Some aspects of the gastrointestinal microflora of germfree mice associated with cultured microfloras

Attempts are described to 'normalize' germfree mice by association with 3, 21 and 71 different intestinal bacterial cultures isolated from mice with an SPF flora. Germfree mice associated naturally with an SPF flora served as controls. Vital bacterial counts were determined by aerobic and anaerobic culture. Stomach and small intestine contained fewer bacteria per gram than caecum and large intestine. Aerobic vital counts from caecum and large intestine were higher in the experimental groups than in control mice. The aerobic and anaerobic flora in stomach and small intestine comprised mainly Gram-positive non-fusiform shaped rods. In the caecum and colon Gram-positive cocci predominated in the aerobic culture while in the anaerobic culture fusiform-shaped rods were prominent. Scanning electron microscopy of oesophagus, ileum, caecum and faeces demonstrated colonization of the oesophageal epithelium only after association with 71 bacterial strains; the filamentous bacteria present in the ileum of SPF mice were not found in the experimental groups and caecum and faeces contained mainly fusiform-shaped bacteria. Non-bacterial matter decreased in the caecum and faeces with increase in the complexity of the flora.     

Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice

Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1). They were still 50-80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1). The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1). Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.     

Bacterial flora in the digestive tract of cattle. I. Comparison of nonselective culture medium and changes in fecal bacterial flora with age.

Studies were made on nonselective culture medium and the method of culture for the investigation of the bacterial flora in the digestive tract of cattle. With their results, further studies were done to clarify changes in the fecal bacterial flora in eight calves less than 6 months of age with the lapse of time. Three roll-tube media were used in the gas jet method. They were modified VL agar (VL medium), rumen fluid glucose cellobiose agar (RGCA medium), and Medium 10 (M 10). Moreover, glucose liver blood agar (BL medium) was used in the anaerobic jar method. In this method the steel wool method was applied after the substitution of carbon dioxide. Of the four media used, VL medium was proved to be the most efficient. It was followed by RGCA medium and M 10. BL medium was much less efficient than any other medium. When the fecal bacterial flora was examined in calves for changes with the advance in age, the total bacterial count and the enteric bacterial count decreased in the second half of the experimental period. The lactobacillary group count remained almost at a constant level of 7 approximately 9 (logarithmic value) per gram in breast-fed calves, but decreased to a level of 5 (logarithmic value) per gram in bottle-fed calves at about 2 months of age or later. The streptococcal group count showed no particular tendency to change. When the organic acid contents of the feces were estimated in calves in every stage of growth, the amount of total organic acids and that of propionic acid were larger in bottle-fed than in breast-fed calves.     

Microflora of raw cacao beans

Whole non-moldy cacao beans from 14 sources cultured on Czapek's agar with six percent NaCl yielded species ofAspergillus glaucus group,Mucor pusillus and unidentified yeast and bacteria. The average number of bacterial colonies from the 14 sources of non-moldy cacao beans was 32.7 million per gram. The average number of colonies of filamentous fungi in dilution cultures was 57,000 colonies per gram. Yeast colonies were present in the amount of 15.8 million colonies per gram. The moldy beans analyzed contained fungi and bacterial counts in the billions; also, yeast was not isolated from the moldy samples.Paper Number 3170 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina 27607.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.     

Endogenous infection in mice with streptozotocin-induced diabetes. A feature of bacterial translocation

Slc:ddY mice that received a single intraperitoneal injection of 200 mg/kg streptozotocin (STZ) were examined for persistency of diabetes (changes of indigenous bacterial floras, and bacterial translocation. Significant diabetes (increase in plasma glucose and decrease in insulin) was recognized 2 weeks after the injection, and persisted for 12 weeks. The numbers of aerobic gram-negative bacilli, staphylococci (including micrococci), and streptococci in caecal and oral floras were significantly increased, but the numbers of anaerobic bacteria in caecal flora were hardly changed. Bacterial translocation of indigenous bacteria to the mesenteric lymph node, lung, or kidney was detectable in some mice 2 weeks after the injection. The incidence of bacterial translocation in these STZ-treated mice then increased; infection caused by several organisms, e.g., Klebsiella pneumoniae, Staphylococcus epidermidis, streptococci, or Lactobacillus sp., occurred in lung, liver, spleen, kidneys, and mesenteric lymph node. No indigenous bacteria were cultured from these organs of control mice. This endogenous infection may have been due to the over population of several bacterial strains caused by disruption of indigenous floras along with depression of immunological function.     

