This study shows that the choice of AC significantly influences the results of the selected R. equi in vitro assays. Overall, exposure of R. equi to K2EDTA resulted in inhibition of CFU formation shortly after exposure; this was not observed when other AC were used. This is important as growth inhibition of R. equi is a desirable effect of HIP. Most of the published work evaluating in vitro R. equi growth does not report the type of AC used for plasma collection. Therefore, it is difficult to establish if the effect of the AC was taken into consideration at the time of results interpretation. Failure to do so has led to equivocal efficacy reports in humans . ACD and heparin did not have any significant impact on any of the assays evaluated.
As described for other Gram-negative and positive bacteria [14, 15, 20, 21], direct exposure to K2EDTA in broth resulted in complete inhibition of CFU by 18 h. This is likely the result of the strong Ca2+ and Mg2+ chelating capacity of K2EDTA increasing cell permeability and fragility which may lead to cell lysis . Interestingly, a similar effect was not observed when the other citrate-based AC (Na Citrate and ACD) were used. Citrate-based anticoagulants prevent coagulation by chelating ionized calcium present in the blood to form non-ionized calcium-citrate complexes; however, their chelating effect is weaker than that of K2EDTA, especially for Mg2+ [15, 22]. Heparin, an AC that inhibits coagulation mainly by enhancing the activity of antithrombin III, didn’t show a direct effect on R. equi growth. This was expected as the authors could find no evidence that antithrombin is relevant to bacterial survival.
In addition to the effects seen with K2EDTA, there was a bacteriostatic effect observed the first 12 h post-inoculation with the other plasmas and serum. This is likely the effect of the antimicrobial proteins normally present in these bodily fluids as serum was collected without AC .
Intracellular infections were performed using murine macrophages which have been used to study R. equi infection [10, 13, 24]. Opsonization of R. equi with R. equi-specific antibodies increases microorganism uptake by Fcγ receptors of macrophages and enhances their oxidative burst [19, 25, 26]. Moreover, plasma and serum boost R. equi killing by enhancing phagosome-lysosome fusion . Na Citrate significantly decreased intracellular R. equi growth overtime suggesting that sufficient chelation of calcium to weaken cell wall occurs , although other mechanism of growth inhibition such as hyperosmolarity of the solution [28, 29] and partial complement inhibition  can’t be ruled out. Interestingly, opsonization with plasma collected with K2EDTA resulted in intracellular R. equi growth inhibition but not death in the 72h period studied. EDTA partially decreased bacterial deposition of C5b9, a multimer that mediates bacterial killing on Neisseria meningitidis . Others have shown that EDTA inhibited CR3-mediated binding on RAW264.7 cells, decreasing phagocytosis of the Gram-negative bacterium Borrelia burgdorferi . The exact mechanism for the lack of R. equi death inside macrophages in our study remains to be determined.
There were no significant differences in the number of infected cells per 300 macrophages or number of macrophages with 10 or more R. equi immediately post-infection suggesting that AC do not affect the initial R. equi uptake by macrophages. The number of macrophages containing 10 or more bacteria after 24 h was significantly lower when EDTA was used. This likely reflects the direct effect of K2EDTA on R. equi growth. Unfortunately, bacterial fluorescence was not reliable past 24 h (data not shown) which limited our ability to evaluate cell replication past this point . Thus, only CFU data are reported for subsequent timepoints.