lntraperitoneal Povidone-iodine in Experimental Canine and Murine Peritonitis
John S. Bolton, MD, New Orleans, Louisiana George H. Bornside, PhD, New Orleans, Louisiana lsidore Cohn, Jr, MD, New Orleans, Louisiana
Diffuse peritonitis remains a formidable problem and is still associated with considerable mortality despite the benefits of proper therapy with surgery, fluids, and antibiotics. Experimental and clinical studies have shown that intraperitoneal administration of antibiotics, such as kanamycin and cephalothin, is effective [l-3]. Some investigators believe that the use of alternative agents, if available, would help diminish problems related to antibiotic toxicity and the development of antibiotic-resistant hospital strains of bacteria [4]. One such alternative agent is the antiseptic, povidone-iodine, a water-soluble complex of polyvinylpyrrolidone and iodine. Polymeric polyvinylpyrrolidone is a carrier and solubilizing agent for iodine, which is slowly released from povidone-iodine as free iodine. Povidone-iodine is more bactericidal and less toxic than aqueous solutions of free iodine and provides approximately 1 per cent of iodine. During World War I, Alexander Fleming showed antiseptics to be unsuitable as therapeutic agents in septic wounds [5]. The introduction of penicillin during World War II was the death knell for further consideration of a therapeutic role for antiseptic agents. During the Vietnam War, nevertheless, antiseptics were used to improve accepted therapy. Povidone-iodine solution, specifically, was used to irrigate battle wounds and was instilled into the abdominal cavity after hemostasis. Several investiga-
tors have obtained good results with povidone-iodine solutions for peritoneal and mediastinal lavage [6,7], intraperitoneal instillation for experimental peritonitis [8,9], and the prevention of postoperative wound infection [10,11]. Recent work in our laboratory, however, has shown that intraperitoneal instillation of nonlethal doses of povidone-iodine solution into dogs 24 hours after the cecum was strangulated was fatal within the succeeding 24 hours, whereas control strangulated dogs treated with saline solution survived an average of 96 hours [8]. These results do not warrant recommendation of povidone-iodine for use in peritonitis and emphasize the difference between an active, diffuse peritonitis [S] and one treated soon after insult to the peritoneal cavity [4,9]. In this paper, we report further studies with the canine model and examine the quantitative bacteriology of canine peritoneal fluid after the instillation of povidpne-iodine. In addition, we assess the bactericidal effect of povidone-iodine against intraperitoneal populations of Escherichia coli in mice soon after challenge with lethal doses of this microorganism. These experimental models provided a means of examining the antibacterial activity of povidone-iodine in dogs during an active, established peritonitis and in mice in the absence of stresses due to surgical trauma and prolonged physiologic insult.
From the Department of Surgery, Louisiana State University Medical Canter, New Orleans. Louisiana. Reprint requests should be addressed to George H. Bornside, PhD, Department of Surgery, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, Louisiana 70112.
Material and Methods
780
Appendicitis-Peritonitis in Dogs. Peritonitis was produced in 17 adult, mongrel dogs of both sexes with a mean
The American
Journal of Surgery
lntraperitoneal
weight of 23.3 kg (range 17 to 29). The dogs received no antibiotics before the procedure, were fasted the night before operation, and received I.0 ml/kg of 3 per cent sodium pentobarbital intravenously for anesthesia. The cecum was identified and its blood supply and peritoneal attachments were divided. The base of the cecum was ligated with umbilical tape. A 16 gauge, 20 cm flexible catheter was sutured to the colon adjacent to the appendiceal pouch. The abdomen was closed with 3-O silk sutures, the catheter was brought out through the midline, and the skin was closed with 3-O nylon. Postoperatively, dogs were given 5 ml/kg of castor oil through an orogastric tube [8]. The dogs were allocated randomly either to a control or a treatment group. Twenty-four hours after ligation, control dogs received 100 ml of saline solution through the catheter; treated dogs received 2 ml/kg of povidone-iodine (Betadine@ solution, Purdue Frederick Co., Norwalk, CT) diluted to a constant 100 ml with saline solution [8]. Peritoneal fluid for bacteriologic culture was drawn from the catheter at 6,12, and 24 hours after treatment (given 24 hours after ligation of the cecum) or immediately at death, or both. Blood for culture was