Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Distribution of bifidobacteria in the gastrointestinal tract of calves

Development of gastrointestinal microflora of calves with special reference to bifidobacteria was investigated; fecal bacteria were enumerated in calves aged 3 days to 7 weeks. Bacteria were detected by using selective media, bifidobacteria using modified TPY agar with an addition of mupirocin and acetic acid and by fluorescence in situ hybridization (FISH). Bifidobacteria were dominant group of fecal flora of calves after 7 d of life, constituting 10 % of total bacterial counts. The highest bacterial concentrations were observed in rumen, cecum, and colon, the lowest in abomasum and duodenum. Bifidobacteria and lactobacilli exhibited the highest survival ability during stomach passage and dominated in all parts of the digestive tract. Bifidobacteria counts determined by FISH were significantly higher than those provided by cultivation. Modified TPY agar was highly selective and suitable for bifidobacteria isolation but FISH was shown to be a more precise method for their enumeration. Our results show that gastrointestinal microflora of calves in the milk-feeding period is similar to breast-fed infants with respect to the occurrence of bifidobacteria as a dominant bacterial group. The use of Bifidobacterium strains offers a promising way for providing beneficial effectors for calves in the milk-feeding period.     

Tannin-protein complex-degrading enterobacteria isolated from the alimentary tracts of koalas and a selective medium for their enumeration.

Tannin-protein complex (T-PC)-degrading enterobacteria (T-PCDE) were isolated from the feces and from a layer of bacteria attached to the cecal wall of koalas. The T-PCDE were facultatively anaerobic, gram-negative, pleomorphic, nonmotile bacilli. The bacteria were also oxidase and catalase negative and resistant to vancomycin, reduced nitrates to nitrites, and grew on MacConkey agar. Growth on tannin-treated agar media showed a distinctive clear zone around the colony. From these observations, a selective agar plate medium (vancomycin- and tannin-treated Wilkins-Chalgren anaerobe agar) was developed to enumerate T-PCDE isolated from the feces of koalas. This medium was highly selective in the enumeration of the fecal T-PCDE and inhibited the growth of concomitant T-PC-degrading Streptococcus bovis. The T-PCDE were isolated from 10 of the 12 captive koalas studied; in 8 of these 10 koalas, the facultatively anaerobic bacterial flora was dominated (more than 60%) by T-PCDE. Viable numbers of T-PCDE were, in most of the animals, much larger (more than 100 times) than the numbers of T-PC-degrading S. bovis, suggesting that T-PCDE played a more active role in digesting T-PC in the alimentary tracts of koalas.     

Tannin-protein complex-degrading enterobacteria isolated from the alimentary tracts of koalas and a selective medium for their enumeration.

Tannin-protein complex (T-PC)-degrading enterobacteria (T-PCDE) were isolated from the feces and from a layer of bacteria attached to the cecal wall of koalas. The T-PCDE were facultatively anaerobic, gram-negative, pleomorphic, nonmotile bacilli. The bacteria were also oxidase and catalase negative and resistant to vancomycin, reduced nitrates to nitrites, and grew on MacConkey agar. Growth on tannin-treated agar media showed a distinctive clear zone around the colony. From these observations, a selective agar plate medium (vancomycin- and tannin-treated Wilkins-Chalgren anaerobe agar) was developed to enumerate T-PCDE isolated from the feces of koalas. This medium was highly selective in the enumeration of the fecal T-PCDE and inhibited the growth of concomitant T-PC-degrading Streptococcus bovis. The T-PCDE were isolated from 10 of the 12 captive koalas studied; in 8 of these 10 koalas, the facultatively anaerobic bacterial flora was dominated (more than 60%) by T-PCDE. Viable numbers of T-PCDE were, in most of the animals, much larger (more than 100 times) than the numbers of T-PC-degrading S. bovis, suggesting that T-PCDE played a more active role in digesting T-PC in the alimentary tracts of koalas.     

