Dehydrogenase Patterns in the Study of Bacteroidaceae

Enzyme patterns were obtained by starch-gel electrophoresis of cell-free extracts from organisms in the family Bacteroidaceae. Esterases, phosphatases, and lactic and succinic dehydrogenases were detected but offered no possibility for classification purposes. Alpha-glycerophosphate dehydrogenase and 6-phosphogluconate dehydrogenase were not detected. Zymograms of malic, glutamic, and glucose-6-phosphate dehydrogenases separated Bacteroides species from Sphaerophorus and Fusobacterium species. Malic dehydrogenase and glucose-6-phosphate dehydrogenase were found in Bacteroides but not Sphaerophorus and Fusobacterium. These dehydrogenase zymograms placed three gram-negative, nonsporeforming anaerobic rod isolates with the Bacteroides species. A close correlation was found between the classification of Bacteroidaceae by zymogram analysis and a numerical taxonomy scheme previously published from this laboratory.     

Genome sequence of Peptoniphilus rhinitidis 1-13T, an anaerobic coccus strain isolated from clinical specimens

A new Peptoniphilus species has been isolated from samples from a patient who was scheduled for endoscopic sinus surgery for chronic rhinosinusitis. The isolate, Peptoniphilus rhinitidis 1-13(T) (KCTC 5985(T)), can use peptone as a sole carbon source and produce butyrate as a metabolic end product. This is the first report of the draft genome sequence of a novel species in the genus Peptoniphilus within the group of Gram-positive anaerobic cocci.     

Gram-Negative Anaerobes in the Intestinal Flora of Pigs

A differential count of the gram-negative, anaerobic, non sporeforming bacteria in different segments of the pig intestinal tract was performed in 2 groups of pigs. Seventy-seven strains were identified, belonging to 6 groups: Sphaerophorus necrophorus, anhemolytic Sphaerophorus spp., Bacteroides (Eggerthella) spp., Fusobactenum spp., Veillonella/Acidaminococcus spp., and Peptostreptococcus elsdenii. The characters of these groups are described, and quantitative data on their occurrence in a group of normal porkers and a group of pigs with experimental swine dysentery are given.     

Effect of Bile and Desoxycholate on Gram-Negative Anaerobic Bacteria

The bile tests for characterizing gram-negative anaerobic bacilli were reevaluated in prereduced anaerobically sterilized peptone-yeast-glucose broth, in thioglycollate broth, and on blood agar plates. Blood agar plates were unsatisfactory. The combination of 20% bile with 0.1% desoxycholate inhibited Fusobacterium, Bacteroides melaninogenicus, and B. oralis and sometimes Sphaerophorus necrophorus, but not B. fragilis or other Sphaerophorus species studied. Ten per cent bile with 0.05% desoxycholate was less satisfactory. There was no significant difference between fresh and commercial powdered bile. Desoxycholate (0.1% in thioglycollate broth) inhibited B. fragilis, Fusobacterium, B. melaninogenicus, B. oralis, and S. necrophorus, but not S. varius or S. mortiferus/S. ridiculosus. The bile and desoxycholate tests are simple to perform and helpful for characterization and classification of gram-negative anaerobic bacilli.     

Isolation and characterization of a Gram-positive polyphosphate-accumulating bacterium

A Gram-positive polyphosphate-accumulating bacterium was isolated from phosphate-removal activated sludge using pyruvate-supplemented agar plates. The isolate was oval or coccobacilli (0.4-0.7 x 0.5-1.0 mm) that occurred singly, in pairs or irregular clumps. Polyphosphate granules in the cells were observed by toluidine blue staining. The pure culture of the isolate rapidly took up phosphate (9.2 mg-P/g-dry weight) in the 3-h aerobic incubation without organic substrates, after anaerobic incubation with organic substrates containing casamino acids. When acetate was the sole carbon source in the anaerobic incubation, the isolate did not remove phosphate. These physiological features of the isolate were similar to those of Microlunatus phosphovorus. However, unlike M. phosphovorus the P-removal ability of the isolate was relatively low and was not accelerated by repeating the anaerobic/aerobic incubation cycles. Phylogenetic analysis and comparison of several characteristics showed that the isolate was identified as Tetrasphaera elongata which was recently proposed as a new polyphosphate-accumulating species isolated from activated sludge. As the isolate contained menaquinone (MK)-8(H(4)) as the predominant isoprenoid ubiquinone, it may be significantly responsible for phosphate removal, because MK-8(H(4)) has reportedly been found in fairly high proportions in many phosphate-removing activated sludges.     

