Qualitative and Quantitative Relationships of the Fecal Flora in Cirrhotic Patients with Portal Systemic Encephalopathy and Following Portacaval Anastomosis

Vol. 59, No.1

GASTROENTEROLOGY

Copyright © 1970 by The Williams & Wilkins Co.

Printed in U.S.A.

QUALITATIVE AND QUANTITATIVE RELATIONSHIPS OF THE FECAL FLORA IN CIRRHOTIC PATIENTS WITH PORTAL SYSTEMIC ENCEPHALOPATHY AND FOLLOWING PORTACAVAL ANASTOMOSIS MARTIN H. FLocH, M.D., JuLIAN KATZ, M.D. , AND HAROLD 0. CoNN, M.D.

Gastroenterology Sections and Departments of Medicine, Yale University School of Medicine and West Haven Veterans Administration Hospital, New Haven, Connecticut

Quantitative and qualitative aerobic and anaerobic bacteriological techniques were employed to evaluate the fecal microflora of normal subjects and cirrhotic patients with and without portacaval anastamosis and portal systemic encephalopathy. fu all groups of subjects total anaerobes significantly outnumbered total aerobes. Bacteroides species were the most common organisms and anaerobes encountered, while total aerobic and anaerobic lactobaccilli were the next most frequent. The two most common aerobes were coliforms and streptococci in that order. The only quantitative difference between microflora from normal and cirrhotic subjects was a slight but significant (P < 0.05) increase in total anaerobes and bacteroides in cirrhotic subjects. The major qualitative difference was a greater variation in aerobic recoveries in cirrhotic subjects in whom streptococci were often as numerous as coliform. No statistically significant differences were noted when cirrhotic patients with and without portal systemic encephalopathy or portacaval anastamosis were compared. These findings confirm previous observations on the aerobic flora in cirrhotic subjects and elaborate upon the distribution of the larger anaerobic bacterial population. The major bacterial urease producers, anaerobic bacteroides, aerobic coliforms, and aerobic and anaerobic streptococcal organisms were present in cirrhotic subjects in approximately the same numbers as in normal subjects. Similarly the nonurease producing, and often therapeutically employed lactobacilli were present in abundant amounts and in the same proportions in patients with or without portal systemic encephalopathy.

Although the importance of the intestinal bacteria flora in portal systemic encephalopathy (PSE) is well recognized, 1 the precise role of the individual groups of bacteria or their qualitative and quantita-

tive interrelationships is not known. Bacterial ureases hydrolyze urea and may be responsible for hyperammonemia and PSE in cirrhotic patients with azotemia. 2 Similarly, amino acid oxidases produced by bacteria have been assumed to be re-

Received November 20, 1969. Accepted January 10, 1970. Address requests for reprints to: Dr. Martin H . Floch, Department of Medicine , Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510. This work was supported in part by the Irwin Strasburger Memorial Medical Foundation, the Stratfield Fund, Philips Duphar-Philips Roxane Lab-

oratories, Warner-Lambert Research Laboratories, and by Grant 07817 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States Public Health Service. The authors wish to thank Dr. Howard Spiro for his support of this project and Mr. William Gershengoren and Miss Sylvia Diamond for their technical assistance. 70

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sponsible for the release of ammonia from protein, blood, or amino acids in the gastrointestinal tract. 3 Bacterial production of nonammoniacal substances such as amines, 4 by the action of bacterial decarboxylases on amino acids or phenols5 also has been considered as a possible cause of hepatic coma. 6 It is known that aerobic coliform and streptococcal organisms and anaerobic bacteroides species are intestinal sources of these enzymes. 7 Despite the obvious importance of the bacterial flora in cirrhosis, few studies of the basic ecology in cirrhosis have been performed, 8 - 11 and in none have the more rigorous cultural methods now available for the recovery of anaerobic organisms, which constitute the majority of the intestinal flora, been applied. The present studies were undertaken to compare the qualitative and quantitative recovery of aerobic and anaerobic organisms in the colon of normal subjects and of cirrhotic patients with particular reference to the presence or absence of PSE.

