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Plasmid labeling confirms bacterial translocation in pancreatitis
Plasmid Labeling Confirms Bacterial Translocation in Pancreatitis George B. Kazantsev, MD, David W. Hecht, MD, Robert Rao, MD, Ihor J. Fedorak, MD, Paolo Gattuso, Kenneth Thompson, Phn, Goldte Djuricin, BS, Richard A. Prinz, MD, ~aywood, Illinois
To examine whether the gut is a source of infection in acute pancreatitis,bacterial translocationand alterationsof intestinalmicroeeology and morphology were studied in 16 dogs. Dogs were colonized with a strain of Escherichiu coli (E. coli 6938K) bearing the plasmid pUC%K, which confets hanamycin resistance.In eight dogs (group I), pancreatitiswas induced by sodhun taurocholate/trypsin injection. Eight other dogs (group II) underwent laparotomy only. The pancreas, mesentericlymph nodes, peritoneal fluid, liver, and spleen were harvested 7 days later for culturing and histologic analysis. Identificationof E. coli 6938K was accomplished by phumid DNA analysis. Group I dogs had severe pancreatitisand iscbemicchanges in small bowel mucosa. Croup II dogs had no changes. Transkation to the pancreas occurred in five dogs and to mesentericlymph nodes in six dogs with pancreatitis.No translocationoccurred in group II dogs (p <0.05). In addition to E. coli 6938#, other gram-negative kanamycin-resistantspecies were isolated, inchuling E. coli (other than 6938K) and Enterobucter cloacue. Knteric origin of these strains was confirmed by antibiography and pbrsmid DNA analysis. No overgrowth of cecal gram-negative bacteria was found. This study suggests that the gut is a primary source of i&&ion in pancreatitisand that ischemicdamage of intestinal mucosa may promote bacterial Wan&cation.
MD,
W fection has become the major cause of morbidity and mortality in patients with acute necrotizing pancre-
ith advances in resuscitation and critical care, in-
atitis [Z-3]. The pathogenesis of these pancreatic infections remains obscure. However, recent studies suggest that translocation of intestinal bacteria may be ,a mechanism for secondary contamination of pancreatic necrosis [4,5]. The purpose of this study was to determine if the indigenous enteric flora is a primary source of infection of acutely inflamed pancreatic tissue. Bacterial translocation was studied in dogs with severe acute pancreatitis. Prior to the induction of pancreatitis, dogs were~colonized with a specific strain of Escherichiu coli labeled with a nontransferable plasmid pUC4K. This plasmid confers kanamycin resistance and can be identified by plasmid DNA analysis [a. Intestinal microecology and, morphology were also analyzed to determine possible factors that might promote translocation. MATERIAL AND METHODS Animal preparation: Adult mongrel dogs weighing 18 to 28 kg were observed for at least 1 week prior to beginning the experiment. A wild strain of E. cdi (6938) was isolated from the feces of one of the dogs, (# 6938) and transformed with the plasmid pUC4K (Bethesda Research Laboratories, Gaithersburg, MD) by,using previously described methods [ 71. Transformed kanamycinresistant bacteria (referred to herein as E. coli 6938K) were used for all subsequent experiments. Intestinal decontamination and recolonisation with E co&6938K: On 2 successive days while under
From the Departments of Surgery (GBK, RR, IJF, GD, RAP), Medicine (DWH), Pathology (PG), and Microbiology (KT), Loyola University Medical Center, Maywood, Illinois. Requests for reprints should be addressed to Richard A. Prinz, MD, Department of General Surgery, Rush University, 1653 West Congress, Chicago, Illinois 60612. Presented at the 34th Annual Meeting of The society for Surgery of the Alimentary Tract, Boston, Massachusetts, May 17-19,1993.
light general anesthesia (sodium thiamylal 15 mg/kg, intravenously), the dogs were given 160 mg of gentamicin and 250 mg of vancomycin orally to suppress the indige nous enteric flora. The dogs then received an overnight Luria-broth [8] culture of E. coli 6938K (approximately lo9 colony-forming units) mixed with food. For the rest of the experiment, drinking water was supplemented with 0.5 g/L kanamycin sulfate. Stool samples were cultured daily on McConkey agar (Difco, Detroit, MI), supplemented with 100 pg/mL kanamycin. Colonization was considered established when cultures were positive for 3 successive days. All stool isolates were saved for future analysis. Acute pancreatitiszAll procedures were performed under general anesthesia (sodium thiamylal, 30 mg/kg intravenously for induction; halothane/nitrous oxide for maintenance). Acute pancreatitis was induced by extraduodenal injection of 1 mL/kg of 4% sodium taurocholate with 3,000 IU/kg trypsin into the pancreatic duct under pressure of 200 mm Hg. The right lobe of the pancreas was made ischemic by ligating its blood supply. Sterile cotton swabs of the abdominal cavity were cul-
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Figure 1. A map of pUC4K plasmid. Digestion with FM yields 1.28-kb Kan’ fragment and 2.68-kb LacZ/Amp’ fragment.
tured to detect inadvertent bacterial contamination during the procedure. Experimental design: After colonization was established, the 16 dogs were randomly assigned to 1 of 2 groups. Eight dogs (group I) underwent laparotomy and induction of acute pancreatitis. The other eight dogs (group II) had laparotomy only. Complete blood cell counts (CBC) and plasma amylase levels were measured before the operation and on days 3 and 7 postoperatively. Blood cultures were performed every 24 hours (tryptic soy agar, Difco). The dogs were killed on the seventh postoperative day with 30 mL of saturated potassium chloride solution. Testing for bacterial transloeation: Dogs were anaesthetized as described previously. All procedures were performed under strict aseptic conditions. On entering the peritoneal cavity, 1 mL of free fluid was obtained for culture. If there was no fluid, exposed viscera were rubbed with cotton swabs that were cultured. Mesenteric lymph nodes (MLN) and portions of the liver and spleen were harvested using different sets of sterile instruments. The pancreas was completely excised and divided into 8 to 10 parts that weighed approximately 5 g each. Each part was processed separately. Organs were homogenized in 5 mL of sterile L-broth. Aliquots of the homogenate (0.1 mL) were plated in duplicate on Wilkins-Chalgren (Difco) agar, supplemented with 100 pg/mL gentamicin for detection of anaerobic bacteria. Plates were incubated at 37’C anaerobically and examined at 48 and 96 hours. Other portions of the homogenate (0.5 mL) were added to 5 mL of sterile L-broth, incubated at 37’C in a shaking waterbath for 18 hours, and then qualitatively plated in duplicate on the following media: phenylethyl alcohol (PEA) agar supplemented with 5% sheep blood (BBL, Becton Dickinson, San Jose, CA) for isolation of aerobic and facultative gram-positive bacteria, McConkey agar (BBL) for isolation of aerobic and facultative gram-negative bacteria, and McConkey agar (Difco) supplemented with 100 pg/mL kanamycin for selective isolation of E. coli 6938K or other kanamycin-resistant flora. Plates were incubated aerobically at 37°C and examined at 24 hours.