withdrawn by venipuncture at the same intervals and injected directly into culture bottles. Two additional appendicitis-peritonitis dogs were treated with povidone alone to assess its contribution to survival. A 9 per cent solution of povidone (polyvinylpyrrolidone-IO, Sigma Chemical Co., St. Louis, MO) was instilled at a dose of 2 ml/kg diluted to 100 ml with saline solution. Nine normal dogs were used to study the in vivo conversion of free iodine to iodide. The 2 ml/kg dose of povidone-iodine solution was injected intraperitoneally through a small laparotomy incision. Animals were killed at I/4,1/2, 1, 2, 3, and 6 hours afterward, and peritoneal fluid and urine were tested qualitatively for iodine and iodide by standard methods. Development of a blue color in the presence of starch indicated free iodine. Iodide was indicated by the development of a blue color in the presence of starch after the addition of 0.05 M KI03 to acidified specimens. Canine peritoneal fluids were cultured immediately upon collection. Serial lOO-fold dilutions were prepared from 0.1 ml of each, and 0.1 ml of appropriate dilutions was seeded upon the surface of plates of solid media and spread with a wire loop. For aerobic culture, plates of blood agar medium (5 per cent human blood in brain-heart infusion agar containing 4 per cent agar) and MacConkey agar were inoculated and examined after 24 and 48 hours at 37°C. For anaerobic culture, plates of blood agar medium and Rogosa SL agar were inoculated and incubated in Brewer jars in an atmosphere of 95 per cent Hz and 5 per cent COs for at least 48 hours at 37 “C before being examined and subcultured. Tubes of brain-heart infusion broth, fluid thioglycollate medium and cooked meat medium were inoculated from the initial dilution of each specimen. These allowed both aerobic and anaerobic bacteria to grow, and were studied as a check on the bacteria isolated from sur-
Volume 137, June 1979
Povidone-iodine
face cultures. Aerobic isolates were streaked to eosinmethylene blue agar to identify coliforms and were subcultured to heart infusion brot,h, from which gram-stained smears were prepared. Isolates from anaerobic plates were streaked to a blood agar plate for aerobic incubation to veriffi that the isolate was an anaerobe. Bacteria were identified to the genus level, and all bacterial counts were transformed to logarithms to the base 10 for ease of statistical analysis. Immediate Monobacterial Peritonitis in Mice. The Ancar strain of Escherichia coli, previously used to demonstrate that the infection-enhancing effect of hemoglobin was due to ferric ions, was used with ferric ammonium citrate. Synergistic mixtures of E. coli and either hemoglobin or ferric ammonium citrate allow bacterial multiplication intraperitoneally, and at death mice that have been challenged harbor numbers of E. coli adequate to provide lethal amounts of endotoxin [12]. Bacteria were harvested by centrifugation from overnight cultures incubated at 37°C on a rotary platform shaker in 50 ml of Trypticase Soy Broth (BBL), washed twice with saline solution, and adjusted to read 56 per cent transmittance at 610 nm in a Coleman spectrophotometer. A suspension of fresh bacteria was prepared for each day’s work, and an aliquot was diluted 1:lOO with a 0.2 per cent solution of ferric ammonium citrate (purified brown pearls, Mallinckrodt Chemical Co., St. Louis, MO), which contains 34 mg per cent iron. These preparations contained log 6.88 f 0.37 bacteria/ml (mean f standard deviation) and were injected intraperitoneally at a dose of 20 ml/kg body weight into female, random-bred, albino mice of the CD-1 strain (Charles River Breeding Laboratories, Wilmington, MA). Mice were observed after 24 and 48 hours because mortality did not occur beyond 48 hours. A second intraperitoneal injection (20 ml/kg body weight) of either saline solution or a I:10 dilution in saline of Betadine solution was given either immediately or at 1, 2, or 3 hours after challenge with E. coli plus ferric ammonium citrate. Available iodine content was checked routinely by the standard sodium thiosulfate calorimetric titration. Intraperitoneal populations of bacteria after these injections were studied in mice killed by ether anesthesia immediately upon injection and after l/2, 3, 6, 12, 18, and 24 hours. Four ml of saline solution were injected into the peritoneal cavity of killed mice, and saline aspirates were collected [13]. A sample of each aspirate was serially diluted in sterile saline solution, and 0.1 ml of appropriate dilutions was seeded upon the surface of plates of Trypticase Soy Agar (BBL) in triplicate. Colonies of E. coli were counted after incubation of plates at S7”C for 24 hours.