Methods Patients. Aerobic and anaerobic cultures were obtained from the stools of 20 healthy adults, ranging in age from 21 to 58 years (mean, 35.4 ± 2.4), who worked or lived in the hospital environment and served as the control group. Seventeen male patients with Laennec's cirrhosis, 5 with and 12 without portacaval anastomoses (PCS), who ranged in age from 31 to 55 years (mean, 57.1 ± 2.8), constituted the experimental group. These patients had been admitted to the West Haven Veterans Administration Hospital between March 1966 and June 1968. One had last received neomycin 8 days before the stool culture was performed; none of the others had received antibiotic therapy during the 2 weeks preceding stool culture. The diagnosis of cirrhosis had been established histologically in each patient. All had been evaluated by complete physical examination, with special attention given to the features of liver disease, barium esophagram, esophagoscopy, and the standard battery of liver function tests, and by studies to determine the presence and degree of portal hyPertension. Six patients had portal systemic encephalopathy and had been admitted for evaluation and therapy of this problem. Eight of the other

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11 patients had esophageal varices. All except 1 had grossly abnormal ammonia tolerance tests indicative of extensive portal systemic collateral circulation. None of the patients exhibited severe hepatic decompensation at the time of examination. Bacteriological techniques. The detailed methods and media employed have been described elsewhere. 12 ' 13 Briefly, serial dilutions were made from stool specimens freshly collected in sterile containers. Three hundred thirty-three initial cultures were performed on nine selective media from specimens obtained from the 37 subjects. These were followed by identifying techniques 12 in order to quantitatively and qualitatively identify total aerobes, total anaerobes, bacteroides species, clostridia species, veillonella species, total anaerobic plus microaerophilic lactobacilli species (including the bifidobacterium), anaerobic and aerobic streptococci, coliforms, and pathogenic species. Colony counts were made at the highest dilutions demonstrating growth on the selected media and then converted into logarithms to the base 10. All of the results were recorded in the logarithm number of organisms per gram of wet stool. The standard Student's t-test was used for comparison of groups of bacteria and analysis of variance for significance of individual bacterial variation. 14

Results Qualitative bacterial recoveries. The spectrum of organisms recovered was similar in the normal subjects and cirrhotic patients. There were no differences between cirrhotic patients with or without portal systemic encephalopathy or between patients with or without portacaval shunts. The percentages of positive recoveries for individual bacteria are recorded in table 1. Bacteroides species, lactobacilli species, and coliforms were recovered in all "pecimens, but streptococcal (enterococcal) organisms were not found in 2 cirrhotic patients and 2 control subjects. Either Escherichia coli or Klebsiella aerogenes were identified in all subjects. E. coli was most frequently the predominate coliform. Streptococcus faecalis and group N streptococci were isolated with equal frequency from both groups of subjects. There was no difference in the spectrum of gram-negative aerobes or entero-

FLOCH ET AL.

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cocci isolated from control or cirrhotic patients. Recoveries of clostridia species, diphtheroids, and veillonella species were erratic in both groups and there was no statistically valid difference, although the percentage of recoveries for these organisms appear varied (table 1). Quantitative bacterial relationships. The relative proportions of total growth 1. Percentage of subjects from whom specific bacterial groups were recovered

TABLE

Cirrhotic patients

Bacterial group

Normal subjects

,.. Coliforms Streptococci . . . . . . . . . . . Lactobacilli Bacteroides Clostridia . . . . . . . . . . . . . Diphtheroids Veillonellae •

•

•

•

•

•

•

•

0

•

100 88 100 100 12 23 23

'

100 90 100 100 30 15 25

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and of individual bacteria in both the control and cirrhotic groups were remark. ably similar except for occasional individ. ual variations such as a relatively low bacteroides count in 1 normal subject. Figure 1 demonstrates the ranges and means of recovery of the most commonly recovered bacteria for the 20 normal sub. jects and the entire group of 17 cirrhotic patients. Total anaerobic organisms were signifi. cantly more common than total aerobic bacteria (P < 0.001) in both groups. The most common anaerobic organism was Bacteroides which averaged 9.3 ± 0.23 to the log10 organisms per gram of stool in cirrhotic patients and 8.9 + 0.24 in normal controls. Anaerobic lactobacilli averaged 8.6 + 0.22 in cirrhotic patients and 7.9 ± 0.3 in control subjects, and were next most frequent. Coliforms were the most common aerobes recovered in control subjects (6.8 ± 0.2). In individual cirrhotic patients, however, streptococci (mean, 6.1 ± 0.61)

12

10

8

6

4

2

~NORMAL ~CIRRHOSIS

FIG. 1. Mean and ranges of bacterial colony counts for normal and cirrhotic subjects. The entire bar reW sents the range of counts, the center line within it the mean, and the dotted or cross-hatched area ±2 so.

