. Most of the work in this field has been performed after 2010 using specific extraction kits and lysis protocols [66, 67, 74–76].

The main drawback of DNA-based approaches is that they do not detect only live bacterial cells and may be affected by the presence of dead cells as well as by eDNA [77]. An efficient and widely used approach to this problem is to treat samples with propidium monoazide before DNA extraction [66]. The dye penetrates the cells via damaged walls/membranes and binds their DNA; after photo-activation, binding to the nucleic acids prevents DNA polymerase binding, hence DNA amplification in PCR assays. Since live cells commonly have an intact wall, they are not affected by the dye and only their DNA is detected. The same objective can be achieved with other treatments such as ethidium monoazide and DNase [6, 77].

Despite some drawbacks, DNA-based methods provide additional valuable information to the cell culture results when investigating and monitoring *P. aeruginosa* colonization dynamics in the CF lung [78]. Accordingly, extensive metagenomic studies of the CF microbiota have highlighted that persistent cells play a major role in infection chronicization and that persistence is favored by alterations in bacterial gene expression [79], further stressing the value of molecular techniques in routine diagnostics [80, 81].

Another useful technique capable of providing direct bacterial quantification is flow cytometry. Although it has mostly been employed to investigate bacterial physiology and metabolic responses [82], efforts to optimize its quantification ability have made it suitable for some diagnostic applications [83]. In particular, flow cytometry analysis and imaging now enable detection and enumeration of non-culturable and intracellular *P. aeruginosa* cells [84, 85].

Other approaches to detect the whole microbial community of CF lung have also been developed and in the last years indirect detection has been also achieved by metabolomic methods targeting specific bacterial metabolites as pathogen footprints [86].

#### **4.2 Evidence of the presence of VBNC** *P. aeruginosa* **in CF sputum**

The presence of VBNC *P. aeruginosa* cells in the CF lung and in particular their role in infection recurrence are highly controversial. However, the induction of VBNC cells in the CF lung environment currently seems to be the most likely explanation for the failure of infection eradication in the presence of a negative microbiological diagnosis [6].

The first reports of pathogen persistence in patients with negative sputum cultures, published by Schelstraete and Deschaght and colleagues [87], described the swift reappearance of the same *P. aeruginosa* strain, after a brief interval of ostensible resolution, in patients treated with eradication therapy. Deschaght and co-workers [66] subsequently demonstrated that the pathogen could be detected by qPCR much earlier than by culture assays and that qPCR was able to detect a high percentage (62%) of non-culturable *P. aeruginosa* cells in sputum samples from patients who had received the first week of antibiotic treatment. A discrepancy between culture-based and culture-independent methods has also been reported by Le Gall [75] and Héry-Arnaud [76] who advanced the hypothesis of a shift of bacterial cells to a non-culturable state. A positive qPCR assay preceding a positive culture has also been described by McCulloch and colleagues [74] and, more recently, by Boutin and co-workers [88].

Our group has carried out extensive work to identify and quantify VBNC *P. aeruginosa* cells in CF sputum [6]. Combining two previously published *ecfX*-targeting primers we obtained a new amplicon (145 bp) suitable for qPCR. Testing of the new primer pair against a panel of 115 *P. aeruginosa* strains of different origins and other Gram-negative bacterial species failed to elicit a cross-reaction, confirming the species specificity of the selected target. Moreover – even though the use of a single target gene cannot exclude false-negative results due to target mutations [68] – the *ecfX* sequence yielded a positive PCR result in 111/115 (96.6%) of the *P. aeruginosa* strains and the use of a second target gene (*gyrB*) did not lead to an increase of *P. aeruginosa* detection ([89] unpublished data).

