Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

Rodriguez, Michelle; Paul, Zubin; Wood, Charles E.; Rice, Kelly C.; Triplett, Eric W.

doi:10.3389/fmicb.2017.02491

Cited by 13 publications

(13 citation statements)

References 7 publications

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“…Bacterial strains, listed in Table S 1 , were maintained as stocks in −80 °C. The plasmid pSGFPS1 37 was used to create GFP expressing S. aureus 8325-4, using trimethoprim (25 µg/mL) supplemented cultures when needed. Tryptic Soy Broth (TSB) and Brain Heart Infusion broth (BHI) were prepared according to suppliers’ instruction (Sigma-Aldrich, Sweden).…”

Section: Methodsmentioning

confidence: 99%

Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time

Butina

Tomac

Choong

et al. 2020

npj Biofilms Microbiomes

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Methods for bacterial detection are needed to advance the infection research and diagnostics. Based on conformation-sensitive fluorescent tracer molecules, optotracing was recently established for dynamic detection and visualization of structural amyloids and polysaccharides in the biofilm matrix of gram-negative bacteria. Here, we extend the use of optotracing for detection of gram-positive bacteria, focussing on the clinically relevant opportunistic human pathogen Staphylococcus aureus. We identify a donor-acceptor-donor-type optotracer, whose binding-induced fluorescence enables real-time detection, quantification, and visualization of S. aureus in monoculture and when mixed with gram-negative Salmonella Enteritidis. An algorithm-based automated high-throughput screen of 1920 S. aureus transposon mutants recognized the cell envelope as the binding target, which was corroborated by super-resolution microscopy of bacterial cells and spectroscopic analysis of purified cell wall components. The binding event was essentially governed by hydrophobic interactions, which permitted custom-designed tuning of the binding selectivity towards S. aureus versus Enterococcus faecalis by appropriate selection of buffer conditions. Collectively this work demonstrates optotracing as an enabling technology relevant for any field of basic and applied research, where visualization and detection of S. aureus is needed.

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Section: Methodsmentioning

confidence: 99%

Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time

Butina

Tomac

Choong

et al. 2020

npj Biofilms Microbiomes

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“…Nevertheless, the use of EGFP-expressing S. aureus strains may be easier, more specific and more accurate than live labelling of bacteria with fluorescent dyes or cell-permeable nuclear probes [15,19]. Although only EGFP was tested in this study, the pBSU101 plasmid gives opportunity to replace the egfp gene by another gene coding for other fluorescent proteins such as yellow fluorescent protein or mCherry that have been found suitable to label different S. aureus laboratory strains (RN 4220, SH1000 and SH1001) [20,21].…”

Section: Discussionmentioning

confidence: 99%

Construction and validation of EGFP-expressingStaphylococcus aureusclinical strains for adhesion and internalization assays on epithelial cells

Charaoui-Boukerzaza

Rigaill

et al. 2019

Preprint

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22Background: Staphylococcus aureus is both a major pathogen and a commensal bacterium in 23 humans. It is able to adhere at the surface of epithelial cells of the anterior nares and can trigger 24 its internalization inside these non-professional phagocytic cells. To better understand the 25 interactions of clinical isolates with keratinocytes in the anterior nares, we developed and 26 validated a one-step protocol expressing enhanced green fluorescent protein (EGFP) in S. 27 aureus clinical strains with the aim to study adhesion to and internalization into mammalian 28 cells. 29Methods: Twenty S. aureus clinical isolates belonging to clonal complexes 5, 8, 30, 45, 398 30 were selected for one-step transformation protocol with the EGFP-encoding plasmid pBSU101. 31 EGFP expression was analysed by flow cytometry and confocal microscopy. Wild type and 32 isogenic EGFP-expressing strains were compared for adhesion and internalization levels by 33 using the HaCaT cell model. 34Results: Transformation was achieved in all the S. aureus strains regardless of their genetic 35 background. The flow cytometry analysis showed that the mean proportion of EGFP-expressing 36 bacteria was 97.2% (± 2.1) after 4h of incubation. Adhesion and internalization levels were 37 similar in wild-type and isogenic EGFP-expressing S. aureus strains. Confocal laser scanning 38 microscopy confirmed that EGFP-expressing S. aureus bacteria could be easily identified inside 39HaCaT keratinocytes. 40 Conclusion:This study reports an efficient protocol for expressing EGFP in S. aureus clinical 41 strains and demonstrates that these EGFP-expressing strains are suitable for adhesion and 42 internalization assays using HaCaT cells, which allows to perform static and dynamic in vitro 43 studies of S. aureus colonization. 44

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“…First, leukocyte count and differentiation were measured using the Cell-Dyn automatic hematology analyzer. Then Staphylococcus Aureus strain expressing green fluorescent protein (S. Aureus-GFP) [47] was added to samples with a bacteria-to-phagocytes ratio (multiplicity of infection [MOI]) of 1 and 10 based on measured phagocyte numbers. The whole blood with bacteria was shaken for 40 minutes at 37<C. After 20 and 40 minutes, part of the sample was removed and put on ice.…”

Section: Prospective Analysismentioning

confidence: 99%

Fragile neutrophils in surgical patients: A phenomenon associated with critical illness

et al. 2020

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Leukocyte viability (determined by e.g. propidium iodide [PI] staining) is automatically measured by hematology analyzers to check for delayed bench time. Incidental findings in fresh blood samples revealed the existence of leukocytes with decreased viability in critically ill surgical patients. Not much is known about these cells and their functional and/or clinical implications. Therefore, we investigated the incidence of decreased leukocyte viability, the implications for leukocyte functioning and its relation with clinical outcomes. An automated alarm was set in a routine hematology analyzer (Cell-Dyn Sapphire) for the presence of non-viable leukocytes characterized by increased fluorescence in the PI-channel (FL3:630 ±30nm). Patients with non-viable leukocytes were prospectively included and blood samples were drawn to investigate leukocyte viability in detail and to investigate leukocyte functioning (phagocytosis and responsiveness to a bacterial stimulus). Then, a retrospective analysis was conducted to investigate the incidence of fragile neutrophils in the circulation and clinical outcomes of surgical patients with fragile neutrophils hospitalized between 2013-2017. A high FL3 signal was either caused by 1) neutrophil autofluorescence which was considered false positive, or by 2) actual non-viable PI-positive neutrophils in the blood sample. These two causes could be distinguished using automatically generated data from the hematology analyzer. The non-viable (PI-positive) neutrophils proved to be viable (PInegative) in non-lysed blood samples, and were therefore referred to as 'fragile neutrophils'. Overall leukocyte functioning was not impaired in patients with fragile neutrophils. Of the 11 872 retrospectively included surgical patients, 75 (0.63%) were identified to have fragile neutrophils during hospitalization. Of all patients with fragile neutrophils, 75.7% developed an infection, 70.3% were admitted to the ICU and 31.3% died during hospitalization. In conclusion, fragile neutrophils occur in the circulation of critically ill surgical patients. These cells can be automatically detected during routine blood analyses and are an indicator of critical illness.

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Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

Cited by 13 publications

References 7 publications

Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time

Optotracing for selective fluorescence-based detection, visualization and quantification of live S. aureus in real-time

Construction and validation of EGFP-expressingStaphylococcus aureusclinical strains for adhesion and internalization assays on epithelial cells

Fragile neutrophils in surgical patients: A phenomenon associated with critical illness

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