Aliquots (0.5 mL) of the homogenate were serially diluted in D-PBS and quantitatively plated on plain and kanamycin-supplemented McConkey agar. Plates were incubated aerobically at 37°C for 24 hours. Cecal bacteria were enumerated as the number of colony-forming units (CFUs) per gram of cecum (wet weight). Bacterial identification and plasmid analysis: All bacteria isolated from stool as well as those isolated from harvested organs were identified by standard techniques (AMS bioMeriex Vitek, Hazelwood, MO), and their antibiotic sensitivity patterns were determined. E. coli 6938K was initially identified by its characteristic antibiography. Final identification of this strain was accomplished by confirming the presence of plasmid pUC4K. Plasmid DNA was purified by an alkaline lysis method [8,9] and subjected to restriction digest with endonuclease PstI (New England Biolabs, Beverly, MA) (Figure 1). DNA fragments were separated by electrophoresis through horizontal 0.6% agarose gel, stained with ethidium bromide, and photographed by using Polaroid type 667 film. Plasmid DNA from gram-negative bacteria other than E. coli 6938K was purified by the same method and digested with several restriction enzymes including PstI, BumHI, Hin&II, and HincII (New England Biolabs) in an attempt to obtain a distinctive plasmid profile. Analysis of intestinal morphology and statistical analysis: Paraffin-embedded sections of jejunum, ileum,
and cecum were stained with hematoxylin and eosin and analyzed by light microscopy. Translocation incidences (discontinuous data) were evaluated by x2 analysis. Continuous data were analyzed by analysis of variance (ANOVA), followed by Duncan’s Multiple Range Test. Probabilities less than 0.05 were considered significant. RESULTS Colonization:
Ceca were excised, weighed, and homogenized in 50 mL of Dulbecco’s phosphate-buffered saline (D-PBS).
Bowel decontamination and subsequent colonization with E. coli 6938K were accomplished in all dogs. Surveillance stool cultures showed that, although E. coli 6938K was always a predominant organism, two to three additional kanamycin-resistant bacterial species could also be recovered after 3 to 4 days of kanamycin treatment. These species included E. coli (other than E. coli 6938K), Enterobacter cloacae, Proteus mirabilis, and Pseudomonas species. Acute pancreatitis: All group I dogs developed clinical signs of severe pancreatitis. Laboratory tests showed significant hyperamylasemia and leukocytosis on both the third and the seventh postoperative days (Figures 2 and 3). There was no early mortality. Group II dogs did not exhibit any distress. They had mild leukocytosis on the third postoperative day that usually normalized by the seventh day. Plasma amylase levels remained normal. At death, the pancreas in group I dogs looked enlarged and swollen with visible areas of hemorrhage and fat necrosis. Histologic examination revealed severe hemorrhagic pancreatitis with extensive parenchymal and fat necrosis and polymorphonuclear leukocytic infiltration of the stroma (Figure 4). Bacterial translocation: Infection was not seen in
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Enumeration
of viable gram-negative
cecal bacte-
ria:
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5000 m m
l
T
4000
2 3 G
Poncrsotitis Laparotomy
lp
3000
: :! g
2000
<
1000
0
0
3
7
OAIS
FIgwe 2. Plasma amylase levels, mean f SEM, in dogs with acute pancreatitisand laparotomyalone.
any group II dogs. In dogs with acute pancreatitis, E. coli 6938K was found in the MLN, the pancreas, and the peritoneal fluid (Table I). DNA gel electrophoresis confirmed the identity of these bacteria as 6938K (Figure 5, lanes 6 to 13). Other enteric bacteria isolated in addition to E. coli 6938K included a different strain(s) of E. coli, E. cloacae, and Enterococcus sp. All gram-negative strains were kanamycin resistant and were also previously detected by surveillance stool cultures during the experiment. To confirm their enteric origin, antibiograms and the plasmid profiles of the tissue isolates, along with those of corresponding stool isolates, were analyzed. The strains of E. coli other than 6938K had identical antibiograms but demonstrated no plasmid DNA. Thus, absolute confirmation that these were exactly the same strains was not possible. All E. cloacae isolates had identical antibiograms and contained plasmid DNA that appeared identical by plasmid electrophoresis profile. This suggests that they were the same strains (Figure 6). Digestion of plasmid DNA with multiple enzymes did not yield a distinctive pattern. Three dogs had persistently positive blood cultures beginning on the third postoperative day. Two dogs had Staphylococcus species, and one had Clostridium perfringens. In each dog with positive blood cultures, the same organisms with identical antibiograms were also isolated from the pancreas. Gram-negative bacteremia was not observed, and no other anaerobes were isolated from blood or tissues. Overall, enteric organisms were isolated from MLN in 15% and from the pancreas in 63% of the dogs with pancreatitis versus no infection in group II dogs (p <0.05) (Table II). In four dogs, MLN cultures were monomicrobial (E. coli 6938K, E. coli, Enterococcus sp., and E. cloacae). In two other dogs, at least two different bacterial species were recovered from the MLN. Cultures of pancreas were polymicrobial in all five dogs. THE AMERICAN
_ 3. White blood cell counts(WBC), mean f SEM, in dogs with acute pancreatitisand laparotomyalone.
rlgure
FIgwe 4. Pencreas from the dog with acute panaeetltis. Widespreadpemwlpd and fat necrosiswlth amdated neutrophilic infiltrate(hematoxylinand eosin, 100X).
Cecal population: E. coli 6938K was the primary gram-negative organism in ceca (80% to 100% of the gram-negative flora). The population levels were similar in group I and group II: 8.3 f 5.7 X lo7 and 5.7 f 3.9 X lo7 CFU/g, respectively. Intestinal morphology: In dogs with pancreatitis, the jejunal and ileal mucosa appeared severely damaged. The surface epithelium was denuded, and there was an extensive lymphoplasmacytic infiltration of the lamina propria (Figures 7 and 8). Group II dogs had no such pathologic changes. The cecal mucosa appeared normal in both groups.
COMMENTS Secondary pancreatic infections occur in 2% to 10% of patients with acute pancreatitis and are associated with a mortality of 16% to 40% with surgical therapy [10,11]. Since the vast majority of bacterial species involved in
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TABLE I Translocation of Different Bacterlal Species In Eight Dogs With Acute Pancreatitis Source of Isolation Mesenteric Lymph Nodes
Dog 1. 2.
Pancreas
Liver
Spleen
-
-
Enterobacter cloacae
Enterobacter cloacae
Enterobacter cloacae Enterococcus Enterobacter cloacae
Escherichia co/i
Escherichia co/i Staphylococcus sp Escberichia co/i 6938K Staphylococcus sp
-
-
-
-
-
-
-
Clostridium
Escherichia co/i 6938K
4. 5. 6.
Enterococcus Enterobacter
cloacae
Escherichia
co/i
Enterobacter cloacae Escherichia co/i 6938K Escherichia co/i -
Escherichia co/i 6938K Enterobacter
Enterobacter
cloacae
cloacae
-
Escherichia co/i Clostfidium
Blood -
Escherichia co/i 6938K Enterococcus Escherichia co/i 6938K
3.
7. 8.
