OBJECTIVETo detect anaerobic bacteria Clostridium sp. and Bacteroides fragilis in intrahepatic stones by molecular genetic method.METHODSDNA was extracted from 59 stone samples and subjected to polymerase chain reaction (PCR) amplification targeting the 16S rRNA gene of Clostridium sp. and the glutamine synthetase gene of Bacteroides fragilis. Single-strand conformational polymorphism (SSCP) analysis was performed to identify the Clostridium sp.RESULTS16S rRNA gene sequences for Clostridium sp. were identified in 49 stones (83%, 49/59). The two most common groups were detected in 19 (41%) and 17 (37%) of the 46 samples using SSPC analysis, and 25/59 (42%) stones were tested positive for Bacteroides fragilis.CONCLUSIONSAnaerobes such as Clostridium sp. and Bacteroides fragilis present in intrahepatic stones and may play a role in stone formation. PCR is a useful technique to detect fastidious pathogens, which are difficult to culture. SSCP of PCR products is a rapid method in differentiating bacterial species.

Background: Late stent blockage is a major complication of endoscopic stent insertion for malignant obstructive jaundice. Stents block as a result of bacterial infection and biofilm formation. We report a randomized but unblinded study using an animal model to evaluate the effect of prophylactic ciprofloxacin, which selectively suppresses gram-negative bacteria, in an attempt to prolong stent patency.Method: Ten adult cats underwent surgical implantation of 5F polyethylene stents through common bile duct strictures created around the choledochotomy, with the tip of the stent left in the duodenum, Five animals received intravenous ciprofloxacin perioperatively and were then maintained on oral ciprofloxacin 25 mg twice daily. Control cats were not given antibiotics. The animals were sacrificed when signs of biliary obstruction or cholangitis persisted for more than 3 days or at the end of the 16-week study period. Stents were removed and examined for patency. In addition, the stents were cultured to recover the adherent bacteria.Results: All control animals developed stent blockage within 4 weeks. Two ciprofloxacin-treated cats developed stent blockage at 21 and 42 days, respectively, whereas the other 3 cats had patent stents for the entire study period. There was a significant difference between the median stent patency of 112 days for the ciprofloxacin group versus 16 days for the control group (p < 0.02). Bacteriologic cultures showed that the bile and blocked stents from the control group had predominantly gram-negative bacteria, whereas the bile and stents of the ciprofloxacin group had gram-positive and anaerobic bacteria, with an absence of gram-negative bacteria (p < 0.01).Conclusion: Ciprofloxacin prophylaxis eliminates gram-negative bacterial infection in bile and minimizes sludge formation and may have a potential benefit in delaying stent blockage.

BACKGROUND:Bacterial adherence and biofilm formation are important factors in the blockage of biliary stents. Clinical studies with oral antibiotic prophylaxis to prevent stent blockage have produced conflicting results. The aim of this study was to evaluate the in vitro effect of single antibiotic (ciprofloxacin, ceftazidime, or ampicillin) treatment on adherence of Escherichia coli and Enterococcus to plastic stents. METHODS:Selected clinical isolates of E coli and Enterococcus were perfused through a modified Robbins device containing segments of polyethylene stents. The stents were removed daily and the number of bacteria attached was measured. The effect of antibiotic treatment on bacterial adherence was tested by the perfusion of individual antibiotics into separate modified Robbins devices using a side-arm adaptor and the results were compared with saline controls. RESULTS:Compared with the saline controls, ciprofloxacin and ceftazidime caused a 10- to 100-fold reduction in the number of E coli attached to the stents, whereas ampicillin had no effect on adherence of E coli. Ampicillin caused a 5- to 10-fold reduction in Enterococcus adherence but there was no change with ceftazidime. Sustained reduction in E coli adherence was observed with prolonged ciprofloxacin perfusion. CONCLUSION:Timely treatment with appropriate antibiotics reduced bacterial adherence in vitro and may be potentially beneficial in the prevention of stent blockage.