Resdts Experimental Canine Peritonitis. Four of the five appendicitis-peritonitis dogs treated with saline solution (controls) survived 85.5 f 25.3 hours (mean f standard deviation); one lived 14 days and was then killed. Twelve dogs were treated with povidone-
781
Bolton
et al
TABLE I Bacterial Counts of Peritoneal Fluids After Povidone-iodine Treatment of Dogs With Appendicitis-peritonitis Povidoneiodine
Microorganism Enterobacteria
12 Hours
24 Hours
Death
+
2.66 f 3.70 (5) 2.03 f 2.69 (10)
4.66 f 3.46 (4) 3.13 f 3.16 (7)
2.30 f 3.28 (5) 4.76 f 0.35 (2)
+ +
4.76 2.07 4.23 3.38
5.49 1.73 4.53 4.11
5.83 1.00 6.96 4.89
+
5.49 f 3.24 (5) 1.46 f 2.20 (10)’
3.56 f 3.46 (4) 4.06 f 3.35 (7)
3.96 f 3.32 (5) 6.14 f 1.07 (2)
4.50 f 3.82 (12)
+
6.50 f 2.65 (5) 4.82 f 2.23 (10)
7.05 f 1.18 (4) 4.24 f 2.54 (7)
8.61 f 1.49 (5) 5.17 f 0.87 (2)
5.24 f
+
5.86 f 2.79 (5) 3.65 f 2.70 (10)
5.65 f 1.84 (4) 4.98 f 2.67 (7)
7.23 f 6.36 f
6.30 f 2.54 (12)
Streptococci Clostridia
6 Hours
Bacteroides Total aerobes Total anaerobes
f f f f
4.39 2.72 3.00 2.56
(5) (10) (5) (10)
f 3.74 (4) f 2.47 (7)’ f 1.47 (4) f 2.08 (7)
f 1.34 (5) f 0 (2)” f 1.20 (5) f 2.25 (2)
1.35 (5) 1.39 (2)
3.51 f
1.90 (12)
3.65 f 2.93 (12) 4.84 f 2.33 (12)
1.85 (12)
Arithmetic means of loglO/ml f standard deviation: the number of specimens cultured is indicated in parentheses. p
John S. Bolton, MD, New Orleans, Louisiana George H. Bornside, PhD, New Orleans, Louisiana lsidore Cohn, Jr, MD, New Orleans, Louisiana
Diffuse peritonitis remains a formidable problem and is still associated with considerable mortality despite the benefits of proper therapy with surgery, fluids, and antibiotics. Experimental and clinical studies have shown that intraperitoneal administration of antibiotics, such as kanamycin and cephalothin, is effective [l-3]. Some investigators believe that the use of alternative agents, if available, would help diminish problems related to antibiotic toxicity and the development of antibiotic-resistant hospital strains of bacteria [4]. One such alternative agent is the antiseptic, povidone-iodine, a water-soluble complex of polyvinylpyrrolidone and iodine. Polymeric polyvinylpyrrolidone is a carrier and solubilizing agent for iodine, which is slowly released from povidone-iodine as free iodine. Povidone-iodine is more bactericidal and less toxic than aqueous solutions of free iodine and provides approximately 1 per cent of iodine. During World War I, Alexander Fleming showed antiseptics to be unsuitable as therapeutic agents in septic wounds [5]. The introduction of penicillin during World War II was the death knell for further consideration of a therapeutic role for antiseptic agents. During the Vietnam War, nevertheless, antiseptics were used to improve accepted therapy. Povidone-iodine solution, specifically, was used to irrigate battle wounds and was instilled into the abdominal cavity after hemostasis. Several investiga-
tors have obtained good results with povidone-iodine solutions for peritoneal and mediastinal lavage [6,7], intraperitoneal instillation for experimental peritonitis [8,9], and the prevention of postoperative wound infection [10,11]. Recent work in our laboratory, however, has shown that intraperitoneal instillation of nonlethal doses of povidone-iodine solution into dogs 24 hours after the cecum was strangulated was fatal within the succeeding 24 hours, whereas control strangulated dogs treated with saline solution survived an average of 96 hours [8]. These results do not warrant recommendation of povidone-iodine for use in peritonitis and emphasize the difference between an active, diffuse peritonitis [S] and one treated soon after insult to the peritoneal cavity [4,9]. In this paper, we report further studies with the canine model and examine the quantitative bacteriology of canine peritoneal fluid after the instillation of povidpne-iodine. In addition, we assess the bactericidal effect of povidone-iodine against intraperitoneal populations of Escherichia coli in mice soon after challenge with lethal doses of this microorganism. These experimental models provided a means of examining the antibacterial activity of povidone-iodine in dogs during an active, established peritonitis and in mice in the absence of stresses due to surgical trauma and prolonged physiologic insult.