Total DNA was extracted from CF sputum samples using the QIAamp DNA kit (Qiagen, Hilden, Germany) and qPCR assays were performed using a SYBR Green reaction format. The sensitivity of the protocol combining DNA extraction and qPCR was determined by testing *P. aeruginosa*-free sputum samples inoculated with

**41**

**Figure 3.**

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

serial dilutions of log phase *P. aeruginosa* cultures. Protocol sensitivity was 70 cells/ ml, which is comparable to the sensitivity of TaqMan probe-based qPCR assays [90]. Its limit of detection, determined by amplifying serial dilutions of a purified *ecfX* amplicon, was 5.2 x 10−9 ng/reaction, corresponding to about 140 cells/ml in

eDNA interference was excluded by treating samples with DNase I (18 U) before DNA extraction. Preliminary assays were performed using *P. aeruginosa*-free sputum samples inoculated with 10% live and 90% dead *P. aeruginosa* cultures. DNasetreated and untreated aliquots were processed using an in-house crude extraction

The qPCR counts of DNase-treated aliquots always matched the live cell quota

We performed the same procedure in 88 CF sputum samples from 55 patients. The qPCR and culture-based counts were largely comparable (i.e. 78.41% of all samples, 43.18% culture-negative and 35.23% culture-positive). Notably, the absence of samples that were simultaneously culture-positive and qPCR-negative excluded false negatives. The most interesting results were those where the qPCR count exceeded the culture-based count (11.40% of samples) and those where culture-negative samples showed a qPCR-positive result (10.23%). Given eDNA exclusion by DNase treatment and DNA extraction procedure, the discrepancy was held to reflect the presence of VBNC *P. aeruginosa* cells, in line with data reported by Deschaght [66], Le Gall [75], McCulloch [74] and Boutin [88]. Crucially, 1 and 3 months after the PCR-positive results, the cultures turned

*(Modified from Ref. [6]) Detection of live* P. aeruginosa *cells in DNase-treated sputum samples.* P. aeruginosa *abundance was quantified by qPCR in CF sputum samples inoculated with 10% live and 90% dead cultures with/without DNase I pretreatment. DNA was extracted with a crude extraction procedure or with the* 

*QIAlamp extraction kit. The qPCR counts were compared to the whole bacterial inoculum.*

(10%) of the *P. aeruginosa* inoculum. As regards the untreated aliquots, they corresponded to the whole *P. aeruginosa* load (100%) when qPCR was performed on crude extracts, whereas qPCR performed on DNA extracted with the QIAamp kit yielded counts that were comparable to those obtained after DNase pretreatment. This is likely due to the fact that the eDNA of dead *P. aeruginosa* cells was too damaged to be efficiently bound and retained in the extraction column. It can thus be assumed that DNA extraction with suitable commercial kits – whether alone or combined with DNase treatment – excluded eDNA and provided reliable

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

procedure or the QIAamp extraction kit (**Figure 3**).

quantification of live bacterial cells.

positive in 2 patients.

the original samples.

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

serial dilutions of log phase *P. aeruginosa* cultures. Protocol sensitivity was 70 cells/ ml, which is comparable to the sensitivity of TaqMan probe-based qPCR assays [90]. Its limit of detection, determined by amplifying serial dilutions of a purified *ecfX* amplicon, was 5.2 x 10−9 ng/reaction, corresponding to about 140 cells/ml in the original samples.

eDNA interference was excluded by treating samples with DNase I (18 U) before DNA extraction. Preliminary assays were performed using *P. aeruginosa*-free sputum samples inoculated with 10% live and 90% dead *P. aeruginosa* cultures. DNasetreated and untreated aliquots were processed using an in-house crude extraction procedure or the QIAamp extraction kit (**Figure 3**).

The qPCR counts of DNase-treated aliquots always matched the live cell quota (10%) of the *P. aeruginosa* inoculum. As regards the untreated aliquots, they corresponded to the whole *P. aeruginosa* load (100%) when qPCR was performed on crude extracts, whereas qPCR performed on DNA extracted with the QIAamp kit yielded counts that were comparable to those obtained after DNase pretreatment. This is likely due to the fact that the eDNA of dead *P. aeruginosa* cells was too damaged to be efficiently bound and retained in the extraction column. It can thus be assumed that DNA extraction with suitable commercial kits – whether alone or combined with DNase treatment – excluded eDNA and provided reliable quantification of live bacterial cells.

We performed the same procedure in 88 CF sputum samples from 55 patients.

