Radial and axial variations of bacteria within the cecum and proximal colon of guinea pigs revealed by PCR-DGGE

We investigated the local variability of bacterial species within the lumen of the cecum and proximal colon of guinea pigs by using PCR-denaturing gradient gel electrophoresis (DGGE). The DGGE banding profiles revealed radial and axial variation of the bacteria. This variation of bacteria within the large intestinal lumen might be of physiological importance for the degradation of dietary fiber and interaction between bacteria and the mucosal immune system.     

Ascorbic acid catabolism by gut microflora: studies in germ-free and conventional guinea pigs

The role of gut microflora in ascorbic acid catabolism was investigated in both conventional and germ-free guinea pigs. In vitro studies demonstrated extensive degradation of the vitamin by fresh feces, cecal, and colonic contents of conventional guinea pigs. Direct injection of [1-¹⁴C] ascorbic acid into the cecum of conventional guinea pigs in vivo yielded a 70% recovery of the label as respiratory ¹⁴CO₂ within 6 hr compared with only 5% recovery following injection into the virtually sterile peritoneum in a comparable group of conventional guinea pigs. Thus, ascorbic acid not absorbed prior to reaching the lower gastrointestinal tract stands to be extensively decarboxylated by microflora in the cecum. In a companion study of germ-free guinea pigs, 10% of an administered dose of [1-¹⁴C] ascorbic acid was expired as ¹⁴CO₂ within 36 hr post-injection following intraperitoneal injection compared with 16% recovery in a matched group of conventional animals injected at the same site. Results of this series of studies suggest that hepatic decarboxylation and gut microflora, in tandem, contribute to ascorbic acid decarboxylation in this species.     

Studies of mixed anaerobic infections involving Bacteroides gingivalis

The infectiousness of several combinations of bacteria containing Bacteroides gingivalis and other bacteria associated with periodontal diseases was evaluated by subcutaneous injection to guinea pigs. Among the seven mixtures studied, only one permitted the development of an infection easily transmissible to a second guinea pig; this bacterial mixture was composed of B. gingivalis, Fusobacterium nucleatum, Eubacterium saburreum, and Capnocytophaga ochracea (formerly Bacteroides ochraceus). Owing to its ability to synthesize many lytic enzymes and potentially cytotoxic products, B. gingivalis represented the most virulent species of the mixture. Results from in vitro studies suggest that the development of B. gingivalis in the infected animal depends on the growth of C. ochracea. Succinic acid produced in large amount by C. ochracea seems to be one of the growth factors used by B. gingivalis.     

Naturally occurring Tyzzer's disease and intestinal spirochetosis in guinea pigs

Bacillus piliformis infection (Tyzzer's disease) occurred in two young guinea pigs, causing unthriftiness and diarrhea which resulted in death. There was necrosis and inflammation of the ileum, cecum, and colon. Intestinal epithelial cells contained organisms resembling Bacillus piliformis. Spirochetes were found in the cecum and colon, mainly in crypts. Acute diarrhea occurred in another guinea pig which became cachetic and was killed. Histologically, large numbers of spirochetes were present in the wall of both the cecum and colon, and they were associated with severe necrosis and inflammation. Bacillus pilformis was not found in this animal.     

Microbiological Quality of Protein Feed Supplements Produced by Rendering Plants

Samples of protein feed supplements produced by rendering plants were examined for salmonellae, total aerobic bacterial counts, coliform counts, and enterococci. Isolations of salmonellae were more frequent from products with high counts; however, 6% of the samples with total counts of less than 1,000 per gram and 14% of the samples with coliform counts of less than 1 per gram contained salmonellae. Serotypes of Escherichia coli which have been associated with disease in domestic animals and poultry were also isolated from products. Although the distribution of serotypes of salmonellae isolated from environmental swabs and flies was similar to that isolated from products, the isolation of several serotypes from flies which had not been isolated in plants suggested that flies may be a potential source of contamination.     

Polypeptide antibiotic 26a from Bacillus subtilis. IV. Evaluation of biological in vivo activity.

The studies performed with the antibiotic 26a, which has a close resemblance to bacitracin family polypeptide antibiotics, revealed a prominent therapeutic and protective actions. The antibiotic is characterized by a moderately low toxicity, lack of cytotoxic and cytostatic effects on several tissue cultures, and therapeutic effectiveness in lethal streptococcal infections in mice, and corynebacterial infections in guinea pigs.     