From the Department of Surgery, Louisiana State University Medical Canter, New Orleans. Louisiana. Reprint requests should be addressed to George H. Bornside, PhD, Department of Surgery, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, Louisiana 70112.
Material and Methods
780
Appendicitis-Peritonitis in Dogs. Peritonitis was produced in 17 adult, mongrel dogs of both sexes with a mean
The American
Journal of Surgery
lntraperitoneal
weight of 23.3 kg (range 17 to 29). The dogs received no antibiotics before the procedure, were fasted the night before operation, and received I.0 ml/kg of 3 per cent sodium pentobarbital intravenously for anesthesia. The cecum was identified and its blood supply and peritoneal attachments were divided. The base of the cecum was ligated with umbilical tape. A 16 gauge, 20 cm flexible catheter was sutured to the colon adjacent to the appendiceal pouch. The abdomen was closed with 3-O silk sutures, the catheter was brought out through the midline, and the skin was closed with 3-O nylon. Postoperatively, dogs were given 5 ml/kg of castor oil through an orogastric tube [8]. The dogs were allocated randomly either to a control or a treatment group. Twenty-four hours after ligation, control dogs received 100 ml of saline solution through the catheter; treated dogs received 2 ml/kg of povidone-iodine (Betadine@ solution, Purdue Frederick Co., Norwalk, CT) diluted to a constant 100 ml with saline solution [8]. Peritoneal fluid for bacteriologic culture was drawn from the catheter at 6,12, and 24 hours after treatment (given 24 hours after ligation of the cecum) or immediately at death, or both. Blood for culture was withdrawn by venipuncture at the same intervals and injected directly into culture bottles. Two additional appendicitis-peritonitis dogs were treated with povidone alone to assess its contribution to survival. A 9 per cent solution of povidone (polyvinylpyrrolidone-IO, Sigma Chemical Co., St. Louis, MO) was instilled at a dose of 2 ml/kg diluted to 100 ml with saline solution. Nine normal dogs were used to study the in vivo conversion of free iodine to iodide. The 2 ml/kg dose of povidone-iodine solution was injected intraperitoneally through a small laparotomy incision. Animals were killed at I/4,1/2, 1, 2, 3, and 6 hours afterward, and peritoneal fluid and urine were tested qualitatively for iodine and iodide by standard methods. Development of a blue color in the presence of starch indicated free iodine. Iodide was indicated by the development of a blue color in the presence of starch after the addition of 0.05 M KI03 to acidified specimens. Canine peritoneal fluids were cultured immediately upon collection. Serial lOO-fold dilutions were prepared from 0.1 ml of each, and 0.1 ml of appropriate dilutions was seeded upon the surface of plates of solid media and spread with a wire loop. For aerobic culture, plates of blood agar medium (5 per cent human blood in brain-heart infusion agar containing 4 per cent agar) and MacConkey agar were inoculated and examined after 24 and 48 hours at 37°C. For anaerobic culture, plates of blood agar medium and Rogosa SL agar were inoculated and incubated in Brewer jars in an atmosphere of 95 per cent Hz and 5 per cent COs for at least 48 hours at 37 “C before being examined and subcultured. Tubes of brain-heart infusion broth, fluid thioglycollate medium and cooked meat medium were inoculated from the initial dilution of each specimen. These allowed both aerobic and anaerobic bacteria to grow, and were studied as a check on the bacteria isolated from sur-
Volume 137, June 1979
Povidone-iodine