Comparison of bacterial flora and enzymatic activity in faeces of infants and calves

Sixty-four breast-fed infants and 23 calves were investigated for bacteria and enzymatic activity in their faecal samples. The bacteria were measured using cultivation and fluorescence in situ hybridization. Enzymatic activity was also examined. Forty-seven (64%) infants and all the calves had high numbers of bifidobacteria (usually >9 log CFU g-1) in their faeces, but 17 infants (36%) did not have a detectable amount of the bacteria. Most of the bifidobacteria-negative infants had significant quantities of clostridia in their faecal flora. While the infants did not have significantly higher counts of bifidobacteria, the samples from calves contained significantly (P<0.05) more coliform bacteria and lactobacilli. There were also significant differences in their enzymatic activities. Bifidobacteria-positive samples had a greater alpha-glucosidase activity, while bifidobacteria-negative samples had a lower activity of alpha-galactosidase, and calf samples had the highest beta-glucuronidase activity. A significant increase in bifidobacteria in calf faeces between days 3 and 7 was accompanied by a decrease in Escherichia coli. Our results show that the faecal flora of calves is similar to that of infants with regard to the occurrence of bifidobacteria as a dominant bacterial group.     

Effect of high-fiber and high-oil diets on the fecal flora of swine.

Six pairs of pigs were fed a basal diet, a high-fiber diet, and a diet high in corn oil in different sequences to minimize the carry-over effect of diet. After 2 months on each diet, a fecal specimen from each pig was cultured on nonselective medium in roll tubes. Fifty colonies were randomly selected from each fecal sample, and isolates were characterized to identify a representative cross section of the fecal flora. The bacterial composition of the fecal flora differed between basal and high-fiber diets (P = 0.002) and between high-fiber and high-oil diets (P = 0.015). However, the floras were not significantly different between the basal and the high-oil diets (P = 0.135), nor were the floras of the 12 individual pigs (each on all three diets) statistically different (P = 0.103). Only 14 of the 160 observed taxa have been detected in the human fecal flora, and only 159 of 1,871 total isolates (8.5%) were members of described species. The most common isolate was a Streptococcus species similar to that reported by Robinson et al. (I. M. Robinson, S. C. Whipp, J. A. Bucklin, and M. J. Allison, Appl. Environ. Microbiol. 48:964-969, 1984), which was found in 34 of 36 samples and which represented 27.5% of all isolates. Lactobacillus, Fusobacterium, Eubacterium, Bacteroides, and Peptostreptococcus species were the next most common bacteria. Escherichia coli represented 1.7% of all fecal isolates, which is somewhat higher than the 0.1 to 0.6% observed in human feces cultured similarly with prereduced anaerobically sterilized media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rifampin-Blood-Agar as a Selective Medium for the Isolation of Certain Anaerobic Bacteria

A selective medium which allows detection of relatively small numbers of Fusobacterium varium in fecal specimens is described. Blood-agar containing 50 mug of rifampin per ml inhibits the growth of many species of Bacteriodes and of F. fusi-forme/nucleatum but allows good growth of F. varium and most strains of F. mortiferum. Quantitative cultures of 11 fecal specimens were done on rifampin and other selective and nonselective media. F. varium was recovered in counts of 10(6) and 10(7) per gram from two specimens on rifampin only. A third specimen yielded 10(10)F. varium on several media, including rifampin. Some Eubacterium and Clostridium species also grew on rifampin, and these ordinarily were distinguished from the Fusobacterium by colony morphology. This medium is of value in fecal flora studies and should be useful with other kinds of specimens where mixtures of organisms are common.     

Study of the bacterial flora of a non-carbonated natural mineral water.

Natural mineral water from a UK spring was monitored at various stages after it was pumped from the ground, through to bottling and during shelf life before consumption. Samples were collected in commercial PVC bottles, in PVC bottles previously sterilized and hand-filled and in glass bottles. The bacterial flora was counted on plate count agar (PCA) and on PCA diluted 10 times (PCA/10). The predominant bacteria were identified to genus level. Growth rates and nutrient types of isolates were determined by the nutrient-tolerance test (NT). The plate counts at the pre-bottling stage were low. During storage larger numbers of bacteria grew in glass than PVC bottles; the largest number grew in PVC bottles filled by hand. Most of the pigmented bacteria isolated were oligocarbotolerant.     

