Gut microflora in the pathogenesis of the complications of cirrhosis

Best Practice & Research Clinical Gastroenterology Vol. 18, No. 2, pp. 353 –372, 2004 doi:10.1053/ybega.2004.442, available online at http://www.sciencedirect.com

10 Gut microflora in the pathogenesis of the complications of cirrhosis Guadalupe Garcia-Tsao*

MD

Professor of Medicine Gastroenterology Service, VA Connecticut Healthcare System and Section of Digestive Diseases, Yale University School of Medicine, 333 Cedar Street-1080 LMP, P.O. Box 3333, New Haven, CT 06520-8019, USA

Reiner Wiest

MD

Assistant Professor of Medicine Department of Internal Medicine, University of Regensburg, Regensburg, Germany

The gut flora plays an important role in the pathogenesis of the complications of cirrhosis. Cirrhotic patients are prone to develop bacterial infections, mainly the ‘spontaneous‘ infection of ascites or spontaneous bacterial peritonitis. Other complications of cirrhosis, such as variceal haemorrhage and ascites, occur mostly or solely as a consequence of portal hypertension. Portal pressure increases initially as a consequence of an increased intrahepatic resistance but, once collaterals have formed, high portal pressure is maintained by an increased splanchnic blood inflow secondary to vasodilatation. Splanchnic vasodilatation is the initiating event in the hyperdynamic circulatory state that aggravates the complications of cirrhosis. The gut flora plays a role in both the development of infections and in the hyperdynamic circulatory state of cirrhosis and, although less prominently, it also plays a role in the pathogenesis of hepatic encephalopathy. This chapter presents evidence regarding gut flora and its modification in the pathogenesis and management of these complications of cirrhosis. Key words: cirrhosis; gut flora; spontaneous bacterial peritonitis; hyperdynamic circulation; hepatic encephalopathy; bacterial translocation; intestinal bacterial overgrowth; intestinal permeability.

Cirrhosis represents the end-stage of any chronic liver disease. The gut flora has been implicated in the pathogenesis of alcoholic and non-alcoholic steatohepatitis, frequent causes of cirrhosis. However, the most important role of the gut flora in chronic liver disease relates to its contribution to the pathogenesis of the complications of cirrhosis. These complications are common to cirrhosis of all eatiologies and account for the high morbidity, mortality and healthcare costs associated with cirrhosis. Therefore, this chapter focuses on the role the gut flora plays in the pathogenesis of the complications of cirrhosis. * Corresponding author. Tel.: þ1-203-737-6060; Fax: þ1-203-785-7273. E-mail address: [email protected] (G. Garcia-Tsao). 1521-6918/$ - see front matter Q 2003 Elsevier Ltd. All rights reserved.

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Two major syndromes result from cirrhosis: portal hypertension and hepatic insufficiency. Cirrhosis can remain compensated for many years prior to the development of decompensation, marked by the development of any of the following complications: jaundice, variceal haemorrhage, ascites or encephalopathy. Except for jaundice, which occurs as a result of hepatic insufficiency, all other complications of cirrhosis occur mostly or solely as a consequence of portal hypertension.1 Additionally, cirrhotic patients are prone to develop bacterial infections, mainly the ‘spontaneous’ infection of ascites or spontaneous bacterial peritonitis (SBP). Cirrhosis leads to both an increase in hepatic sinusoidal pressure and an increase in portal pressure gradient, that is, the pressure difference between the portal vein and the systemic veins. Pressure in any vascular system results from the interaction between resistance to flow and/or blood flow into the system. Portal pressure increases initially as a consequence of an increased intrahepatic resistance but, once collaterals have formed, high portal pressure is maintained by an increased splanchnic blood inflow secondary to vasodilatation. Splanchnic vasodilatation is the initiating event in the hyperdynamic circulatory state that aggravates the complications of cirrhosis.2 The gut flora plays a role in the development of infections and also in the hyperdynamic circulatory state of cirrhosis and, although less prominently, it also plays a role in the pathogenesis of hepatic encephalopathy (HE).

THE GUT FLORA AND SPONTANEOUS INFECTIONS IN CIRRHOSIS The incidence of infections and risk factors Bacterial infections are a known complication of cirrhosis, with a reported incidence that ranges between 15 and 47%.3 – 5 More recent, larger, prospective series, report bacterial infection rates in cirrhotic patients (either at the time of admission or during hospitalization) of 32% (507 of 1567 admissions)6 and 34% (139 of 405 admissions).7 These figures contrast with a general hospital population rate of infection of 5– 7%. In fact, a 15-year population-based study in northern Denmark revealed that the incidence of bacteraemia in 1339 cirrhotic patients was 10.5 times higher than the expected incidence among the background population (Danish citizens older than 20 years living in the same area).8 Two factors are predictive of the development of bacterial infections in cirrhosis: the severity of the liver disease and gastrointestinal (GI) haemorrhage. Patients with decompensated cirrhosis have been consistently shown to develop infections at a higher rate compared to compensated cirrhotic patients.3,5 In prospective series, the incidence of bacterial infections in cirrhotic patients with GI haemorrhage is about 45%, greater than the 32–34% infection rate in hospitalized cirrhotic patients at large.6,7 In a prospective study, low serum albumin and admission for GI bleeding were the only variables independently predictive of the development of a bacterial infection in cirrhosis.4 The consequences of bacterial infections in cirrhosis Cirrhotic patients who develop an infection have a significantly higher mortality than uninfected patients.5,7,9 Although this could be related to the susceptibility of patients with more severe liver disease to develop infections, a study performed in 405 cirrhotic patients, identified Child C and the occurrence of bacterial infection as independent