face cultures. Aerobic isolates were streaked to eosinmethylene blue agar to identify coliforms and were subcultured to heart infusion brot,h, from which gram-stained smears were prepared. Isolates from anaerobic plates were streaked to a blood agar plate for aerobic incubation to veriffi that the isolate was an anaerobe. Bacteria were identified to the genus level, and all bacterial counts were transformed to logarithms to the base 10 for ease of statistical analysis. Immediate Monobacterial Peritonitis in Mice. The Ancar strain of Escherichia coli, previously used to demonstrate that the infection-enhancing effect of hemoglobin was due to ferric ions, was used with ferric ammonium citrate. Synergistic mixtures of E. coli and either hemoglobin or ferric ammonium citrate allow bacterial multiplication intraperitoneally, and at death mice that have been challenged harbor numbers of E. coli adequate to provide lethal amounts of endotoxin [12]. Bacteria were harvested by centrifugation from overnight cultures incubated at 37°C on a rotary platform shaker in 50 ml of Trypticase Soy Broth (BBL), washed twice with saline solution, and adjusted to read 56 per cent transmittance at 610 nm in a Coleman spectrophotometer. A suspension of fresh bacteria was prepared for each day’s work, and an aliquot was diluted 1:lOO with a 0.2 per cent solution of ferric ammonium citrate (purified brown pearls, Mallinckrodt Chemical Co., St. Louis, MO), which contains 34 mg per cent iron. These preparations contained log 6.88 f 0.37 bacteria/ml (mean f standard deviation) and were injected intraperitoneally at a dose of 20 ml/kg body weight into female, random-bred, albino mice of the CD-1 strain (Charles River Breeding Laboratories, Wilmington, MA). Mice were observed after 24 and 48 hours because mortality did not occur beyond 48 hours. A second intraperitoneal injection (20 ml/kg body weight) of either saline solution or a I:10 dilution in saline of Betadine solution was given either immediately or at 1, 2, or 3 hours after challenge with E. coli plus ferric ammonium citrate. Available iodine content was checked routinely by the standard sodium thiosulfate calorimetric titration. Intraperitoneal populations of bacteria after these injections were studied in mice killed by ether anesthesia immediately upon injection and after l/2, 3, 6, 12, 18, and 24 hours. Four ml of saline solution were injected into the peritoneal cavity of killed mice, and saline aspirates were collected [13]. A sample of each aspirate was serially diluted in sterile saline solution, and 0.1 ml of appropriate dilutions was seeded upon the surface of plates of Trypticase Soy Agar (BBL) in triplicate. Colonies of E. coli were counted after incubation of plates at S7”C for 24 hours.
Resdts Experimental Canine Peritonitis. Four of the five appendicitis-peritonitis dogs treated with saline solution (controls) survived 85.5 f 25.3 hours (mean f standard deviation); one lived 14 days and was then killed. Twelve dogs were treated with povidone-
781
Bolton
et al
TABLE I Bacterial Counts of Peritoneal Fluids After Povidone-iodine Treatment of Dogs With Appendicitis-peritonitis Povidoneiodine
Microorganism Enterobacteria
12 Hours
24 Hours
Death
+
2.66 f 3.70 (5) 2.03 f 2.69 (10)
4.66 f 3.46 (4) 3.13 f 3.16 (7)
2.30 f 3.28 (5) 4.76 f 0.35 (2)
+ +
4.76 2.07 4.23 3.38
5.49 1.73 4.53 4.11
5.83 1.00 6.96 4.89
+
5.49 f 3.24 (5) 1.46 f 2.20 (10)’
3.56 f 3.46 (4) 4.06 f 3.35 (7)
3.96 f 3.32 (5) 6.14 f 1.07 (2)
4.50 f 3.82 (12)
+
6.50 f 2.65 (5) 4.82 f 2.23 (10)
7.05 f 1.18 (4) 4.24 f 2.54 (7)
8.61 f 1.49 (5) 5.17 f 0.87 (2)
5.24 f
+
5.86 f 2.79 (5) 3.65 f 2.70 (10)
5.65 f 1.84 (4) 4.98 f 2.67 (7)
7.23 f 6.36 f
6.30 f 2.54 (12)
Streptococci Clostridia
6 Hours
Bacteroides Total aerobes Total anaerobes
f f f f
4.39 2.72 3.00 2.56
(5) (10) (5) (10)
f 3.74 (4) f 2.47 (7)’ f 1.47 (4) f 2.08 (7)
f 1.34 (5) f 0 (2)” f 1.20 (5) f 2.25 (2)
1.35 (5) 1.39 (2)
3.51 f
1.90 (12)
3.65 f 2.93 (12) 4.84 f 2.33 (12)
1.85 (12)
Arithmetic means of loglO/ml f standard deviation: the number of specimens cultured is indicated in parentheses. p
