A real-time impedance based method to assess Rhodococcus equi virulence.

Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP) is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains

Infection of macrophages with <i>R. equi</i> affects host cell size and morphology.

<p>Macrophage monolayers were infected with ATTO 488 labeled virulent <i>R. equi</i> 103+ or attenuated <i>R. equi</i> 103− strains at increasing multiplicities (MOI) of infection. Monolayers were fixed and labelled 24 h post-infection. The cell size (panel A) and cell roundness (panel B) of infected cells were compared to control cells (not infected, indicated as MOI = 0). Green bars: no intracellular bacteria; blue bars: 1 to 5 intracellular bacteria; red bars: more than 5 intracellular bacteria. +: <i>R. equi</i> 103+; −: <i>R. equi</i> 103−. Error bars denote the standard deviation of the mean.</p

Time-dependent Cell Response Profile (TCRP) of infected macrophage monolayers.

<p>Monolayers were infected with a virulent (<i>R. equi</i> 103+; top panel) or with an avirulent (<i>R. equi</i> 103−; bottom panel) strain of <i>R. equi</i> at increasing multiplicities (MOI) of infection. The TCRP was normalized against the TCRP of a non-infected macrophage monolayer.</p

Intracellular growth of virulent and attenuated <i>R. equi</i>.

<p>J774A.1 monolayers were infected with virulent (♦, <i>R. equi</i> 103+; ∇, <i>R. equi</i> Δ35000) and attenuated (□, <i>R. equi</i> 103−; •, <i>R. equi</i> Δ<i>vapA</i>) <i>R. equi</i> strains. Following a 1 hour incubation to allow phagocytosis, monolayers were washed and treated with vancomycin to kill remaining extra cellular bacteria. Intracellular bacteria were enumerated via real time qPCR of the <i>R. equi</i> 16S rRNA gene. Results are expressed as fold change in bacterial numbers relative to t = 0 (h) values. Error bars denote the standard deviation of the mean.</p

The morphology of macrophages changes following infection with <i>R. equi</i>.

<p>J774A.1 monolayers were infected with ATTO 488 labelled virulent <i>R. equi</i> 103+ (green). Monolayers were fixed 24 h post-infection; the actin cytoskeleton and cell nuclei were stained with Texas Red Phalloidin (red) and Hoechst 33258 (blue), respectively. Panel A: non-infected monolayers, panel B: monolayers infected with <i>R. equi</i> 103+. Arrows indicate the change in cell shape following infection with <i>R. equi</i>. The length of the white bar is 40 µm.</p

Bacterial strains, plasmids and oligonucleotides used in this study.

<p>Bacterial strains, plasmids and oligonucleotides used in this study.</p

Macrophages in infected monolayers are not homogeneous with respect to the number of intracellular <i>R. equi</i> they harbour.

<p>Macrophage monolayers were infected with ATTO 488 labeled virulent (<i>R. equi</i> 103+; top panel) or attenuated (<i>R. equi</i> 103−; bottom panel) strains of <i>R. equi</i> at increasing multiplicities (MOI) of infection. Monolayers were fixed and labelled 24 h post-infection. High content image analysis using Columbus software was employed to enumerate infected macrophages and to determine the number of intracellular <i>R. equi</i>. Shown are the number of macrophages that contained no intracellular bacteria (grey), between 1 and 5 bacteria (white) or more than 5 bacteria (black).</p

Key TCRP parameters of macrophage monolayers infected with virulent or attenuated <i>R. equi</i> strains.

<p>Shown are the maximum cell indices (Ci) values (panel A) and the the C_i inflection times (panel B) following infection of J774A.1 macrophage monolayers with virulent (♦, <i>R. equi</i> 103+; ∇, <i>R. equi</i> Δ35000) and attenuated (□, <i>R. equi</i> 103−; •, <i>R. equi</i> Δ<i>vapA</i>) <i>R. equi</i> strains at increasing multiplicity of infection. Values are the means ± SD.</p