Establishment of a mucin-degrading intestinal microflora during the first two years of human life.

The establishment of a mucin-degrading intestinal microflora was followed in 30 healthy Swedish children by agar gel electrophoresis of fecal samples taken at 0, 1, 3, 6, 9, 12, 15, 18, 21, and 24 months of age. At 3 months, 18 children showed a limited degree of mucin degradation, while 12 children showed no degradation. In children exclusively breast-fed for at least 4 months, mucin degradation was initiated significantly later than in children who received formula, with or without breast milk, by 1 month of age. Complete degradation of mucin was seen in 21 children after 1 year of age and in all children at 2 years. A positive correlation between increasing age and increased degradation of mucin was found between birth and 1 month and between 6 and 9 months, after standardization for the period of exposure to foods other than breast milk.

Intestinal microbial conversion of cholesterol to coprostanol in man. Influence of antibiotics.

The intestinal microbial conversion of cholesterol to coprostanol has been measured in groups of healthy subjects before, during and after they received the antibiotics ampicillin, bacitracin, clindamycin, co-trimoxazole, doxycycline, erythromycin, metronidazole, nalidixic acid, ofloxacin or vancomycin orally for 6 days. Before they received antibiotics, the subjects demonstrated two distinct patterns of cholesterol conversion. One pattern was characterised by extensive conversion of cholesterol, the other by little or no conversion. Intake of bacitracin, clindamycin, erythromycin, metronidazole and vancomycin significantly reduced the conversion to coprostanol. In the groups receiving ampicillin or doxycycline, marked reductions were found in most of the subjects. No alterations were found in the groups receiving co-trimoxazole, nalidixic acid or ofloxacin. In 6 subjects no conversion of cholesterol to coprostanol was found up to 5 weeks after the end of the antibiotic intake. We conclude that orally given antibiotics may cause alterations in the intestinal conversion of cholesterol, reflecting changes in the anaerobic, Gram-positive component of the gut flora.

Enteral nutrition and the function of the intestinal microflora in healthy adults.

An oral feeding formula was given to 5 healthy volunteers for 8 days. Faecal samples were collected before, during and after the feeding period. The effect of enteral nutrition (EN) on the following seven intestinal microflora-associated characteristics (MACs) was studied: formation of urobilinogen, coprostanol and deoxycholic acid, degradation of mucin and beta-aspartylglycine, faecal tryptic activity, and production of short-chain fatty acids (SCFAs). None of the microbial functions studied were lost during the study. The urobilinogen level increased during EN (P < 0.05) but it seems reasonable to assume that this was a concentration effect due to a decrease in stool mass. The concentration of SCFAs decreased during EN (P < 0.05) and this reflects the absence of dietary fibre in the feed used.

The function of the intestinal microflora in patients with ulcerative colitis before and after colectomy.

The function of the intestinal microflora was studied in patients with ulcerative colitis before and after colectomy. The following six microflora-associated characteristics (MACs) were investigated: formation of coprostanol and urobilinogen; degradation of mucin, water-soluble protein, and beta-aspartylglycine; and presence of faecal tryptic activity. In 12 unoperated patients without sulphasalazine as maintenance therapy the six MACs were similar to those in normal subjects. In 12 unoperated patients receiving sulphasalazine the formation of coprostanol and urobilinogen was significantly lower (p less than 0.01 and p less than 0.001, respectively) and the level of faecal tryptic activity was significantly higher (p less than 0.01) than in normal subjects. The functional capacity of the microflora in operated patients treated by colectomy combined with one of four surgical procedures (ileorectal anastomosis, ileoanal anastomosis with pelvic pouch, Kock's continent ileostomy, or conventional ileostomy) was disturbed with regard to all six MACs. The disturbance was most pronounced in patients with conventional ileostomy.

Development of five metabolic activities associated with the intestinal microflora of healthy infants.