Peritoneal Fluid
Staphylococcus
sp
Staphylococcus
sp
-
-
Clostridium
TABLE II Total Incidence of Bacterial Translocation in Dogs With Acute Pancreatltis and Laparotomy Alone Site of Translocation
lp
Group
Mesenteric Lymph Nodes
Pancreas
Peritoneal Fluid
Liver
Spleen
Blood
Pancreatitis (n = 8) Laparotomy (n = 8)
6* 0
5* 0
3 0
2 0
2 0
3 0
co.05
by ~2analysis.
pancreatic sepsis are common enteric organisms, the gut has been a suspected source of indigenous contamination. The mechanism by which the enteric flora reach the pancreas has not been clearly elucidated. Transmural migration and hematogenous or lymphatic spread have been proposed as possible routes [22,13], but direct evidence linking any of these means has been lacking. Recent studies have implicated bacterial translocation [Z4] in the pathogenesis of secondary pancreatic infections. Runkel et al [4] recovered viable enteric bacteria from the MLN in 100% of rats with mechanically induced pancreatitis and from distant sites such as blood, liver, and spleen in 30%. Isaji et al [5] found positive cultures of enteric organisms in the blood, ascites, or splenic tissue of 12% of mice with diet-induced pancreatitis. However, only 1 of 12 rats in the study by Runkel and associates [4] Figure 5. Agarose gel eleclrophoresis of plasmid DNA obtained had pancreatic infection, and only 5% of mice had panfrom Escheridia coliisolates. Lane 1: DNA size standard, arrows creatic infection in the study reported by Isaji et al [5]. at left indicate fragment size in kilobases (kb); lane 2: E. m/i The isolation of common enteric organisms from ex6936K (stock strain); lane 4: E. co/i, wild strain (6936) before transformation; lanes 6,8, 10, and 12: undigested DNA from E. traintestinal sites strongly suggests but does not prove co/i isolated from stool, cecum, mesenteric lymph nodes, and bacterial translocation. To confirm the intestinal origin of pancreas, respectively, of the same dog. Lanes 3,5,7,8,11, and isolated bacteria, we have colonized dogs with a plasmid13: P&l digestion of DNA from lanes 2, 4, 8, 8, 10, and 12, labeled strain of E. coli prior to the induction of acute respectively. Arrows on the right indicate 2.68-kb and 1.28-kb fragments of pUC4K plasmid seen in lanes 3, 7,9, 11, and 13. pancreatitis. pUC4K is a non-naturally occurring and
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nontransferable plasmid. It contains an aminoglycoside 3’-phosphotransferase gene (Figure l), which confers resistance to kanamycin and neomycin [6j. DNA digestion with one of several enzymes produces DNA fragments of known molecular weight, which serve as a fingerprint for this plasmid. The composition and population level of the intestinal flora is an important factor in bacterial translocation [I5]. In our model, continuous kanamycin pressure was employed to eliminate all kanamycin-sensitive coliforms and replace them with kanamycin-resistant E. coli 6938K. This treatment should not alter either the grampositive aerobic or the strict anaerobic bacterial population of the gut. Consequently, this model should closely mimic the real-life clinical situation. Since our dogs were not kept under strict barrier conditions, it is not surprising that they were colonized by other kanamycin-resistant bacteria in addition to E. coli 6938K. In 60?&of our dogs, pancreatic necrosis was infected with enteric flora. Isolation of E. coli 6938K from the pancreas and MLN in animals previously colonized with this organism is substantial evidence for the gut being a primary source of these infections. The enteric origin of other gram-negative tissue isolates was also confirmed on the basis of the results of their antibiographics and plasmid profile analyses, along with those of the corresponding strains isolated from stool. Plasmid profile analysis has been shown to be very helpful in determining the identity of bacteria and has been used successfully to investigate outbreaks of nosocomial infections [ Id,1 71. The obvious drawback of this method is that it is not useful when bacteria are free of plasmids. Although this is uncommon for antibiotic-resistant bacteria, it was the case for the E. coli recovered from MLN and pancreas in two of our dogs. Plasmid fingerprinting was not applicable for identifying this particular strain; however, its enteric origin was confirmed on the basis of antibiographic analysis. The plasmid profiles of the E. cloucae strains obtained by stool cultures completely matched the profiles of those isolated from pancreas, MLN, and peritoneal fluid, suggesting that they were the same strains. An overgrowth of gram-negative enteric flora was not observed in our dogs with acute pancreatitis as was reported earlier by Runkel et al [4]. In their study, a notable increase in the gram-negative bacterial population was found along with a dramatic reduction of intestinal motility 48 hours after the induction of pancreatitis. However, these alterations were already less prominent after 96 hours. We studied bacterial microecology 7 days after the induction of pancreatitis. By that time, the intestinal motility as well as the bacterial population level may have returned to normal. The physical and functional integrity of the intestinal mucosa is the principal barrier preventing bacterial translocation in the normal host [I8]. The morphologic alterations of the small bowel mucosa in our dogs with acute pancreatitis are of particular importance in this context. Although the exact mechanism responsible for these morphologic changes is not clear from our study, intestinal
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5.19
3.05 1.64 1.02 Flgure 6. Agarose gel electrophoresisof undigestedplasmidDNA
chcae isolates. Lam, 1: DNA size obtainedfrom Ehfohcfw standard(kb); lanes 2 to g: E. cloacae isolatedfrom stool (2, 6), cecum (3,7), masentericlymphnodes(4), peritonealfluid(8). and pancreas (5,Q) of two differentdogs.
F@ure 7. Heal mucosa from the controldog. Villous projections lined by intact columnar epitfwlium (hematoxylin and eosin, 100X).
Figure 8. Neal mueoss fromthe dog with acute panwatitis. Extensive denudationof the mucosal swface epithellum with marked stromal lymphoplasmacyticinfiltration(hnatoxylin and eosin, 100X).