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predictors of mortality.7 Although about a third of patients die from sepsis, renal dysfunction is the most important predictor of mortality in patients with SBP.10 – 14 Current evidence also suggests that infection predisposes to recurrent variceal hemorrhage15 and is associated with failure to control variceal haemorrhage.16,17 Sources and type of bacterial infection in cirrhosis Older studies assessing the aetiology and types of bacterial infections in cirrhotic patients showed that the most common infections were community-acquired, mainly urinary-tract infections, SBP and pneumonia, 70– 80% of which were caused by Gramnegative bacilli (GNB), mainly Escherichia coli, suggesting that the gut was the main source of bacteria. The spectrum of bacteria causing infection in cirrhosis in more recent series show a significantly higher rate of Gram-positive cocci infections, probably due to an increase in the number of therapeutic invasive procedures6 and to the use of chronic antibiotic prophylaxis.18 – 20 However, the most common infections, SBP and urinary-tract infection, are still caused mainly by GNB.6 Bacterial translocation in cirrhosis Bacterial translocation (BT) is defined as the migration of viable microorganisms from the intestinal lumen to mesenteric lymph nodes (MLN) and other extraintestinal organs and sites. The phenomenon of BT increases in conditions associated with a high risk of infections by GNB and multiple organ failure such as haemorrhagic shock, intestinal obstruction, major burn injury and serious trauma.21 Given the predominance of GNB isolated from ascites22, BT has been postulated as the main mechanism in the pathogenesis of SBP. The presence of bacteraemia in half of the cases of SBP and the occurrence of cases of isolated bacteraemia in cirrhotic patients without an obvious primary focus of infection (spontaneous bacteraemia), suggest that bacteria gain access to the systemic circulation prior to infecting the peritoneal fluid. BT in experimental cirrhosis In experimental studies, BT is defined as a positive MLN bacteriological culture. Initial studies of BT performed in animals with pre-hepatic portal hypertension (portal vein ligation) showed that, although in acute portal hypertension (i.e. 2 days after portal vein ligation) over 80% of rats demonstrated BT (compared to only a third of controls), in the chronic setting (i.e. 15 days after ligation), a setting akin to patients with cirrhosis, BTwas not different between portal hypertensive animals and controls.23 Conversely, a study performed in rats with CCl4-induced cirrhosis demonstrated that 56% of cirrhotic rats with ascites had BT, while BT did not occur in cirrhotic rats without ascites or in normal rats.24 Animals with ascites had lower serum albumin and higher bilirubin levels, i.e. a poorer liver function, compared to non-ascitic cirrhotic animals. Several studies confirm that BT to MLN occurs in about 50% of cirrhotic rats with ascites, compared to 0 –10% rates in normal rats, and that most organisms isolated are members of the family Enterobacteriaceae, with E. coli being the predominant organism.25 The lack of significantly increased translocation in portal hypertensive animals with normal livers and in cirrhotic animals without ascites suggests that portal hypertension is not an important factor in the pathogenesis of BT in cirrhosis and that liver insufficiency is a key factor. This contention is supported by studies in animals with galactosamine-induced liver failure, an experimental model that does not develop

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portal hypertension, in which BT was detected in all liver failure animals at 24 and 48 hours compared to only 16% at 24 hours in controls.26 Immunofluorescence studies have shown larger numbers of bacteria in caecal and colonic tissues compared to stomach and small intestine, suggesting that the preferred site for BT might be the caecum or colon.27 Accordingly, histological abnormalities, including immunostaining for E. coli, have been more prominent in the caecum of cirrhotic animals with ascites.24 Two studies, one in endotoxin-induced BT28 and another in cirrhotic rats29, showed mucosal injury mainly in the ileum and caecum but not in the jejunum. However, it has been postulated that the lower GI tract, given the constant presence of bacteria, is more efficient in eliminating translocating bacteria compared to the upper GI tract. In fact, inoculation of equal concentrations of E. coli into small or large bowel demonstrated higher rates of translocation with small bowel inoculation, indicating that the threshold for BT is lower in the small bowel.30 This is further supported by a study that showed that lower BT rates in cisapride-treated animals were related to lower jejunal bacterial counts and not to lower caecal counts.31 BT in cirrhotic patients Most studies of BT in humans have been performed in non-cirrhotic patients subjected to elective or emergency laparotomy. In these patients, BT to MLN ranges between 4 and 59%, with highest rates observed in patients with intestinal obstruction, Crohn’s disease and organ donors and with the most common organisms being members of the family Enterobacteriaceae, mainly E. coli.25 However, in cirrhotic patients, BT has either been shown to be absent32 or not different from non-cirrhotic controls (, 10% rate).33 In the largest study by Cirera et al33, BT to MLN was higher in Child C cirrhotic patients (4/13 or 30%), compared to Child B (3/37 or 8%) and Child A (1/29 or 3%) patients. Perioperative antibiotic prophylaxis almost certainly underestimates the prevalence of BT in these studies. To circumvent this problem, levels of tumour necrosis factor (TNF)-a were measured in MLN as a surrogate for BT and were found to be higher in cirrhotic patients than in controls, particularly in those with ascites.34 Patients with high levels of TNF had a higher Child-Pugh score and were the only ones to develop bacterial infections during the first month post-transplant. Once in MLN, it is assumed that bacteria go through lymphatics and into the systemic circulation. Because translocation to MLN is so difficult to assess in humans, and because most episodes of systemic bacterial seeding remain undetected, a recent study looked at bacterial DNA (by the polymerase-chain reaction) and detected it in blood and ascites of nine of 28 cirrhotic patients who had negative ascites and blood bacteriological cultures.35 The similarity between sequences from ascites and blood indicated that the DNA present in both locations originated from a single clone and probably represented single-clone episodes of BT and systemic seeding. However, contrary to results from the TNF-a study, there were no differences in the severity of liver disease between patients with and without bacterial DNA and therefore the significance of these findings is uncertain. Factors that facilitate BT in cirrhosis Three factors have been implicated in the development of BT21, all of which have been found to be present in cirrhosis: impaired immunity, intestinal bacterial overgrowth and increased intestinal permeability.