The qPCR and culture-based counts were largely comparable (i.e. 78.41% of all samples, 43.18% culture-negative and 35.23% culture-positive). Notably, the absence of samples that were simultaneously culture-positive and qPCR-negative excluded false negatives. The most interesting results were those where the qPCR count exceeded the culture-based count (11.40% of samples) and those where culture-negative samples showed a qPCR-positive result (10.23%). Given eDNA exclusion by DNase treatment and DNA extraction procedure, the discrepancy was held to reflect the presence of VBNC *P. aeruginosa* cells, in line with data reported by Deschaght [66], Le Gall [75], McCulloch [74] and Boutin [88]. Crucially, 1 and 3 months after the PCR-positive results, the cultures turned positive in 2 patients.

#### **Figure 3.**

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

non-culturable and intracellular *P. aeruginosa* cells [84, 85].

**4.2 Evidence of the presence of VBNC** *P. aeruginosa* **in CF sputum**

niques in routine diagnostics [80, 81].

footprints [86].

microbiological diagnosis [6].

recently, by Boutin and co-workers [88].

*aeruginosa* detection ([89] unpublished data).

Despite some drawbacks, DNA-based methods provide additional valuable information to the cell culture results when investigating and monitoring *P. aeruginosa* colonization dynamics in the CF lung [78]. Accordingly, extensive metagenomic studies of the CF microbiota have highlighted that persistent cells play a major role in infection chronicization and that persistence is favored by alterations in bacterial gene expression [79], further stressing the value of molecular tech-

Another useful technique capable of providing direct bacterial quantification is flow cytometry. Although it has mostly been employed to investigate bacterial physiology and metabolic responses [82], efforts to optimize its quantification ability have made it suitable for some diagnostic applications [83]. In particular, flow cytometry analysis and imaging now enable detection and enumeration of

Other approaches to detect the whole microbial community of CF lung have also been developed and in the last years indirect detection has been also achieved by metabolomic methods targeting specific bacterial metabolites as pathogen

The presence of VBNC *P. aeruginosa* cells in the CF lung and in particular their role in infection recurrence are highly controversial. However, the induction of VBNC cells in the CF lung environment currently seems to be the most likely explanation for the failure of infection eradication in the presence of a negative

The first reports of pathogen persistence in patients with negative sputum cultures, published by Schelstraete and Deschaght and colleagues [87], described the swift reappearance of the same *P. aeruginosa* strain, after a brief interval of ostensible resolution, in patients treated with eradication therapy. Deschaght and co-workers [66] subsequently demonstrated that the pathogen could be detected by qPCR much earlier than by culture assays and that qPCR was able to detect a high percentage (62%) of non-culturable *P. aeruginosa* cells in sputum samples from patients who had received the first week of antibiotic treatment. A discrepancy between culture-based and culture-independent methods has also been reported by Le Gall [75] and Héry-Arnaud [76] who advanced the hypothesis of a shift of bacterial cells to a non-culturable state. A positive qPCR assay preceding a positive culture has also been described by McCulloch and colleagues [74] and, more

Our group has carried out extensive work to identify and quantify VBNC *P. aeruginosa* cells in CF sputum [6]. Combining two previously published *ecfX*-targeting primers we obtained a new amplicon (145 bp) suitable for qPCR. Testing of the new primer pair against a panel of 115 *P. aeruginosa* strains of different origins and other Gram-negative bacterial species failed to elicit a cross-reaction, confirming the species specificity of the selected target. Moreover – even though the use of a single target gene cannot exclude false-negative results due to target mutations [68] – the *ecfX* sequence yielded a positive PCR result in 111/115 (96.6%) of the *P. aeruginosa* strains and the use of a second target gene (*gyrB*) did not lead to an increase of *P.* 

Total DNA was extracted from CF sputum samples using the QIAamp DNA kit (Qiagen, Hilden, Germany) and qPCR assays were performed using a SYBR Green reaction format. The sensitivity of the protocol combining DNA extraction and qPCR was determined by testing *P. aeruginosa*-free sputum samples inoculated with

**40**

*(Modified from Ref. [6]) Detection of live* P. aeruginosa *cells in DNase-treated sputum samples.* P. aeruginosa *abundance was quantified by qPCR in CF sputum samples inoculated with 10% live and 90% dead cultures with/without DNase I pretreatment. DNA was extracted with a crude extraction procedure or with the QIAlamp extraction kit. The qPCR counts were compared to the whole bacterial inoculum.*
