PMID- 36246234
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20221019
IS  - 1664-302X (Print)
IS  - 1664-302X (Electronic)
IS  - 1664-302X (Linking)
VI  - 13
DP  - 2022
TI  - Spectral imaging and nucleic acid mimics fluorescence in situ hybridization 
      (SI-NAM-FISH) for multiplex detection of clinical pathogens.
PG  - 976639
LID - 10.3389/fmicb.2022.976639 [doi]
LID - 976639
AB  - The application of nucleic acid mimics (NAMs), such as locked nucleic acid (LNA) 
      and 2'-O-methyl-RNA (2'OMe), has improved the performance of fluorescence in situ 
      hybridization (FISH) methods for the detection/location of clinical pathogens 
      since they provide design versatility and thermodynamic control. However, an 
      important limitation of FISH techniques is the low number of distinguishable 
      targets. The use of filters in fluorescence image acquisition limits the number 
      of fluorochromes that can be simultaneously differentiated. Recent advances in 
      fluorescence spectral image acquisition have allowed the unambiguous 
      identification of several microorganisms in a single sample. In this work, we 
      aimed to combine NAM-FISH and spectral image analysis to develop and validate a 
      new FISH variant, the spectral imaging-NAM-FISH (SI-NAM-FISH), that allows a 
      multiplexed, robust and rapid detection of clinical pathogens. In the first 
      stage, to implement/validate the method, we have selected seven fluorochromes 
      with distinct spectral properties and seven bacterial species (Pseudomonas 
      aeruginosa, Citrobacter freundii, Staphylococcus aureus, Enterococcus faecalis, 
      Klebsiella pneumoniae, Escherichia coli, and Acinetobacter calcoaceticus). As a 
      strong variation in fluorescence intensities is found between species and between 
      fluorochromes, seven versions of a EUB LNA/2'OMe probe, each conjugated to one of 
      seven fluorochromes, were used to rank species/fluorochromes by FISH and then 
      optimize species/fluorochrome pairing. Then, final validation tests were 
      performed using mixed populations to evaluate the potential of the technique for 
      separating/quantifying the different targets. Overall, validation tests with 
      different proportions of bacteria labeled with the respective fluorochrome have 
      shown the ability of the method to correctly distinguish the species.
CI  - Copyright (c) 2022 Azevedo, Fernandes, Faria, Silvestre, Nieder, Lou, Wengel, 
      Almeida and Azevedo.
FAU - Azevedo, Andreia S
AU  - Azevedo AS
AD  - LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and 
      Energy, Faculty of Engineering, University of Porto, Porto, Portugal.
AD  - ALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, 
      University of Porto, Porto, Portugal.
AD  - i3S-Instituto de Investigacao e Inovacao em Saude, Universidade do Porto, Porto, 
      Portugal.
AD  - IPATIMUP-Instituto de Patologia e Imunologia Molecular, Universidade do Porto, 
      Porto, Portugal.
FAU - Fernandes, Ricardo M
AU  - Fernandes RM
AD  - LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and 
      Energy, Faculty of Engineering, University of Porto, Porto, Portugal.
AD  - ALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, 
      University of Porto, Porto, Portugal.
AD  - INIAV, IP-National Institute for Agrarian and Veterinary Research, Vila Do Conde, 
      Portugal.
FAU - Faria, Ana R
AU  - Faria AR
AD  - INL International Iberian Nanotechnology Laboratory, Av Mestre Jose Veiga s/n, 
      Braga, Portugal.
FAU - Silvestre, Oscar F
AU  - Silvestre OF
AD  - INL International Iberian Nanotechnology Laboratory, Av Mestre Jose Veiga s/n, 
      Braga, Portugal.
FAU - Nieder, Jana B
AU  - Nieder JB
AD  - INL International Iberian Nanotechnology Laboratory, Av Mestre Jose Veiga s/n, 
      Braga, Portugal.
FAU - Lou, Chenguang
AU  - Lou C
AD  - Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and 
      Pharmacy, University of Southern Denmark, Campusvej 55, Odense, Denmark.
FAU - Wengel, Jesper
AU  - Wengel J
AD  - Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry and 
      Pharmacy, University of Southern Denmark, Campusvej 55, Odense, Denmark.
FAU - Almeida, Carina
AU  - Almeida C
AD  - LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and 
      Energy, Faculty of Engineering, University of Porto, Porto, Portugal.
AD  - ALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, 
      University of Porto, Porto, Portugal.
AD  - INIAV, IP-National Institute for Agrarian and Veterinary Research, Vila Do Conde, 
      Portugal.
FAU - Azevedo, Nuno F
AU  - Azevedo NF
AD  - LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and 
      Energy, Faculty of Engineering, University of Porto, Porto, Portugal.
AD  - ALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, 
      University of Porto, Porto, Portugal.
LA  - eng
PT  - Journal Article
DEP - 20220929
PL  - Switzerland
TA  - Front Microbiol
JT  - Frontiers in microbiology
JID - 101548977
PMC - PMC9557775
OTO - NOTNLM
OT  - bacteria
OT  - clinical pathogens
OT  - epifluorescence microscope
OT  - fluorescence in situ hybridization
OT  - multiplex detection
OT  - nucleic acid mimics
COIS- The authors declare that the research was conducted in the absence of any 
      commercial or financial relationships that could be construed as a potential 
      conflict of interest.
EDAT- 2022/10/18 06:00
MHDA- 2022/10/18 06:01
PMCR- 2022/09/29
CRDT- 2022/10/17 04:28
PHST- 2022/06/23 00:00 [received]
PHST- 2022/09/08 00:00 [accepted]
PHST- 2022/10/17 04:28 [entrez]
PHST- 2022/10/18 06:00 [pubmed]
PHST- 2022/10/18 06:01 [medline]
PHST- 2022/09/29 00:00 [pmc-release]
AID - 10.3389/fmicb.2022.976639 [doi]
PST - epublish
SO  - Front Microbiol. 2022 Sep 29;13:976639. doi: 10.3389/fmicb.2022.976639. 
      eCollection 2022.