Adenosine Triphosphate and Deoxyribonucleic Acid in the Alimentary Tract of Cattle Fed Different Nitrogen Sources

Total microbial cell counts and the content of deoxyribonucleic acid (DNA) and adenosine triphosphate (ATP) were determined in ruminal and abomasal digesta and in faeces from heifers fed a basal diet of barley and straw supplemented with urea, casein, soybean-protein concentrate or feather meal. The concentration of ATP in the rumen content varied independently of the DNA content and total cell count, but dependently upon the nitrogen sources, the highest ATP/DNA ratio being obtained with casein. The ATP/DNA ratio in faeces was only one-tenth of that found in the digesta of the rumen and abomasum, indicating either an extremely low level of activity of the microbial cells or more probably a very large number of dead organisms. It is suggested that DNA and ATP might be useful indices of the microbial status in terms of biomass and metabolic activity. The total cell count should still be included in routine studies to determine the proportion of protozoa to bacteria.     

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.     

Establishment in the piglet gut of lactobacilli capable of degrading mixed-linked beta-glucans

The establishment in piglets of lactobacilli with ability to degrade mixed-linked (1----3), (1----4) beta-D-glucans was studied in faeces from 15 animals. The piglets had free access to creep feed with an estimated content of 2% beta-D-glucans from 5 days of age. On days 3 and 35, ca log 8 cfu/g of beta-glucan-degrading lactobacilli were found in a majority of the samples. On days 7, 14 and 21 such bacteria were only found occasionally. During establishment of the microflora in the neonate, the faecal lactobacilli of the piglet seemed related to those of the sow. Later, the metabolic activity of the lactobacilli in piglet faeces showed a connection to the composition of the diet. The possible relation of these bacteria to occurrence of beta-glucanases attributed to be endogenous in the pig is discussed.     

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.     

Microbiology of Drycleaning

An appreciable number of bacteria on contaminated fabric survived modern drycleaning procedures. Various stages in the process, especially steam pressing, reduced the total number of bacteria, but viable organisms were found on certain areas of garments even after pressing. A significant number of bacteria were redeposited on clean fabric during the washing of ordinary soiled garments in drycleaning units. These bacteria included gram-positive cocci, diphtheroid bacilli, and gram-positive sporeformers. Gram-negative bacilli were seldom found, although some gram-negative bacilli survived drycleaning. The redeposited organisms apparently came mainly from other garments in the same loads, as few bacteria were isolated from the filtered solvent used for washing. The number of bacteria in the drycleaning washwheel was highest shortly after the beginning of the wash, and decreased, with the exchange of solvent in the wheel, to a low level at the end. Although it appears that in most cases several factors combine to reduce to a low level the numbers of bacteria on articles cleaned in a well-operated drycleaning plant, it would seem that under certain conditions pathogenic microorganisms could be disseminated by drycleaning.     

Microbiology of Drycleaning

An appreciable number of bacteria on contaminated fabric survived modern drycleaning procedures. Various stages in the process, especially steam pressing, reduced the total number of bacteria, but viable organisms were found on certain areas of garments even after pressing. A significant number of bacteria were redeposited on clean fabric during the washing of ordinary soiled garments in drycleaning units. These bacteria included gram-positive cocci, diphtheroid bacilli, and gram-positive sporeformers. Gram-negative bacilli were seldom found, although some gram-negative bacilli survived drycleaning. The redeposited organisms apparently came mainly from other garments in the same loads, as few bacteria were isolated from the filtered solvent used for washing. The number of bacteria in the drycleaning washwheel was highest shortly after the beginning of the wash, and decreased, with the exchange of solvent in the wheel, to a low level at the end. Although it appears that in most cases several factors combine to reduce to a low level the numbers of bacteria on articles cleaned in a well-operated drycleaning plant, it would seem that under certain conditions pathogenic microorganisms could be disseminated by drycleaning.     