Temperature Effects on Legionella pneumophila Killing by and Multiplication in Phagocytes of Guinea Pigs

We examined the effects of temperature on the interaction between Legionella pneumophila and phagocytes of guinea pigs. The body temperatures of guinea pigs infected with a sublethal dose (1.2 × 10⁴ CFU) or a lethal dose (1.0 × 10⁵ CFU) of L. pneumophila elevated from 38.4±0.15 C to 40.2±0.42 C or 40.3 ± 0.62 C, respectively. The intracellular bacterial killing by and bacterial proliferation in the phagocytes were examined at 33, 37, 40, and 42 C, using in vitro culture systems of peritoneal macrophages or polymorphonuclear leukocytes (PMN) of guinea pigs. In all the macrophages incubated at different temperatures, significant intracellular bacterial killings were observed at 4 hr after in vitro phagocytosis. After 24 hr of incubation, there was about a 100-fold increase of CFU and the number reached a maximum after 48 hr of incubation in the macrophages incubated at 42 C as well as 37 and 40 C, suggesting that macrophages support the intracellular bacterial growth in hyperthermia. In the PMN, L. pneumophila CFU 4 hr or 12 hr after the infection were significantly lower at 42 C than those at 37 C (P<0.05), indicating that the bactericidal capacity of PMN was enhanced at 42 C compared to 37 C. However, in all the PMN incubated at different temperatures, there were about 10-fold increases of CFU 24 hr after the infection, suggesting that PMN as well as macrophages support intracellular bacterial growth in hyperthermia. The extracellular bacterial growth was examined at 33, 37, 40, and 42 C in buffered yeast extract (BYE) broth or RPMI 1640 medium containing 50% guinea pig serum as a permissive or non-permissive liquid medium for the bacterial growth, respectively. Inhibition of bacterial growth in BYE broth at 42 C, and a decrease of CFU in RPMI 1640 medium containing 50% guinea pig serum at 42 C were observed. In conclusion, hyperthermia may be beneficial by restricting extracellular bacterial survival, but it exerts no beneficial effect on the restriction of intracellular bacterial growth in phagocytes, though PMN showed enhanced initial killing at 42 C. These results suggest that fever, or hyperthermia itself, may not largely contribute as a nonspecific host defense early in the course of legionellosis.     

Naturally occurring Bacillus piliformis infection (Tyzzer's disease) in guinea pigs

Two juvenile male Hartley guinea pigs (Cavia porcellus) were found dead 36 hours after receipt from a commercial source. Both guinea pigs had dependent, subcutaneous edema and excess serous fluid in their thoracic and abdominal cavities. Their livers were mottled and the cecal walls were reddened focally. Histopathologic exam revealed nests of Bacillus piliformis within the absorptive epithelial cells of the ileum, cecum and colon. Vegetative organisms and spore-like structures were observed in the cytoplasm of intestinal epithelial cells by electron microscopy. A diagnosis of Tyzzer's disease was made.     

Enteric Bacterial Growth Rates in River Water

Enteric bacteria, including stocked strains of pathogenic species and organisms naturally present in the stream, were capable of growth in a chemostat with autoclaved river water taken 750 m below a sewage outfall. Maximal specific growth rates for all organisms occurred at 30 C, whereas culture generation times ranged between 33.3 and 116 hr. Of the six laboratory strains of enteric species used, Escherichia coli and Enterobacter aerogenes grew at generation times of 34.5 and 33.3 hr, respectively, while the remaining Proteus, Arizona, Salmonella, and Shigella spp. reproduced at a rate two to three times slower than the coliforms. Little or no growth occurred in the water at incubation temperatures of 20 and 5 C, and death was observed for Salmonella senftenberg at 20 and 5 C and for E. aerogenes and Proteus rettgeri at 5 C. When enteric bacteria naturally present in the river water were employed in similar experiments, coliform bacteria demonstrated a generation time of approximately 116 hr, whereas fecal coliforms failed to grow. Growth of the bacteria from the river demonstrated a periodicity of approximately 100 hr, which suggests that much of the growth of these organisms in the chemostat may be on the glass surfaces. This phenomenon, however, was not observed with any of the stocked enteric species. Neither the stock cultures nor the aquatic strains were capable of growth in autoclaved river water taken above the sewage outfall at the three temperatures tested.     

Survival of bacteria in carcasses.

Bacteria injected into the bloodstream of guinea pigs shortly before death decreased in number in carcass tissues for about 1 h after death. If initial bacterial numbers were sufficiently low, all bacteria were eliminated, and carcass tissues were sterile 24 h after death. Carcass tissue sterility was maintained with an initial density of Clostridium perfringens or Salmonella typhimurium of 20 cells per g or with an initial density of the other species examined of several hundred cells per gram. With larger numbers of strict and facultative anaerobes, growth commenced after 3 h in carcasses incubated at 30 degrees C. Spores of C. perfringens were killed over the same period as vegetative cells, but growth did not commence until 8 h after death. Bactericidal activity in carcass tissues must therefore be taken into account in evaluating the significance of reports of deep-tissue contamination of carcasses from meat animals.     

Survival of bacteria in carcasses.

Bacteria injected into the bloodstream of guinea pigs shortly before death decreased in number in carcass tissues for about 1 h after death. If initial bacterial numbers were sufficiently low, all bacteria were eliminated, and carcass tissues were sterile 24 h after death. Carcass tissue sterility was maintained with an initial density of Clostridium perfringens or Salmonella typhimurium of 20 cells per g or with an initial density of the other species examined of several hundred cells per gram. With larger numbers of strict and facultative anaerobes, growth commenced after 3 h in carcasses incubated at 30 degrees C. Spores of C. perfringens were killed over the same period as vegetative cells, but growth did not commence until 8 h after death. Bactericidal activity in carcass tissues must therefore be taken into account in evaluating the significance of reports of deep-tissue contamination of carcasses from meat animals.     

