Cellulolytic bacteria from pig large intestine

An anaerobic, cellulose-degrading, gram-negative rod and a gram-positive coccus, identified as Bacteroides succinogenes and Ruminococcus flavefaciens, respectively, were isolated from pig fecal samples. These organisms were isolated from cellulolytic most-probable-number dilutions which represented 4 or 6% of the viable bacterial count when pigs were fed a low- or high-fiber diet, respectively.     

Utilization of amino acids by bacteria from the pig small intestine

This study determined the utilization of amino acids (AA) by bacteria from the lumen of the pig small intestine. Digesta samples from different segments of the small intestine were inoculated into media containing 10 mmol/L each of select AA (l-lysine, l-threonine, l-arginine, l-glutamate, l-histidine, l-leucine, l-isoleucine, l-valine, l-proline, l-methionine, l-phenylalanine or l-tryptophan) and incubated for 24 h. The previous 24-h culture served as an inoculum for a subsequent 24-h subculture during each of 30 subcultures. Results of the in vitro cultivation experiment indicated that the 24-h disappearance rates for lysine, arginine, threonine, glutamate, leucine, isoleucine, valine or histidine were 50–90% in the duodenum, jejunum or ileum groups. After 30 subcultures, the 24-h disappearance rates for lysine, threonine, arginine or glutamate remained greater than 50%. The denaturing gradient gel electrophoresis analysis showed that Streptococcus sp., Mitsuokella sp., and Megasphaera elsdenii-like bacteria were predominant in subcultures for utilizing lysine, threonine, arginine and glutamate. In contrast, Klebsiella sp. was not a major user of arginine or glutamate. Furthermore, analysis of AA composition and the incorporation of AA into polypeptides indicated that protein synthesis was a major pathway for AA metabolism in all the bacteria studied. The current work identified the possible predominant bacterial species responsible for AA metabolism in the pig small intestine. The findings provide a new framework for future studies to characterize the metabolic fate of AA in intestinal microbes and define their nutritional significance for both animals and humans.     

Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20 mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/l sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.     

Metabolism of select amino acids in bacteria from the pig small intestine

This study investigated the metabolism of select amino acids (AA) in bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the jejunum and ileum of pigs. Cells were incubated at 37°C for 3 h in anaerobic media containing 0.5–5 mM select AA plus [U-¹⁴C]-labeled tracers to determine their decarboxylation and incorporation into bacterial protein. Results showed that all types of bacteria rapidly utilized glutamine, lysine, arginine and threonine. However, rates of the utilization of AA by pure cultures of E. coli and Klebsiella sp. were greater than those for mixed bacterial cultures or Streptococcus sp. The oxidation of lysine, threonine and arginine accounted for 10% of their utilization in these pure bacterial cultures, but values were either higher or lower in mixed bacterial cultures depending on AA, bacterial species and the gut segment (e.g., 15% for lysine in jejunal and ileal mixed bacteria; 5.5 and 0.3% for threonine in jejunal mixed bacteria and ileal mixed bacteria, respectively; and 20% for arginine in ileal mixed bacteria). Percentages of AA used for bacterial protein synthesis were 50–70% for leucine, 25% for threonine, proline and methionine, 15% for lysine and arginine and 10% for glutamine. These results indicate diverse metabolism of AA in small-intestinal bacteria in a species- and gut compartment-dependent manner. This diversity may contribute to AA homeostasis in the gut. The findings have important implications for both animal and human nutrition, as well as their health and well-beings.     

Cellulolytic activity of luminous bacteria

Summary The luminous bacteriaVibrio harveyi andV. fischeri degrade cellulose. Cellulase activity was high when carboxymethyl cellulose and cellobiose were used as substrates but low with cellulose powder. The role of these microbes in the digestion of food material in fish gut is also discussed.

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Resumen Las bacterias luminosasVibrio harveyi yV. fischeri degradan celulosa. La actividad celulásica fue alta cuando se utilizó Carboxy-metil-celulosa o celobiosa como sustrato y baja cuando se utilizó polvo de celulosa. Se discute el papel de estos microorganismos en la digestión intestinal de los alimentos en peces.

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Résumé Les bactéries lumineusesVibrio harveyi etV. fischeri dégradent la cellulose. L'activité cellulolytique était élevée lorsque la carboxméthylcellulose et la cellobiose servaient de substrats, mais elle était faible avec la poudre de cellulose. On discute également de rôle de ces microorganismes dans la digestion du matériau alimentaire.