Survival of Human Dental Plaque Flora in Various Transport Media

Dental plaque samples from (i) subjects with no apparent oral disease, (ii) mentally retarded subjects with periodontal disease, and (iii) subjects with active caries were collected in three transport media viz. a dithiothreitol poised balanced mineral salt solution designated as reduced transport fluid (RTF), VMG II, and modified Stuart medium (SBL). The samples were dispersed by sonic treatment, diluted in the respective medium in which they were collected, and cultured on MM10 sucrose agar. The efficiency of the transport media in the survival of dental plaque flora was determined by comparing the quantitative recovery (expressed as percentage of the initial viable count) from the specimens stored for various lengths of time. The data showed a great variation in the recovery of the oral bacterial flora from the plaque samples. VMG II and SBL served better than RTF as storage media for non-disease-associated dental plaque cultured under strict anaerobic conditions. Recoveries of bacteria from periodontal plaque specimens stored in RTF were higher than SBL and VMG II under identical conditions. The organisms present in the carious plaque samples appeared to survive much better in RTF and VMG II than in SBL as determined by conventional anaerobic culturing technique. However, VMG II showed a higher recovery of organisms from these specimens with an increase in the storage period, suggesting multiplication of the plaque flora. RTF did not allow the growth of oral bacterial flora under all experimental conditions. On the basis of the relative performance of these media it is suggested that RTF is a satisfactory medium for the transport of oral bacteria present in the samples.     

Cecal and fecal bacterial flora of the Mongolian gerbil and the chinchilla.

The Mongolian gerbil is being increasingly used as a laboratory animal and as a pet. Both chinchillas and gerbils are used as animal models for otitis media and other otic research. Previously, only incomplete information was available regarding the indigenous bacterial flora of the lower intestinal tracts of these coprophagic animals. Using the strict anaerobic methodology of the Virginia Polytechnic Institute Anaerobe Laboratory, we studied the predominant bacterial flora of the cecum and fecal pellets of the gerbil and the chinchilla and the bacterial flora of digesta pellets in the proximal colon. We found species of the following anaerobic genera in high dilutions of gerbil fecal pellets: Bifidobacterium, Clostridium, Propionibacterium, Lactobacillus, and Bacteroides. Only lactobacilli were found in high dilutions of digesta from the upper colon, although the cecum yielded Peptostreptococcus, Bifidobacterium, Clostridium, Lactobacillus, Propionibacterium, and Bacteroides species from high dilutions of cecal contents. The facultatively anaerobic and aerobic flora isolated consisted of species of Bacillus, Streptococcus, Staphylococcus, Acinetobacter, Alcaligenes, Escherichia, Pasteurella, and Pseudomonas plus several unidentifiable organisms. Species of Bifidobacterium, Bacteroides, Eubacterium, and anaerobic Lactobacillus were isolated from chinchillas.     

The intestinal microflora of infants: fecal flora of infants with vitamin K deficiency

The fecal flora of 10 infants with vitamin K deficiency (VKD) and 10 healthy infants was examined. All the infants were breast-fed. In the infants with VKD, the total counts (P less than 0.01) and the numbers of bifidobacteria (P less than 0.001) were lower than in the healthy infants, whereas bacteroides, veillonella and enterococci were present in greater numbers in the feces of infants with VKD. The incidence of the Bacteroides fragilis group was higher (P less than 0.05) in the infants with VKD than in the healthy infants. A significant reduction (P less than 0.05) in Bifidobacterium breve was shown in the infants with VKD. The data emphasize the abnormal flora in infants with VKD.     