Background: Biliary sludge which forms as a result of bacterial adherence and biofilm formation in the biliary system is a recognized cause of blockage of plastic stents. Bacteriological cultures of sludge have revealed a mixed infection with gram-positive and gram-negative bacteria. Animal studies have shown that prophylactic ciprofloxacin, which selectively suppress gram-negative bacteria, results in prolonged stent patency despite colonization of the stents by gram-positive bacteria.Methods: We tested a possible synergistic effect between gram-negative and gram-positive bacteria in adherence and biofilm formation on plastic stents. Clinical isolates of Escherichia coli and Enterococcus were cultured in separate chemostats to achieve a steady growth. Adherence of the two bacteria on plastic stent surface were tested separately by perfusing infected bile with the respective bacteria through different modified Robbins devices containing 10F polyethylene stent pieces up to 4 days. In a second experiment, Enterococcus was perfused through stent pieces precolonized with E. coli for 24 hours. The stent pieces were then removed daily and analyzed by bacteriologic culture and scanning electron microscopy for bacterial adherence and biofilm formation.Results: Gram-negative E. coli were more adherent than gram-positive Enterococcus. Precolonization with E. coli facilitates subsequent attachment of Enterococcus.Conclusions: We concluded that there is a synergistic effect between grampositive and gram-negative bacteria in adherence and biofilm formation.

The mesenteric hyperemia induced by intraduodenal application of hydrochloric acid (HCl) is mediated in part by capsaicin-sensitive afferent nerves. Antagonist of capsaicin-sensitive receptors (capsazepine) and blocker of capsaicin-sensitive cation channels (ruthenium red) have been described. We employed these tools to dissect the mechanism of regulation of mesenteric hyperemia induced by intraduodenal administration of HCl. Subcutaneous 100 μmolkg capsazepine or intraduodenal 0.1% ruthenium red was administered to pentobarbital anesthetized rats. Then, 2.5 mlkg of 640 μM capsaicin or 0.1 N HCl was administered intraduodenally. The mesenteric hyperemic responses were recorded. The results demonstrated that in a dose that decreased the mesenteric hyperemia induced by intraduodenal capsaicin, capsazepine failed to attenuate the mesenteric vasodilatory effect of intraduodenal HCl. Ruthenium red significantly attenuated the mesenteric hyperemia after intraduodenal capsaicin and HCl. These in vivo data provide the first functional evidence for the existence of capsazepine-sensitive capsaicin receptors and cation channel complexes in the rat duodenal and intestinal mucosa. The capsaicin- and HCl-sensitive receptors are unlikely to be functionally identical in these locations. The ruthenium redsensitive cation channels appear to mediate the capsaicin- and HCl-induced mesenteric hyperemia.

Adenosine triphosphate-dependent potassium (K+ATP) channels in several types of vascular smooth muscles mediate the vasodilation induced by calcitonin gene-related peptide (CGRP). Upon stimulation, primary afferent nerve terminals in the gastric mucosa release CGRP which mediates a protective hyperemia. We tested the hypothesis that a potassium channel blocker aggravates gastric mucosal injury by impairing afferent nerve-mediated hyperemia in the gastric mucosa. Rats were treated with K+ATP channel blocker, glybenclamide (20 mg/kg intravenously). Intragastric added ethanol (0.15 N HCl, 15% ethanol) and intragastric capsaicin (160 microM) were also administered. Glybenclamide aggravated the acidified ethanol-induced mucosal injury, and attenuated the mucosal hyperemia (hydrogen gas clearance) induced by intragastric acidified ethanol and intragastric capsaicin. These findings suggest for the first time that K+ATP channels modulate primary afferent nerve-mediated mucosal defense mechanisms in the gastric mucosa.

