| Abstract | Background: Routine culture-based diagnosis of Pseudomonas aeruginosa lung infection in Cystic Fibrosis (CF) patients can be hampered by the phenotypic variability of the microorganism, including its transition to a Viable But Non-Culturable (VBNC) state. The aim of this study was to validate an ecfX-targeting qPCR protocol developed to detect all viable P. aeruginosa bacteria and to identify VBNC forms in CF sputum samples. Methods: The study involved 115 P. aeruginosa strains of different origins and 10 non-P. aeruginosa strains and 88 CF sputum samples, 41 Culture-Positive (CP) and 47 Culture-Negative (CN). Spiking assays were performed using scalar dilutions of a mixture of live and dead P. aeruginosa ATCC 9027 and a pooled P. aeruginosa-free sputum batch. Total DNA from sputum samples was extracted by a commercial kit, whereas a crude extract was obtained from the broth cultures. Extracellular DNA (eDNA) interference was evaluated by comparing the qPCR counts obtained from DNase-treated and untreated aliquots of the same samples. The statistical significance of the results was assessed by the Wilcoxon test and Student's t test. Results: The newly-developed qPCR protocol identified 96.6% of the P. aeruginosa isolates; no amplification was obtained with strains belonging to different species. Spiking assays supported protocol reliability, since counts always matched the amount of live bacteria, thus excluding the interference of dead cells and eDNA. The protocol sensitivity threshold was 70 cells/ml of the original sample. Moreover, qPCR detected P. aeruginosa in 9/47 CN samples and showed higher bacterial counts compared with the culture method in 10/41 CP samples. Conclusions: Our findings demonstrate the reliability of the newly-developed qPCR protocol and further highlight the need for harnessing a non-culture approach to achieve an accurate microbiological diagnosis of P. aeruginosa CF lung infection and a greater understanding of its evolution. |
| Text | 420944 2018 10.1186/s12879 018 3612 9 Scopus 2 s2.0 85059230625 Cystic fibrosis; Lung infection; Pseudomonas aeruginosa; Viable but non culturable bacterial forms; qPCR Detection of viable but non culturable Pseudomonas aeruginosa in cystic fibrosis by qPCR A validation study Mangiaterra G.; Amiri M.; Di Cesare A.; Pasquaroli S.; Manso E.; Cirilli N.; Citterio B.; Vignaroli C.; Biavasco F. Department of Life and Environmental Sciences, Polytechnic University of Marche, via Brecce Bianche, Ancona, 60131, , Italy; Department of Earth, Environmental and Life Sciences, University of Genoa, Corso Europa, 26, Genoa, 16132, , Italy; Microbiology Laboratory, Azienda Ospedaliero Universitaria, Ospedali Riuniti Umberto I G.M. Lancisi G. Salesi, Ancona, , Italy; Mother Child Department, Cystic Fibrosis Referral Care Center, United Hospitals, Ancona, , Italy; Department of Biomolecular Sciences Sect. Biotechnology, University of Urbino Carlo Bo, Urbino, , Italy Background Routine culture based diagnosis of Pseudomonas aeruginosa lung infection in Cystic Fibrosis CF patients can be hampered by the phenotypic variability of the microorganism, including its transition to a Viable But Non Culturable VBNC state. The aim of this study was to validate an ecfX targeting qPCR protocol developed to detect all viable P. aeruginosa bacteria and to identify VBNC forms in CF sputum samples. Methods The study involved 115 P. aeruginosa strains of different origins and 10 non P. aeruginosa strains and 88 CF sputum samples, 41 Culture Positive CP and 47 Culture Negative CN . Spiking assays were performed using scalar dilutions of a mixture of live and dead P. aeruginosa ATCC 9027 and a pooled P. aeruginosa free sputum batch. Total DNA from sputum samples was extracted by a commercial kit, whereas a crude extract was obtained from the broth cultures. Extracellular DNA eDNA interference was evaluated by comparing the qPCR counts obtained from DNase treated and untreated aliquots of the same samples. The statistical significance of the results was assessed by the Wilcoxon test and Student s t test. Results The newly developed qPCR protocol identified 96.6% of the P. aeruginosa isolates; no amplification was obtained with strains belonging to different species. Spiking assays supported protocol reliability, since counts always matched the amount of live bacteria, thus excluding the interference of dead cells and eDNA. The protocol sensitivity threshold was 70 cells/ml of the original sample. Moreover, qPCR detected P. aeruginosa in 9/47 CN samples and showed higher bacterial counts compared with the culture method in 10/41 CP samples. Conclusions Our findings demonstrate the reliability of the newly developed qPCR protocol and further highlight the need for harnessing a non culture approach to achieve an accurate microbiological diagnosis of P. aeruginosa CF lung infection and a greater understanding of its evolution. 18 Published version http //www.scopus.com/record/display.url eid=2 s2.0 85059230625 origin=inward Articolo in rivista BioMed Central, 1471 2334 BMC infectious diseases Online BMC infectious diseases Online BMC infect. dis. Online BMC infectious diseases Online BioMed Central infectious diseases Online Infectious diseases Online andrea.dicesare DI CESARE ANDREA |