The establishment of a functionally active intestinal flora was followed in 17 healthy Swedish children from birth up to 6 months of age. Utilizing gas chromatography, spectrophotometry, and gel electrophoresis, feces were analyzed on certain biochemical markers that reflect the action of the intestinal flora in vivo. The establishment of the following five flora-related functions was investigated: production of short chain fatty acids (SCFAs), degradation of mucin, conversion of bilirubin to urobilinogen and of cholesterol to coprostanol, and inactivation of fecal tryptic activity (FTA). Production of SCFAs was the first function to be established, followed by bilirubin conversion and mucin degradation. No child showed conversion of cholesterol. The values of FTA were lower than in adults. This study indicates that the establishment of a functionally active flora is a slow process and that some functions are almost fully established before other functions have started to develop. Environmental factors, such as the diet, seem to be of importance. In general, the functions seem to develop slower in those children receiving breast milk exclusively than in those receiving formula supplements.

Total parenteral nutrition and the function of the intestinal microflora in Crohn's disease.

The effect of total parenteral nutrition (TPN) on the following six intestinal microflora-associated characteristics (MACs) was studied in patients with Crohn's disease: faecal tryptic activity (FTA), formation of coprostanol, urobilinogen, and deoxycholic acid, and degradation of mucin and beta-aspartylglycine. The FTA showed high levels before TPN, in accordance with previous findings, and decreased during TPN. Formation of coprostanol, urobilinogen, and deoxycholic acid was reduced in some patients, whereas no changes were found in the mucin and beta-aspartylglycine degradation.

Establishment of a biochemically active intestinal ecosystem in ex-germfree rats.

A time course study for the establishment of some biochemical microbial intestinal functions was undertaken in ex-germfree rats conventionalized, i.e., colonized with conventional flora, in three different ways: untreated (group 1); contact with visitor rats (group 2); inoculated with intestinal contents from conventional rats (group 3). The first two groups of rats were inoculated with the intestinal contents from conventional rats after being out of the germfree isolators for 4 weeks. The biochemical parameters studied were degradation of mucin, inactivation of tryptic activity, conversion of cholesterol to coprostanol and of bilirubin to urobilinogen, degradation of beta-aspartylglycine, and formation of short-chain fatty acids. The results showed that the way in which the microbes were introduced and the microbial biochemical functions themselves were of importance. In several cases, social contacts, i.e., contact with visitor rats, were just as effective for the functionally adequate establishment of microbial intestinal functions as was inoculation with intestinal contents from conventional rats. Some of the biochemical parameters studied were established after a few days, whereas the establishment of others was markedly delayed. When inoculated after 4 weeks, all rats in the first two groups were colonized with conventional flora within 1 week. The results indicate that the model system described is suitable when studying buildup mechanisms in intestinal ecosystem(s).

Influence of antibiotics on intestinal mucin in healthy subjects.

To determine the effect on microbial breakdown of intestinal mucin healthy volunteers were treated orally with ten different antibiotics. The most pronounced effects were seen after administration of bacitracin, clindamycin or vancomycin: the electrophoretic mucin pattern in faeces changed from a normal conventional pattern to a specific pattern similar to that found in germ-free rats. Disturbed patterns were also observed in some of the subjects treated with ampicillin, doxycycline, erythromycin, metronidazole or nalidixic acid. In most cases the electrophoretic mucin pattern normalized within five weeks after the end of treatment. There were no effects on the microbial breakdown of intestinal mucin in the groups treated with ofloxacin or trimethoprim/sulfamethoxazole. Thus, administration of antimicrobial drugs in clinically recommended doses may cause long-term disturbances in one microflora-associated characteristic, the breakdown of intestinal mucin.

Influence of antibiotics on the faecal excretion of bile pigments in healthy subjects.

