Lorenzen, Jan

Rasmussen, Anne K. I.

Thomsen, Trine Rolighed

Wolff, Tine Yding

English

    Aalborg UniversitetDetection of Bacterial Gene Expression by mRNA PNA FISHA New User Friendly and Rapid Molecular TestRasmussen, Anne K. I.; Wolff, Tine Yding; Thomsen, Trine Rolighed; Lorenzen, JanPublication date:2012Document VersionEarly version, also known as pre-printLink to publication from Aalborg UniversityCitation for published version (APA):Rasmussen, A. K. I., Wolff, T. Y., Thomsen, T. R., & Lorenzen, J. (2012). Detection of Bacterial GeneExpression by mRNA PNA FISH: A New User Friendly and Rapid Molecular Test. Poster presented at 14thInternational Symposium on Microbial Ecology, Copenhagen, Denmark.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: December 26, 2020Detection of Bacterial Gene Expression by mRNA PNA FISH –  A New User Friendly and Rapid Molecular Test Anne K I Rasmussen1, Tine Yding Wolff2, Trine Rolighed Thomsen2,3, Jan Lorenzen2 1:AdvanDx, 2:The Danish Technological Institute, 3: Aalborg University      Methods so far  Introduction Conclusion • Fluorescence in situ hybridization (FISH) is primarily used for microbial species identification.  • Bacterial mRNA molecules which hold information on expressed functional genes e.g. antibiotic resistance have a much shorter half-life (often only few minutes) than rRNA and are generally much less abundant (1% or less of rRNA abundance).   • Therefore, so far it has only been possible to target and visualize microbial mRNA using very laborious protocols.   Objective The aim was to develop a PNA FISH protocol to actually visualize the mRNA molecules responsible for the antibiotic resistance (mecA) in methicillin resistant Staphylococcus aureus (MRSA) in an end-user friendly and rapid assay format (2 hrs) and combine this with species identity.   Results Results   This work was supported by the Ministry of Science Technology and Innovation. We thank Masumeh Chavoshi, Helle Andersen, Allan Mortensen, Berit Kummerfeldt for valuable technical assistance.  ACKNOWLEDGEMENTS . • The mecA PNA FISH assay was positive for all strains that exhibited baseline values above 10-15 mecA copies/10.000 16S rRNA copies.  • The assay showed 100% (13/13) sensitivity and 100% specificity (14/14) for identification of MRSA directly from S. aureus-positive blood culture bottles. Validation As a benchmark for the mRNA PNA FISH assay reverse transcriptase quantitative PCR (RT-qPCR) measurements of both 16S rRNA and mecA mRNA were used.    Induction in tube – 30 minutes Add blood culture from ventilation needle to the tube  Add pre-mixed cefoxitin/TSB solution to the tube.  Incubate at 35±2⁰C for 40 min.   Fixation – 10 minutes Add 10 μL of induced blood culture to the well on the microscope slide. Add fixation solution to each well on microscope slide and mix gently to    emulsify. Heat slides at minimum 80⁰C for 2 min. and transfer slides to PNA FISH Workstation pre-heated to 55⁰C±1⁰C. Add 100 μL (or fill slides well) 100% MeOH to the slide well. Incubate slides for 5 min. on PNA FISH Workstation at 55⁰C±1⁰C .    Hybridization – 30 min. Same as current PNA FISH   Rinse, Stringent Wash, Mount and Examination – 35-40 min. Same as current PNA FISH for Gram-negative rods.      • The mRNA PNA FISH technique is a new molecular test that provides a phenotypic antibiotic resistance answer.   • Great potential for coupling species identity to expression of selected genes on single cell level.  • Can shed light on heterogeneities in gene expression for instance in complex and stratified microbial systems.   • The method has great potential in different clinical applications but also in industrial and environmental settings. b c a A                                                            B              C Fig 1: Uninduced MRSA show strain dependent mecA expression in PNA FISH. Red (negative): Strains that do not express the mecA gene without induction. Green (positive): Strains that express the mecA gene constitutively. Blue (neg/pos): Strains that both show individual cells with  and without mecA expression. All strains were mecA positive upon induction with the antibiotic cefoxitin.  Fig 2: Simultaneous determination of species and gene expression. The 16S rRNA of S. aureus was targeted with a red probes and mecA mRNA with green probes.   Panel A: mecA mRNA in a sample with only methicillin resistant S. aureus (MRSA).          Panel B: Simultaneous detection oin a mixture of MRSA and methicillin susceptible S. aureus (MSSA) using both probes. MRSA appear yellow, because MRSA bind both the red probes for S. aureus and the green probes for mecA, while MSSA appear red, because these only bind the red species identification probe.       Panel C: Both types of probes are applied, but in this instance on a mixture of MRSA (yellow) and methicillin resistant coagulase negative staphylococci (green). The latter expresses the mecA-gene (green probe) but because they are not S. aureus they do not bind the red species identification probe (16S rRNA).  ISME Copenhagen 2012 Poster number 233A  

Detection of Bacterial Gene Expression by mRNA PNA FISH:A New User Friendly and Rapid Molecular Test

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Detection of Bacterial Gene Expression by mRNA PNA FISH:A New User Friendly and Rapid Molecular Test

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