We tested the hypothesis that in the rat duodenum and intestine the mesenteric hyperemia due to beta-adrenoceptor stimulation is mediated by capsaicin-sensitive afferent nerves. Superior mesenteric artery blood flow was measured by pulsed Doppler flowmetry in the anesthetized rat. Functional ablation of afferent nerves was accomplished by subcutaneous 125 mg/kg capsaicin pretreatment 10 to 14 days before blood flow studies. Blockade of capsaicin-sensitive cation-selective ion channels of the duodenal and intestinal mucosal afferent nerves was achieved by intraduodenal 0.1% ruthenium red given 15 min prior to the intraduodenal administration of 5 mg/kg isoproterenol. Functional ablation of the afferent nerves and blockade of the capsaicin-sensitive cation-selective ion channels alone or in combination resulted in a significant reduction of mesenteric hyperemia induced by intraduodenal isoproterenol. These data support the hypothesis that beta-adrenoceptor stimulation by intraduodenal isoproterenol induces mesenteric hyperemia in part through afferent nerves in the rat duodenal and intestinal mucosa. The results suggest for the first time a link between beta-adrenoceptor function and peripheral capsaicin-sensitive afferent nerve-mediated mechanism in the rat gut.

The high incidence of peptic ulcer disease despite decreased acid secretion in the elderly suggests an impairment of mucosal defense mechanism with aging. Stimulation of the intestinal mucosal afferent nerves by intraduodenal application of capsaicin or hydrochloric acid (HCl) increases superior mesenteric artery (SMA) blood flow and protects the duodenal mucosa against deep damage. We tested the hypothesis that the intestinal hyperemia induced by intraduodenal capsaicin or HCL is significantly reduced in older (12 months) rats compared with younger (2 months) rats. Mesenteric blood flow was measured by pulsed Doppler flowmetry in anesthetized rats with the flow probe around the SMA. Two milliliters per kilogram of 160 microM capsaicin or 0.1 N HCl administered intraduodenally increased SMA blood flow significantly in both age groups. The peak response in SMA blood flow, however, was significantly smaller in the older rats than in the younger rats. These observations support the hypothesis that impairment of afferent nerve function occurs with aging in the rat intestine.

The efficacy of fleroxacin as therapy for experimentally induced catheter-associated urinary tract infection (CAUTI) was examined. A rabbit model of CAUTI using a closed urinary catheter drainage system and the mutant strain of Escherichia coli (WE 6933) were used to examine three dosage regimens (30 mg/kg q8h i.v.; 20 mg/kg q8h i.v.; and 10 mg/kg q8h++i.v.) of fleroxacin administered intravenously for 4 days. Quantitative bacterial counts, urinary concentrations of fleroxacin and desmethylferoxacin, histopathologic changes, and electron microscopic evaluation of catheter-associated biofilm and mucosal biofilm were performed. The results indicated that the bacterial biofilm on the urinary catheter could be eliminated by fleroxacin at 30 mg/kg q8h i.v. and 20 mg/kg q8h i.v. Fleroxacin concentrations in urine exceeded the levels necessary to destroy E. coli. Viable bacteria were eliminated with the third regimen (10 mg/kg q8h i.v.), but electron microscopy demonstrated remnants of bacterial biofilm. Histopathologic changes were significantly reduced in all fleroxacin-treated rabbits, and scanning electron microscopy showed deterioration of the bacterial biofilm on the surface of the Foley catheter in treated animals. These data suggest that fleroxacin may be useful for treating catheter-related infections because these therapeutic dosages limited ascending infections of the urethra and bladder, eliminated catheter-associated biofilms, and killed planktonic bacteria in urine.