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Impaired immunity For translocation to become clinically significant, i.e. for it to lead to SBP or bacteraemia, a failure of local and systemic immune defences should also be present. That is, in a healthy, non-immunocompromised host, translocated bacteria may reach MLN or portal blood but they will usually be phagocytosed and killed prior to multiplication and seeding of systemic blood and other sites. Cirrhosis is accompanied by a decrease in bactericidal activity by phagocytic cells.36 – 39 The presence of ascites indicates further immunosuppression as demonstrated by a study in which a significantly higher rate of bacteraemia occurred after the intratracheal instillation of Streptococcus pneumoniae in cirrhotic rats with ascites compared to cirrhotic animals without ascites or normal controls.40 In this study, cirrhotic rats with ascites were shown to have the lowest serum complement levels, critical elements for bacterial phagocytosis. In cirrhotic patients, serum complement levels are low and correlate with markers of liver synthetic function (serum albumin and prothrombin time), indicating decreased hepatic synthesis of complement as a cause of these deficiencies.41,42 In a prospective study of 77 patients with alcoholic cirrhosis, low serum concentrations of C3 and decompensated cirrhosis were independent predictors of infection and mortality.42 Importantly, cirrhosis is accompanied by an impaired reticuloendothelial system (RES) activity. The RES is the main defensive system against bacteraemia and other infections acquired through a haematogenous route. Most of the RES activity is located in the liver where Kupffer cells (tissue macrophages) are the major components. In cirrhosis, RES activity is impaired because of porto-systemic shunting that bypasses the liver (thereby escaping the action of the RES) and because of an impaired phagocytic activity of Kupffer cells. It has been shown that cirrhotic patients with a decreased RES activity develop spontaneous bacteraemia and SBP at a higher rate than patients with normal RES activity.43 Bypassing the RES through porto-systemic shunting is not only an important mechanism that explains failure to clear portal or systemic bacteria in cirrhosis, but it would also explain failure to clear other bacterial products such as endotoxins and cytokines. The even higher risk of infection in cirrhotic patients with GI haemorrhage is thought to be secondary to multiple factors, among them a further decrease in RES activity44,45 and a higher BT rate.46 Intestinal bacterial overgrowth (IBO) Experimental studies demonstrate that rats with cirrhosis, ascites and BT have significantly higher rates of IBO compared to animals without BT.31,47 Importantly, in the absence of IBO (that is, with bacterial counts within two standard deviations from the mean bacterial count of normal rats), BT occurs rarely (0– 11%) and at rates comparable to those in normal rats. However, because BT does not occur in up to half the animals with IBO, it appears that IBO is necessary but not sufficient for BT to occur and that another factor, perhaps a decreased immunity, plays the most important role. In humans, IBO has been shown to be more prevalent in cirrhotic patients than in healthy controls, particularly in those with more severe liver disease48,49 and in those with a prior history of SBP.50 These studies have raised concerns regarding the use of breath tests in the diagnosis of IBO, but studies in which IBO is assessed by quantitative culture of jejunal aspirates have also shown high IBO rates of 43%31 and 61%51 in cirrhotic patients. Interestingly, in one of these studies51, development of SBP did not correlate with IBO but did correlate with ascitic fluid protein (marker of decreased

