The effect of Escherichia coli heat-stable toxin on the trans-epithelial intestinal elimination of ciprofloxacin in the rabbit

Albert Musafijaa, Yuri Shtelmana, Jacob Ramonb, Moshe Volkc, Shlomo Segeva and Ethan Rubinsteina,*

a The Infectious Diseases Unit, b Department of Urology, Sheba Medical Center, Tel Hashomer 52621, Tel Aviv University School of Medicine; c Central Laboratories, Ministry of Health, Jerusalem, Israel


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The effect of Escherichia coli heat-stable toxin (ST) on the intestinal trans-epithelial elimina-tion of ciprofloxacin was studied in the rabbit model. Following a systemic 27 mg/kg dose, excreted amounts of ciprofloxacin during the 2 h experiment were: 9.3 (± 4.8); 12.5 (± 5.2) and 0.3 (± 0.1) µg/cm2 surface of intestinal wall in ST-challenged jejunal, ileal and caecal loops, respectively. In control loops the corresponding amounts were: 5.0 (± 2.8), 6.5 (± 3.4) and 0.3 (± 0.1) µg/cm2, respectively. The trans-epithelial elimination of ciprofloxacin was significantly higher in ST-challenged small bowel loops than in controls while caecal elimination was negligible.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The intestinal tract has been recognized as an important pathway in eliminating fluoroquinolones, with biliary and trans-epithelial secretion being the most important routes.1 The effect of intestinal infections on this pathway is important as ciprofloxacin is frequently used to treat enteric infections.2 Certain Escherichia coli strains produce heat-stable enterotoxins (STs). STs are small peptides that act on the intestinal epithelium through stimulation of the guanylate cyclase system without actually penetrating the cell.3,4 The action of ST differs from the action of E. coli heat-labile toxin and cholera toxin, which penetrate the intestinal cell and activate adenylate cyclase to produce cAMP, causing water and electrolyte secretion.4

The objective of the present study was to assess the effect of E. coli ST on the trans-epithelial elimination of ciprofloxacin in the intestinal tract of the rabbit.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The study design was approved by the animal welfare committee of the Sheba Medical Centre.

Fifteen New Zealand White albino male rabbits, weighing c. 3.0 kg were used (purchased from the Lowenstein Farm, Yokneam, Israel). Following a 24 h fast, but with free access to water, animals were anaesthetized with a combination of diazepam (Teva, Petach-Tikva, Israel) 3 mg/kg and ketamine (Parke Davis, Pontypool, UK) 25 mg/kg. A midline abdominal incision was performed and the jejunum, ileum and caecum identified. In each section, two loops c. 5 cm long were created. In each segment, one loop was used for challenge with ST while the other adjacent loop served as control. ST loops were marked with coloured suture material.

E. coli ST (obtained from the Central Laboratory of the Ministry of Health, Jerusalem, Israel) was produced by the strain E. coli E 6397/5 (e). The concentration of ST was determined by an ELISA method. The activity of the ST was assayed in suckling mice. Toxin with an OD of 0.1, in suckling mice, yielded a ratio of 0.15 between the weight of challenged intestine and the whole body weight.5 Four batches with different concentrations of ST were used. Lower concentrations had no effect whereas higher doses caused a toxic state of the rabbit within a few hours and death within 18 h.

Eighteen hours following the introduction of ST into the intestinal loop, animals that tolerated the highly concentrated toxin well were re-anaesthetized, catheters were introduced into the femoral artery and jugular vein and blood was obtained at time 0. Thereafter, ciprofloxacin 27 mg/kg, dissolved in phosphate-buffered saline pH 7.3 (Unipath, Denver, CO, USA), was administered via the jugular vein over a 10 min period. The abdomen was then reopened and intestinal fluid samples were obtained from each loop, through careful aspiration of the loop's content with a 2 mL syringe, along with blood samples obtained from the femoral artery at 15 min intervals for 2 h. The experiment ended by killing the animal with iv administration of sodium barbital (Sanofi, Amsterdam, The Netherlands). The intestine was excised, the loops isolated and their mucosal surface measured with a planimeter. The concentrations of ciprofloxacin in blood and intestinal fluid were measured by bioassay using Bacillus subtilis on tryptic soya agar pH 9.0.6 The sensitivity of the assay was 0.1 mg/L in both serum and intestinal fluid.