     

Cellulolytic and non-cellulolytic bacteria in rat gastrointestinal tracts.

Lactobacillus and Bifidobacterium species were the predominant organisms isolated from small intestinal (jejunal) contents of rats, and lactic acid was the only organic acid detected. The numbers of cellulolytic bacteria in small intestines were low (approximately 10(3)/g). The fermentation in ceca was different from that in intestines, as, in addition to small amounts of lactic acid, high concentrations of volatile fatty acids were detected. The mixed cecal microflora was able to digest cellulose (pebble-milled Whatman no. 1) and cabbage. High numbers of cellulolytic bacteria were found (0.5 X 10(8) to 12.2 X 10(8)/g; 6% of total viable bacteria). The predominant celluloytic organism isolated was Bacteroides succinogenes. Ruminococcus flavifaciens was isolated from a few animals. The kinds and numbers of the predominant non-cellulolytic organisms isolated from rat ceca were similar to those described by previous workers.     

Nutritionally limited pectinolytic bacteria from the human intestine.

A selective procedure was used to isolate pectinolytic intestinal bacteria from human subjects. The three isolates with the greatest pectinolytic activity utilized pectin and a few related compounds as fermentable substrates for growth but did not utilize any other compound tested. Thus, their substrate utilization pattern was markedly different from that of previously described intestinal pectinolytic isolates. The three isolates are representatives of a nutritionally defined group of bacteria for which the term pectinophilic is proposed.     

Glucose and lactate catabolism by bacteria of the pig large intestine and sheep rumen as assessed by 13C nuclear magnetic resonance.

The fermentation of [1-13C] glucose and [3-13C]lactate by bacteria isolated from sheep rumen and pig large intestine was compared by the nuclear magnetic resonance (NMR) technique. Washed cell suspensions were incubated directly in the NMR spectrometer and spectra were recorded every 10 min after injection of the labelled substrates. The results showed large differences in the fermentation patterns between rumen and hindgut bacteria. The latter pattern indicated a greater ability for formation and fermentation of lactate than that of the rumen. Moreover, with both substrates the amount of propionate formed via the acrylate pathway was always greater with hindgut than with rumen bacteria, 50% and 20% of the total, respectively.     

Unique cholecystokinin peptides isolated from guinea pig intestine

Fractionation on Sephadex G50 gel of methanol extracts of guinea pig intestine reveals two molecular forms of cholecystokinin (CCK) of about equal abundance. One elutes at the position of CCK8 while the other elutes at a position intermediate between CCK33 and CCK8. Purification and sequencing of these peptides identify them as CCK8 and CCK22, respectively. Guinea pig CCK8 differs from other mammalian CCK octapeptides isolated thus far in that there is a valine substituted for methionine at position 6 from the C-terminus. In addition to the substitution in CCK8, serine is substituted for asparagine in position 22, glycine for serine in position 19, and asparagine for serine in position 15 from the C-terminus compared to the pig sequence. HPLC separation on a C18 column yields two peaks each of CCK8 and of CCK22 in pig intestinal tissue obtained from a commercial supplier. The two CCK8 peptides have identical amino acid sequences as do the two CCK22 peptides. The CCK22 peptides are equally bioactive in the guinea pig pancreatic acinar cell assay but are about 10-fold less potent than synthetic CCK8(s). One of the guinea pig CCK8 peptides is fully bioactive whereas the other is about 50-fold less potent compared to synthetic CCK8(s).     

Microvillus membrane vesicles from pig small intestine. Purity and lipid composition

Microvillus membrane vesicles from pig small intestine were isolated by a method based on hypotonic lysis, Mg2+-aggregation of contaminants and differential centrifugation. The purity of the membrane vesicles were established by measuring the activity of marker enzymes and the RNA and DNA content. The membranes were found free of contamination by other subcellular membrane fragments, except for a minor contamination with basolateral plasma membranes. The lipid composition was established and, based on weight percentage, the membrane contained neutral lipids, phospholipids, neutral glycolipids and gangliosides in the weight ratio of 18 : 50 : 29 : 2%. The amount of individual phospholipids and glycolipids were quantitated. Phosphatidylethanolamine, -choline, -serine, -inositol and sphingomyelin made up 17, 17, 6, 5 and 5%, respectively of the total lipid. The major glycolipids were two monohexosylceramides containing glucose and galactose as the carbohydrate component, a dihexosylceramide containing galactose as the only carbohydrate component and two pentahexosylceramides containing fucose, galactose, glucose and hexosamine (either N-acetylglucosamine or N-acetylgalactosamine) in the molar ratio of 1 : 2 : 1 : 1.     