Clostridium saccharogumia sp. nov. and Lactonifactor longoviformis gen. nov., sp. nov., two novel human faecal bacteria involved in the conversion of the dietary phytoestrogen secoisolariciresinol diglucoside

Two anaerobic bacteria involved in the conversion of the plant lignan secoisolariciresinol diglucoside were isolated from faeces of a healthy male adult. The first isolate, strain SDG-Mt85-3Db, was a mesophilic strictly anaerobic Gram-positive helically coiled rod. Based on 16S r RNA gene sequence analysis, its nearest relatives were Clostridium cocleatum (96.7% similarity) and Clostridium ramosum (96.6%). In contrast to these species, the isolate was devoid of alpha-galactosidase and -glucosidase and did not grow on maltose, melibiose, raffinose, rhamnose and trehalose. The hypothesis that strain SDG-Mt85-3Db represents a new bacterial species of the Clostridium cluster XVIII was confirmed by DNA-DNA hybridisation experiments. The G+C content of DNA of strain SDG-Mt85-3Db (30.7+/-0.8 mol%) was comparable with that of Clostridium butyricum, the type species of the genus Clostridium. The name Clostridium saccharogumia is proposed for strain SDG-Mt85-3Db (=DSM 17460T=CCUG 51486T). The second isolate, strain ED-Mt61/PYG-s6, was a mesophilic strictly anaerobic Gram-positive regular rod. Based on 16S rRNA gene sequence analysis, its nearest relatives were Clostridium amygdalinum (93.3%), Clostridium saccharolyticum (93.1%) and Ruminococcus productus (93.0%). The isolate differed from these species in its ability to dehydrogenate enterodiol. It also possessed alpha-arabinosidase and -galactosidase and had a higher G+C content of DNA (48.0 mol%). According to these findings, it is proposed to create a novel genus, Lactonifactor, and a novel species, Lactonifactor longoviformis, to accommodate strain ED-Mt61/PYG-s6. The type strain is DSM 17459T (=CCUG 51487T).     

Tryptophanase in Diverse Bacterial Species

The distribution of tryptophanase was studied. The highest observed specific activity, mumoles per minute per milligram (dry weight) cells, is given in parentheses after each species. Tryptophanase was inducible and repressible in Escherichia coli (.914), Paracolobactrum coliforme (.210), Proteus vulgaris (.146), Aeromonas liquefaciens (.030), Photobacterium harveyi (.035), Sphaerophorus varius (.021), Bacteroides sp. (.048), and Corynebacterium acnes (.042). The enzyme was constitutive and nonrepressible in Bacillus alvei (.013), and was inducible but not repressible by glucose in Micrococcus aerogenes (.036). Indole-positive bacteria were found in fecal or intestinal samples from a variety of animals among the mammals, reptiles, insects, molluscs, fish, crustaceans, and amphibians.     

Quantitative Studies on Some of the Gram-negative Anaerobic Bacteria in the Pig Alimentary Tract

S ummary . A medium suitable for isolating some groups of Gram-negative anaerobic bacteria from the pig alimentary tract is described. Colony counts were made of Gram-negative anaerobes in the gut of pigs varying in age from 1 day to 6 months. The genera Bacteroides, Veillonella, Sphaerophorus and Peptostreptococcus were recognized on the basis of morphology. The Bacteroides spp. were of two morphological types: a coccobacillus, present only in pigs up to 18 days of age and a bacillus, present in all the pigs with the exception of those between the ages of 43 and 67 days. Veillonellae were absent from the gut of pigs up to 11 days of age and thereafter were usually the predominant anaerobic flora. Sphaerophorus spp. were not isolated until the pigs reached 43 days of age. Peptostreptococci were isolated only occasionally.     