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ischemia is likely to play a pivotal role. Acute pancreatitis is known to cause severe volume depletion with reduction of cardiac output and intestinal blood flow [ 19,201, which may lead to ischemic or ischemic/reperfusion type of injury of the intestinal mucosa. Bacterial translocation is a likely result from this severe compromise of gut barrier function. In the normal host, intestinal bacteria are presumably translocating in small numbers and are constantly being killed by immunocompetent cells [ 181. Hence, the status of the immune system is a major determinant of whether translocated bacteria are eliminated or allowed to spread systemically and contaminate distant organs. The immune status was not studied in our dogs. However, acute pancreatitis is associated with severe depression of the reticuloendothelial system [21]. Nonspecific immunostimulation with levamisole reportedly decreases the incidence of pancreatic infection [22]. Enhancement of host immunity may provide a means to prevent and/or treat pancreatic sepsis. In summary, our study provides substantial evidence for the gut being a primary source of infection of acutely inflamed pancreatic tissue. Failure of intestinal barrier mechanisms, bacterial overgrowth due to impaired intestinal motility, and a depression of the immune system possibly create a situation in which the patient with acute pancreatitis continuously contaminates himself or herself with indigenous enteric organisms. Although it is uncertain whether this is the only route for bacterial infection, protection of the intestinal mucosa from ischemic damage, selective bowel decontamination, and immunostimulation are promising future approaches to prevent infection secondary to acute pancreatitis and to decrease the mortality from this devastating disease. REFERENCES 1. Frey CP, Bradley EL, Beger HG. Progress in acute pancreatitis. Surg Gynecol Obstet 1988; 167: 282-6. 2. Lumsden A, Bradley EL. Secondary pancreatic infections. Surg Gynecol Obstet 1990; 170: 459-67. 3. Buggy BP, Nostrant TT. Lethal pancreatitis. Am J Gastrcenterol 1983; 78: 810-4. 4. Runkel NSF, Moody FG, Smith GS, Rodriguez LF, LaRocco MT, Miller TA. The role of the gut in the development of sepsis in acute pancreatitis. J Surg Res 1991; 51: 18-23. 5. Isaji S, Suzuki M, Frey CF, Ruebner B, Carlson J. Role of bacterial infection in diet-induced acute pancreatitis in mice. Int J Pancreatol 1992; 11: 49-57. 6. Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 1982; 19: 259-68. 7. Hanahan D. Studies of transformation of Escherichiu coli with plasmids. J Mol Biol 1983; 166: 557-80. 8. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Springs Harbor, NY: Cold Springs Harbor Laboratory, 1989. 9. Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 1979; 7: 1513-23. 10. Becker JM, Pemberton JH, DiMagno EP, Ilstrup DM, McIlrath DC, Dozios RR. Prognostic factors in pancreatic abscess. Surgery 1984; 96: 455-61. 206
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11. Bassi C, Vesentini S, Nifosi F, et al. Pancreatic abscess and other pus-harboring collections related to pancreatitis: a review of 108 cases. World J Surg 1990; 14: 505-12. 12. Warshaw AL. Pancreatic abscess. N Engl J Med 1972; 287: 1234-6. 13. Miller TA, Lindenauer M, Frey CF, Stanley JC. Pancreatic abscess. Arch Surg 1974; 108: 545-51. 14. Berg RD, Garlington AW. Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model. Infect Immun 1979; 23: 403-11. 15. Berg RD. Inhibition of Escherichia coli translocation from the gastrointestinal tract by normal cecal flora in gnotobiotic or antibiotic-decontaminated mice. Infect Immun 1980; 29: 1073-81. 16. Parisi JT, Hecht DW. Plasmid profiles in epidemiologic studies of infections by Staphylococcus epidermidis. J Infect Dis 1980; 141: 637-43. 17. Schaberg DR, Tomkins LS, Falkow S. Use of agarose gel electrophoresis of plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli. J Clin Microbial 1981; 13: 1105-8. 18. Deitch EA. Bacterial translocation of the gut flora. J Trauma 1990; 30: S184-9. 19. Trapnell JE. Pathophysiology of acute pancreatitis. World J Surg 1981; 5: 319-27. 20. Wulff K, Sjostrom B. Influence of acute pancreatitis on central hemodynamic, regional blood flow distribution and arteriovenous shunting in the dog. Eur Surg Res 1974; 6: 354-63. 21. Larvin M, Switala SF, McMahon MJ. Impaired clearance of circulating macromolecular enzyme inhibitor complexes during severe acute pancreatitis: an important aspect of pathogenesis? Digestion 1987; 38: 32-3. 22. Widdison AL, Karanjia ND, Alvarez C, Reber HA. Influence of levamisole on pancreatic infection in acute pancreatitis. Am J Surg 1991; 163: 100-4.
DISCUSSION Charles F. Frey (Sacramento, CA): Since you demonstrated that translocation of bacteria does occur from the gut to the lymph nodes to the pancreas, would you speculate on the source of these organisms? Have you considered the possibility that there are differences in species that may account for some of the microbiologic and mucosal lesions seen in the small bowel? The portal vein in dogs does have viable bacteria, which is uncommon in either man or pig. In our experience, with dogs as opposed to pigs, there is a much higher incidence of infection in the dog model involving the pancreas and the peritoneal fluid, particularly with Escherichia coli and Clostridium sp, than in the pig model. It is also been noted in the hemorrhagic shock model of the dog that there is a very high incidence of mucosal lesions in the small bowel. On the other hand, the incidence of mucosal lesions is much lower in the pig. Would you speculate as to the best approach to prevent infection in the necrotic pancreas, either by antibiotics, immunostimulation, or mucosal cytoprotection? George Kazantsev: We don’t know the exact origin of Staphylococcus sp we have found in blood and the pancreas in two of our dogs, but it’s likely to be a bloodborne infection. This observation correlates with human data in which Staphylococcus sp occur in approximately 30% of cases. Translocation is not the only mechanism of
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infection, and there may be more than one mechanism. Since we did not obtain cultures of the portal vein, I cannot comment on the incidence of portal bacteria in our dogs. The mucosal lesions were severe, and we did not observe these lesions in our control dogs. The usual approach to preventing infection would be manipulation of the enteric microflora by giving nonabsorbable antibiotics and selectively decontaminating the gut, then enhancing the immune defense. We have had some encouraging results using granulocyte colony-stimulating factor in dogs. The third approach would be to try to protect the gut from mucosal injury, but since the mechanism of this injury remains unclear, it’s uncertain what would be beneficial, except fluid resuscitation. Edward L. Bradley III (Atlanta, GA): We can now state absolutely that with this plasmid technology, the bacteria do indeed come from the intestine. There is the very important question of the bacterial pathway to the pancreas. How does the pancreas physically become infected? This is going to be very important for therapy. Whether bacteria go through the lymphatics, through the peritoneal fluid, or through the blood will affect exactly how we use prophylaxis in these patients in an attempt to prevent pancreatic infection. One of the observations in your paper that you did not have time to present was a loss of the small intestinal barrier but not the colonic barrier. Are you, in fact, suggesting that the site of entry for the bacteria is through the damaged small bowel intestinal mucosa? If you are making that suggestion, would you give us the data showing that you found plasmid-labeled E. coli in the small intestine itself? It would be very important to evaluate and determine the pathway of the bacteria to the pancreas. George Kazantsev: In addition to the cecum, qualitative cultures from ileum and jejunum confirmed that the labeled bacteria were present. E. coli 6938K were present in ileum in all control and experimental dogs and in jejunum in five of eight control dogs and six of eight experimental dogs. This suggests that any of these segments of gastrointestinal tract could be involved in translocation but does not allow us to identify the exact site of translocation. The reason the cecum appeared normal
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while the terminal ileum was severely damaged is unclear. One possibility is the dependence of the terminal ileum on glutamine and the high concentration of xanthine oxidase that is involved in ischemic reperfusion type of injury. That could make the small bowel more vulnerable to stress than the colon. We studied the small intestinal microecology and the status of the intestinal mucosa 7 days after pancreatitis was induced, but we don’t know how the small intestinal mucosa or colonic mucosa would look during the acute stage of disease. It may have sustained ischemic injury and then repaired it. Another experiment would be necessary to look at it day by day after the induction of pancreatitis. Frank G. Moody (Houston, TX): In our work with the rat, we have found a very tight link between bacterial overgrowth and translocation. In your cecal counts, you just did the plasmid counts quantitatively, but you didn’t do total bacterial quantitation? George Kazantsev: We counted total gram-negative bacteria. Frank C. Moody: Not all the bacteria. George Kazantsev: No. Just the gentamicin-resistant bacteria, but not aerobic gram-positive and anaerobic bacteria. Frank G. Moody: What were these animals like, especially the two that had the plasmid translocation? Why didn’t they all experience translocation? Were they given supportive intravenous infusions? Did they receive analgesics? Morphine, for example, is a significant agent of translocation. Give us a little bit more detail about the animals that experienced translocation and their general well-being. George Kazantsev: Clinically, they all looked the same, except for two animals who had fever the day of death. These two animals had large peripancreatic abscesses with collection of pus and tissue sequester in the lesser sac. All other animals with positive pancreatic cultures looked grossly normal. Unidentified participant: You mean these animals were normal with the pancreatitis and pus? George Kazantsev: No. Those with pancreatic abscesses had fever the day of death. Three other animals had no fever and looked grossly normal so that clinically they didn’t appear infected.