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FECAL FLORA IN CIRRHOTIC PATIENTS

were often as common as the coliforms (mean, 6.8 ± 0.41), although the mean was smaller. Statistical analyses. Although analysis of variance of the groups of bacteria substantiated the validity of the techniques (F = 8.8 in control subjects and 5.6 in cirrhotic patients; P < 0.01), statistical analysis revealed no significant variations between the control group, the cirrhotic patients, or the cirrhotic patients with portacaval anastomosis. The greater recovery of total anaerobic organisms in cirrhotic patients than in control subjects (9.8 ± 0.14 versus 9.3 ± 0.16) is of uncertain significance. Although these differences were probably significant (P < 0.05), the total number of bacteroides recovered was also greater in cirrhotic patients. Neither the total number nor the mean number of any other group of organisms was significantly more common in the cirrhotic patients than in the control subjects. Great variation in specific organisms and in total counts occurred in individual subjects in both control and cirrhotic groups. Although the bacterial pattern was stable in individual subjects as noted by others, 12 ' 1 5 variation from subject to subject was readily apparent. Discussion This investigation failed to demonstrate any gross qualitative or quantitative abnormalities in the bacterial stool pattern of cirrhotic subjects or in pertinent subgroups such as patients with PSE or with portacaval anastomoses. Phear and Ruedner in their studies 15 years ago found no significant differences between the basic aerobic stool bacteria of cirrhotic patients and normal subjects. 9 Recent advances in the recovery of anaerobic bacteria from the intestinal tract, have demonstrated the numerical predominance of the anaerobic intestinal flora. 16 We assumed when this investigation was undertaken that if differences did exist between cirrhotic and noncirrhotic patients, these differences would be found in the anaerobic flora. Our findings, however, confirm those of Phear and Ruedner 9 and in addi-

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tion demonstrate that the anaerobic fecal flora of the cirrhotic patient does not differ appeciably from the normal. Although no striking abnormalities were found in the fecal flora from cirrhotic patients, there were several interesting differences from normal values. The total number of anaerobic organisms was slightly, but significantly, greater in cirrhotic patients than in normal subjects (P < 0.05}. This difference was largely a consequence of the greater number of bacteroides species in the cirrhotic patients (P < 0.05). Except in occasional subjects the predominant group of organisms in both cirrhotic and normal subjects was 17 19 bacteroides. In both the older studies 16 20 21 and in most recent reports, ' ' bacteroides have been found as the predominant organism in normal subjects, independent of the medium used. We detected very high counts of total lactobacilli species which ran as high as 109 organisms in both control and cirrhotic subjects. Lactobacilli, which include both microaerophilic and strictly anaerobic organisms (the so-called bifidus form), are generally regarded as the second most common group of organisms in feces. Our high recovery of lactobacilli probably reflects the use of the G medium of Schaedler and co-workers, 13 which we have shown to recover greater numbers of lactobacilli than the conventional media used for this purpose. 12 Cultures of lactobacilli, by virtue of their inability to make urease, have been used in the treatment of PSE in hope of displacing putrefactive organisms. 2 2 ' 23 Certainly there is no dearth of lactobacilli in cirrhotic stools, although it must be emphasized that our techniques assessed the entire species of lactobacilli and made no attempt to quantitate subspecies. In cirrhotic patients streptococci frequently challenged coliform bacteria as the most common group of aerobic organisms. Although both coliform and streptococcal organisms were recovered in all patients in both groups, the greatest fluctuations in daily recoveries occurred in these organisms. The factors responsible for these fluctuations are not known but

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FLOCH ET AL.

