J. W. Costerton

TESTING THE SUSCEPTIBILITY OF BACTERIA IN BIOFILMS TO ANTIBACTERIAL AGENTS

In this minireview, we summarize recent studies of the interaction of bacteria in biofilms with antibacterial agents and we discuss the parameters that should be considered in the development of experimental protocols for study of the efficacy of antibacterial agents against pathogenic bacteria in biofilm.

Defense system in the biliary tract against bacterial infection

Bacteria can invade the biliary tract by ascending from the duodenum and via the hematogenous route from the hepatic portal venous blood. The sphincter of Oddi, situated at the junction of the biliary tract and the upper gastrointestinal tract, forms an effective mechanical barrier to duodenal reflux and ascending bacterial infection. Conversely, Kupffer cells and the tight junctions between hepatocytes help prevent bacteria and toxic metabolites from entering the hepatobiliary system from the portal circulation. The continuous flushing action of bile and the bacteriostatic effects of bile salts keeps the biliary tract sterile under normal conditions. Secretory immunoglobulin A (sIgA), the predominant immunoglobulin in the bile, and mucus excreted by the biliary epithelium probably function as antiadherence factors, preventing microbial colonization. When barrier mechanisms break down, as in surgical or endoscopic sphincterotomy and with insertion of biliary stents, pathogenic bacteria enter the biliary system at high concentrations and take up residence on any foreign bodies. Intrabiliary pressure is a key factor in the development of cholangitis. Chronic biliary obstruction raises the intrabiliary pressure. This adversely influences the defensive mechanisms such as the tight junctions, Kupffer cell functions, bile flow, and sIgA production in the system, resulting in a higher incidence of septicemia and endotoxemia in these patients. Knowledge of biliary defense against infection is still quite primitive. Unclear are the roles of sIgA in the bile, mechanism of bacterial adhesion to the biliary epithelium, Kupffer cell function in biliary obstruction, and the antimicrobial activity of bile salts.

Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system

Pseudomonas aeruginosa was cultivated at low growth rates under iron-limiting conditions on acrylic tiles. Biofilm cells exhibited increased tobramycin resistance compared with that of planktonic cells, and in old biofilms were more resistant than were cells in young biofilms. However, on suspension of the biofilm bacteria, glycocalyx-mediated resistance was lost.

Ascending infection of the biliary tract after surgical sphincterotomy and biliary stenting

It has been widely accepted that there is an ascending route of bacterial infection of the biliary tract but there is a lack of direct evidence. This hypothesis was tested in an animal experiment using the cat as an animal model. The implantation of biliary stents and surgical sphincterotomy were performed in these animals, with sham controls. Stents bypassing the sphincter of Oddi with the tip in the duodenal lumen were colonized by duodenal micro‐organisms and the biliary tract was heavily contaminated. Blockage of these stents resulted in biliary obstruction. Stents implanted within the common bile duct, proximal to the sphincter were largely unaffected by biofilm formation. After surgical sphincterotomy the biliary tract was also contaminated but, in the absence of obstruction, the animals did not develop any symptoms. It was concluded that ascending infection by duodenal biliary reflux, via the sphincter of Oddi, is an important route of infection in the biliary system.

Bacterial biofilm, brown pigment stone and blockage of biliary stents

Abstract Bacterial pathogens gain access into the biliary system by descending via the portal venous circulation or ascending through the sphincter of Oddi in duodenal‐biliary reflux. Bacteria thrive as glycocalyx‐enclosed microcolonies, coalescing to form an adherent biofilm. The establishment of biofilm is a key event in the formation of biliary sludge and pigment gallstones, and the blockage of biliary stents. The biofilm mode of growth is very effective because it provides bacteria with a measure of protection from antibacterial agents and phagocytic leucocytes. Calcification of the matrix confers further protection for the micro‐organisms living inside the biofilm. To date, attempts to prevent blockage of biliary stent have employed physical methods by using large self‐expandable stents and stents without side hole. Incorporation of antibiotics within stents has not been successful presumably because bacteria once living in their biofilm are quite resistant to antimicrobial agents. Even the most toxic bile salts have no effect on the biofilm bacteria. Yet, hydrophobic bile salts reduce bacterial adhesion on biomaterial, suggesting that incorporation of such bile salts might prevent the formation of bacterial biofilm.