Degradation of pig gastric and colonic mucins by bacteria isolated from the pig colon.

Mucin degradation was studied with one Clostridium (RS42) and two Bacteroides (RS2 and RS13) strains isolated from the pig colon mucosa. Mucins from pig colon and stomach were prepared in their subunit forms for use as growth substrates, and the loss of the individual sugars from the mucins was measured after bacterial growth. Colonic mucin was more resistant to degradation than gastric mucin. The strains differed in their competence in degrading the mucins. Carbohydrate plus sulfate removal from gastric mucin varied from 63 to 76% for RS2, 37 to 46% for RS13, and 37 to 53% for RS42. All three strains removed more fucose (67 to 87%) and less sulfate (22 to 63%) than the average carbohydrate plus sulfate loss. Under the same conditions of growth, a mixed pig fecal culture removed 78% of sulfate and 96% of each sugar. Of the two major glycoprotein types present in the subunit pig gastric mucin preparation (R. A. Stanley, S. P. Lee, and A. M. Roberton, Biochim. Biophys. Acta 760:262-269, 1983), the less highly sulfated mucin was more susceptible to RS42 degradation. The degradation of gastric mucin by RS2 was not affected by glucose or high sulfate concentrations in the growth medium. The results show that the three strains of colon bacteria are capable of significant hydrolysis of mucin carbohydrate and that the extent of degradation seen with pure cultures is determined in part by the subunit glycoprotein type(s) present in the mucin.     

Flagellated coryneform bacteria from the intestine of earthworms

Summary Representative strains of distinctly flagellated, but doubtfully motile, coryneform bacteria were isolated from the intestine of Indian earthworms. Their morphological, cultural, physiological and nutritional characters are described, and their taxonomic position discussed.     

Nutritionally limited pectinolytic bacteria from the human intestine

A selective procedure was used to isolate pectinolytic intestinal bacteria from human subjects. The three isolates with the greatest pectinolytic activity utilized pectin and a few related compounds as fermentable substrates for growth but did not utilize any other compound tested. Thus, their substrate utilization pattern was markedly different from that of previously described intestinal pectinolytic isolates. The three isolates are representatives of a nutritionally defined group of bacteria for which the term pectinophilic is proposed.     

Deglycosylation by trifluoromethanesulphonic acid of endopeptidase-24.11 purified from pig kidney and intestine.

Endopeptidase-24.11, an integral microvillar membrane enzyme, exists in differently glycosylated forms when purified from pig kidney and intestine [Fulcher, Chaplin & Kenny (1983) Biochem. J. 215, 317-323]. When these glycoproteins, and another form of the kidney enzyme prepared from the Yucatan dwarf strain of piglet, were treated, under controlled conditions, with trifluoromethanesulphonic acid, the proteins were freed of carbohydrate and all had the same apparent subunit Mr (77 000) even though the untreated forms varied from Mr 89 000 to Mr 95 000.     

Succinate accumulation in pig large intestine during antibiotic-associated diarrhea and the constitution of succinate-producing flora

Succinate was the major organic acid detected in the hindgut content of pigs suffering from antibiotic-associated diarrhea. Antibiotic-associated diarrhea was induced by an oral dose of polymyxin B sulfate (3,000,000 units/day) or an intramuscular injection of enrofloxacin (0.6 g enrofloxacin/day). In the large intestine of enrofloxacin-treated pigs, Gram-negative facultative anaerobic rods phylogenetically related to Escherichia coli and Gram-positive facultative anaerobic non-spore-forming rods phylogenetically related to Lactobacilli were isolated as succinate producers. Succinate-producing Lactobacilli were only isolated as the succinate producer in polymyxin B sulfate-treated pigs. In contrast to antibiotic-associated diarrhea pigs, bacteria belonging to Bacteroidaceae, Fusobacteria, and Enterobacteriaceae were detected as succinate producers in a non-treated pig. In antibiotic-associated diarrhea conditions, antibiotic-resistant Enterobacteria, E. coli in particular, and Lactobacilli may contribute to an abnormal succinate accumulation and may affect water absorption in the hindgut that relates to an expression of antibiotic-associated diarrhea.