Bacteria that grow in association with medical devices always form slime enclosed biofilms, within which they are protected, to a large extent, from the bactericidal activity of chemical biocides and antibiotics. Mature biofilms (> 7 days) are demonstrably resistant to 500-5,000 times the concentrations of these agents than are necessary to kill free floating (planktonic) cells of the same organism. The authors have discovered that this well established inherent resistance of biofilm bacteria to antibacterial agents can be completely obviated if these agents are applied to these adherent populations within an electric field. The killing of biofilm bacteria by antibiotics can be dramatically enhanced by relatively weak electric fields (1.5 V/cm and 15 microA/cm2) that, in themselves, have no deleterious effects on these slime protected populations adherent to plastic or metal surfaces. This bioelectric technology can readily be used to enhance the preimplantation sterilization of medical devices by biocides. The authors suggest that it may also be used to control biofilm formation and consequent infection by electrically enhanced perioperative antibiotic prophylaxis and by electrically enhanced penetration of antibiotics to kill the biofilm bacteria that form the inherently resistant nidus of chronic device related infections.

It has been suggested that bacteria in the intestine gain access into the biliary tract by entering the portal-venous blood. We have tested the hypothesis of hematogenous infection of the biliary system in cats. The animals were treated in three different groups: group A (no biliary obstruction), group B (acute biliary obstruction) and group C (chronic biliary obstruction). A mutant strain of Escherichia coli was infused into the splenic vein of cats at three different dosages (10(7), 10(5) and 10(3)) with sham controls. In the unobstructed biliary system, the mutant E. coli was isolated from the bile 30 min and 90 min after the infusion of 10(7) and 10(5) E. coli, respectively. No bacteria were found in the bile with the infusion of 10(3) E. coli and in the control animals. Bile flow was significantly reduced with the infusion of bacteria. The biliary excretion of E. coli in group B was similar to that in group A. In group C, the bile output in the first hour was very high but declined rapidly. E. coli was excreted into the bile at all three dosages of infusion after 30 min. Histological sections of the liver showed that the infused bacteria entered the sinusoidal blood and that some were phagocytosed by Kupffer cells. The portal-venous blood was considered an important route of bacterial invasion into the biliary system, and the penetration of bacteria was facilitated in biliary obstruction.

We have used a catheterized rabbit model to test a newly developed catheter drainage system that incorporates a microbicidal outlet tube. In these tests, this new system was compared with a currently marketed closed drainage system, with daily challenge at the outlet tube using an auxotrophically marked uropathogenic strain of Escherichia coli. In the control series (silicone catheter connected to a standard drainage bag via a tamper-evidence seal) the challenge organism developed upstream biofilm colonization of the luminal surfaces of the catheter and drainage system that was complete in 4.6 +/- 2.1 days in 11 animals, while in 1 animal a rapid overgrowth of the system with Streptococcus faecalis developed. The presence of organisms other than the marked E. coli strain on the luminal surfaces of these control catheters showed that down stream colonization was also operative. In the test catheter drainage series, the microbicidal outlet tube insert precluded upstream colonization by the challenge organism in 12 of 16 catheters and significantly delayed (7.2 +/- 1.0 days) this colonization in the remaining 4 animals. When upstream colonization by exogenous bacteria was precluded by this effective device, downstream colonization of the drainage system by mixed populations of bacteria that probably migrated into the bladder via the extraluminal urethral route developed in some animals (6 of 16).

We simulated touch contamination of peritoneal dialysis fluids perfused through an in vitro system with a modified Robbins' device (MRD) and Y-connecting tubings, to study the pathogenesis of bacterial biofilm (BB) growth on Tenckhoff catheter (TC) discs. The spike ends of Y-connecting sets were dipped in a suspension of freshly cultured cells of Staphylococcus epidermidis (3 X 10(8) cfu/ml), and connected to 2 litres of 0.5% dianeal solution which was perfused through the MRD with plugs containing TC discs. Four simulated clinical exchanges were performed with or without prior flushing and/or bleach treatment of the Y sets. Control experiments were done with fresh Dianeal solution with no contamination, flushing, or bleach treatment. BB growth on the TC discs was examined by scanning electron microscopy (SEM) and transmission electron microscopy and quantitated by routine culture of scrapings from the discs. We noted that touch contamination of dialysis fluids via the spike ends of the connecting sets can generate dense BB growth on TC discs in this experimental system (62 +/- 8% by SEM and 10.2 +/- 8.3 X 10(3) cfu/ml by culture). This growth of BB was significantly reduced by flushing the Y set with sterile Dianeal solution (24.3 +/- 3% by SEM and 5.7 +/- 3.5 X 10(1) cfu/ml by culture) and was absent by bleach treatment. We conclude that although bleach treatment of Y sets can prevent BB growth, the 'flushing' procedure alone can significantly reduce BB growth on TC from touch contamination of dialysate fluid.