require careful evaluation since both groups of organisms are urease producers. Although the fecal flora is involved in the pathogenesis of a number of complications of cirrhosis such as spontaneous enteric bacteremia24 - 28 or PSE, the bacteria are probably not the primary defect. The enteric bacteremia and peritonitis that occur in cirrhosis do not seem to result from any unusual pathogenecity of the bacteria involved, but rather from an abnormality of the normal host defenses in cirrhosis. It is probable that intestinal organisms frequently traverse the intestinal mucosa but are almost immediately removed by the local lymphatics or from the portal blood by the hepatic reticuloendothelial filter. 27 In cirrhosis, the presence of portal systemic shunting around and through the liver permits the prolonged cycling of bacteria that enter the bloodstream. Indeed, pneumococcal peritonitis and bacteremia, probably of nonenteric origin, also occur in cirrhosis~ (also H. 0 . Conn, unpublished data). Perhaps no gross abno'"malities in the qualitative or quantitative bacterial pattern exist in cirrhosis. The activity of bacterial enzymes for example, may not be solely dependent upon the absolute number of bacteria. This concept, however, need not exclude the possibility that bacteria may play a variety of roles in the production, potentiation, and complications of cirrhosis. Studies in germ-free animals have suggested that intestinal bacteria, or their products, may be injurious to the liver and may even be pathogenetically important in the development of dietary cirrhosis. 30 - 3 J In a similar vein, Carey suggested that altered metabolism of the bile acids in cirrhosis, aided by the bacterial conversion in the intestine of excessive amounts of chenodeoxycholic acid to lithocholic acid, a potent hepatotoxin, perpetuate liver damage. 34 Others have postulated that the bacterial decarboxylation of the histidine to histamine, a potent stimulus for gastric acid production, may account for the high incidence of peptic ulcerative disease in cirrhotic patients. 3 5

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It is possible that the fecal flora may not accurately reflect the bacterial pattern at some higher, critical level in the intestinal tract and that the occurrence of significant growth in the small bowel in cirrhotic subjects would lend support to these hypotheses. Intestinal bacteria have been found in greater numbers in the jejunum of cirrhotic patients than of normal subjects. 8 It is conceivable that special conditions in the upper intestinal tract of cirrhotic patients may permit the selective growth of strains of organisms which may be especially deleterious. Further, careful aerobic and anaerobic qualitative and quantitative investigations of the bacterial flora of the upper gastrointestinal tract m cirrhotic patients are needed. REFERENCES 1. Silen W, Harper HA, Mawdsley DL, et al: Effect

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23 . Read AE, McCarthy CF, Heaton KW, et al: Lactobacillus acidophilus (Enpac) in treatment of hepatic encephalopathy. Brit Med J 1:12671269, 1966 24. Tisdale WA: Spontaneous colon bacillus bacteremia in Laennec's cirrhosis. Gastroenterology 40: 141- 148, 1961 25. Careli J, Platteborse R: Septicemie portocave cirrhosis du foie et septicemie a colibacille. Sem Hop Paris 34:472-487, 1958 26. Martin WJ, Spittel JA, Morlock JA, et al: Severe liver disease complicated by bacteremia due to Gram-negative bacilli. Arch Intern Med (Chicago) 98:8-15, 1956 27. Conn HO: Spontaneous peritonitis and bacteremia in Laennec's cirrhosis caused by enteric organisms. A relatively common but rarely recognized syndrome. Ann Intern Med 60:568-580, 1964 28. Kerr DNS, Pierson DT, Reed AE: Infection of acitic fluid in patients with hepatic cirrhosis. Gut 4:394-398, 1963 29. Epstein M, Calia FM, Gabuzda GJ: Pneumococcal peritonitis in patients with postnecrotic cirrhotic. New Eng J Med 278:69-73, 1968 30. Luckey TD, Reyniers JA, Gyorgy E , et a!: Germfree animals and liver necrosis. Ann NY Acad Sci 57:932-935, 1954 31. Ruttenberg AM, Sonnenbrock E, Koven I, et al: The role of intestinal bacteria in the development of dietary cirrhosis in rats. J Exp Med 106: 1-13, 1957 32. Broitman SA, Gottlieb LP, Zamcheck N: Influence of neomycin and ingested endotoxin in the pathogenesis of choline deficiency cirrhosis in the adult rat. J Exp Med 113:633-647, 1964 33. Salmon WD, Newberne DM : Effective antibiotics, sulphonomides and nitrofuran on development of hepatic cirrhosis in colon deficient rats. J Nutr 76:483-494, 1962 34. Carey JB Jr, Wilson ID, Zaki FG, et a!: The metabolism of bile acids with specific reference to liver injury. Medicine (Bait) 45:461-470, 1966 35. Clarke JS: Influence of the liver upon gastric secretion. Arner J Med 29:740-747, 1960