Quantitative assessment of individual populations within polymicrobial biofilms

Abstract

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.

Biofilm model systems for root canal disinfection: a literature review

“…DNA extraction is another critical step, especially in a multispecies sample, since cell lysis is not equal for all the microbial cells in the sample (Lopes et al . ).…”

Time-Dependent Changes in Morphostructural Properties and Relative Abundances of Contributors in Pleurotus ostreatus/Pseudomonas alcaliphila Mixed Biofilms

“…The estimation of the relative contribution of the two partners in the makeup of a dual biofilm is very important to explain both the structural and functional properties of the system (Lopes et al, 2018). To this aim, the approach of colony counting has been used for fungal quantification in dual biofilms involving dimorphic species, such as C. glabrata and C. albicans in association with either Streptococcus mutans (Pereira-Cenci et al, 2008) or Staphylococcus aureus (Zago et al, 2015).…”

“…In a recent study aimed at comparing five different assay methods for the measurement of fungal growth, namely colony counting, determination of ergosterol or beta-N-acetylhexosaminidase activity, quantitative PCR and microscopic spore counting, the first method exhibited a coefficient of variation of replicated samples (CV r ) that was twice as high the other methods (Mensah-Attipoe et al, 2016). Moreover, marked discrepancies between plate count and molecular tools, relying on qPCR of 16S rRNA and peptide nucleic acid probe fluorescence in situ hybridization (PNA-FISH), were even observed in quantifying populations inhabiting dual and three-membered bacterial biofilms (Lopes et al, 2018). The same authors suggested that at least two quantification techniques might be needed to gain reliable estimates of the relative abundances of the biofilm’s contributors.…”

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation

“…For counting of the biofilm-associated cells, the biofilm was first washed three times with 1.0% NaCl, then scraped out from the microtiter well, and resuspended into 1.0% NaCl as described by Leuck et al 25 and Lopes et al 26 The bacterial suspension was serially 10-fold diluted with 1.0% NaCl and appropriate dilutions were plated. TSA-NaCl plates were used for plating in the experiments with single culture, whereas CHROMagar for Vibrio and CHROMagar for E. coli were used in the experiments with mixed culture.…”