Alteration in the endogenous intestinal flora of Swiss Webster mice by experimental Angiostrongylus costaricensis infection

The association between worm infections and bacterial diseases has only recently been emphasized. This study examined the effect of experimental Angiostrongylus costaricensis infection on endogenous intestinal flora of Swiss Webster mice. Eight mice aging six weeks were selected for this experiment. Four were infected with A. costaricensis and the other four were used as controls. Twenty eight days after the worm infection, all mice in both groups were sacrificed and samples of the contents of the ileum and colon were obtained and cultured for aerobic and anaerobic bacteria. In the mice infected with A. costaricensis there was a significant increase in the number of bacteria of the endogenous intestinal flora, accompanied by a decrease in the number of Peptostreptococcus spp. This alteration in the intestinal flora of mice infected by the nematode may help to understand some bacterial infections described in humans.     

Tritium distribution in newborn mice after providing mother mice with drinking water containing tritiated thymidine

Throughout gestation pregnant mice received drinking water which contained [methyl-3H]thymidine (18.5 kBq/ml). The newborn mice were divided into two groups. One group was nursed by their own mothers, which were further supplied with tritiated thymidine until 4 weeks after delivery (Experiment I). The other group was nursed by "nonradioactive mothers" which were given no tritiated thymidine (Experiment II). Tritium incorporation into the small molecular components of the acid-soluble fraction, lipid, RNA, DNA, and protein was analyzed for the newborn mice at various ages. In Experiment II, total radioactivity per gram tissue decreased initially after birth with a half life of 2.5-2.9 days in spleen, liver, intestine, stomach, thymus, lung, kidney, heart, and brain. At about 2 weeks after birth, a slower component of tritium elimination due mainly to the DNA-bound tritium appeared. Specific activity of DNA at birth was organ specific, highest in heart and lowest in thymus. Cumulative absorbed dose in various organs was estimated for the first 4 weeks after birth based upon an assumption that total and DNA-bound tritium are uniformly distributed. The result showed that organ specificity of dose accumulation is obvious for DNA-bound tritium, highest in spleen (1.15 mGy) and lowest in brain (0.13 mGy). It was also shown that the tritium supply from mother's milk is of minor importance for dose accumulation of DNA-bound tritium in the cell nuclei of organs of suckling mice.     

Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 x 10(-2) M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 microgram per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.     

A Note on the Flora and Fauna in the Rumen of Steers Fed a Feedlot Bloat-provoking Ration and the Effect of Penicillin

A study was made of the predominant culturable bacteria and ciliate protozoa present in the rumen of two steers that were regularly bloating on a pelleted ratio containing 22% alfalfa meal, 16% soybean oil meal, 61% barley, and 1% common salt. The ruminal microorganisms in the two animals differed as indicated by a high total culture count of bacteria, an almost complete absence of ciliate protozoa, a low pH, and a difference in the proportions of presumptively identified predominant bacterial groups in one animal (steer 26) as compared with the other (steer 32). The first exposure of the animals to procaine penicillin (75 or 150 mg per day on 2 successive days) resulted in an abnormal ruminal flora 31 hr after the first treatment as indicated by drastic drops in total and cellulolytic bacterial counts and a change in the proportions of predominant bacterial groups. The animals refused feed for 32 to 48 hr after the first treatment. After feed consumption resumed, further treatment with 75 mg penicillin on 4 successive days did not appear to greatly alter the flora and did not result in feed refusal in animal 32. In animal 26, amounts of penicillin progressing from 50 to 200 mg per day did not result in feed refusals and observations on rumen ingesta samples during this period indicated a decrease in total bacterial count, a great increase in numbers of ciliate protozoa, a higher pH, and a change in the proportions of predominant bacterial groups so that the ruminal picture was much more similar to that of animal 32 than to its own during the pre-penicillin period. Bloat was not relieved except during the period of feed refusal.

The results indicate that the ruminal flora rapidly adapts to penicillin and that bloat of the feedlot type can occur in animals with widely differing numbers and kinds of bacteria and protozoa. Feedlot bloat does not appear to be correlated with the occurrence or numbers of any of the individual predominant groups of bacteria cultured.

     

High incidence of scrapie induced by repeated injections of subinfectious prion doses

To clarify the mechanisms leading to the development of Creutzfeldt-Jakob disease in some recipients of pituitary-derived human growth hormone (hGH), we investigated the effects of repeated injections of low prion doses in mice. The injections were performed, as in hGH-treated children, by a peripheral route at short intervals and for an extended period. Twelve groups of 24 mice were intraperitoneally inoculated one, two, or five times per week for 200 days with 2 x 10(-5) to 2 x 10(-8) dilutions of brain homogenate containing the mouse-adapted C506M3 scrapie strain. Sixteen control mice were injected once a week for 200 days with a 2 x 10(-4) dilution of normal brain homogenate. Of mice injected in a single challenge with a scrapie inoculum of a 2 x 10(-4), 2 x 10(-5), or 2 x 10(-6) dilution, 2/10, 1/10, and 0/10 animals developed scrapie, respectively. Control mice remained healthy. One hundred thirty-five of 135 mice injected with repeated prion doses of a 2 x 10(-5) or 2 x 10(-6) dilution succumbed to scrapie. Of mice injected with repeated scrapie doses of a 2 x 10(-7) or 2 x 10(-8) dilution, 52/59 and 38/67 animals died of scrapie, respectively. A high incidence of scrapie was observed in mice receiving repeated doses at low infectivity, whereas there was no disease in mice that were injected once with the same doses. Repeated injections of low prion doses thus constitute a risk for development of prion disease even if the same total dose inoculated in a single challenge does not induce the disease.     