Simple Medium for the Selective Isolation of Bacteroides and Related Organisms, and Their Occurrence in Sewage

A medium composed of 0.009% sodium azide, 0.07% sodium deoxycholate, and 0.0007% ethyl violet in Brain Heart Infusion Agar (Difco) and a process of incubation in an atmosphere of 90% N(2) and 10% CO(2) for the selective isolation of certain members of the intestinal bacteroides are described. The medium appears to select predominantly members of the genus Bacteroides and a few of the genus Sphaerophorus. A survey of the occurrence of these organisms in sewage and various stages of sewage treatment indicates that they survive complete sewage treatment in low numbers and that their rate of decline parallels that of the coliforms. Large numbers were recovered from sludge digestion tanks, suggesting a possible role in the anaerobic breakdown of organic matter.     

Effect of Exposure to Hyperoxic, Hypobaric, and Hyperbaric Environments on Concentrations of Selected Aerobic and Anaerobic Fecal Flora of Mice

Alterations in selected aerobic and anaerobic fecal microflora of the mouse were determined during exposure to hyperoxic and normoxic hypo- and hyperbaric environments. Examination of fecal cultures obtained during exposure for 6 weeks to either 60 or 77% oxygen concentration at 1 atmosphere absolute revealed little alteration in the aerobic or anaerobic flora. There appeared to be only a retardation in the reduction of the Klebsiella-Enterobacter flora which normally occurs after weaning. During exposure to hypobaric environments (100% O₂, 0.2 atmosphere absolute), significant alterations in concentrations of Escherichia coli, slow lactose fermenters, Klebsiella-Enterobacter, and enterococci were found in some instances. All alterations were toward increased concentrations. Variations in concentrations of different colony types of obligately anaerobic gram-positive (anGPR) and gram-negative (anGNR) rods cultured during the same experiments also occurred. One colony type of anGPR appeared to decrease while a second type increased in numbers. Concentrations of three colony types of anGNR were generally, but not always, increased. During hyperbaric exposure (2.8% O₂, 7.5 atmospheres absolute), increased concentrations of Klebsiella-Enterobacter, E. coli, slow lactose fermenters and enterococci were also noted. Changes in numbers of both colony types of anGPR, when occurring, were in the direction of lower numbers. Alteration in numbers of anGNR were in both directions but were more frequent in the direction of higher numbers. After return to normal air for 4 weeks of either hypo- or hyperbaric exposure, fecal concentrations of all organisms tended to revert toward control values with the exception of the anGPR which remained in lower concentrations after termination of the hyperbaric exposure. These observations indicate that, despite the great variation in the fecal flora among individual mice, it is possible to discover the effects induced by altered gaseous environments.     

Cooperative formation of omega-muricholic acid by intestinal microorganisms.

Three anaerobic bacteria, isolated from the ceca of rats and mice, converted, through a concerted mechanism, beta-muricholic acid, the predominant bile acid in germfree rats, into omega-muricholic acid. One isolate was a Eubacterium lentum strain; the second and third isolates were tentatively identified as atypical Fusobacterium sp. strains. The conversion of beta-muricholic acid into omega-muricholic acid proceeded in two steps: E. lentum oxidized the 6 beta-hydroxyl group of beta-muricholic acid to a 6-oxo group, which was reduced by either of the two other species to a 6 alpha-hydroxyl group, yielding omega-muricholic acid. This transformation occurred both in vitro and in gnotobiotic rats. Monoassociation of germfree rats with the E. lentum strain gave rise to an unidentified fecal bile acid, probably a derivative of beta-muricholic acid having a double bond in the side chain.     