We have evaluated the effects of 10 antibiotics, given orally for 6 days to healthy subjects, on faecal excretion of urobilinogen. Intake of bacitracin, vancomycin, clindamycin, erythromycin, and ampicillin resulted in a pronounced suppression of the faecal excretion of urobilinogen (p less than 0.05). Intake of doxycycline, metronidazole, nalidixic acid, ofloxacin, and trimethoprim/sulphamethoxazole had no significant effect. The effects of three antibiotics-ampicillin, clindamycin, and metronidazole--on faecal excretion of conjugated bilirubin were similarly evaluated. Intake of clindamycin led to a marked increase of conjugated bilirubin (p less than 0.05) in the faeces, and the pattern of separated azopigment derivatives of the bilirubin conjugates became altered. Intake of ampicillin and metronidazole resulted in far less alterations in faecal conjugated bilirubin, although a significant change was observed in the subjects receiving metronidazole (p less than 0.05). The differences between the antibiotics with regard to altered intestinal bile pigment metabolism may be due to differences in antimicrobial spectra and/or intestinal concentrations of the drugs. Our findings indicate that orally taken antibiotics may cause a suppression of the microbial deconjugation of conjugated bilirubin and urobilinogen formation, respectively. This may reflect a pronounced disturbance of the intestinal microflora.

Influence of oral intake of seven different antibiotics on faecal short-chain fatty acid excretion in healthy subjects.

Faecal excretion of short-chain fatty acids (SCFAs) has been measured by gas chromatography in groups of six or seven healthy subjects before, during, and after they received the antibiotics bacitracin, co-trimoxazol, doxycycline, erythromycin, nalidixic acid, ofloxazin, or vancomycin orally for 6 days. Intake of bacitracin and vancomycin had pronounced effects on faecal SCFAs excretion and reduced median total concentration of SCFAs from 105.4 mmol/kg to 21.8 mmol/kg and from 69.3 mmol/kg to 19.4 mmol/kg, respectively (p less than 0.05). Erythromycin had moderate effects on the faecal SCFAs excretion, whereas small or no changes were seen during intake of co-trimoxazol, doxycycline, nalidixic acid, and ofloxacin. 2-Methylbutyric acid, a SCFA not previously seen in human faeces, was found in the faeces of all subjects (median concentration before intake of antibiotic, 1.3 mmol/kg). Bacitracin, erythromycin, nalidixic acid, and vancomycin were detected in high concentrations in faeces during therapy, whereas trimethoprim, doxycycline, and ofloxacin were found in relatively low concentrations. In conclusion, some, but not all, peroral antimicrobials induce changes in faecal SCFAs, most likely reflecting changes in the colonic ecosystem.

Isolation and characterization of a mucin-degrading strain of Peptostreptococcus from rat intestinal tract.

A mucin-degrading microorganism was isolated from the intestinal tract by serial sectioning from the serosal side of the caecum wall from a conventional rat. The ability of degrading the intestinal water-soluble mucin was present both in vivo after monocontamination of germ-free rats and in vitro, when adding the microbe to Mucin medium. The morphology, Gram-positive cocci single or in short chains and the very weak biochemical activities allow us to place this strain in the species Peptostreptococcus micros.

Influence of ampicillin, clindamycin, and metronidazole on faecal excretion of short-chain fatty acids in healthy subjects.

The faecal excretion of short-chain fatty acids (SCFAs) has been measured in groups of six healthy subjects before, during, and after they received the antibiotics clindamycin, ampicillin, or metronidazole perorally for 6 days. Intake of clindamycin reduced the median total concentration of SCFAs from 62.9 mmol/kg faeces (wet weight) to 7.3 mmol/kg (p less than 0.05). During therapy the relative amounts of acetic acid increased from 50% to 90% of the total concentration (p less than 0.05). Ampicillin reduced the median SCFAs concentration from 62.4 mmol/kg to 47.8 mmol/kg (p less than 0.05), whereas metronidazole did not change the SCFAs concentrations significantly. The SCFAs concentrations returned to normal within 5 weeks after the treatment in all subjects. Clindamycin was detected in high concentrations in faeces during therapy. Ampicillin was detected in only one faecal sample, which was from the only subject in the ampicillin group without detectable beta-lactamase activity in faeces. Metronidazole could not be detected in faeces from any subjects receiving this drug. Clindamycin and ampicillin, but not metronidazole, induce pronounced changes in faecal SCFAs, most likely reflecting severe changes in the colonic ecosystem. An antibiotic's influence on the colonic microflora may in part depend on its antimicrobial spectrum and the concentration of antimicrobially active drug in the gut.

Influence of peroral antibiotics upon the biotransformatory activity of the intestinal microflora in healthy subjects.

