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Optimizing locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH) procedure for bacterial detection.

ABSTRACT: Despite the successful application of LNA/2'OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2'OMe hybridization step-hybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria (Enterococcus faecalis and Staphylococcus epidermidis), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2'OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2'OMe and 64% of GC content, should be use in initial optimization of new LNA/2'OMe-FISH protocols.

SUBMITTER: Azevedo AS

PROVIDER: S-EPMC6544301 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Optimizing locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH) procedure for bacterial detection.

Azevedo Andreia S AS Sousa Inês M IM Fernandes Ricardo M RM Azevedo Nuno F NF Almeida Carina C

PloS one 20190531 5

Despite the successful application of LNA/2'OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2'OMe hybridization step-hybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed fo ...[more]

PMID: 31150460

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Project description:BackgroundOur current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data.Methodology/principal findingsWe employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm(2)) for 48 h biofilm: E. coli 2,1 × 10(8) (± 2,4 × 10(7)); L. monocytogenes 6,8 × 10(7) (± 9,4 × 10(6)); and S. enterica 1,4 × 10(6) (± 4,1 × 10(5))]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica.SignificanceWhile PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.

Dataset Information

Optimizing locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH) procedure for bacterial detection.

Publications

Optimizing locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH) procedure for bacterial detection.

Similar Datasets

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