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local immunity) and with serum bilirubin (marker of poor liver function), underscoring the importance of a decreased immune status as the main factor in the pathogenesis of BT in cirrhosis. IBO in cirrhosis has been related to a delayed intestinal transit time that has been shown to occur in cirrhotic rats, particularly in those with BT47 and in patients with cirrhosis52, particularly in those with more severe liver disease.53 Interestingly, the administration of octreotide, which results in a further delay in intestinal transit time, was not associated with an increase in BT.54 Intestinal permeability Cirrhosis is associated with structural and functional alterations in intestinal mucosa that may increase permeability to bacteria. While structural changes such as intestinal mucosal congestion, submucosal inflammation and oedema have been observed in cirrhosis24,46,55 it is uncertain whether these are the cause or the result of BT. Experimental cirrhosis results in oxidative stress of the mucosa of the small intestine as determined by increased xanthine oxidase activity and altered antioxidant status, increased lipid peroxidation of the brush border membranes and abnormal intestinal transport.56 These are changes similar to those described in endotoxin-induced damage, in which BT is also increased.28 The association of cirrhosis with oxidative damage of the intestinal mucosa is further supported in another study in which malonandehyde, a marker of lipid peroxidation, was found to be significantly higher in ileal and caecal (but not in jejunal) mucosa of cirrhotic rats, mainly in those with ascites and BT.29 However, even in the presence of mucosal injury, the strain of bacteria (pathogenic versus non-pathogenic) appears to be a more important determinant of BT.57 Regarding functional changes, intestinal permeability has been shown to be increased in cirrhotic rats with ascites, particularly in those with BT.47,58 However, while BT occurred in 13/15 (87%) cirrhotic rats with both IBO and increased intestinal permeability, it did not occur in any of six animals with increased intestinal permeability alone.47 Furthermore, elimination of IBO alone, without changes in intestinal permeability, led to a decrease in BT. This further suggests that IBO is more important than increased gut permeability in promoting BT. Studies of intestinal permeability in patients with cirrhosis using differential sugar absorption are controversial. While some show no differences in permeability between cirrhotic patients and controls59, a more recent study showed a decrease in mannitol excretion in cirrhotic patients, particularly in those who had spontaneous bacteraemia or SBP within 10 days of the test.60 As sepsis itself can induce intestinal permeability changes, these results are difficult to interpret. BT in the pathogenesis of bacterial infections in cirrhosis Although there is evidence of an association between BT and SBP, it is still unclear whether the relationship is causal or circumstantial. In experimental studies, cirrhotic rats with positive ascites cultures have all had concurrent positive MLN cultures, often with the same organism, suggesting a causal relationship. In fact, a study that typed the DNA of isolated organisms showed an identity rate of 80% in five cases in which bacteria were isolated from both MLN and ascites.61 However, studies in humans are not as clear. In fact, although most human studies show that BT correlates with the development of a post-operative infection,

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the majority of patients with evidence of BT do not develop a related infection.25 Furthermore, in patients who develop an infection, organisms cultured from MLN rarely correlate with those causing post-operative infection. In cirrhotic patients, BT was not only unable to predict post-operative sepsis but was actually associated with a lower rate of post-operative infection33. Human studies are confounded by methodological issues, including the fact that only one lymph node is cultured and that patients with cirrhosis receive perioperative antibiotics. More compelling evidence regarding a causal relationship between BT and development of bacterial infections in cirrhosis comes from studies showing that selective intestinal decontamination by the use of oral non-absorbable antibiotics decreases the development of SBP and other spontaneous infections in cirrhotic patients.62 – 65

GUT FLORA AND THE HYPERDYNAMIC CIRCULATORY STATE (HCS) IN CIRRHOSIS Consequences of HCS in cirrhosis Vasodilatation, and the subsequent development of the HCS, lead to a worsening of all complications of cirrhosis.2 Although the increased portal pressure gradient per se leads to the formation of gastro-oesophageal varices, it is the increased flow through them, a result of the hyperdynamic splanchnic circulation, that leads to their growth and eventual rupture. Another frequent complication of cirrhosis, ascites, results not only from an increased sinusoidal pressure but also from sodium retention that, in turn, results from vasodilatation and activation of neurohumoral systems. The hepatorenal syndrome results from severe peripheral vasodilatation that leads to renal vasoconstriction. The hepatopulmonary syndrome results from severe pulmonary arterial vasodilatation leading to shunting and hypoxaemia. HE is a consequence, not only of shunting of toxins through porto-systemic collaterals, but also of brain oedema secondary to brain arterial vasodilatation. Splanchnic hyperaemia impairs rolling, adherence and migration of phagocytic cells in mesenteric venules contributing to the impaired immune response in cirrhosis and may possibly contribute to the development of infections.66 Finally, the severity of arterial hypotension and activation of neurohumoral systems have been shown to be a strong and independent predictor of survival in cirrhosis.67 Pathophysiology of the hyperdynamic circulatory state (HCS) The HCS is characterized by low vascular resistance and mean arterial pressure and by increased heart rate, cardiac output and regional blood flow.2 The exact pathophysiology of the HCS is still controversial; however, peripheral and predominantly splanchnic arterial vasodilatation induces arterial underfilling and hence, relative hypovolaemia and reduction in arterial pressure, leading to activation of neurohumoral systems (renin –angiotensin –aldosterone, sympathetic nervous system, antidiuretic hormone) by stimulation of arterial baroreceptors and cardiopulmonary volume receptors. Mediators from these systems lead to sodium and water retention by the kidneys, increasing plasma volume. The expansion in plasma volume and the corresponding increase in blood volume are essential for the full expression of the HCS.2

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Although several vasodilators, such as prostacyclin, adrenomedullin, calcitonin-generelated peptide, substance P and glucagon have been related to the pathogenesis of HCS, nitric oxide (NO) appears to be the key vasodilator responsible for the haemodynamic abnormalities of cirrhosis.68 This is most clearly shown in experiments in which non-specific inhibition of NO synthesis leads to an almost complete normalization of splanchnic haemodynamics and to prevention of the HCS.69,70 NO is synthesized by different isoforms of NO synthases (NOS); endothelial (e) NOS and neuronal (n) NOS are expressed constitutively whereas inducible (i) NOS is expressed after induction by lipopolysaccharides, endotoxins and cytokines. As early as 1991, Vallance and Moncada had put forward the hypothesis that gutderived endotoxins and the resultant increase in inflammatory cytokines induced the expression of iNOS in vessel walls and led to sustained NO production, vasodilatation and HCS.71 Endotoxaemia is present in cirrhosis and has been shown to correlate with the severity of liver disease72,73 and with serum NO-metabolite levels.74,75 Also, endotoxaemia in cirrhosis is associated with a lower systemic vascular resistance and a higher cardiac output.72 The importance of endotoxins in the development of HCS is further supported by a recent study in which the HCS was found to be more marked in a subgroup of patients with cirrhosis and ascites who had high levels of LPS-bindingprotein, a protein that is synthesized in the liver in response to endotoxin.76 The importance of cytokines, specifically TNF-a, in the development of the HCS is evidenced by studies which show that TNF-a inhibitors ameliorate HCS in cirrhotic rats.77

