In all experiments, cells were infected at a multiplicity of infection (MOI) = 5. Bacteria RG7420 were added to the apical chamber of the inserts, and monolayers were incubated at 37 °C for 1 h with agitation. Following incubation, all basolateral medium was removed and replaced
with prewarmed DMEM minus Pen-Strep. The monolayers were incubated at 37 °C for a further hour with agitation, and the basolateral medium was again collected. The basolateral medium was serially diluted and plated on LBN. TER was measured at 0-, 1- and 2-h time points. All LBN plates were incubated at 37 °C overnight. Colony-forming units (CFU) were counted for each experimental parameter. Data were analysed using Graph Pad Prism® software. All experiments were carried out three times in duplicate or triplicate, unless otherwise stated. Data are expressed as mean ± standard error of mean (SEM). Significances check details of differences between mean values were assessed using the two-tailed, unpaired Student’s t-test or one-way anova with Tukey post hoc test where
appropriate, with significance set as P < 0.05 *, P < 0.01 **, P < 0.001 ***. The passage of V. parahaemolyticus across the M cell-like co-culture model vs. Caco-2 monolayers was investigated. First, the translocation of fluorescently labelled particles across both the monolayers and co-cultures was investigated to confirm in vitro induction of the M-like phenotype in the latter. After 2 h, an 18- and 16-fold increase in 1- and 0.5-μm particle transcytosis, respectively, were observed in the co-cultures, when compared to transcytosis across the Caco-2 monolayer (Fig. 1a). HSP90 These values are in agreement with the reported value of 14-fold for similarly sized particles in former studies (Martinez-Argudo et al., 2007). These data highlight the significant sampling ability of M-like cells, thereby confirming the successful induction of the co-culture model in vitro. Examination of the transcytosis
of V. parahaemolyticus indicated an increase in bacterial translocation across the co-cultures compared to the Caco-2 monolayers following 1 and 2 h of infection (Fig. 1b). One hour postinfection, transcytosed bacterial numbers were 3.3-fold higher in the co-cultures with a 3.2-fold difference between Caco-2 monolayers and co-cultures observed following 2 h of infection. Comparison of translocation of V. parahaemolyticus across the co-culture and control demonstrated a 5.7- and 5.8-fold increase, respectively, between the 1- and 2-h time points. The data indicate that while V. parahaemolyticus has the ability to translocate across both the control and co-culture models 1 h postinfection with a dramatic increase in translocation observed 2 h postinfection, it translocates across the co-culture model in significantly increased numbers when compared to passage across the Caco-2 monolayer.