Pseudomonas aeruginosa virulence expression is directly activated by morphine and is capable of causing lethal gut-derived sepsis in mice during chronic morphine administration

Abstract

Objective: This study was designed to examine the effect of morphine administration on the intestinal mucus barrier and determine its direct effect on the virulence and lethality of Pseudomonas aeruginosa, one of the most frequent pathogens to colonize the gut of critically ill patients.

Background data: Surgical injury is associated with significant exposure of host tissues to morphine from both endogenous release and its use as a potent analgesic agent. Morphine use in surgical patients exposed to extreme physiologic stress is well established to result in increased infection risk. Although morphine is a known immunosuppressant, whether it directly induces virulence expression and lethality in microbes that colonize the human gut remains unknown.

Methods: Mice were implanted with a slow release morphine or placebo pellet with and without intestinal inoculation of P. aeruginosa created by direct cecal injection. Mucus production and epithelial integrity was assessed in cecal tissue via Alcian blue staining and histologic analysis. In vivo and in vitro P. aeruginosa virulence expression was examined using reporter strains tagged to the epithelial barrier disrupting protein PA-I lectin. P. aeruginosa chemotaxis toward morphine was also assayed in vitro. Finally, the direct effect of morphine to induce PA-I lectin expression was determined in the absence and presence of methylnaltrexone, a μ opioid receptor antagonist.

Results: Mice intestinally inoculated with P. aeruginosa and implanted with a morphine pellet demonstrated significant suppression of intestinal mucus, disrupted intestinal epithelium, and enhanced mortality; whereas exposure of mice to either systemic morphine or intestinal P. aeruginosa alone enhanced intestinal mucus without mortality, suggesting a shift in P. aeruginosa during morphine exposure to a mucus suppressing, barrier disrupting, and lethal phenotype. Direct exposure of P. aeruginosa to morphine in vitro confirmed that morphine can transform P. aeruginosa to a more virulent phenotype that is attenuated in part by methylnaltrexone.

Conclusions: Morphine administration shifts intestinal P. aeruginosa to express a virulent phenotype and may play a role in its ability to causes lethal gut-derived sepsis in a susceptible host.

Figure 1. Synergistic effect of morphine administration and P. aeruginosa intestinal injection on mice mortality

25 mg of placebo pellet or morphine pellet were implanted in neck of C57BL6 with or without simultaneous direct cecal injection of 200 μl of 10⁷ CFU/ml P. aeruginosa PAO1. Data are presented as Kaplan- Mayer survival curves. n=10/group, p< 0.001.

Figure 2. Histology of ileal-cecal intestinal tissue sections

(A–D), H&E staining; (A'–D'), alcial blue staining. (A, A') mice, implanted with placebo pellet. (B, B') mice, implanted with morphine pellet. (C, C') mice, implanted with placebo pellet and intestinal inoculation with P. aeruginosa PAO1, 200 μl of 10⁷ CFU/ml via direct puncture. Increased amount of goblet cells in crypts is shown by arrow. (D, D') mice, implanted with morphine pellet and injected P. aeruginosa PAO1 similar to C. Focal superficial necrosis (shown by black arrows on panel D, C') and the appearance of lymophocytic infiltration (shown by yellow arrows) are seen. Images were taken with Zeiss Axioskop using a 20 × objective. (E), Quantitative assessment of intestinal mucus abundance represented by % of blue stained mucus/field using ACIS software (Automated Cellular Imaging Software) n=40 fields of evaluation from 4 mice/group, *p<0.05 Placebo vs morphine; **p<0.05 Placebo vs Placebo + P. aeruginosa; ***p<0.001 Placebo vs morphine + P. aeruginosa). (F, G), Production of (F) IL-6 and (G) IL-10 in serum and cecum. (H) QRT-PCR to detect P. aeruginosa PAO1 in cecal lumen, intestinal epithelium, and blood of mice 36 hours following P. aeruginosa inoculation into the cecum. •, mouse 1; ∘, mouse 2; ▾, mouse 3; and Δ, mouse 4.

Figure 3. Effect of morphine administration on the intestinal retention of P. aeruginosa

(A) Distribution of constitutive bioluminescent P. aeruginosa XEN41 following direct ileal inoculation by intestinal region at varying time points in mice implanted with placebo (P) or morphine (M) pellets. (B) Photon counts of P. aeruginosa XEN41 following 6, 9 and 14 hours after direct ileal inoculation by intestinal region in mice implanted with placebo (∘) or morphine (•) pellets. Data are expressed as mean ±SEM.

Figure 4. P. aeruginosa recognizes morphine

(A) PA-IL reporter strain demonstrating enhanced PA-IL expression detected by red fluorescence, and clumping formation (precursor to biofilm development) shown by red arrows. (B,B') Exposure to mu opioid receptor antagonists MNTX attenuates the PA-I lectin inducing effect of morphine. (B), dynamic tracking of PA-IL expression, (B'), PA-IL expression following 4 hrs of culture represented as % of control. (C,C') Enhanced expression of PA-IL is seen in P. aeruginosa adherent to the intestinal epithelium in mice implanted with (C') morphine containing pellet but not (C) placebo pellet. (D,D') P. aeruginosa accumulating at the peripheral rim and on the agarose droplet containing (D') morphine but not (D) blank agarose. (E–E") P. aeruginosa migrates to the morphine agarose droplet as seen in images captured at (E) 10 min and (E') 50 min after P. aeruginosa inoculation on the blank non- morphine containing agarose droplet (E") Bacterial counts inside a rectangle of 5 μm width.