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To examine whether the gut is a source of infection in acute pancreatitis,bacterial translocationand alterationsof intestinalmicroeeology and morphology were studied in 16 dogs. Dogs were colonized with a strain of Escherichiu coli (E. coli 6938K) bearing the plasmid pUC%K, which confets hanamycin resistance.In eight dogs (group I), pancreatitiswas induced by sodhun taurocholate/trypsin injection. Eight other dogs (group II) underwent laparotomy only. The pancreas, mesentericlymph nodes, peritoneal fluid, liver, and spleen were harvested 7 days later for culturing and histologic analysis. Identificationof E. coli 6938K was accomplished by phumid DNA analysis. Group I dogs had severe pancreatitisand iscbemicchanges in small bowel mucosa. Croup II dogs had no changes. Transkation to the pancreas occurred in five dogs and to mesentericlymph nodes in six dogs with pancreatitis.No translocationoccurred in group II dogs (p <0.05). In addition to E. coli 6938#, other gram-negative kanamycin-resistantspecies were isolated, inchuling E. coli (other than 6938K) and Enterobucter cloacue. Knteric origin of these strains was confirmed by antibiography and pbrsmid DNA analysis. No overgrowth of cecal gram-negative bacteria was found. This study suggests that the gut is a primary source of i&&ion in pancreatitisand that ischemicdamage of intestinal mucosa may promote bacterial Wan&cation.
MD,
W fection has become the major cause of morbidity and mortality in patients with acute necrotizing pancre-
ith advances in resuscitation and critical care, in-
atitis [Z-3]. The pathogenesis of these pancreatic infections remains obscure. However, recent studies suggest that translocation of intestinal bacteria may be ,a mechanism for secondary contamination of pancreatic necrosis [4,5]. The purpose of this study was to determine if the indigenous enteric flora is a primary source of infection of acutely inflamed pancreatic tissue. Bacterial translocation was studied in dogs with severe acute pancreatitis. Prior to the induction of pancreatitis, dogs were~colonized with a specific strain of Escherichiu coli labeled with a nontransferable plasmid pUC4K. This plasmid confers kanamycin resistance and can be identified by plasmid DNA analysis [a. Intestinal microecology and, morphology were also analyzed to determine possible factors that might promote translocation. MATERIAL AND METHODS Animal preparation: Adult mongrel dogs weighing 18 to 28 kg were observed for at least 1 week prior to beginning the experiment. A wild strain of E. cdi (6938) was isolated from the feces of one of the dogs, (# 6938) and transformed with the plasmid pUC4K (Bethesda Research Laboratories, Gaithersburg, MD) by,using previously described methods [ 71. Transformed kanamycinresistant bacteria (referred to herein as E. coli 6938K) were used for all subsequent experiments. Intestinal decontamination and recolonisation with E co&6938K: On 2 successive days while under
From the Departments of Surgery (GBK, RR, IJF, GD, RAP), Medicine (DWH), Pathology (PG), and Microbiology (KT), Loyola University Medical Center, Maywood, Illinois. Requests for reprints should be addressed to Richard A. Prinz, MD, Department of General Surgery, Rush University, 1653 West Congress, Chicago, Illinois 60612. Presented at the 34th Annual Meeting of The society for Surgery of the Alimentary Tract, Boston, Massachusetts, May 17-19,1993.
light general anesthesia (sodium thiamylal 15 mg/kg, intravenously), the dogs were given 160 mg of gentamicin and 250 mg of vancomycin orally to suppress the indige nous enteric flora. The dogs then received an overnight Luria-broth [8] culture of E. coli 6938K (approximately lo9 colony-forming units) mixed with food. For the rest of the experiment, drinking water was supplemented with 0.5 g/L kanamycin sulfate. Stool samples were cultured daily on McConkey agar (Difco, Detroit, MI), supplemented with 100 pg/mL kanamycin. Colonization was considered established when cultures were positive for 3 successive days. All stool isolates were saved for future analysis. Acute pancreatitiszAll procedures were performed under general anesthesia (sodium thiamylal, 30 mg/kg intravenously for induction; halothane/nitrous oxide for maintenance). Acute pancreatitis was induced by extraduodenal injection of 1 mL/kg of 4% sodium taurocholate with 3,000 IU/kg trypsin into the pancreatic duct under pressure of 200 mm Hg. The right lobe of the pancreas was made ischemic by ligating its blood supply. Sterile cotton swabs of the abdominal cavity were cul-
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Figure 1. A map of pUC4K plasmid. Digestion with FM yields 1.28-kb Kan’ fragment and 2.68-kb LacZ/Amp’ fragment.
tured to detect inadvertent bacterial contamination during the procedure. Experimental design: After colonization was established, the 16 dogs were randomly assigned to 1 of 2 groups. Eight dogs (group I) underwent laparotomy and induction of acute pancreatitis. The other eight dogs (group II) had laparotomy only. Complete blood cell counts (CBC) and plasma amylase levels were measured before the operation and on days 3 and 7 postoperatively. Blood cultures were performed every 24 hours (tryptic soy agar, Difco). The dogs were killed on the seventh postoperative day with 30 mL of saturated potassium chloride solution. Testing for bacterial transloeation: Dogs were anaesthetized as described previously. All procedures were performed under strict aseptic conditions. On entering the peritoneal cavity, 1 mL of free fluid was obtained for culture. If there was no fluid, exposed viscera were rubbed with cotton swabs that were cultured. Mesenteric lymph nodes (MLN) and portions of the liver and spleen were harvested using different sets of sterile instruments. The pancreas was completely excised and divided into 8 to 10 parts that weighed approximately 5 g each. Each part was processed separately. Organs were homogenized in 5 mL of sterile L-broth. Aliquots of the homogenate (0.1 mL) were plated in duplicate on Wilkins-Chalgren (Difco) agar, supplemented with 100 pg/mL gentamicin for detection of anaerobic bacteria. Plates were incubated at 37’C anaerobically and examined at 48 and 96 hours. Other portions of the homogenate (0.5 mL) were added to 5 mL of sterile L-broth, incubated at 37’C in a shaking waterbath for 18 hours, and then qualitatively plated in duplicate on the following media: phenylethyl alcohol (PEA) agar supplemented with 5% sheep blood (BBL, Becton Dickinson, San Jose, CA) for isolation of aerobic and facultative gram-positive bacteria, McConkey agar (BBL) for isolation of aerobic and facultative gram-negative bacteria, and McConkey agar (Difco) supplemented with 100 pg/mL kanamycin for selective isolation of E. coli 6938K or other kanamycin-resistant flora. Plates were incubated aerobically at 37°C and examined at 24 hours.