Biochemical and taxonomic characterization of bacteria associated with the crucifer root maggot (Delia radicum)

The crucifer root maggot, Delia radicum, is an important pest of cruciferous crops; however, little is known about its digestive biochemistry or resident gut microbiota. A culturing approach was used to survey the types of micro organisms associated with eggs, midgut, and faeces of larvae feeding on rutabaga. All bacteria isolated from the midgut and faecal materials were Gram-negative bacilli. Nine types of culturable bacteria were identified within the midgut based on analysis of 60 kDa chaperonin sequences and were generally gamma-Proteobacteria, primarily Enterobacteriaceae. Carbohydrate utilization patterns, select biochemical pathways, and hydrolytic enzymes were examined using the API(R) system for each of the nine groups, revealing an exceptionally broad metabolic and hydrolytic potential. These studies suggest that resident alimentary tract microorganisms have the potential to contribute to host nutrition directly as a food source as well as by providing increased digestive potential.     

Contribution of the microflora to proteolysis in the human large intestine

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p -nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis -type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.     

Contribution of the microflora to proteolysis in the human large intestine

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p-nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis-type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.     

Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment

The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development.To detect bacilli in tissues, a new combination staining method was optimized using auramine O and rhodamine B for staining acid-fast bacilli, hematoxylin QS for staining tissue and DAPI for staining nuclei. Bacillary location was studied in three animal models used in-house for TB drug evaluations: C57BL/6 mice, immunocompromised GKO mice and guinea pigs. In both mouse models, the bacilli were found primarily intracellularly in inflammatory lesions at most stages of disease, except for late stage GKO mice, which showed significant necrosis and extracellular bacilli after 25 days of infection. This is also the time when hypoxia was initially visualized in GKO mice by 2-piminidazole. In guinea pigs, the majority of bacteria in lungs are extracellular organisms in necrotic lesions and only few, if any, were ever visualized in inflammatory lesions. Following drug treatment in mice a homogenous bacillary reduction across lung granulomas was observed, whereas in guinea pigs the remaining extracellular bacilli persisted in lesions with residual necrosis. In summary, differences in pathogenesis between animal models infected with M. tuberculosis result in various granulomatous lesion types, which affect the location, environment and state of bacilli. The majority of M. tuberculosis bacilli in an advanced disease state were found to be extracellular in necrotic lesions with an acellular rim of residual necrosis. Drug development should be designed to target this bacillary population and should evaluate drug regimens in the appropriate animal models.     

THE VALUE OF THE ASSOCIATION ESCHERICHIEAE-GROUP D STREPTOCOCCI IN THE DIAGNOSIS OF CONTAMINATION IN FOODS

SUMMARY: The tests for faecal contamination in foods, based on 'indicator species', should be reconsidered for the following reasons. First, in Europe, Escherichia coli is regarded as a specific index of such contamination; but, apparently through the use of antibiotics, the proportion of human faeces containing Klebsiella has risen from 5.2% in 1947 to 48.4% in 1956–7. Moreover, E. coli and other coli-aerogenes organisms may be absent from the gut of certain animals, notably the pig. Second, while the usual methods of counting coli-aerogenes bacteria do not permit the isolation of strains which ferment lactose slowly or not at all (i.e. paracolons), these occur in 35.6% of samples of human faeces. Further, the recommended methods of detecting such strains are not wholly satisfactory. Third, the origin of the coli-aerogenes bacteria is uncertain.
It is thus necessary to consider other organisms which normally inhabit the intestinal tract of humans and other animals. As to Clostridium perfringens , its source is doubtful, for it may come from faeces or from soil, and many foods contain only soil strains. The group D streptococci, on the other hand, are excellent indicators of faecal contamination. They are constant or frequent in the intestines of man and animals, and often more numerous there than coli-aerogenes bacteria. Their specificity as an index of faecal contamination is high and their investigation is easy with the selective media now available. Associated with the coli-aerogenes bacteria, they justify the diagnosis of faecal contamination in a foodstuff.     