The gut as a source of bacteria, bacterial products, endotoxins and cytokines in cirrhosis Bacteria colonizing the gut represent a large reservoir of microbial products, such as lipopolysaccharides, endotoxins and other bacterial wall fragments capable of inducing cytokine and NO synthesis. If these products cross the gut mucosa they will be delivered directly into the splanchnic circulation. Haemodynamic alterations at the level of the splanchnic circulation are key in the development of HCS, as evidenced by the following facts: (a) haemodynamic changes in the splanchnic circulation precede systemic abnormalities; (b) the hyperdynamic splanchnic circulation is a major contributing factor in maintaining and aggravating portal hypertension; and (c) increases in regional blood flow observed in the HCS are particularly prominent in splanchnic organs. Therefore, a gut origin of products capable of inducing NO synthesis has been postulated as an important mechanism in the development of HCS in cirrhosis. The finding of higher portal than systemic vein concentrations of NO in human cirrhosis support this contention.78 The definition of BT has relied upon the culture of viable bacteria in MLN and this is particularly relevant in associating BT with the development of bacterial infections. However, BT is also linked to the passage of bacterial products such as endotoxin from non-viable bacteria and for the passage of mediators such as cytokines that can induce NO synthesis and thereby the circulatory alterations of cirrhosis. BT can be considered the primary event in the genesis of endotoxaemia in cirrhosis. While endotoxaemia is present in cirrhotic rats with BT, only low and—in most cases—negligible concentrations of endotoxin are detectable in cirrhotic rats without BT.79 The higher endotoxin levels in splanchnic blood of cirrhotic animals and the strong correlation between MLN and blood endotoxin support a gut origin of

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endotoxin. This is further supported by the finding, in patients subjected to shunt surgery, of higher portal than peripheral venous endotoxin levels.80 BT may render the gut a ‘cytokine-releasing’ organ even in conditions in which BT is limited to MLN. Gut-associated-lymphatic-tissue (GALT) has been shown to produce and release TNF in response to BT even in the absence of portal or systemic spread of bacteria.81,82 Additionally, gut flora appear to modulate gut cytokine production because IBO is also associated with an increased production of cytokines by the gut.83 In experimental cirrhosis, TNF-a levels in MLN have been reported to be elevated only in the presence of BT and to correlate closely with TNF-a serum levels.84 Although the relationship between BT and TNF levels has not been established in human cirrhosis, TNF-a in MLN has been shown to be higher in cirrhotic patients than in controls and to correlate with plasma TNF-a levels ðr ¼ 0:56Þ:34 This suggests that lymph node-derived TNF-a contributes to the associated systemic levels, for example, via drainage into the systemic circulation. Interestingly, patients with high TNF-a levels had a tendency for greater haemodynamic disturbances (lower systemic vascular resistance and higher cardiac index and portal pressure).34 Repeated episodes of BT may have a ‘priming’ effect on polymorphonuclear cells, leading to the abnormally increased cytokine response to endotoxin observed in cirrhosis.85,86 Bacterial translocation in the pathogenesis of the HCS of cirrhosis Recent studies performed in cirrhotic rats with ascites exemplify the sequence of events from BT, endotoxins, cytokines, NO overproduction and the development of HCS. In the first of these studies84, cirrhotic rats with ascites were divided according to the presence or absence of BT. While all cirrhotic rats with ascites demonstrated in vitro splanchnic vascular hypo-responsiveness to vasoconstrictors (the experimental hallmark of HCS) and a lower mean arterial pressure than normal rats, these abnormalities were significantly greater in cirrhotic rats with BT. Haemodynamic changes were closely related to an increase in the production of TNF-a and NO. Therefore, this study demonstrates that BT leads to further derangement of the already altered circulatory state of cirrhosis. These findings were supported by an additional study79 which showed that BTwas associated with local MLN and systemic appearance of endotoxin that correlated with increases in serum NO levels. Although TNF-a and endotoxins are well known stimulators of iNOS, both studies showed that NO was eNOS-derived, not iNOS-derived. Another pathway by which TNF-a and LPS can lead to NO overproduction, which was actually shown to occur in this model79,84, is through stimulation of GTP-cyclohydrolase and synthesis of tetrahydrobiopterin, which, in turn, leads to activation of eNOS (not iNOS). The sequence from BT ! endotoxins ! cytokines ! NO overproduction ! HCS is difficult to test in humans given methodological limitations, essentially a lack of a surrogate marker for BT. One could argue that, rather than being the cause of BT, HCS could lead to BT. In fact, the hyperdynamic splanchnic circulation has been associated with a defective inflammatory response that would favour BT66, and NO overproduction may damage the intestinal epithelium87 – 89, also favouring BT. Nonetheless, more compelling evidence regarding the relationship between BT, endotoxaemia and aggravation of HCS in cirrhosis comes from studies showing that selective decontamination of the gut using norfloxacin attenuates the development of HCS.76,90,91 In an animal model of cirrhosis from chronic bile duct ligation, norfloxacin significantly reduced BT due to GNB and ameliorated peripheral and pulmonary