Bicarbonate, chloride, sodium and protein concentrations in the intestinal fluid were measured according to conventional clinical biochemical methods.

The linearity between serum ciprofloxacin concentrations and (i) ciprofloxacin concentrations in the intestinal fluid (ii) excreted amount of ciprofloxacin per cm2 of intestinal wall were expressed graphically by plotting serum AUC (over the 2 h period) against the corresponding intestinal AUCs.

The paired Student's t-test was used for statistical analysis.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Peak serum ciprofloxacin concentration was 18.0 (± 10.4) mg/L at 15 min, following administration of a ciprofloxacin dose of 27 mg/kg, decreasing gradually to 5.3 (± 5.1) mg/L at 120 min.

In intestinal loops challenged with ST, accumulated fluid volumes versus control loops (calculated per cm2 of intestinal mucosa) during the 2 h experiment were: 1.4 (± 1.0) versus 1.0 (± 0.9) mL in the jejunum; 1.5 (± 1.4) versus 1.2 (± 1.0) mL in the ileum and 0.2 (± 0.2) versus 0.2 (± 0.2) mL in the caecum. Concentrations of Na+, Cl, HCO3– and protein were also not significantly different between ST-challenged and control loops with the exception of Na+, which was significantly higher in ST-challenged jejunal loops compared with the control (0.196 versus 0.95 mEq/L; P <= 0.05).

Mean intestinal concentrations of ciprofloxacin (ST versus control) were: 6.9 (± 1.7) versus 4.0 (± 1.4) mg/L in the jejunum; 8.3 (± 1.4) versus 6.1 (± 1.2) mg/L in the ileum and 0.7 (± 0.3) versus 1.2 (± 0.6) mg/L in the caecum.

Total amounts of ciprofloxacin excreted in the intestinal loops (calculated per cm2 of intestinal mucosa) (ST versus control) were: 9.3 (± 4.8) versus 5.0 (± 2.8) µg/cm2 in the jejunum; 12.5 (± 5.2) versus 6.5 (± 3.4) µg/cm2 in the ileum and 0.3 (± 0.02) versus 0.3 (± 0.02) µg/cm2 in the caecum (Figure 1Go).



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Figure 1. Ciprofloxacin intestinal excretion (calculated per cm2 of intestinal mucosa) in ST-challenged ({square}) and control ({blacksquare}) intestinal loops (mean ± S.D.).

 
The relationships between serum AUC and intestinal fluid AUCs are shown in Figure 2Go. The relationship between serum ciprofloxacin AUC and the amounts of ciprofloxacin excreted into the loops (either total amount or amount per cm2) were essentially the same as shown in Figure 2Go.



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Figure 2. Relation between serum ciprofloxacin AUC and intestinal loops AUCs in control and ST-challenged loops over 2 h. Symbols: {circ}, caecum ST; {square}, caecum control; {diamondsuit}, jejunum control; {triangleup}, jejunum ST; •, ileum control; {blacksquare}, ileum ST.

 
In the present investigation we have demonstrated that the elimination of ciprofloxacin in the intestinal tract is increased in the presence of E. coli ST. The effects of ST in the jejunum and ileum were characterized by c. 50% increase in the secretion of ciprofloxacin in these sections (P < 0.05), while the effect of ST in the caecum was negligible. The effect of ST was also characterized by significantly increased fluid secretion into the jejunal and ileal loops (c. 40–50% more than in control loops) and by significantly higher Na+ concentration in the jejunal loop. These results are in contrast to results obtained in the same model when heat-labile cholera toxin (CT) was used. CT did not affect the concentration of ciprofloxacin in the intestinal fluid, while it increased the amount secreted per cm2 by c. 50%.1 Under ST, as shown here, both the concentration and the amount of ciprofloxacin excreted increased by c. 50%.