Numerical Classification of Bacteria

Sixty three organisms selected from 12 genera of bacteria were subjected to numerical analysis. The purpose of this work is to examine the relationships among 38 coryneform bacteria included in the test organisms by two coding methods—Sneath’s and Lockhart’s systems—, and to compare the results with conventional classification. In both cases of codification, five groups and one or two single item(s) were found in the resultant classifications. Different codings brought, however, a few distinct differences in some groups, especially in a group of sporogenic bacilli or lactic-acid bacteria. So far as the present work concerns, the result obtained on Lockhart’s coding rather than that obtained on Sneath’s coding resembled the conventional classification. The taxonomic positions of corynebacteria were quite different from those of the conventional classification, regardless of which coding method was applied.

Though animal corynebacteria have conventionally been considered to occupy the taxonomic position neighboring to genera Arthrobacter and Cellulomonas and regarded to be the nucleus of so-called “coryneform bacteria,’ the present work showed that many of the corynebacteria are akin to certain mycobacteria rather than to the organisms belonging to the above two genera.     

Studies on the Significance of Three Bacillus species to the Ensiling Process

The activities of Bacillus coagulans, Bacillus licheniformis and Bacillus polymyxa were examined in a defined environment prepared to simulate a grass with low contents of dry matter and water-soluble carbohydrates. All of these organisms produced lactic and acetic acids, and their growth was not suppressed by these fermentation products nor by low pH. The acid content of silages made by inoculation with lactic acid bacteria alone and in combination with each of the bacilli was greater than in the silages containing each of the bacilli alone. The acid content of the silages with lactic acid bacteria was not increased by the presence of bacilli. It is concluded that bacilli can compete with lactic acid bacteria for substrate but contribute little to the fermentation and in this context should be discouraged.     

The role of the gut flora in the metabolism of cyclamate

1. [(14)C]Cyclamate was not metabolized when incubated with the liver, spleen, kidney or blood of rats of rabbits kept on a cyclamate-containing diet, and that had become converters of cyclamate into cyclohexylamine. 2. [(14)C]Cyclamate was converted into cyclohexylamine when incubated under anaerobic conditions with the contents of the caecum, colon or rectum or with the faeces of cyclamate-pretreated rats. Similar results were obtained with cyclamate-pretreated rabbits. With cyclamate-pretreated guinea pigs, which did not readily convert cyclamate into cyclohexylamine, the colon contents showed only low activity in this respect. 3. The faeces of a human converter of [(14)C]cyclamate into cyclohexylamine were also very active, but became less active when cyclamate was removed from his diet. 4. On subculturing the organisms from the contents of the colon and rectum of rats, the ability to convert cyclamate into cyclohexylamine was lost during three subcultures, but the loss of the activity was considerably decreased by subculturing in the presence of cyclamate. 5. Incubation of rat faeces in broths containing cyclamate increased their ability to metabolize cyclamate, but similar treatment of rabbit and human faeces suppressed this activity. 6. When rats are kept on a cyclamate diet the number of clostridia in the faeces increased considerably. In human dietary cyclamate did not appear to alter the counts of various faecal micro-organisms. 7. The gut organisms that appear to develop the ability to convert cyclamate into cyclohexylamine are clostridia in rats, enterobacteria in rabbits and enterococci in man. 8. [(14)C]Cyclohexylamine injected into the caecum or colon of rats is readily absorbed and excreted in the urine. 9. It appears that on continued intake of cyclamate the gut flora develop the ability to convert cyclamate into cyclohexylamine, which is then absorbed and excreted mainly in the urine, although a small proportion is metabolized to other compounds.     

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.     

