In CF lung infection, the possible shift of
P. aeruginosa to a dormant, non-culturable form [
4] can hamper pathogen eradication, leading to recurrent episodes of relapse [
19]. The urgent need for a reliable routine technique capable of detecting all the different forms of
P. aeruginosa is thus widely acknowledged. However, none of the protocols devised to date has proved entirely satisfactory, due to suboptimal specificity and sensitivity [
20] and to their inability to discriminate between live and dead cells or to exclude the possible interference of eDNA in bacterial counts [
11].
In this study, we described a new real-time PCR targeting
ecfX, which amplifies a 145 bp sequence, and demonstrated that it reliably detected and quantified
P. aeruginosa in CF sputum samples. Notably, none of the previously described
ecfX-targeting qPCR protocols [
7,
15,
21], used alone, has provided the desired specificity and sensitivity, possibly because they targeted different sequences in the same gene. The SYBR Green
ecfX-targeting PCR described herein provided several major advantages compared with routine culture, including faster (a few hours) and reliable pathogen detection and a very high sensitivity threshold (70 cells/ml vs. 100 CFU/ml) that was comparable to the one reported for probe-based assays [
22]. With regard to DNA purity and abundance, spiking assays performed with live and dead cells after excluding the presence of PCR inhibitors, showed that qPCR counts matched the amount of the live inoculum. Conservation of the target sequence was supported by its detection in all the CP samples analysed and in 96.6% of the
P. aeruginosa strains isolated from samples of different origins; on the other hand, the 19
ecfX copies contained in the
P. aeruginosa genome make mutational events affecting all of them unlikely. In addition, the absence of cross-reaction with similar bacterial species supports protocol specificity. These data, together with our previous report of real-time PCRs targeting the
P. aeruginosa genes
gyrB and
oprL [
23], highlight the efficiency of the qPCR protocol described here, which reliably detected
P. aeruginosa by targeting a single gene. Experiments are currently under way to achieve further improvements, both by designing new primer pairs inside the 145 bp amplicon and by developing an
ecfX Taqman probe; sequencing of 23S rDNA amplicons is also being performed to confirm
P. aeruginosa identification by the newly-developed PCR protocol [
24].
eDNA detection is a longstanding limitation in qPCR assays. Treatment of biofilm samples with propidium or ethidium monoazide before DNA extraction [
25] has proved unsatisfactory [
26]. The DNase I digestion assays performed in the present study provided similar qPCR counts of both digested and undigested aliquots of spiked sputum samples and demonstrated that the counts corresponded with the live bacterial population. These findings, coupled with the results obtained in untreated and DNase-treated aliquots of 26 clinical CF sputum samples, suggest that the DNA extraction procedure from sputum samples efficiently excluded eDNA as well as DNA from dead bacterial cells, which may be too small and/or too damaged to be retained by the DNA purification column.
The discrepancy between the culture-based and the qPCR approach, i.e.
P. aeruginosa detection in 9/47 CN samples and the greater bacterial abundance found in 10/41 CP samples, supports the ability of the proposed PCR protocol to provide a fast and reliable diagnosis and suggests that VBNC forms of
P. aeruginosa may be found in CF samples, in line with earlier works [
4,
5,
23,
27]. Moreover, the ability of antibiotics to induce a VBNC state in biofilm-growing
Staphylococcus aureus [
28] further supports the notion that a VBNC
P. aeruginosa population may survive in the CF pulmonary biofilm subjected to extended and repeated antibiotic treatment [
19].