Detection and discrimination of biofilm populations using locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH)

Andreia S Azevedo, Carina Almeida, Bruno Pereira, Pedro Madureira, Jesper Wengel, Nuno F Azevedo

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Multispecies biofilms are the dominant form in nature. The application of fluorescence in situ hybridization (FISH)-based techniques to the discrimination of biofilm populations might contribute to the understanding of microorganism interactions in these structures, and might allow the development of efficient strategies to prevent or minimize biofilm-associated diseases. This work presents the first study that develops, optimizes and validates a multiplex FISH procedure using locked nucleic acid (LNA) and 2′-O-methyl RNA (2′OMe) oligonucleotides probes for the in vitro discrimination within mixed populations. As a case study, Escherichia coli, the major cause of urinary tract infections (UTIs), and three other atypical colonizers of urinary catheters (Delftia tsuruhatensis, Achromobacter xylosoxidans and Burkholderia fungorum) with unproven pathogenic potential, were selected. Specific probes for these species were designed and optimized for specific hybridization in multiplex experiments. Results showed that the LNA/2′OMe-FISH method performed well in multiplex experiments and presented a good correlation with total and cultivability counts, regardless of the cells physiological state. In fact, the method was also able to report variations of viable but non-cultivable populations. Further analysis of mixed biofilm structures by confocal laser scanning microscopy provided a clear discrimination in three dimensions between the location of the different populations.
Original languageEnglish
JournalBiochemical Engineering Journal
Volume104
Issue number15 December
Pages (from-to)64-73
Number of pages10
ISSN1369-703X
DOIs
Publication statusPublished - 2015

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Biofilms
Fluorescence
RNA
Oligonucleotide Probes
Catheters
Microorganisms
Escherichia coli
Microscopic examination
Experiments
Scanning
locked nucleic acid
Lasers

Cite this

Azevedo, Andreia S ; Almeida, Carina ; Pereira, Bruno ; Madureira, Pedro ; Wengel, Jesper ; Azevedo, Nuno F. / Detection and discrimination of biofilm populations using locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH). In: Biochemical Engineering Journal. 2015 ; Vol. 104, No. 15 December. pp. 64-73.
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abstract = "Multispecies biofilms are the dominant form in nature. The application of fluorescence in situ hybridization (FISH)-based techniques to the discrimination of biofilm populations might contribute to the understanding of microorganism interactions in these structures, and might allow the development of efficient strategies to prevent or minimize biofilm-associated diseases. This work presents the first study that develops, optimizes and validates a multiplex FISH procedure using locked nucleic acid (LNA) and 2′-O-methyl RNA (2′OMe) oligonucleotides probes for the in vitro discrimination within mixed populations. As a case study, Escherichia coli, the major cause of urinary tract infections (UTIs), and three other atypical colonizers of urinary catheters (Delftia tsuruhatensis, Achromobacter xylosoxidans and Burkholderia fungorum) with unproven pathogenic potential, were selected. Specific probes for these species were designed and optimized for specific hybridization in multiplex experiments. Results showed that the LNA/2′OMe-FISH method performed well in multiplex experiments and presented a good correlation with total and cultivability counts, regardless of the cells physiological state. In fact, the method was also able to report variations of viable but non-cultivable populations. Further analysis of mixed biofilm structures by confocal laser scanning microscopy provided a clear discrimination in three dimensions between the location of the different populations.",
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Detection and discrimination of biofilm populations using locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH). / Azevedo, Andreia S; Almeida, Carina; Pereira, Bruno; Madureira, Pedro; Wengel, Jesper; Azevedo, Nuno F.

In: Biochemical Engineering Journal, Vol. 104, No. 15 December, 2015, p. 64-73.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Detection and discrimination of biofilm populations using locked nucleic acid/2'-O-methyl-RNA fluorescence in situ hybridization (LNA/2'OMe-FISH)

AU - Azevedo, Andreia S

AU - Almeida, Carina

AU - Pereira, Bruno

AU - Madureira, Pedro

AU - Wengel, Jesper

AU - Azevedo, Nuno F

PY - 2015

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N2 - Multispecies biofilms are the dominant form in nature. The application of fluorescence in situ hybridization (FISH)-based techniques to the discrimination of biofilm populations might contribute to the understanding of microorganism interactions in these structures, and might allow the development of efficient strategies to prevent or minimize biofilm-associated diseases. This work presents the first study that develops, optimizes and validates a multiplex FISH procedure using locked nucleic acid (LNA) and 2′-O-methyl RNA (2′OMe) oligonucleotides probes for the in vitro discrimination within mixed populations. As a case study, Escherichia coli, the major cause of urinary tract infections (UTIs), and three other atypical colonizers of urinary catheters (Delftia tsuruhatensis, Achromobacter xylosoxidans and Burkholderia fungorum) with unproven pathogenic potential, were selected. Specific probes for these species were designed and optimized for specific hybridization in multiplex experiments. Results showed that the LNA/2′OMe-FISH method performed well in multiplex experiments and presented a good correlation with total and cultivability counts, regardless of the cells physiological state. In fact, the method was also able to report variations of viable but non-cultivable populations. Further analysis of mixed biofilm structures by confocal laser scanning microscopy provided a clear discrimination in three dimensions between the location of the different populations.

AB - Multispecies biofilms are the dominant form in nature. The application of fluorescence in situ hybridization (FISH)-based techniques to the discrimination of biofilm populations might contribute to the understanding of microorganism interactions in these structures, and might allow the development of efficient strategies to prevent or minimize biofilm-associated diseases. This work presents the first study that develops, optimizes and validates a multiplex FISH procedure using locked nucleic acid (LNA) and 2′-O-methyl RNA (2′OMe) oligonucleotides probes for the in vitro discrimination within mixed populations. As a case study, Escherichia coli, the major cause of urinary tract infections (UTIs), and three other atypical colonizers of urinary catheters (Delftia tsuruhatensis, Achromobacter xylosoxidans and Burkholderia fungorum) with unproven pathogenic potential, were selected. Specific probes for these species were designed and optimized for specific hybridization in multiplex experiments. Results showed that the LNA/2′OMe-FISH method performed well in multiplex experiments and presented a good correlation with total and cultivability counts, regardless of the cells physiological state. In fact, the method was also able to report variations of viable but non-cultivable populations. Further analysis of mixed biofilm structures by confocal laser scanning microscopy provided a clear discrimination in three dimensions between the location of the different populations.

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