Destruction by Anaerobic Mesophilic and Thermophilic Digestion of Viruses and Indicator Bacteria Indigenous to Domestic Sludges

In raw sludges and in mesophilically and thermophilically digested anaerobic sludges, large variations in numbers of viruses occurred over narrow ranges of numbers of fecal coliforms, total coliforms, and fecal streptococci, demonstrating that the bacteria were poor quantitative reflectors of the numbers of the viruses detected. Mesophilic and thermophilic digestion of anaerobic sludges destroyed all three indicator bacteria more rapidly than such digestion destroyed the viruses. The relative rates for the destruction of viruses, fecal coliforms, and fecal streptococci in the digested sludges were consistent over the 17-month study. Fecal coliforms were 7 to 8 times more sensitive than the viruses to mesophilic digestion and 9 to 10 times more sensitive to thermophilic digestion. Total coliforms were even more sensitive. The rates at which fecal streptococci were destroyed by mesophilic and thermophilic digestion of anaerobic sludges approached those at which the viruses were destroyed by those processes; this suggested that the rates at which fecal streptococci in sludges are destroyed by those processes may serve as useful indicators for the rates at which viruses in sludges are destroyed by those processes.     

Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs

The microbiota of the small intestine is poorly known because of difficulties in sampling. In this study, we examined whether the organisms cultured from the jejunum and feces resemble each other. Small-intestinal fluid samples were collected from 22 beagle dogs with a permanent jejunal fistula in parallel with fecal samples. In addition, corresponding samples from seven of the dogs were collected during a 4-week period (days 4, 10, 14, and 28) to examine the stability of the microbiota. In the jejunal samples, aerobic/facultative and anaerobic bacteria were equally represented, whereas anaerobes dominated in the fecal samples. Despite lower numbers of bacteria in the jejunum (range, 10(2) to 10(6) CFU/g) than in feces (range, 10(8) to 10(11) CFU/g), some microbial groups were more prevalent in the small intestine: staphylococci, 64% versus 36%; nonfermentative gram-negative rods, 27% versus 9%; and yeasts, 27% versus 5%, respectively. In contrast, part of the fecal dominant microbiota (bile-resistant Bacteroides spp., Clostridium hiranonis-like organisms, and lactobacilli) was practically absent in the jejunum. Many species were seldom isolated simultaneously from both sample types, regardless of their overall prevalence. In conclusion, the small intestine contains a few bacterial species at a time with vastly fluctuating counts, opposite to the results obtained for the colon, where the major bacterial groups remain relatively constant over time. Qualitative and quantitative differences between the corresponding jejunal and fecal samples indicate the inability of fecal samples to represent the microbiotas present in the upper gut.     

Comparison of fecal microflora of elderly persons in rural and urban areas of Japan

The Fecal microflora of 15 healthy elderly persons with a median age of 84 years in a rural area whose inhabitants tend to be long-lived (Yuzurihara-area, Uenohara, Yamanashi Prefecture) was compared with the microflora of individuals with a median age of 68 years in an urban area (Tokyo). The diet of the elderly persons in the Yuzurihara area is characterized by a high intake of dietary fiber. Total numbers of anaerobic bacteria were significantly smaller in the elderly persons in the Yuzurihara area than those in the Tokyo area. A significantly large number of bifidobacteria, but not of lecithinase-negative clostridia, was observed in the elderly persons in the Yuzurihara area. Large numbers and high incidences of bacilli and lecithinase-positive clostridia (mainly Clostridium perfringens) were found in the elderly persons in the Tokyo area. Twenty-five genera and over 81 species were isolated from the elderly persons in the Yuzurihara area, and 25 genera and over 92 species were isolated from the elderly persons in the Tokyo area. Furthermore, significantly larger numbers of Bifidobacterium adolescentis and Fusobacterium mortiferum strains were found in the Yuzurihara group, but significant reductions in the Bacteroides buccae-oris group, B. thetaiotaomicron, Bacteroides spp., C. coccoides, C. paraputrificum, and Clostridium spp. were observed in the same group. A significantly higher isolation rate of Bacillus subtilis was observed in the elderly persons in the Tokyo area. The difference in the fecal microflora between elderly persons in Yuzurihara and those in the Tokyo area might be due to a difference in the intake of dietary fiber.     