Intestinal bacteria antagonistic to Clostridium difficile in mice

Overgrowth by Clostridium difficile has been reported in conventional mice injected intraperitoneally with ampicillin. In this study, we aimed to determine which types of indigenous intestinal bacteria were eliminated by ampicillin to allow overgrowth by C. difficile. C. difficile overgrowth was associated with a decrease in the numbers of lactobacilli, an increase in bacteroidaceae and a slight decrease in the frequency of isolation of fusiform-shaped bacteria (clostridia). C. difficile cytotoxin was detected in caeca from mice in which the numbers of C. difficile were greater than 10(5) per gram of faeces. Gnotobiotic mice were inoculated with various groups of intestinal anaerobes to determine which members of the indigenous flora would antagonize C. difficile. Gnotobiotic mice inoculated with three strains of lactobacilli, 37 strains of bacteroides or 46 strains of clostridia isolated from limited-flora mice were unable to eliminate C. difficile. C. difficile was eliminated, however, from the gastrointestinal tracts of gnotobiotic mice inoculated with whole faeces or chloroform-treated faeces from conventional mice or whole faeces from limited-flora mice containing only clostridia.     

Colonization resistance against Pseudomonas aeruginosa in gnotobiotic mice

Gnotobiotic (GB) mice were colonized with various groups of intestinal bacteria to determine which members of the indigenous flora would exert colonization resistance against Pseudomonas aeruginosa. P. aeruginosa was cultured from the faeces at levels of 10(3)-10(4) cells/g in GB mice inoculated with either the combination of bacteroides and clostridia obtained from conventional (CV) mice or the combination of bacteroides, lactobacilli and clostridia obtained from limited flora mice. The combination of lactobacilli and clostridia from CV mice also did not eliminate P. aeruginosa from GB mice. However, P. aeruginosa was not detected in the faeces of GB mice by 14 days after inoculation with the combination of bacteroides, lactobacilli and clostridia obtained from CV mice. Thus, a complex indigenous flora consisting of bacteroides, lactobacilli and certain clostridia obtained from CV mice but not clostridia obtained from limited flora mice is required to exert complete colonization resistance against P. aeruginosa in GB mice.     

Response of the Extract-Release Volume and Water-Holding Capacity Phenomena to Microbiologically Spoiled Beef and Aged Beef

The aging of ground beef was effected by storing in gas-impermeable, sterile plastic bags with incubation at 7 and 15 C. Control meat from the same preparations was wrapped in aluminum foil and stored at the same temperature. In three experiments where control meat was tested, aged meat did not attain a log bacterial number of ca. 8.4 per gram until an average of 6 days after this level was reached in control meats. This degree of difference was shown in values for both extract-release volume (ERV) and water-holding capacity. The previously reported ERV value of around 25, which was found to correspond to an average log bacterial number of ca. 8.5 per gram for ground beef allowed to spoil in aluminum foil and freezer paper, was approximated for aged meats, which required an average of 9.7 days to attain this number compared with 4.1 days for unaged meats. Plate count methods indicated the predominant flora of aged beef to be gram-negative, facultatively psychrophilic rods.     

Microbiological Deterioration of Frozen Parfried Potatoes upon Holding After Thawing

Frozen parfried potatoes were thawed and stored at 55, 45, and 34 F (12.8, 7.2, 1.1 C). Significant changes in flavor and texture did not occur at these temperatures until the total bacterial count exceeded 100 million per gram. These sensory changes were produced after 4, 8, and 20 days of storage at 55, 45, and 34 F, respectively. Detectable color change appeared sooner and probably was not of microbial origin. It is unlikely that any health hazard exists under the range of conditions studied. Nevertheless, it seems undesirable to market food with such a high bacterial count. At half the storage periods given above, the count did not exceed 100,000 per gram.     