Aliquots (0.5 mL) of the homogenate were serially diluted in D-PBS and quantitatively plated on plain and kanamycin-supplemented McConkey agar. Plates were incubated aerobically at 37°C for 24 hours. Cecal bacteria were enumerated as the number of colony-forming units (CFUs) per gram of cecum (wet weight). Bacterial identification and plasmid analysis: All bacteria isolated from stool as well as those isolated from harvested organs were identified by standard techniques (AMS bioMeriex Vitek, Hazelwood, MO), and their antibiotic sensitivity patterns were determined. E. coli 6938K was initially identified by its characteristic antibiography. Final identification of this strain was accomplished by confirming the presence of plasmid pUC4K. Plasmid DNA was purified by an alkaline lysis method [8,9] and subjected to restriction digest with endonuclease PstI (New England Biolabs, Beverly, MA) (Figure 1). DNA fragments were separated by electrophoresis through horizontal 0.6% agarose gel, stained with ethidium bromide, and photographed by using Polaroid type 667 film. Plasmid DNA from gram-negative bacteria other than E. coli 6938K was purified by the same method and digested with several restriction enzymes including PstI, BumHI, Hin&II, and HincII (New England Biolabs) in an attempt to obtain a distinctive plasmid profile. Analysis of intestinal morphology and statistical analysis: Paraffin-embedded sections of jejunum, ileum,
and cecum were stained with hematoxylin and eosin and analyzed by light microscopy. Translocation incidences (discontinuous data) were evaluated by x2 analysis. Continuous data were analyzed by analysis of variance (ANOVA), followed by Duncan’s Multiple Range Test. Probabilities less than 0.05 were considered significant. RESULTS Colonization:
Ceca were excised, weighed, and homogenized in 50 mL of Dulbecco’s phosphate-buffered saline (D-PBS).
Bowel decontamination and subsequent colonization with E. coli 6938K were accomplished in all dogs. Surveillance stool cultures showed that, although E. coli 6938K was always a predominant organism, two to three additional kanamycin-resistant bacterial species could also be recovered after 3 to 4 days of kanamycin treatment. These species included E. coli (other than E. coli 6938K), Enterobacter cloacae, Proteus mirabilis, and Pseudomonas species. Acute pancreatitis: All group I dogs developed clinical signs of severe pancreatitis. Laboratory tests showed significant hyperamylasemia and leukocytosis on both the third and the seventh postoperative days (Figures 2 and 3). There was no early mortality. Group II dogs did not exhibit any distress. They had mild leukocytosis on the third postoperative day that usually normalized by the seventh day. Plasma amylase levels remained normal. At death, the pancreas in group I dogs looked enlarged and swollen with visible areas of hemorrhage and fat necrosis. Histologic examination revealed severe hemorrhagic pancreatitis with extensive parenchymal and fat necrosis and polymorphonuclear leukocytic infiltration of the stroma (Figure 4). Bacterial translocation: Infection was not seen in
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167
Enumeration
of viable gram-negative
cecal bacte-
ria:
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5000 m m
l
T
4000
2 3 G
Poncrsotitis Laparotomy
lp
3000
: :! g
2000
<
1000
0
0
3
7
OAIS
FIgwe 2. Plasma amylase levels, mean f SEM, in dogs with acute pancreatitisand laparotomyalone.
any group II dogs. In dogs with acute pancreatitis, E. coli 6938K was found in the MLN, the pancreas, and the peritoneal fluid (Table I). DNA gel electrophoresis confirmed the identity of these bacteria as 6938K (Figure 5, lanes 6 to 13). Other enteric bacteria isolated in addition to E. coli 6938K included a different strain(s) of E. coli, E. cloacae, and Enterococcus sp. All gram-negative strains were kanamycin resistant and were also previously detected by surveillance stool cultures during the experiment. To confirm their enteric origin, antibiograms and the plasmid profiles of the tissue isolates, along with those of corresponding stool isolates, were analyzed. The strains of E. coli other than 6938K had identical antibiograms but demonstrated no plasmid DNA. Thus, absolute confirmation that these were exactly the same strains was not possible. All E. cloacae isolates had identical antibiograms and contained plasmid DNA that appeared identical by plasmid electrophoresis profile. This suggests that they were the same strains (Figure 6). Digestion of plasmid DNA with multiple enzymes did not yield a distinctive pattern. Three dogs had persistently positive blood cultures beginning on the third postoperative day. Two dogs had Staphylococcus species, and one had Clostridium perfringens. In each dog with positive blood cultures, the same organisms with identical antibiograms were also isolated from the pancreas. Gram-negative bacteremia was not observed, and no other anaerobes were isolated from blood or tissues. Overall, enteric organisms were isolated from MLN in 15% and from the pancreas in 63% of the dogs with pancreatitis versus no infection in group II dogs (p <0.05) (Table II). In four dogs, MLN cultures were monomicrobial (E. coli 6938K, E. coli, Enterococcus sp., and E. cloacae). In two other dogs, at least two different bacterial species were recovered from the MLN. Cultures of pancreas were polymicrobial in all five dogs. THE AMERICAN
_ 3. White blood cell counts(WBC), mean f SEM, in dogs with acute pancreatitisand laparotomyalone.
rlgure
FIgwe 4. Pencreas from the dog with acute panaeetltis. Widespreadpemwlpd and fat necrosiswlth amdated neutrophilic infiltrate(hematoxylinand eosin, 100X).
Cecal population: E. coli 6938K was the primary gram-negative organism in ceca (80% to 100% of the gram-negative flora). The population levels were similar in group I and group II: 8.3 f 5.7 X lo7 and 5.7 f 3.9 X lo7 CFU/g, respectively. Intestinal morphology: In dogs with pancreatitis, the jejunal and ileal mucosa appeared severely damaged. The surface epithelium was denuded, and there was an extensive lymphoplasmacytic infiltration of the lamina propria (Figures 7 and 8). Group II dogs had no such pathologic changes. The cecal mucosa appeared normal in both groups.
COMMENTS Secondary pancreatic infections occur in 2% to 10% of patients with acute pancreatitis and are associated with a mortality of 16% to 40% with surgical therapy [10,11]. Since the vast majority of bacterial species involved in
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TABLE I Translocation of Different Bacterlal Species In Eight Dogs With Acute Pancreatitis Source of Isolation Mesenteric Lymph Nodes
Dog 1. 2.
Pancreas
Liver
Spleen
-
-
Enterobacter cloacae
Enterobacter cloacae
Enterobacter cloacae Enterococcus Enterobacter cloacae
Escherichia co/i
Escherichia co/i Staphylococcus sp Escberichia co/i 6938K Staphylococcus sp
-
-
-
-
-
-
-
Clostridium
Escherichia co/i 6938K
4. 5. 6.
Enterococcus Enterobacter
cloacae
Escherichia
co/i
Enterobacter cloacae Escherichia co/i 6938K Escherichia co/i -
Escherichia co/i 6938K Enterobacter
Enterobacter
cloacae
cloacae
-
Escherichia co/i Clostfidium
Blood -
Escherichia co/i 6938K Enterococcus Escherichia co/i 6938K
3.
7. 8.