Comparison of fecal microflora of elderly persons in rural and urban areas of Japan.

The Fecal microflora of 15 healthy elderly persons with a median age of 84 years in a rural area whose inhabitants tend to be long-lived (Yuzurihara-area, Uenohara, Yamanashi Prefecture) was compared with the microflora of individuals with a median age of 68 years in an urban area (Tokyo). The diet of the elderly persons in the Yuzurihara area is characterized by a high intake of dietary fiber. Total numbers of anaerobic bacteria were significantly smaller in the elderly persons in the Yuzurihara area than those in the Tokyo area. A significantly large number of bifidobacteria, but not of lecithinase-negative clostridia, was observed in the elderly persons in the Yuzurihara area. Large numbers and high incidences of bacilli and lecithinase-positive clostridia (mainly Clostridium perfringens) were found in the elderly persons in the Tokyo area. Twenty-five genera and over 81 species were isolated from the elderly persons in the Yuzurihara area, and 25 genera and over 92 species were isolated from the elderly persons in the Tokyo area. Furthermore, significantly larger numbers of Bifidobacterium adolescentis and Fusobacterium mortiferum strains were found in the Yuzurihara group, but significant reductions in the Bacteroides buccae-oris group, B. thetaiotaomicron, Bacteroides spp., C. coccoides, C. paraputrificum, and Clostridium spp. were observed in the same group. A significantly higher isolation rate of Bacillus subtilis was observed in the elderly persons in the Tokyo area. The difference in the fecal microflora between elderly persons in Yuzurihara and those in the Tokyo area might be due to a difference in the intake of dietary fiber.     

Constitutive and Inducible Aerobic and Anaerobic Stress Proteins in the Echinochloa Complex and Rice

Anaerobic stress resulted in a change in the protein accumulation patterns in shoots of several Echinochloa (barnyard grass) species and Oryza sativa (L.) (rice) as resolved by two-dimensional gel electrophoresis. Of the six Echinochloa species investigated, E. phyllopogon (Stev.) Koss, E. muricata (Beauv.) Fern, E. oryzoides (Ard.) Fritsch Clayton, and E. crus-galli (L.) Beauv. are tolerant of anaerobiosis and germinate in the absence of oxygen, as does rice. In contrast, E. crus-pavonis (H.B.K.) Schult and E. colonum (L.) Link are intolerant and do not germinate without oxygen. Computer analysis of the protein patterns from the four tolerant species and rice indicated that the anaerobic response is of five classes: class 1 proteins, enhanced under anaerobiosis (9 to 13 polypeptides ranging from 16-68 kD); class 2 proteins, unique to anaerobiosis (1 to 5 polypeptides ranging from 17-69 kD); class 3 proteins, remained constant under aerobiosis and anaerobiosis; class 4 proteins, prominent only in air and repressed under anoxia (3 to 7 polypeptides ranging from 19-45 kD); and class 5 proteins, unique to aerobiosis (1 to 4 polypeptides ranging from 18-63 kD). In the intolerant species, E. colonum and E. crus-pavonis, no polypeptides were enhanced or repressed under anoxia (class 1 and class 4, respectively), whereas in the tolerant Echinochloa species and rice, a total of at least 9 to 13 anaerobic stress proteins and 4 to 7 "aerobic" proteins were noted. Immunoblotting identified two of the major anaerobic stress proteins as fructose-1,6-bisphosphate aldolase and pyruvate decarboxylase. Based on the differential response of the intolerant species to anaerobiosis, we suggest that another set of genes, whose products may not necessarily be among the major anaerobic stress polypeptides, might confer tolerance in Echinochloa under prolonged anaerobic stress.     