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vasodilatation.91 A placebo-controlled, cross-over study performed in nine cirrhotic patients showed an increased baseline forearm blood flow (a measure of peripheral vasodilatation) that normalized after a 4-week course of oral norfloxacin.90 Furthermore, the increased response to an NO inhibitor observed at baseline normalized after norfloxacin, suggesting that most of the vasodilatation in cirrhotic patients was due to an increase in NO. In the previously mentioned study in which LPSbinding protein (LBP) was used as a marker of endotoxaemia, a 4-week course of oral norfloxacin significantly increased systemic vascular resistance and decreased cardiac index, that is, ameliorated HCS76 in patients with high LBP. It is important to stress that the involvement of enteric bacterial products in increasing NO synthesis and worsening HCS is probably limited to a subset of decompensated cirrhotic patients with advanced liver disease because norfloxacin did not modify NO levels or improve haemodynamics in patients with ascites and normal LBP levels.

MANIPULATION OF THE GUT FLORA, EFFECT ON BT AND EFFECT ON DEVELOPMENT OF INFECTIONS AND HCS Selective intestinal decontamination in prevention of BT The use of antibiotics that will selectively eliminate GNB (selective intestinal decontamination or SID) should be effective in eliminating IBO and BT. Long-term administration of orally administered norfloxacin, a poorly absorbed quinolone, has been shown to produce a marked reduction in GNB from the faecal flora of cirrhotic patients without significant effects on Gram-positive cocci or anaerobic bacteria.64 Experimental studies using oral norfloxacin or trimethoprim/sulfamethoxazole have shown a decrease in BT46 or a decrease in translocation due to GNB in animals receiving the antibiotic.91,92 However, other studies have shown no effect on BT93,94 and even studies that have shown an effect on BT have been unable to demonstrate a decrease in ascites infection. Oral non-absorbable or poorly absorbed antibiotics (i.e. SID) have been used in6 cirrhotic patients in different clinical settings. Even though several randomized trials have investigated the efficacy of antibiotics in preventing infections in cirrhotic patients with GI haemorrhage, most of them use systemic antibiotics,95 only two of them used short-term SID62,63 and both demonstrated a significant reduction in infection rate in treated patients. In a double-blind, placebo-controlled study, continuous oral norfloxacin (long-term SID) was shown to significantly decrease the 1-year probability of developing recurrent SBP from 68% (in the placebo group) to 20% (in the norfloxacin group)64, which was even more obvious for the probability of developing SBP caused by GNB that was reduced from 60 to 3%. Two randomized studies analysed the effect of SID in the prevention of the first episode of SBP in patients with low ascites protein65,96, demonstrating a decrease in the rate of SBP in norfloxacin-treated patients, albeit not statistically significant in the only placebo-controlled trial.65 The above is evidence that SID is effective in the prophylaxis of bacterial infections in cirrhosis. However, long-term prophylaxis is not ideal as it has been clearly associated with the development of quinolone-resistant infections.96 In a recent study, 65% of GNB isolated from patients on long-term norfloxacin prophylaxis were resistant to quinolones, while this occurred in only 29% of GNB isolated from patients not on prophylaxis.6 Interestingly, GNB isolated from patients

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on prophylaxis also had a significantly higher rate of trimethoprim – sulfamethoxazole resistance (68 versus 44%). It is therefore important to find non-antibiotic methods to eliminate BT and prevent infections.

Prokinetics IBO is facilitated by alterations in intestinal motility, and one of the purported mechanisms for the altered motility in cirrhosis is an increased adrenergic activity. An experimental study using propranolol, a b-adrenergic blocker, showed that it significantly shortened intestinal transit time in cirrhotic rats and decreased IBO and BT.47 However, no differences in the rate of ascites infection were observed between treatment groups. Cisapride, an intestinal prokinetic, has been used in two experimental studies, both of which demonstrated that cisapride-treated animals had decreases in intestinal transit time, IBO and BT.31,58 Two studies in cirrhotic patients show that a 6-month course of oral cisapride decreases orocaecal transit time and eliminates IBO in 80% of patients with IBO at baseline.31,97 Although neither experimental studies nor studies in humans have shown a decrease in SBP or other infections in treated subjects, one of them showed that 2/10 patients on placebo developed SBP and urinary infection while infections do not seem to have occurred in patients on antibiotics or cisapride.97 These results are encouraging and should stimulate further studies using prokinetics.