Nitrogen metabolism by the microbial flora of the rabbit caecum

The dense microbial flora of the rabbit caecum consisted chiefly of bacteria (10(11)/g) with small numbers of yeast cells (10(6)/g). Using strictly anaerobic technique, 23% of the direct microscopic cell count was cultivated and 55% of the cultivatable bacteria utilized ammonia as the sole source of nitrogen. Ureolytic bacteria were isolated from the caecal lumen and mucosa and were identified as Bacteroides vulgatus, Clostridium clostridiiforme, Bacillus spp. and Staphylococcus spp. Ammonia assimilation by the bacterial flora of the caecum was by incorporation into alpha-oxoglutarate catalysed by NADPH-linked glutamate dehydrogenase.     

Safety evaluation of Bifidobacterium pseudocatenulatum G4 as assessed in BALB/c mice

AIMS: To assess the safety of Bifidobacterium pseudocatenulatum G4 in BALB/c mice that involves examination of bacterial translocation, changes in the internal organs and histology of the intestinal lining. METHODS AND RESULTS: Forty male BALB/c mice were randomly assigned into five groups (n = 8). Three groups were orally fed with 50 microl of three different concentrations of B. pseudocatenulatum G4 (2 x 10(4), 1 x 10(8) and 1 x 10(11) CFU day(-1)) for 4 weeks. One group was orally administered with 50 microl of 1 x 10(8) CFU B. longum BB536 per day for 4 weeks and last group was used as a nonbifidobacterial treatment control, which received 50 microl of skim milk. The administered strains did not affect the general health of mice and incapable of carrying out translocation to blood or liver. There were no significant differences in the internal organ (liver, heart, kidney and spleen) indices, serum enzymes of liver (aspartate aminotransferase, alkaline phosphate, alanine aminotransferase) and kidney (urea and creatinine) and histology (villi height, crypts height, mucosa thickness and epithelial cell height) of caecum, ileum and colon. CONCLUSION: Administration of high dose of up to 1 x 10(11) CFU B. pseudocatenulatum G4 per day to mice did not show any health threatening symptoms. SIGNIFICANCE AND IMPACT OF THE STUDY: Bifidobacterium pseudocatenulatum G4 is none pathogenic to BALB/c mice and could be safe probiotic for human consumption.     

Detection of Corynebacterium kutscheri in the faeces of subclinically infected mice

Mice were infected experimentally and subclinically with Corynebacterium kutscheri to recover the organism from mice faeces. The faeces were then cultured using selective furazolidone-nalidixic acid-colimycin agar. The number of C. kutscheri per gram of fresh faeces varied from mouse to mouse, but once established in the intestine, the organism was excreted in the faeces for at least five months. Viable bacteria were detected in most of the faecal samples, including those stored in the animal room for five days. The number of organisms in the stored faeces decreased gradually but did not differ significantly from those in the fresh faeces until they had been stored for more than three days. Many infected mice excreted between 10(4.77) and 10(5.37) colony forming units (CFU) of C. kutscheri per day in their faeces, and one mouse even excreted 10(3.74) CFU at eight weeks postinfection. These values showed little daily variation. Our present study showed that subclinically infected mice discharged the organism continuously and persistently in their faeces. Therefore, faecal samples would be useful for monitoring infection with C. kutscheri in living mice in a manner that is not stressful for the animals.     

蜂胶酊对金黄色葡萄球菌性阴道炎疗效观察

目的探讨蜂胶酊对小鼠阴道内致病菌抑制及调理阴道菌群的作用。方法通过感染金黄色葡萄球菌建立小鼠阴道炎模型,用10％蜂胶酊冲洗治疗去除金黄色葡萄球菌在小鼠阴道的定植。倾注培养法（37℃,48h）计数阴道分泌物的细菌总数,镜下观察阴道黏膜炎症程度。结果蜂胶酊治疗组小鼠阴道内细菌的数量明显较对照组数量减少（P〈0．05）,治疗组黏膜炎症的治愈程度显著好于对照组。结论蜂胶酊对小鼠金黄色葡萄球菌性阴道炎有较好的治疗效果。     

Anaerobic bacteria from the large intestine of mice.

Anaerobic bacteria from the colon of laboratory mice were enumerated and isolated using strict anaerobic techniques. Direct microscopic counts revealed 4.4 X 10(10) organisms in each gram (wet weight) of colon contents. Actual cultural counts averaged 3.2 X 10(10) organisms, which was 73% of the direct microscopic count. The tentatively identified genera were Bacteroides, Eubacterium, Fusobacterium, Lactobacillus, Peptostreptococcus, and Propionibacterium. Strains of Fusobacterium, Lactobacillus, Peptostreptococcus, and Propionibacterium were biochemically homogeneous. Strains of Bacteroides and Eubacterium, on the other hand, were biochemically heterogeneous and were subdivided into several distinct groups. The data indicate that many of the isolates are different from previously described species of the respective genera and may belong to new species.