We compared a newly developed closed urinary drainage system incorporating a retrograde contamination guard (RCG) with the conventional closed drainage system. The new system contains a solid bactericide (povidone iodine) pellet enclosed in a porous cartridge at the drain port of the urine collection bag. A catheterised rabbit model was used. The urine drainage bags were challenged daily for 8 days with an auxotrophically marked uropathogenic strain of Escherichia coli at the outlet tube. The bag urine was infected with the marked organism in 10 of the 11 rabbits in the control group (conventional collection bags) in 3.8 +/- 1.03 days and in only 1 of the 13 rabbits fitted with the RCG. The RCG effectively prevents contamination of the urine drainage bag and thus may play an important role in reducing the incidence of nosocomial urinary tract infections.

We have used modern techniques of direct microscopic examination and quantitative bacterial recovery to show the existence of a route of bacterial colonization along the external and internal surfaces of Tenckhoff catheters implanted in experimental animals. The external route of progressive bacterial colonization extends from the cutaneous exit site through the dacron cuff and into the peritoneum. Bacterial growth along this route consists primarily of glycocalyx enclosed bacterial biofilms adherent to catheter and tissue surfaces, and this surface colonization may or may not give rise to peritoneal infection in which free-living bacteria are found in the peritoneal fluid. The rate of this progressive bacterial colonization depends on the degree of bacterial contamination of the exit site at the time of implantation. Exit site sterilization (hibitane) delays the process while inoculation with rabbit skin strains of Staphylococcus epidermidis accelerates it. Even with optimal implantation techniques, bacterial colonization proceeds via this subcutaneous route so that most Tenckhoff catheter surfaces are covered with a bacterial biofilm, consisting predominantly of gram positive cocci, within three weeks after the implantation of these devices. The rate of bacterial biofilm development on both surfaces of these Tenckhoff catheters, the bacterial colonization of peritoneal tissues, and the dissemination of bacteria into the peritoneal fluid are all significantly accelerated by dialysis in this experimental animal model of continuous ambulatory peritoneal dialysis (CAPD).

We have developed an extracorporeal system for investigating in vitro the biofilm-adherent bacterial microcolonies (BABM) that grow on Tenckhoff catheters (TC), to study peritonitis in patients undergoing continuous ambulatory peritoneal dialysis (CAPD). A modified Robbins' device, attached to sampling plugs with TC discs and connected to the dialysate via a peristaltic pump, is run for 24 h; scrapings from pairs of TC discs are processed for assessment of viable BABM, one of each pair for culture by routine microbiology techniques and the other for examination by scanning and transmission electron microscopy (EM). No colonization was noted with fresh dialysis solutions and spent dialysates from patients without clinical peritonits; but, when bacterial suspensions were added to aliquots of the same dialysates, BABM were noted on both culture and EM. In a study of 4 patients on CAPD treatment, who had clinically evident peritonitis, routine cultures of spent dialysate were positive in only 2, but BABM were found in cultures and EM preparations of disc scrapings in all 4 cases. We conclude from these preliminary findings that this extracorporeal system is reliable, and well suited for studying the role of BABM in CAPD-associated peritonitis in vitro.