Probiotics Bacteriotherapy with Lactobacillus has been reported to correct bacterial overgrowth, stabilize mucosal barrier function, and decrease BT in rat models of acute liver injury and failure. Therefore, studies have been performed in animals with portal hypertension and cirrhosis using different lactobacilli-fermented diets. In a study using Lactobacillus acidophilus- and Lactobacillus GG-fermented milk in rats with acute pre-hepatic portal hypertension, BT was not significantly different between animals that received Lactobacillus (82%) and those that received placebo (75%).98 In another study in rats with CCl4-induced cirrhosis and ascites, BT rates were not significantly different between rats that received Lactobacillus GG and those that received milk alone (93 versus 84%, respectively), despite demonstration of caecal colonization by Lactobacillus in 90% of treated animals94. Importantly, ascites infection rates were also no different among groups and BT of Lactobacillus was observed in 24% of rats treated, underlining the importance of the severity of mucosal barrier dysfunction in cirrhosis with ascites. The addition of fibre to Lactobacilli appears to be effective in patients with liver disease. In a prospective randomized trial, patients who received fibre in addition to living Lactobacillus plantarum 299 had a significantly lower rate of post-operative bacterial infections (13%) compared to patients who received SID alone, in whom the rate of infection was surprisingly high at 48%.99 Additionally, the infection rate was not significantly different from patients who received inactivated Lactobacilli and fibre (34%). As it is a cheap and feasible alternative to SID, further studies should evaluate the effect of this combination in other cirrhotic populations.

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Other Antioxidants ^ probiotics An experimental study in rats with CCL4-induced cirrhosis showed that intestinal enterobacteria and enterococci, BT, and ileal malonaldehyde levels were significantly lower in cirrhotic rats treated with antioxidants alone or in combination with Lactobacillus johnsonii La1 compared to cirrhotic rats receiving water.100 Only rats treated with antioxidants plus L. johnsonii La1 showed a decrease in endotoxaemia with respect to cirrhotic rats receiving water. Unfortunately, the effectiveness of L. johnsonii La1 alone was not evaluated, neither was the potential beneficial effect of antioxidants on CCl4-induced liver damage. Bile acids Bile acids are bacteriostatic and may contribute to the sterility of the content of the small intestine in health; however, they appear to inhibit growth—particularly of anaerobic bacteria. The absence of bile in the intestine has been shown to promote BT101; however, this could be attributed to malnutrition.102 In cirrhosis there is a decrease in bile acid secretion, and a recent study was performed using orally administered conjugated bile acids, cholylsarcosine and cholylglycine, in ascitic cirrhotic rats.103 Total ileal bacterial content was six-fold higher in ascitic cirrhotic rats than in healthy rats. Administration of conjugated bile acid reduced bacterial content to normal levels, reduced the rate of BT (cholylsarcosine, 33%; cholylglycine, 26 versus placebo, 66%) and reduced endotoxaemia. These compelling findings require further exploration in humans.

THE GUT FLORA AND HEPATIC ENCEPHALOPATHY HE is a complication of cirrhosis considered a reversible metabolic encephalopathy. It occurs as a result of both liver insufficiency and increased portal-systemic shunting of gut-derived nitrogenous compounds and toxins. Bacterial infections are a common precipitant not only of acute but also chronic HE.104 Ammonia is the main nitrogenous compound implicated in the pathogenesis of HE. Ammonia is generated in both the small bowel (from the effects of glutaminase on glutamine) and large intestine (from urease activity of the colonic flora). Ureaseproducing bacteria in the colon include anaerobic bacteroides, aerobic coliforms, and aerobic and anaerobic streptococcal organisms. It appears that ammonia generation through glutaminase is more important because (a) HE can still occur in germ-free animals105 and (b) neomycin, an antibiotic used in HE with the objective of eliminating urease-producing colonic bacteria, also inhibits small bowel glutaminase.106 Once ammonia is absorbed it is metabolized by the liver. However, metabolism of ammonia is altered in cirrhosis because of porto-systemic shunting (portal blood containing ammonia is shunted away from the liver) and because of changes in periportal (site of urea synthesis) and perivenous (glutamine synthesis) hepatocyte function. Other gut-derived toxins implicated in HE are gamma-amino-butyric acid (GABA) and benzodiazepine (BZD)-like substances107,108, both of which may also be produced by specific colonic bacteria.109,110 Interestingly, haemoglobin increases the production of GABA and may contribute to the pathogenesis of HE after gastrointestinal bleeding.111

complications of cirrhosis 365

In the past decade, several studies have implicated gastric infection with the ureaseproducing organism Helicobacter pylori as an additional pathogenic factor in hyperammonaemia, especially in the presence of gastric hypochlorhydria. However, data are still controversial.112 Importantly, H. pylori infection has not been associated with an increase in the frequency or degree of HE after of the transjugular intrahepatic portosystemic shunt insertion, in which porto-systemic shunting is maximal.113 The importance of the intestinal flora in the pathogenesis of HE is supported by studies showing that total colectomy leads to a significant decrease in baseline and protein-induced ammonia production114 and reverses cases of medically intractable HE.115 However, HE recurrence occurs in this setting probably as a result of the colonization of the small bowel.116 Besides elimination of precipitating factors, management of HE currently involves measures that will achieve bowel cleansing and elimination of urease-producing bacteria and include the administration of non-absorbable disaccharides, antibiotics and probiotics. Synthetic non-absorbable disaccharides (e.g. lactulose and lactitol) are not broken down by intestinal disaccharidases and once they reach the colon, bacteria metabolize them, generating organic acids. A lower colonic pH creates an environment hostile to the survival of urease-producing intestinal bacteria and may promote the growth of non-urease-producing lactobacilli117. Additionally, an osmotic cathartic action, improvement in gastrointestinal transit time and increased faecal nitrogen excretion contribute to their beneficial effect. Expectedly, lactose has been shown to have the same effects in lactase-deficient individuals.118 Non-absorbable antibiotics with activity against urease-producting bacteria (e.g. neomycin, paromomycin, metronidazole or rifaximin) also reduce bacterial ammonia production in the colon and are clinically effective.119 A decrease in BZD-like compounds has been observed with rifaximin, supporting the contention that the intestinal flora is at least partially involved in the production of these compounds.120 Another approach used in HE is modification of colonic bacteria by the administration of high oral doses of urease-negative Lactobacillus acidophilus121; however, controlled data do not convincingly show that this approach is efficacious.122 More recently, the long-term oral administration of Enterococcus faecium SF 68, a fermentative, lactic-acid producing, urease-negative bacterium that inhibits replication of several intestinal bacteria, was at least as effective as lactulose in improving both blood ammonia concentrations and number connection tests and hence, treatment of chronic HE.123 It had no adverse effects and, in contrast to lactulose, treatment can be interrupted for 2 weeks without losing the beneficial effects. This approach requires further exploration. A vegetable protein diet appears to also benefit patients with chronic HE through an effect on nitrogen metabolism, mainly accounted for by the increased intake of dietary fibre and increased incorporation and elimination of nitrogen in fecal bacteria.124