Degradation of natural and synthetic polyesters under anaerobic conditions

Often, degradability under anaerobic conditions is desirable for plastics claimed to be biodegradable, e.g. in anaerobic biowaste treatment plants, landfills and in natural anaerobic sediments. The biodegradation of the natural polyesters poly(beta-hydroxybutyrate) (PHB), poly(beta-hydroxybutyrate-co-11.6%-beta-hydroxyvalerate) (PHBV) and the synthetic polyester poly(epsilon-caprolactone) (PCL) was studied in two anaerobic sludges and individual polyester degrading anaerobic strains were isolated, characterized and used for degradation experiments under controlled laboratory conditions. Incubation of PHB and PHBV films in two anaerobic sludges exhibited significant degradation in a time scale of 6-10 weeks monitored by weight loss and biogas formation. In contrast to aerobic conditions, PHB was degraded anaerobically more rapidly than the copolyester PHBV, when tested with either mixed cultures or a single strained isolate. PCL tends to degrade slower than the natural polyesters PHB and PHBV. Four PHB and PCL degrading isolates were taxonomically identified and are obviously new species belonging to the genus Clostridium group I. The depolymerizing enzyme systems of PHB and PCL degrading isolates are supposed to be different. Using one isolated strain in an optimized laboratory degradation test with PHB powder, the degradation time was drastically reduced compared to the degradation in sludges (2 days vs. 6-10 weeks).     

Aerobic and facultatively anaerobic bacteria associated with the gut of Canada geese (Branta canadensis) and whistling swans (Cygnus columbianus columbianus)

Aerobic and facultatively anaerobic bacteria from the intestinal tracts of swans and geese were isolated and characterized as part of a larger study of the microbiological effects of migratory waterfowl on water quality. A total of 356 isolates were identified by using rapid identification methods and classified by using numerical taxonomy. A diverse population of bacteria was recovered from the waterfowl, and representative strains could be classified into 21 phena. The majority of the aerobic, heterotrophic bacteria found in the gut of the waterfowl were species of Enterobacteriaceae. Streptococcus. Lactobacillus, and Bacillus. Unfortunately, the birds that were examined did not harbor significant numbers of any waterfowl-specific bacterial species. Thus, it may not be possible to assess microbiological impact of migratory waterfowl by using and "indicator" species since avian fecal pollution could not be distinguished from animal and human fecal pollution.     

Adult diarrhea rotavirus in Jilin, China

In May 1986, an outbreak of epidemic acute diarrhea occurred in one city and three counties in Yanbian area. The diarrheal cases were seen in all age groups, the majority of the cases were seen in adult age group. No bacterial pathogens were isolated in 22 fecal samples examined, however rotavirus like particles of 52-68 nm in diameter were found in 50% (11/22) of fecal samples by the immunoelectron microscopy using convalescent sera. Examination by the adult diarrhea rotavirus (ADRV) ELISA kit showed positive reaction in 86% (6/7) of fecal extracts, however in all 22 fecal extracts examination using ELISA kit for detection of group A rotavirus showed negative reaction. The PAGE patterns of viral RNAs similar to those of ADRV were seen in 13 out of 22 fecal extracts. The detection rate by PAGE analysis was 59%. Based on the above data, the etiological agent of the epidemic acute diarrhea, which occurred in Yanbian area, was identified to be ADRV.     

Some Morphological and Physiological Characteristics of Gram Negative Anaerobic Bacteria isolated from the Alimentary Tract of the Pig

S ummary . A collection of 100 strains of Gram negative anaerobic bacteria isolated from the alimentary tract of the pig has been divided into four groups on morphological and physiological grounds. The four groups resemble the genera Bacteroides, Sphaerophorus, Veillonella and Peptostreptococcus as described in Bergey's Manual (Breed, Murray & Smith, 1957).