The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Selecting appropriate tools providing reliable quantitative measures of individual populations in biofilms is critical as we now recognize their true polymicrobial and heterogeneous nature. Here, plate count, quantitative real-time polymerase chain reaction (q-PCR) and peptide nucleic acid probe-fluorescence in situ hybridization (PNA-FISH) were employed to quantitate cystic fibrosis multispecies biofilms. Growth of Pseudomonas aeruginosa, Inquilinus limosus and Dolosigranulum pigrum was assessed in dual- and triple-species consortia under oxygen and antibiotic stress. Quantification methods, that were previously optimized and validated in planktonic consortia, were not always in agreement when applied in multispecies biofilms. Discrepancies in culture and molecular outcomes were observed, particularly for triple-species consortia and antibiotic-stressed biofilms. Some differences were observed, such as the higher bacterial counts obtained by q-PCR and/or PNA-FISH (?4 log10 cells/cm2) compared to culture. But the discrepancies between PNA-FISH and q-PCR data (eg D. pigrum limited assessment by q-PCR) demonstrate the effect of biofilm heterogeneity in method's reliability. As the heterogeneity in biofilms is a reflection of a myriad of variables, tailoring an accurate picture of communities? changes is crucial. This work demonstrates that at least two, but preferentially three, quantification techniques are required to obtain reliable measures and take comprehensive analysis of polymicrobial biofilm-associated infections.The authors thank the financial support from the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI-01–0145-FEDER-006684) and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 - Programa Operacional Regional do Norte. This work was also the result of the projects: (i) POCI01-0145-FEDER-006939 (Laboratory for Processing Engineering, Environment, Biotechnology and Energy – UID/EQU/00511/2013) funded by the ERDF, through COMPETE2020 and by national funds, through FCT; (ii) NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, supported by North Portugal Regional Operational Programme (NORTE2020), under the Portugal2020 Partnership Agreement, through the ERDF; (iii) Coded-FISH PTDC/DTP-PIC/4562/2014/16678; (iv) POCI-01-0145-FEDER-029841, through COMPETE2020 - Programa Operacional Competitividade e Internacionalização and by national funds, through FCT). Also, the fellowship of Susana P. Lopes SFRH/BPD/95616/2013 is acknowledged. The authors would also like to thanks to Dr Michael Surette (Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, ON, Canada) for kindly providing the I. limosus strain used in this study.info:eu-repo/semantics/publishedVersio