Sex specificity of myocardial damage in mice fed a purified diet

Both sexes of BALB/c and B6C3F1 mice were divided into test groups and fed either a purified diet (AIN-76A) or a natural ingredient diet (NIH-07) containing graded levels of 2-acetylaminofluorine (2-AAF) for 90 days. A large number of dead or moribund B6C3F1 males fed the AIN diet were removed from the study prematurely. AIN-fed B6C3F1 mice removed early as well as some sacrificed at the end of the study showed myocardial damage with hemorrhage. A much smaller number of BALB/c males fed the AIN diet also exhibited these signs while none of the females from either stock were affected. Mice having these lesions were confined largely to 2 of 5 treatment groups. Increased levels of serum aspartate aminotransferase (GOT) (P less than .01) occurred in the AIN-fed B6C3F1 male mice that were sacrificed, supporting the histopathological observation of myocardial damage. There was no other significant difference in the GOT between diets or 2-AAF doses. No environmental factors could be associated with the problem and microbiological and chemical analyses of the diets showed no convincing evidence of specific pathogenic organisms or nutritional deficiencies that might have caused these lesions. Extended storage (up to 4 months) and one batch of feed in particular seemed to be associated with mice having myocardial damage. These associations were highly strain and sex dependent and suggest that great care must be taken in the manufacture and handling of the diet. Furthermore, it seems likely that the diet may be marginally adequate for some strains of mice and may require modification before it will become generally useful.     

Distribution of spermine in bacilli and lactic acid bacteria

Obligate moderately thermophilic bacilli and obligate moderately thermoacidophilic bacilli contained spermine as the major polyamine in addition to putrescine and spermidine. The identity of spermine was confirmed by thin-layer chromatography and high-performance liquid chromatography before and after treatment with putrescine oxidase. Using these methods, thermospermine and spermine can be separated; thermospermine was not present in these organisms. On the other hand, various facultative thermophiles and mesophilic strains of the genus Bacillus, including alkalophiles and halophiles, lack spermine and other tetraamines. No spermine was detected in several strains of mesophilic or facultative slightly thermophilic lactic acid bacteria, Lactobacillus and Streptococcus.     

Occurrence of bifidobacteria in faeces of calves fed milk or a combined diet

The development of faecal bacteria composition in calves fed milk or a combined diet was investigated from 4 to 21 days of age. On day 7, bifidobacteria in faeces of milk-fed calves already increased from about 7.6 to 9.2 log CFU/g and did not change until the end of the study, whereas in calves fed the combined diet bifidobacteria only moderately increased to 7.9 log CFU/g and decreased slowly until day 21. The counts of bifidobacteria in calves on a combined diet were significantly (p < 0.01) lower compared to those in milk-fed calves. Bifidobacterial counts determined by cultivation or by fluorescence in situ hybridisation (FISH) did not differ significantly. Our results showed that the occurrence of bifidobacteria in calf faeces is highly dependent on the diet composition. Faecal bacteria flora of calves fed exclusively by milk is rich in bifidobacteria, but in calves on a combined diet coliforms dominated.     

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.     

Phage cocktail to control the exponential growth of normal flora in processed sputum specimens grown overnight in liquid medium for rapid TB diagnosis

The mechanical pressure exerted during centrifugation and the chemical pressure experienced when sputum specimens are processed, leave the tubercle bacilli in the sputum unsuitable for rapid detection especially in phage based assays. Thus, growing Mycobacterium tuberculosis in broth, at least overnight, is mandatory for allowing the tubercle bacilli to recoup. During this time the surviving colonizing flora grow faster and overgrow tubercle bacilli interfering with TB diagnosis. In the present study normal flora surviving the action of 4% NaOH was isolated and characterized. Phages capable of killing 14 different species representing this normal flora were isolated from soil and sewage samples and characterized. A novel and bio-friendly approach to treat sputum samples with a cocktail of three phages capable of killing most of the 14 representative organisms and not infecting mycobacteria is explored to control the overgrowth of colonizing bacteria in broth culture. While 26 of the 100 sputum samples processed by modified Petroff's procedure showed growth of colonizing flora on blood agar, all of them when grown in broth overnight showed mixed, confluent growth. The addition of phagebiotics controlled them all, showing a significant reduction in colony forming units but resulting in few discrete colonies in 54 samples. Isolation of phages capable of controlling these surviving organisms and including them in the phagebiotics mixture should lead to the control of colonizing bacteria effectively.