Cooperation between prokaryotic (Lde) and eukaryotic (MRP) efflux transporters in J774 macrophages infected with Listeria monocytogenes: studies with ciprofloxacin and moxifloxacin

Abstract

Antibiotic efflux is observed in both eukaryotic and prokaryotic cells, modulating accumulation and resistance. The present study examines whether eukaryotic and prokaryotic fluoroquinolone transporters can cooperate in the context of an intracellular infection. We have used (i) J774 macrophages (comparing a ciprofloxacin-resistant cell line overexpressing an MRP-like transporter with wild-type cells with basal expression), (ii) Listeria monocytogenes (comparing a clinical isolate [CLIP21369] displaying ciprofloxacin resistance associated with overexpression of the Lde efflux system with a wild-type strain [EGD]), (iii) ciprofloxacin (substrate of both Lde and MRP) and moxifloxacin (nonsubstrate), and (iv) probenecid and reserpine (preferential inhibitors of MRP and Lde, respectively). The ciprofloxacin MICs for EGD were unaffected by reserpine, while those for CLIP21369 were decreased approximately fourfold (and made similar to those of EGD). Neither probenecid nor reserpine affected the moxifloxacin MICs against EGD or CLIP21369. In dose-response studies (0.01x to 100x MIC) in broth, reserpine fully restored the susceptibility of CLIP21369 to ciprofloxacin (no effect on EGD) but did not influence the activity of moxifloxacin. In studies with intracellular bacteria, reserpine, probenecid, and their combination increased the activity of ciprofloxacin in wild-type and ciprofloxacin-resistant macrophages in parallel with an increase in ciprofloxacin accumulation in macrophages for EGD and an increase in accumulation and decrease in MIC (in broth) for CLIP21369. Moxifloxacin accumulation and intracellular activity were consistently not affected by the inhibitors. A bacterial efflux pump may thus actively cooperate with a eukaryotic efflux transporter to reduce the activity of a common substrate (ciprofloxacin) toward an intracellular bacterial target.

FIG. 1.

Killing curves of ciprofloxacin (top) or moxifloxacin (bottom) against L. monocytogenes EGD (left) or CLIP21369 (right). Bacteria in broth were incubated for 5 h in the presence of the antibiotic alone (open symbols) or combined with reserpine (33 μM; 20 mg/liter; closed symbols). The abscissas indicate the extracellular concentrations of the antibiotic in log scale; the ordinates show the changes in CFU (log₁₀) per ml as observed after 5 h of incubation in comparison with the original inocula (horizontal dotted lines). The arrowheads point to the MIC of the strain (open arrowhead, antibiotic alone; closed arrowhead, antibiotic plus reserpine; values are indicated in Table 1). All values are the means of three independent determinations ± standard deviations (when not visible, error bars are smaller than the size of the symbol).

FIG. 2.

Concentration (left) and intracellular activity (right) of ciprofloxacin in J774 wild-type macrophages (top) or ciprofloxacin-resistant macrophages (bottom) exposed to 4.3 mg/liter of the antibiotic alone (C) or combined with reserpine (33 μM; 20 mg/liter [C+R]), probenecid (15 mM; 4.3 g/liter [C+P]), or both inhibitors (C+R+P). CT, control (no drug added). Cellular concentrations were measured in uninfected cells after 5 h of incubation and expressed as ng/mg cell protein; intracellular activities were compared in cells infected by L. monocytogenes EGD (middle panels) or CLIP21369 (right panels) and expressed as the change in CFU (log₁₀) per mg cell protein after 5 h or 24 h of incubation in comparison with the original inocula. Values are the means of three independent determinations ± standard deviations (when not visible, error bars are smaller than the thickness of the bar border).

FIG. 3.

Concentration (left) and intracellular activity (right) of moxifloxacin in J774 wild-type macrophages (top) or ciprofloxacin-resistant macrophages (bottom) exposed for 5 h to 4 mg/liter of the antibiotic alone (M) or combined with reserpine (33 μM; 20 mg/liter [M+R]), probenecid (15 mM; 4.3 g/liter [M+P]), or both inhibitors (M+R+P). CT, control (no drug added). Cellular concentrations were measured in uninfected cells after 5 h of incubation and expressed as ng/mg cell protein; intracellular activity was compared in cells infected by L. monocytogenes EGD (middle panels) or CLIP21369 (right panels) and expressed as the change in CFU (log₁₀) per mg cell protein after 5 h or 24 h of incubation in comparison with the original inocula. Values are the means of three independent determinations ± standard deviations (when not visible, error bars are smaller than the thickness of the bar border).