Optimization of a two‐step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of <i>Staphylococcus aureus</i>

Lawson, Thomas S.; Connally, Russell; Vemulpad, Subramanyam; Piper, James A.

doi:10.1002/jcla.20486

Cited by 5 publications

(5 citation statements)

References 27 publications

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“…To support this observation, a commercially available fluorescent probe (SAU69-Alexa Fluor 488, excitation at 488 nm) was also used as a positive control for comparison staining of the S. aureus cells. The hybridisation was then optimised to maximise fluorescence [21,22,23] and luminescence yield at the lowest background emission for comparison with the LISH labelling with the SAU69 specific DNA–BHHTEGST–Eu 3+ probe. After obtaining the corresponding images, the detection sensitivity or SNR of the luminescent Europium probe (SAU69–BHHTEGST–Eu3+) in comparison with fluorescent probe (SAU69-Alexa Fluor 488) for the staining of S. aureus was investigated.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously reported an improvement to the specificity of fluorescence in situ hybridisation (FISH) detection of S. aureus . This was achieved using a simple and effective permeabilisation technique in conjunction with a conventional fluorescent organic dye, such as Alexa Fluor 488 [21,22,23]. However, imaging the FISH labelled S. aureus under fluorescent microscopy has two major limitations.…”

Section: Introductionmentioning

confidence: 99%

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Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus

et al. 2019

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We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338–BHHTEGST–Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus

et al. 2019

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“…Other studies make use of a combination of enzymes that will act over different components of the cell envelope. For instance, FISH methods for Staphylococcus apply very often a mixture of lysozyme and lysostaphin [15]. Mixtures of mutanolysin/lysozyme or lipase/proteinase K have been applied to mycolic-acid-containing bacteria (e.g.…”

Section: Fixation/permeabilizationmentioning

confidence: 99%

An Introduction to Fluorescence in situ Hybridization in Microorganisms

Almeida

Azevedo

2021

Methods in Molecular Biology

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Fluorescence in situ hybridization (FISH) is a molecular biology technique that enables the localization, quantification and identification of microorganisms in a sample. This technique has found applications in several areas, most notably the environmental, for quantification and diversity assessment of microorganisms, and the clinical, for the rapid diagnostic of infectious agents. The FISH method is based on the hybridization of a fluorescently-labelled nucleic acid probe with a complementary sequence that is present inside the microbial cell, typically in the form of ribosomal RNA (rRNA). In fact, a hybridized cell is typically only detectable because a large number of multiple fluorescent particles (as many as the number of target sequences available) are present inside the cell.In here, we will review the major steps involved in a standard FISH protocol, namely fixation/permeabilization, hybridization, washing and visualization/detection. For each step the major variables/parameters are identified and, subsequently, their impact on the overall hybridization performance is assessed in detail. .

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“…The resulting suspensions were serially diluted, and inoculated onto agar plates, specifically Plate Count Agar (PCA, Liofilchem) to quantify the total number of cultivable cells; Manitol Salt Agar (MSA, Liofilchem) and MacConkey Agar (MCA, Liofilchem) to isolate and selectively quantify S. aureus and E. coli, respectively. After 24 h incubation at 378C, the CFUs were counted and expressed as CFU cm 22 .…”

Section: Cultivable Cell Numbermentioning

confidence: 99%

“…The probe Sau 16S69 (5 0 -Alexa 546-GAAGCAAGCTTCTCGTC CG-3 0 ) detects specifically S. aureus as previously described by Poppert et al (2010) and Lawson et al (2011). 21,22 E. coli-specific probe Col23S1502 (5 0 -Alexa 488-CACGCCTC AGCCTTGATT-3 0 ) was designed based on Azevedo et al's (2015) study. 23 Both probes were purchased from Thermo Fisher Scientific.…”

Section: Fluorescent In Situ Hybridization (Fish)mentioning

confidence: 99%

Staphylococcus aureusandEscherichia colidual‐species biofilms on nanohydroxyapatite loaded with CHX or ZnO nanoparticles

Barros

Grenho

Fontenente

et al. 2016

J Biomedical Materials Res

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Implant-associated infections are caused by surface-adhering microorganisms persisting as biofilms, resistant to host defense and antimicrobial agents. Given the limited efficacy of traditional antibiotics, novel strategies may rely on the prevention of such infections through the design of new biomaterials. In this work, two antimicrobial agents applied to nanohydroxyapatite materials-namely, chlorhexidine digluconate (CHX) and zinc oxide (ZnO) nanoparticles-were compared concerning their ability to avoid single- or dual-species biofilms of Staphylococcus aureus and Escherichia coli. The resulting biofilms were quantified by the enumeration of colony-forming units and examined by confocal microscopy using both Live/Dead staining and bacterial-specific fluorescent in situ hybridization. The sessile population arrangement was also observed by scanning electron microscopy. Both biomaterials showed to be effective in impairing bacterial adhesion and proliferation for either single- or dual-species biofilms. Furthermore, a competitive interaction was observed for dual-species biofilms wherein E. coli exhibited higher proliferative capacity than S. aureus, an inverse behavior from the one observed in single-species biofilms. Therefore, either nanoHA-CHX or nanoHA-ZnO surfaces appear as promising alternatives to antibiotics for the prevention of devices-related infections avoiding the critical risk of antibiotic-resistant strains emergence. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 491-497, 2017.

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Optimization of a two‐step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of Staphylococcus aureus

Abstract: The two-step assay might be used when the FISH signal is weak, bacterial numbers are low or if there is a need to use other reporter molecules.

Cited by 5 publications

References 27 publications

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus

An Introduction to Fluorescence in situ Hybridization in Microorganisms

Staphylococcus aureusandEscherichia colidual‐species biofilms on nanohydroxyapatite loaded with CHX or ZnO nanoparticles

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