SUMMARY SBP is the most common bacterial infection in cirrhotic patients. The HCS, typical haemodynamic alteration of cirrhosis, is characterized by low systemic vascular resistance, increased cardiac index and low blood pressure. It is initiated by splanchnic vasodilatation and it is associated with worsening of the complications of cirrhosis. HE is a metabolic encephalopathy secondary to increased brain levels of nitrogenous

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complications of cirrhosis 367

compounds, mostly ammonia. The passage of bacteria, bacterial products and/or nitrogenous toxic compounds from the gut to the systemic circulation explains these complications (Figure 1). BT is increased in cirrhotic animals, mainly in those with ascites. A defective immune system, intestinal bacterial overgrowth and increased intestinal permeability occur in cirrhosis and facilitate BT (Figure 1). Passage of bacteria/ bacterial products from the MLN and splanchnic circulation to the systemic circulation is facilitated in cirrhosis because of a defective hepatic RES function due to an impairment in its phagocytic activity and to portosystemic shunting. In experimental cirrhosis, BT has been clearly related to ascites infection and to aggravation of

Practice points † gut BT occurs in cirrhotic patients; however, its clinical significance remains to be determined † non-absorbable antibiotics (for selective intestinal decontamination) are useful in the prevention of bacterial infections in cirrhosis. However, their use is associated with the development of infections caused by antibiotic-resistant organisms † long-term antibiotic prophylaxis should be restricted to cirrhotic patients at the highest risk of infection

Research agenda † surrogate markers of BT in cirrhotic patients need to be determined † non-antibiotic methods to decrease Gram-negative organisms in the gut, such as prokinetics and probiotics, should be investigated in clinical trials for the prevention of infections in cirrhosis † the effect of non-absorbable antibiotics (or other methods to decrease BT) on the hyperdynamic circulatory state of cirrhosis needs to be established † the use of probiotics in the treatment of hepatic encephalopathy deserves further study

R Figure 1. Mechanisms of bacterial translocation in cirrhosis and its relationship to the pathogenesis of spontaneous bacterial peritonitis (SBP), the hyperdynamic circulatory state (HCS) and hepatic encephalopathy (HE). Cirrhosis leads to bacterial translocation via different mechanisms: (1) a delayed intestinal transit time, and possibly malnutrition, lead to intestinal bacterial overgrowth, mainly of enteric Gram-negative bacteria; (2) intestinal mucosal oxidative stress augmented by translocation of bacteria, nitric oxide and cytokines such as necrosis factor (TNF) lead to increases in intestinal permeability; and perhaps most importantly, (3) an impairment in intestinal immunity leading to an impaired phagocytic activity. Gut-associated lymphatic tissue (GALT) produces cytokines in response to translocation rendering the gut a cytokine-releasing organ. In cirrhosis, a failure of the hepatic reticuloendothelial system (RES) due to impaired RES phagocytic activity and portosystemic shunting, allow bacteria, bacterial products, endotoxins (LPS), cytokines and neurotoxic nitrogenous compounds such as ammonia, into the systemic circulation. Bacteria, bacterial products (endotoxins) and cytokines induce synthesis of the vasodilator nitric oxide (NO), leading to splanchnic and systemic vasodilatation and thus to the HCS that, in turn, worsens complications of cirrhosis such as variceal haemorrhage. In the presence of ascites and severe liver dysfunction (which correlates with immune dysfunction) circulating bacteria seed ascites, producing SBP. An overt bacterial infection such as SBP leads to further haemodynamic alterations that may result in renal failure. Lastly, gut-derived neurotoxins such as ammonia and benzodiazepines contribute to the development of HE.

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the hyperdynamic circulation. The lack of a non-invasive reliable marker of BT in humans limits the extension of experimental findings to the clinical setting. In cirrhotic patients, elimination of intestinal bacteria through the use of non-absorbable or poorlyabsorbed antibiotics has been shown to prevent SBP, however the development of infections due to antibiotic-resistant organisms should limit their widespread use. Other therapies, such as prokinetics, probiotics and bile acids, have shown some promise in experimental studies and in limited clinical studies but require further investigation in larger controlled trials.

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