Peritoneal Fluid
Staphylococcus
sp
Staphylococcus
sp
-
-
Clostridium
TABLE II Total Incidence of Bacterial Translocation in Dogs With Acute Pancreatltis and Laparotomy Alone Site of Translocation
lp
Group
Mesenteric Lymph Nodes
Pancreas
Peritoneal Fluid
Liver
Spleen
Blood
Pancreatitis (n = 8) Laparotomy (n = 8)
6* 0
5* 0
3 0
2 0
2 0
3 0
co.05
by ~2analysis.
pancreatic sepsis are common enteric organisms, the gut has been a suspected source of indigenous contamination. The mechanism by which the enteric flora reach the pancreas has not been clearly elucidated. Transmural migration and hematogenous or lymphatic spread have been proposed as possible routes [22,13], but direct evidence linking any of these means has been lacking. Recent studies have implicated bacterial translocation [Z4] in the pathogenesis of secondary pancreatic infections. Runkel et al [4] recovered viable enteric bacteria from the MLN in 100% of rats with mechanically induced pancreatitis and from distant sites such as blood, liver, and spleen in 30%. Isaji et al [5] found positive cultures of enteric organisms in the blood, ascites, or splenic tissue of 12% of mice with diet-induced pancreatitis. However, only 1 of 12 rats in the study by Runkel and associates [4] Figure 5. Agarose gel eleclrophoresis of plasmid DNA obtained had pancreatic infection, and only 5% of mice had panfrom Escheridia coliisolates. Lane 1: DNA size standard, arrows creatic infection in the study reported by Isaji et al [5]. at left indicate fragment size in kilobases (kb); lane 2: E. m/i The isolation of common enteric organisms from ex6936K (stock strain); lane 4: E. co/i, wild strain (6936) before transformation; lanes 6,8, 10, and 12: undigested DNA from E. traintestinal sites strongly suggests but does not prove co/i isolated from stool, cecum, mesenteric lymph nodes, and bacterial translocation. To confirm the intestinal origin of pancreas, respectively, of the same dog. Lanes 3,5,7,8,11, and isolated bacteria, we have colonized dogs with a plasmid13: P&l digestion of DNA from lanes 2, 4, 8, 8, 10, and 12, labeled strain of E. coli prior to the induction of acute respectively. Arrows on the right indicate 2.68-kb and 1.28-kb fragments of pUC4K plasmid seen in lanes 3, 7,9, 11, and 13. pancreatitis. pUC4K is a non-naturally occurring and
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nontransferable plasmid. It contains an aminoglycoside 3’-phosphotransferase gene (Figure l), which confers resistance to kanamycin and neomycin [6j. DNA digestion with one of several enzymes produces DNA fragments of known molecular weight, which serve as a fingerprint for this plasmid. The composition and population level of the intestinal flora is an important factor in bacterial translocation [I5]. In our model, continuous kanamycin pressure was employed to eliminate all kanamycin-sensitive coliforms and replace them with kanamycin-resistant E. coli 6938K. This treatment should not alter either the grampositive aerobic or the strict anaerobic bacterial population of the gut. Consequently, this model should closely mimic the real-life clinical situation. Since our dogs were not kept under strict barrier conditions, it is not surprising that they were colonized by other kanamycin-resistant bacteria in addition to E. coli 6938K. In 60?&of our dogs, pancreatic necrosis was infected with enteric flora. Isolation of E. coli 6938K from the pancreas and MLN in animals previously colonized with this organism is substantial evidence for the gut being a primary source of these infections. The enteric origin of other gram-negative tissue isolates was also confirmed on the basis of the results of their antibiographics and plasmid profile analyses, along with those of the corresponding strains isolated from stool. Plasmid profile analysis has been shown to be very helpful in determining the identity of bacteria and has been used successfully to investigate outbreaks of nosocomial infections [ Id,1 71. The obvious drawback of this method is that it is not useful when bacteria are free of plasmids. Although this is uncommon for antibiotic-resistant bacteria, it was the case for the E. coli recovered from MLN and pancreas in two of our dogs. Plasmid fingerprinting was not applicable for identifying this particular strain; however, its enteric origin was confirmed on the basis of antibiographic analysis. The plasmid profiles of the E. cloucae strains obtained by stool cultures completely matched the profiles of those isolated from pancreas, MLN, and peritoneal fluid, suggesting that they were the same strains. An overgrowth of gram-negative enteric flora was not observed in our dogs with acute pancreatitis as was reported earlier by Runkel et al [4]. In their study, a notable increase in the gram-negative bacterial population was found along with a dramatic reduction of intestinal motility 48 hours after the induction of pancreatitis. However, these alterations were already less prominent after 96 hours. We studied bacterial microecology 7 days after the induction of pancreatitis. By that time, the intestinal motility as well as the bacterial population level may have returned to normal. The physical and functional integrity of the intestinal mucosa is the principal barrier preventing bacterial translocation in the normal host [I8]. The morphologic alterations of the small bowel mucosa in our dogs with acute pancreatitis are of particular importance in this context. Although the exact mechanism responsible for these morphologic changes is not clear from our study, intestinal
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5.19
3.05 1.64 1.02 Flgure 6. Agarose gel electrophoresisof undigestedplasmidDNA
chcae isolates. Lam, 1: DNA size obtainedfrom Ehfohcfw standard(kb); lanes 2 to g: E. cloacae isolatedfrom stool (2, 6), cecum (3,7), masentericlymphnodes(4), peritonealfluid(8). and pancreas (5,Q) of two differentdogs.
F@ure 7. Heal mucosa from the controldog. Villous projections lined by intact columnar epitfwlium (hematoxylin and eosin, 100X).
Figure 8. Neal mueoss fromthe dog with acute panwatitis. Extensive denudationof the mucosal swface epithellum with marked stromal lymphoplasmacyticinfiltration(hnatoxylin and eosin, 100X).