Effect of high-fiber and high-oil diets on the fecal flora of swine

Six pairs of pigs were fed a basal diet, a high-fiber diet, and a diet high in corn oil in different sequences to minimize the carry-over effect of diet. After 2 months on each diet, a fecal specimen from each pig was cultured on nonselective medium in roll tubes. Fifty colonies were randomly selected from each fecal sample, and isolates were characterized to identify a representative cross section of the fecal flora. The bacterial composition of the fecal flora differed between basal and high-fiber diets (P = 0.002) and between high-fiber and high-oil diets (P = 0.015). However, the floras were not significantly different between the basal and the high-oil diets (P = 0.135), nor were the floras of the 12 individual pigs (each on all three diets) statistically different (P = 0.103). Only 14 of the 160 observed taxa have been detected in the human fecal flora, and only 159 of 1,871 total isolates (8.5%) were members of described species. The most common isolate was a Streptococcus species similar to that reported by Robinson et al. (I. M. Robinson, S. C. Whipp, J. A. Bucklin, and M. J. Allison, Appl. Environ. Microbiol. 48:964-969, 1984), which was found in 34 of 36 samples and which represented 27.5% of all isolates. Lactobacillus, Fusobacterium, Eubacterium, Bacteroides, and Peptostreptococcus species were the next most common bacteria. Escherichia coli represented 1.7% of all fecal isolates, which is somewhat higher than the 0.1 to 0.6% observed in human feces cultured similarly with prereduced anaerobically sterilized media.(ABSTRACT TRUNCATED AT 250 WORDS)     

PCR-DGGE技术评价抗生素诱导的肠道菌群失调

目的 利用PCR-DGGE技术结合活菌计数评价抗生素引起的肠道菌群失调.方法 SPF级BALB/c雌性小鼠连续4 d灌胃头孢曲松溶液125 mg/ml,0.4 ml/d,运用基于细菌16S DNA的PCR-DGGE技术结合活菌计数分析小鼠肠道菌群变化.结果 活菌计数结果与PCR-DGGE图谱显示,头孢曲松处理3 d后小鼠肠道菌群出现严重失调.结论 PCR-DGGE技术可直观而灵敏地显示抗生素处理过程中肠道菌群的动态变化,适用于评价抗生素引起的肠道菌群失调.     

Studies of the cultivable flora of normal human feces

Highly diluted feces, obtained from healthy adult individuals, was plated on blood-agar plates which were incubated both aerobically and anaerobically. From the anaerobic plates containing 30 to 60 colonies, every colony was subcultured. Nearly all isolates were obtained in pure culture and partially characterized. It was found thatBacteroides species were the most predominant organisms, being present in numbers approximating 10¹⁰ per gram wet weight. Selected bacteria present in lower numbers were determined by plating appropriate dilutions of feces on selective media. It was found that coliforms, streptococci and lactobacilli were regularly present in concentrations of 10⁶ ? 10⁸ organisms per gram wet weight material, whileVeillonella, Streptococcus salivarius, Bacteroides melaninogenicus and staphylococci were present in lower numbers. Fusobacteria were only found in one sample, whileNeisseria were not detected in any of the samples. Wet mounts of fecal material, inspected by darkfield microscopy, did not reveal the presence of spirochetes. Anaerobes outnumbered facultative bacteria by a factor of 40, indicating that the human adult fecal flora is predominantly anaerobic. Total microscopic counts indicate that bacteria comprise approximately 30% of the mass of human feces.     

Fecal Microflora in Healthy Persons in a Preindustrial Region

Procedures for quantitating the fecal microflora of man were described. Special attention was given to criteria for characterizing the culturable aerobic, Micro-aerophilic, and anaerobic bacteria. Three groups of healthy persons were studied: wholly breast-fed infants (2 to 4 month-olds), weanlings (1 to 2 year-olds), and adults. In breast-fed children, bifidobacteria predominate and outnumber by one or several logs all other culturable bacteria. The fecal flora of wholly breast-fed infants is "simpler" and more numerous [10(11) to 10(12) per g (wet weight) of feces than that of the adult 10(2) to 10(11) per g]. In the adult, gram-negative anaerobic bacilli (bacteroides) outnumber all other groups by a factor of 1 log or more. Weanlings receiving an adult-type diet, but still breast-fed, showed a flora intermediate between that of the wholly breast-fed infant and that of the adult, but more similar to the latter. Anaerobes always constitute the predominant component of the culturable flora of children and adults and are always found in large numbers under conditions of health. The aerobes are significantly less numerous, and vary widely in their number and in the frequency with which they appear in feces.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