The effects of ampicillin, clindamycin or metronidazole, given perorally for 6 days to eighteen healthy volunteers, upon the following intestinal microflora-associated characteristics (MACs) were evaluated: breakdown of mucin, formation of coprostanol, hydrolysis of bilirubin conjugates, formation of urobilinogen, and of some short chain fatty acids (SCFAs), presence of beta-aspartylglycine and inactivation of trypsin. Clindamycin markedly influenced the expression of all characteristics, but trypsin and beta-aspartylglycine, resulting in a pattern very much alike what has been found in germ-free animals. Ampicillin caused a significant reduction in total amount of SCFAs (P less than 0.05) and urobilinogen (P less than 0.05) present in the faecal samples. Metronidazole caused a significant reduction in the formation of coprostanol and the deconjugation of bilirubin (P less than 0.05). We conclude that orally given antibiotics may cause major alterations in several parameters reflecting the normal biotransformatory activity of the intestinal microflora, probably caused by severe disturbances in the intestinal ecosystem.

Clindamycin-induced alterations in intestinal microflora-associated characteristics in rats.

Conventional Sprague-Dawley rats were treated with clindamycin, 40 mg/kg/day and 0.04 mg/kg/day, for 5 days. At a dose of 40 mg/kg/day, microflora-associated characteristics (MACs), such as shape, color, and consistency of feces, proteolytic activity, electrophoretic pattern, and cholesterol and bilirubin metabolism were transformed into values like those found in germfree rats: germfree animal characteristics (GACs). The effect on the proteolytic activity lasted longest. It did not disappear until one or two enemas with cecal contents from intact conventional rats were administered. At a dose of 0.04 mg/kg/day, effects on the proteolytic activity and cholesterol metabolism were seen. With the exception of one rat, the effect on proteolytic activity did not disappear until one or two enemas were given. The results indicate that clindamycin, even in very small daily doses, has a profound and long-lasting influence on many intestinal MACs in rats.

Intestinal water-soluble mucins in germfree, exgermfree and conventional animals.

Water-soluble intestinal mucins were investigated in germfree (GF), exgermfree (EXG) and conventional (CONV) rats and in GF and CONV mice. After agar gel electrophoresis, all GF animals had similar specific band patterns demonstrated by PAS and Toluidine Blue. These patterns, never seen in CONV animals, disappeared in GF animals infected either with intestinal contents from CONV rats or mono-infected with a mucin converting microorganism, labelled Peptostreptococcus N. The intestinal microflora seem to have a profound influence on the water-soluble mucins, and specific microorganisms appear to be involved in the conversion of these substances. Any CONV animal with a GF mucin pattern in the faeces must be considered to have a disturbance of the normal intestinal microflora.

Prolonged induction of germfree bile acid pattern in conventional rats by antibiotics.

Male conventional rats have been treated for five days with benzylpenicillin, neomycin, kanamycin, erythromycin, bacitracintneomycin, succihylsulfathiazole or metronidazole. Total fecal bile acids were analyzed in samples collected during periods of three days during the pretreatment period and during the eight weeks following drug treatment. Metronidazole or succinylsulfathiazole had no or minor effects on the conventional bile acid pattern and the "bile acid index" (ratio beta-muricholic acid/deoxycholic acid) remained low. Benzylpenicillin, neomycin or kanamycin induced a germfree bile acid pattern, i.e. increased the relative amounts of alpha-and beta-muricholic acid in feces and eliminated deoxycholic acid and hyodeoxycholic acid from feces. The high bile acid index was normalized within three weeks after termination of drug treatment but the excretion of alpha- and beta-muricholic acid was not normalized until a normal flora had been established by giving an enema with intestinal contents from intact, oncentional rats. Treatment with eythromycin or bacitracintineomycin also produced a germfree bile acid pattern. In these cases, the bile acid index was not back to normal until after five to eight weeks and the excretion of the muricholic acids was not normalized until an enema with intestinal bacteria had been given. It is suggested that these long-lasting effects of antibiotics on the metabolism of bile acids in the intestinal tract should be considered after short-term antibiotic therapy in humans.