A novel flow cytometry-based assay for the quantification of Staphylococcus aureus adhesion to and invasion of eukaryotic cells

Trouillet, Sophie; Rasigade, Jean‐Philippe; Lhoste, Yannick; Ferry, Tristan; Vandenesch, François; Étienne, Jérôme; Laurent, François

doi:10.1016/j.mimet.2011.04.012

Cited by 32 publications

(31 citation statements)

References 17 publications

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“…The intracellular infection of MG-63 cells was performed as described elsewhere with modifications [60]. MG-63 cells were seeded at 50,000 cells/well in 24-well plates and incubated at 37°C with 5% CO 2 for 48 h in culture medium.…”

Section: Methodsmentioning

confidence: 99%

PSMs of Hypervirulent Staphylococcus aureus Act as Intracellular Toxins That Kill Infected Osteoblasts

et al. 2013

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Epidemic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is associated with more severe and acute forms of osteomyelitis than healthcare-associated (HA-) MRSA. Although S. aureus is now recognized as a facultative intracellular pathogen, the contribution of osteoblast invasion by CA-MRSA to the pathogenesis of osteomyelitis is unknown. Using an ex vivo model of intracellular infection of human osteoblasts, we demonstrated that CA-MRSA strains of diverse lineages share an enhanced ability to kill infected osteoblasts compared to HA-MRSA. Cytotoxicity comparisons of CA-MRSA isogenic deletion mutants revealed that phenol-soluble modulins (PSMs), a class of membrane-damaging exoproteins that are expressed at higher levels in CA-MRSA than in HA-MRSA, are involved in this osteoblast killing, whereas other major CA-MRSA virulence determinants, the Panton-Valentine leukocidin and alpha-toxin, are not involved. Similarly, functional agr and sarA regulators, which control the expression of PSMs and alpha-toxin, were required for the expression of the intracellular cytotoxic phenotype by CA-MRSA, whereas the saeRS regulator, which controls the expression of alpha-toxin but not PSMs, had no impact on cytotoxicity. Finally, PSM transcript levels determined by quantitative reverse-transcriptase PCR were significantly higher in CA-MRSA than in HA-MRSA strains and associated with cell damage in MRSA-infected osteoblasts. These findings provide new insights into the pathogenesis of severe CA-MRSA osteomyelitis and unravel a novel virulence strategy of CA-MRSA, based on the invasion and subsequent killing of osteoblasts by PSMs acting as intracellular toxins.

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Section: Methodsmentioning

confidence: 99%

PSMs of Hypervirulent Staphylococcus aureus Act as Intracellular Toxins That Kill Infected Osteoblasts

et al. 2013

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“…The S. aureus 8325-4 laboratory strain, which has been well characterized in its ability to invade osteoblasts [13], was used as a control in each osteoblast infection experiment. Prior to performing the assays, S. epidermis cells were grown overnight (18 h) in a brain heart infusion (BHI) (AES, Bruz, France) aerobically at 36.5°C.…”

Section: Methodsmentioning

confidence: 99%

Staphylococcus epidermidis in Orthopedic Device Infections: The Role of Bacterial Internalization in Human Osteoblasts and Biofilm Formation

Valour¹,

Trouillet‐Assant²,

Rasigade³

et al. 2013

PLoS ONE

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Background Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms.Materials and MethodsOrthopedic device infection S. epidermidis strains (n = 15) were compared to nasal carriage isolates (n = 22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method.ResultsNo difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/−631 and 347+/−431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups.ConclusionOsteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.

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“…The methodologies utilized in order to understand these biological processes have not greatly changed since their early development (16). However, with the advent of fluorescence technology many bacterial infection models have been analyzed using flow cytometry techniques (18)(19)(20)24). MIFC is a powerful technique that can be used for many different applications and is already being used for the analysis of other pathogenic organisms such as Plasmodium falciparum (25,26), Neisseria meningitidis (27), Yersinia pseudotuberculosis (28), Leishmania donovani (29), and Brucella melitensis (30).…”

Section: Discussionmentioning

confidence: 99%

Using multispectral imaging flow cytometry to assess an in vitro intracellular Burkholderia thailandensis infection model

et al. 2016

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The use of in vitro models to understand the interaction of bacteria with host cells is well established. In vitro bacterial infection models are often used to quantify intracellular bacterial load by lysing cell populations and subsequently enumerating the bacteria. Modern established techniques employ the use of fluorescence technologies such as flow cytometry, fluorescent microscopy, and/or confocal microscopy. However, these techniques often lack either the quantification of large data sets (microscopy) or use of gross fluorescence signal which lacks the visual confirmation that can provide additional confidence in data sets. Multispectral imaging flow cytometry (MIFC) is a novel emerging field of technology. This technology captures a bright field and fluorescence image of cells in a flow using a charged coupled device camera. It allows the analysis of tens of thousands of single cell images, making it an extremely powerful technology. Here MIFC was used as an alternative method of analyzing intracellular bacterial infection using Burkholderia thailandensis E555 as a model organism. It has been demonstrated that the data produced using traditional enumeration is comparable to data analyzed using MIFC. It has also been shown that by using MIFC it is possible to generate other data on the dynamics of the infection model rather than viable counts alone. It has been demonstrated that it is possible to inhibit the uptake of bacteria into mammalian cells and identify differences between treated and untreated cell populations. The authors believe this to be the first use of MIFC to analyze a Burkholderia bacterial species during intracellular infection.

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A novel flow cytometry-based assay for the quantification of Staphylococcus aureus adhesion to and invasion of eukaryotic cells

Cited by 32 publications

References 17 publications

PSMs of Hypervirulent Staphylococcus aureus Act as Intracellular Toxins That Kill Infected Osteoblasts

PSMs of Hypervirulent Staphylococcus aureus Act as Intracellular Toxins That Kill Infected Osteoblasts

Staphylococcus epidermidis in Orthopedic Device Infections: The Role of Bacterial Internalization in Human Osteoblasts and Biofilm Formation

Using multispectral imaging flow cytometry to assess an in vitro intracellular Burkholderia thailandensis infection model

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