Study of the bacterial flora of a non-carbonated natural mineral water

Natural mineral water from a UK spring was monitored at various stages after it was pumped from the ground, through to bottling and during shelf life before consumption. Samples were collected in commercial PVC bottles, in PVC bottles previously sterilized and hand-filled and in glass bottles. The bacterial flora was counted on plate count agar (PCA) and on PCA diluted 10 times (PCA/10). The predominant bacteria were identified to genus level. Growth rates and nutrient types of isolates were determined by the nutrient-tolerance test (NT). The plate counts at the prebottling stage were low. During storage larger numbers of bacteria grew in glass than PVC bottles; the largest number grew in PVC bottles filled by hand. Most of the pigmented bacteria isolated were oligocarbotolerant.     

The intestinal microflora of infants: composition of fecal flora in breast-fed and bottle-fed infants

The fecal flora of 35 breast-fed and 35 bottle-fed babies was determined. Bifidobacteria were the predominant fecal bacteria in both groups. Conversely, the counts of most of the other bacteria, such as bacteroides, eubacteria, peptococci, veillonella, clostridia, enterobacteria, streptococci, and bacilli in the bottle-fed group were significantly higher than those in the breast-fed group. The frequencies of occurrence of lecithinase positive clostridia, clostridia-others, pseudomonas and bacilli in the bottle-fed group were significantly higher than those in the breast-fed group. Twenty-one genera and 103 species or biovars of microorganisms were isolated from the feces of the breast-fed group and 20 genera and 97 species or biovars from the bottle-fed group. The organism that showed the highest number and the highest frequency of occurrence in both groups was Bifidobacterium breve. Bifidobacterium infantis, which was formerly the most prevalent Bifidobacterium species in baby feces, was never isolated in this study. Further, the counts and incidences of Clostridium paraputrificum, C. perfringens, and Bacillus subtilis, the counts of C. clostridiiforme, Bacteroides vulgatus, Veillonella parvula, Lactobacillus acidophilus, Escherichia coli, Streptococcus bovis, S. faecalis, and S. faecium and the incidences of C. difficile, C. tertium, and Pseudomonas aeruginosa in the bottle-fed infants were significantly higher than those in the breast-fed infants.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

Cecal and fecal bacterial flora of the Mongolian gerbil and the chinchilla

The Mongolian gerbil is being increasingly used as a laboratory animal and as a pet. Both chinchillas and gerbils are used as animal models for otitis media and other otic research. Previously, only incomplete information was available regarding the indigenous bacterial flora of the lower intestinal tracts of these coprophagic animals. Using the strict anaerobic methodology of the Virginia Polytechnic Institute Anaerobe Laboratory, we studied the predominant bacterial flora of the cecum and fecal pellets of the gerbil and the chinchilla and the bacterial flora of digesta pellets in the proximal colon. We found species of the following anaerobic genera in high dilutions of gerbil fecal pellets: Bifidobacterium, Clostridium, Propionibacterium, Lactobacillus, and Bacteroides. Only lactobacilli were found in high dilutions of digesta from the upper colon, although the cecum yielded Peptostreptococcus, Bifidobacterium, Clostridium, Lactobacillus, Propionibacterium, and Bacteroides species from high dilutions of cecal contents. The facultatively anaerobic and aerobic flora isolated consisted of species of Bacillus, Streptococcus, Staphylococcus, Acinetobacter, Alcaligenes, Escherichia, Pasteurella, and Pseudomonas plus several unidentifiable organisms. Species of Bifidobacterium, Bacteroides, Eubacterium, and anaerobic Lactobacillus were isolated from chinchillas.