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ischemia is likely to play a pivotal role. Acute pancreatitis is known to cause severe volume depletion with reduction of cardiac output and intestinal blood flow [ 19,201, which may lead to ischemic or ischemic/reperfusion type of injury of the intestinal mucosa. Bacterial translocation is a likely result from this severe compromise of gut barrier function. In the normal host, intestinal bacteria are presumably translocating in small numbers and are constantly being killed by immunocompetent cells [ 181. Hence, the status of the immune system is a major determinant of whether translocated bacteria are eliminated or allowed to spread systemically and contaminate distant organs. The immune status was not studied in our dogs. However, acute pancreatitis is associated with severe depression of the reticuloendothelial system [21]. Nonspecific immunostimulation with levamisole reportedly decreases the incidence of pancreatic infection [22]. Enhancement of host immunity may provide a means to prevent and/or treat pancreatic sepsis. In summary, our study provides substantial evidence for the gut being a primary source of infection of acutely inflamed pancreatic tissue. Failure of intestinal barrier mechanisms, bacterial overgrowth due to impaired intestinal motility, and a depression of the immune system possibly create a situation in which the patient with acute pancreatitis continuously contaminates himself or herself with indigenous enteric organisms. Although it is uncertain whether this is the only route for bacterial infection, protection of the intestinal mucosa from ischemic damage, selective bowel decontamination, and immunostimulation are promising future approaches to prevent infection secondary to acute pancreatitis and to decrease the mortality from this devastating disease. REFERENCES 1. Frey CP, Bradley EL, Beger HG. Progress in acute pancreatitis. Surg Gynecol Obstet 1988; 167: 282-6. 2. Lumsden A, Bradley EL. Secondary pancreatic infections. Surg Gynecol Obstet 1990; 170: 459-67. 3. Buggy BP, Nostrant TT. Lethal pancreatitis. Am J Gastrcenterol 1983; 78: 810-4. 4. Runkel NSF, Moody FG, Smith GS, Rodriguez LF, LaRocco MT, Miller TA. The role of the gut in the development of sepsis in acute pancreatitis. J Surg Res 1991; 51: 18-23. 5. Isaji S, Suzuki M, Frey CF, Ruebner B, Carlson J. Role of bacterial infection in diet-induced acute pancreatitis in mice. Int J Pancreatol 1992; 11: 49-57. 6. Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 1982; 19: 259-68. 7. Hanahan D. Studies of transformation of Escherichiu coli with plasmids. J Mol Biol 1983; 166: 557-80. 8. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Springs Harbor, NY: Cold Springs Harbor Laboratory, 1989. 9. Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 1979; 7: 1513-23. 10. Becker JM, Pemberton JH, DiMagno EP, Ilstrup DM, McIlrath DC, Dozios RR. Prognostic factors in pancreatic abscess. Surgery 1984; 96: 455-61. 206
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11. Bassi C, Vesentini S, Nifosi F, et al. Pancreatic abscess and other pus-harboring collections related to pancreatitis: a review of 108 cases. World J Surg 1990; 14: 505-12. 12. Warshaw AL. Pancreatic abscess. N Engl J Med 1972; 287: 1234-6. 13. Miller TA, Lindenauer M, Frey CF, Stanley JC. Pancreatic abscess. Arch Surg 1974; 108: 545-51. 14. Berg RD, Garlington AW. Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model. Infect Immun 1979; 23: 403-11. 15. Berg RD. Inhibition of Escherichia coli translocation from the gastrointestinal tract by normal cecal flora in gnotobiotic or antibiotic-decontaminated mice. Infect Immun 1980; 29: 1073-81. 16. Parisi JT, Hecht DW. Plasmid profiles in epidemiologic studies of infections by Staphylococcus epidermidis. J Infect Dis 1980; 141: 637-43. 17. Schaberg DR, Tomkins LS, Falkow S. Use of agarose gel electrophoresis of plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli. J Clin Microbial 1981; 13: 1105-8. 18. Deitch EA. Bacterial translocation of the gut flora. J Trauma 1990; 30: S184-9. 19. Trapnell JE. Pathophysiology of acute pancreatitis. World J Surg 1981; 5: 319-27. 20. Wulff K, Sjostrom B. Influence of acute pancreatitis on central hemodynamic, regional blood flow distribution and arteriovenous shunting in the dog. Eur Surg Res 1974; 6: 354-63. 21. Larvin M, Switala SF, McMahon MJ. Impaired clearance of circulating macromolecular enzyme inhibitor complexes during severe acute pancreatitis: an important aspect of pathogenesis? Digestion 1987; 38: 32-3. 22. Widdison AL, Karanjia ND, Alvarez C, Reber HA. Influence of levamisole on pancreatic infection in acute pancreatitis. Am J Surg 1991; 163: 100-4.
DISCUSSION Charles F. Frey (Sacramento, CA): Since you demonstrated that translocation of bacteria does occur from the gut to the lymph nodes to the pancreas, would you speculate on the source of these organisms? Have you considered the possibility that there are differences in species that may account for some of the microbiologic and mucosal lesions seen in the small bowel? The portal vein in dogs does have viable bacteria, which is uncommon in either man or pig. In our experience, with dogs as opposed to pigs, there is a much higher incidence of infection in the dog model involving the pancreas and the peritoneal fluid, particularly with Escherichia coli and Clostridium sp, than in the pig model. It is also been noted in the hemorrhagic shock model of the dog that there is a very high incidence of mucosal lesions in the small bowel. On the other hand, the incidence of mucosal lesions is much lower in the pig. Would you speculate as to the best approach to prevent infection in the necrotic pancreas, either by antibiotics, immunostimulation, or mucosal cytoprotection? George Kazantsev: We don’t know the exact origin of Staphylococcus sp we have found in blood and the pancreas in two of our dogs, but it’s likely to be a bloodborne infection. This observation correlates with human data in which Staphylococcus sp occur in approximately 30% of cases. Translocation is not the only mechanism of
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infection, and there may be more than one mechanism. Since we did not obtain cultures of the portal vein, I cannot comment on the incidence of portal bacteria in our dogs. The mucosal lesions were severe, and we did not observe these lesions in our control dogs. The usual approach to preventing infection would be manipulation of the enteric microflora by giving nonabsorbable antibiotics and selectively decontaminating the gut, then enhancing the immune defense. We have had some encouraging results using granulocyte colony-stimulating factor in dogs. The third approach would be to try to protect the gut from mucosal injury, but since the mechanism of this injury remains unclear, it’s uncertain what would be beneficial, except fluid resuscitation. Edward L. Bradley III (Atlanta, GA): We can now state absolutely that with this plasmid technology, the bacteria do indeed come from the intestine. There is the very important question of the bacterial pathway to the pancreas. How does the pancreas physically become infected? This is going to be very important for therapy. Whether bacteria go through the lymphatics, through the peritoneal fluid, or through the blood will affect exactly how we use prophylaxis in these patients in an attempt to prevent pancreatic infection. One of the observations in your paper that you did not have time to present was a loss of the small intestinal barrier but not the colonic barrier. Are you, in fact, suggesting that the site of entry for the bacteria is through the damaged small bowel intestinal mucosa? If you are making that suggestion, would you give us the data showing that you found plasmid-labeled E. coli in the small intestine itself? It would be very important to evaluate and determine the pathway of the bacteria to the pancreas. George Kazantsev: In addition to the cecum, qualitative cultures from ileum and jejunum confirmed that the labeled bacteria were present. E. coli 6938K were present in ileum in all control and experimental dogs and in jejunum in five of eight control dogs and six of eight experimental dogs. This suggests that any of these segments of gastrointestinal tract could be involved in translocation but does not allow us to identify the exact site of translocation. The reason the cecum appeared normal
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while the terminal ileum was severely damaged is unclear. One possibility is the dependence of the terminal ileum on glutamine and the high concentration of xanthine oxidase that is involved in ischemic reperfusion type of injury. That could make the small bowel more vulnerable to stress than the colon. We studied the small intestinal microecology and the status of the intestinal mucosa 7 days after pancreatitis was induced, but we don’t know how the small intestinal mucosa or colonic mucosa would look during the acute stage of disease. It may have sustained ischemic injury and then repaired it. Another experiment would be necessary to look at it day by day after the induction of pancreatitis. Frank G. Moody (Houston, TX): In our work with the rat, we have found a very tight link between bacterial overgrowth and translocation. In your cecal counts, you just did the plasmid counts quantitatively, but you didn’t do total bacterial quantitation? George Kazantsev: We counted total gram-negative bacteria. Frank C. Moody: Not all the bacteria. George Kazantsev: No. Just the gentamicin-resistant bacteria, but not aerobic gram-positive and anaerobic bacteria. Frank G. Moody: What were these animals like, especially the two that had the plasmid translocation? Why didn’t they all experience translocation? Were they given supportive intravenous infusions? Did they receive analgesics? Morphine, for example, is a significant agent of translocation. Give us a little bit more detail about the animals that experienced translocation and their general well-being. George Kazantsev: Clinically, they all looked the same, except for two animals who had fever the day of death. These two animals had large peripancreatic abscesses with collection of pus and tissue sequester in the lesser sac. All other animals with positive pancreatic cultures looked grossly normal. Unidentified participant: You mean these animals were normal with the pancreatitis and pus? George Kazantsev: No. Those with pancreatic abscesses had fever the day of death. Three other animals had no fever and looked grossly normal so that clinically they didn’t appear infected.
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