While the mechanism of excretion of ciprofloxacin under CT appears to be simple diffusion,1 in the presence of ST, as suggested also by the linear relationship between serum AUC and intestinal AUC (Figure 2Go), an additional excretory mechanism is operative. This additional mechanism(s) is probably dependent on ST-induced apoptosis in enteric cells7 and damage to vascular endothelial cells including those in the intestine.8 These effects may enhance the exudation of ciprofloxacin into the intestine, or cause paracellular transport, in ST-challenged intestinal loops.

In previous investigations we have demonstrated that c. 25% of a single parenteral ciprofloxacin dose is eliminated in the small intestine in the rabbit.1 Under the influence of ST, this fraction is expected to increase to 36–40%. This increased excreted fraction may have a role in inducing antimicrobial resistance in the intestinal flora, but may also be the mechanism responsible for the rapid cure of bacterial enteritis obtained by ciprofloxacin therapy.9,10

In summary, we have shown that E. coli ST enhances ciprofloxacin excretion into the small bowel, but not into the caecum of the rabbit. This enhanced excretion may have important clinical implications.


    Notes
 
* Corresponding author. Tel: +972-3-5345389; Fax: +972-3-5347081; E-mail: unit{at}netvision.net.il Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Musafija, A., Barzilai, A., Ramon, J. & Rubinstein, E. (1998). Effect of cholera toxin on intestinal elimination of ciprofloxacin in rabbits. Antimicrobial Agents and Chemotherapy 42, 473–4.[Abstract/Free Full Text]

2 . Dryden, M. S., Gabb, R. J. & Wright, S. K. (1996). Empirical treatment of severe acute community-acquired gastroenteritis with ciprofloxacin. Clinical Infectious Diseases 22, 1019–25.[ISI][Medline]

3 . Field, M., Graf, L. H., Jr, Laird, W. J. & Smith, P. L. (1978). Heat-stable enterotoxin of Escherichia coli: In vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine. Proceedings of the National Academy of Sciences, USA 75, 2800–4.[Abstract]

4 . Field, M., Rao, M. C. & Chang, E. B. (1989). Intestinal electrolyte transport and diarrheal disease. II. New England Journal of Medicine 321, 879–83.[ISI][Medline]

5 . Balows, A. (Ed.) (1991). Manual of Clinical Microbiology, 5th edn, p. 142. American Society of Microbiology, Washington, DC.

6 . Lorian, V. (Ed.) (1986). Antibiotics in Laboratory Medicine, p. 11–24. Williams & Wilkins, Baltimore, MD.

7 . Guichon, A. & Zychlinsky, A. (1997). Clinical isolates of Shigella species induce apoptosis in macrophages. Journal of Infectious Diseases 175, 470–3.[ISI][Medline]

8 . Tesh, V. L., Samuel, J. E., Perera, L. P., Sharefkin, J. B. & O'Brien A. D. (1991). Evaluation of the role of Shiga and Shiga-like toxins in mediating direct damage to human vascular endothelial cells. Journal of Infectious Diseases 164, 344–52.[ISI][Medline]

9 . Talsma, E., Goettsch, W. G., Nieste, H. L., Schrijnemakers, P. M. & Sprenger M. J. (1999). Resistance in Campylobacter species: increased resistance to fluoroquinolones and seasonal variation. Clinical Infectious Diseases 29, 845–8.[ISI][Medline]

10 . Khan, A. W., Seas, C., Dhar, U., Salam, M. A. & Bennish, M. L. (1997). Treatment of shigellosis: V. comparison of azithromycin and ciprofloxacin. Annals of Internal Medicine 126, 697–701.[Abstract/Free Full Text]

Received 14 August 2000; returned 30 October 2000; revised 9 January 2001; accepted 13 February 2001





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