The Sphincter of Oddi is a Boundary for Bacterial Colonisation in the Feline Biliary Tract

The autochthonous microbial flora of the gastrointestinal and biliary system were studied in ten cats using standard bacteriological cultures and electron microscopic techniques. The most common organisms isolated from the gastrointestinal tract were streptococci, anaerobes and coliform bacteria. A spiral organism was identified in the feline stomach and duodenum. The bile and the biliary system were entirely sterile except in one cat. Bacteria were isolated from the sphincter of Oddi. Electron microscopy confirmed that bacteria had colonised in the thick mucous layer in the gastrointestinal tract and the sphincter of Oddi. No bacteria could be found above the sphincter by electron microscopy. The sphincter of Oddi is hence the natural boundary and probably serves as a physical barrier to the upward migration of bacteria from the colonised gastrointestinal tract to the uncolonised biliary system. The cat may be a suitable animal model for bacteriological studies of the biliary system in the human. Keywords: Microbial flora; Biliary tract; Sphincter of Oddi.

Demonstration of transient bacterobilia by foreign body implantation in feline biliary tract.

The biliary tract of cats is known to be free of autochthonous bacteria above the sphincter of Oddi. In this experiment we investigated whether transient bacterobilia occurs in the biliary system under normal conditions. Polyethylene tubes and human cholesterol stones were implanted surgically into the gallbladder of cats. Sham cholecystostomy was performed as control operation. These cats were euthanized at two, six, and 12 weeks, and the implants were removed, cultured, and studied by scanning electron microscopy (SEM). Cultures and SEM also were undertaken for material scraped from the mucosal surface of the biliary tract from these animals. Colonization of bacteria on the polyethylene tubes and the gallstones was found six and 12 weeks after implantation. Adherent bacterial biofilms were demonstrated on the surfaces of these implants. This experiment showed that transient bacterobilia exists in the feline biliary tract. The foreign body implants have facilitated the adhesion of planktonic bacteria in the bile onto their surfaces and have initiated the formation of adherent biofilms within which these bacteria persisted until the system was sampled.

Bacterial biofilms and peritonitis in continuous ambulatory peritoneal dialysis

Abstract Colonization of peritoneal catheters by biofilm bacteria is common in patients undergoing treatment with continuous ambulatory peritoneal dialysis (CAPD) for end stage renal disease. The biofilm matrix generated by the colonized bacteria involves changes of the surface composition by interreaction with host tissue proteins and phagocytic cells. The biofilm therefore represents a state of biodegradation of colonized bacteria, host proteins, cells, dialysis solutions and plastic catheters. Stable biofilm, thus formed, protects the colonized bacteria from the killing effects of antibiotics, antibodies and other host defense molecules. It is postulated that catheter biofilms constitute a bacterial reservoir in patients undergoing CAPD treatment and may produce clinical peritonitis. A method to isolate biofilm bacteria from peritoneal dialysis effluents with the use of a modified Robbins Device is described. Preliminary results indicate that use of this technique can be useful in the identification of a subgroup of CAPD patients at risk of developing antibiotic resistant stable biofilms with recurrent peritonitis and loss of peritoneal catheters.

A new biofilm story in biliary tract infection

Abstract Acute cholangitis is commonly caused by biliary obstruction due to gallstones blocking the bile ducts and to malignancies of the biliary and pancreatic system. The pathogenesis of brown pigment gallstones, blockage of biliary stents, and biliary parasitic infestation are related to the formation of bacterial biofilms. Biliary pathogens, such as Escherichia coli and other enteric organisms, attach themselves to the surface of an ulcerated biliary mucosa or implanted biomaterial to form a bacterial biofilm. Some of these bacteria produce enzymes (β-glucuronidase and phospholipid A 1 ) and glycocalyx material that aid colonization and infestation. Crystals of calcium bilirubinate and other biliary sediments accumulate around the bacterial microcolonies and glycoproteins, forming adherent bacterial biofilms. The thick and often calcified biofilms obstruct the normal bile flow and lead to cholangitis.