Antibiotics conspicuously affect community profiles and richness, but not the density of bacterial cells associated with mucosa in the large and small intestines of mice

The influence of three antibiotics (bacitracin, enrofloxacin, and neomycin sulfate) on the mucosa-associated enteric microbiota and the intestines of mice was examined. Antibiotics caused conspicuous enlargement of ceca and an increase in overall length of the intestine. However, there were no pathologic changes associated with increased cecal size or length of the intestine. Conspicuous reductions in the richness of mucosa-associated bacteria and changes to community profiles within the small (duodenum, proximal jejunum, middle jejunum, distal jejunum, and ileum) and large (cecum, ascending colon, and descending colon) intestine occurred in mice administered antibiotics. Communities in antibiotic-treated mice were dominated by a limited number of Clostridium-like (i.e. clostridial cluster XIVa) and Bacteroides species. The richness of mucosa-associated communities within the small and large intestine increased during the 14-day recovery period. However, community profiles within the large intestine did not return to baseline (i.e. relative to the control). Although antibiotic administration greatly reduced bacterial richness, densities of mucosa-associated bacteria were not reduced correspondingly. These data showed that the antibiotics, bacitracin, enrofloxacin, and neomycin sulfate, administered for 21 days to mice did not sterilize the intestine, but did impart a tremendous and prolonged impact on mucosa-associated bacterial communities throughout the small and large intestine.     

In vitro toxicity of alloxan for guinea pig B cells: comparison with rat B cells

Previous studies have shown that guinea pigs are resistant to the in vivo diabetogenic action of alloxan and that this resistance may be accompanied by a regeneration of B cells in the initial days following administration of the drug. In the studies reported here, we used the measurement of insulin and glucagon released over a 7-day culture period as indices of islet cell viability and examined effects of in vitro exposure to alloxan upon subsequent release of insulin and glucagon from guinea pig (alloxan-resistant) and rat (alloxan-sensitive) islet cell cultures. An alloxan dose-dependent decrease in subsequent insulin release was found. However, whereas the lowest concentration of the drug (1 mM) produced a significant depression in insulin release in rat islet cultures, with maximal depression occurring after exposure to 5 mM alloxan, insulin release from guinea pig cultures was not significantly depressed by 1 or 2 mM alloxan, and 5 mM alloxan treatment produced a submaximal depression. Furthermore, insulin release from guinea pig but not rat cultures increased transiently at between 6 and 18 hr during the first day following exposure to all doses of alloxan. Treatment with high doses of the drug (40 mM or greater) caused the same maximal chronic depression of insulin release for both species. In contrast, glucagon release from cultures of both species was not affected significantly following alloxan treatment. Thus, guinea pig B cells are more resistant than those of the rat to the action of alloxan, but this resistance can be overcome by employing high doses of the drug. Other factions unidentified by the present studies may also be involved in the failure of guinea pigs to develop diabetes following in vivo treatment with alloxan.     

The electron microscopic findings of helicoid bacteria lining the mucosa of the cecum in guinea pigs

This study has revealed that helical bacteria inhabiting the mucous membrane of the cecum of guinea pigs are localized in the parietal zone of the epithelium and can be detected as biological film consisting of many microcolonies. Helical bacteria are attached to the epithelium by insertion of one of the ends of an eukaryotic cell into the space between microvilli without damaging epithelial cells and their microvilli. Helical bacteria have been found to use the "anchor" type of attachment to the epithelium, which ensures the stability of their high population level in the biotope. These microorganisms appear on the mucous membrane of the epithelium, starting from day 15 of the life of guinea pigs. At the period of the transition of suckling guinea pigs to independent nourishment the population of helical bacteria is partially suppressed due to the appearance of bacillary and filamentous forms of bacteria, but later, after the adaptation of the animals to their diet, helical bacteria become normal resident microflora which forms biofilm covering large areas of the mucous membrane and the entrances of crypts of Lieberkühn.     

Effects of physical properties of feed on microbial ecology and survival of Salmonella enterica serovar Typhimurium in the pig gastrointestinal tract

A two-by-two factorial experiment with pigs was conducted to study the effect of feed grinding (fine and coarse) and feed processing (pelleted and nonpelleted) on physicochemical properties, microbial populations, and survival of Salmonella enterica serovar Typhimurium DT12 in the gastrointestinal tracts of pigs. Results demonstrated a strong effect of diet on parameters measured in the stomachs of the pigs, whereas the effect was less in the other parts of the gastrointestinal tract. Pigs fed the coarse nonpelleted (C-NP) diet showed more solid gastric content with higher dry matter content than pigs fed the fine nonpelleted (F-NP), coarse pelleted (C-P), or fine pelleted (F-P) diet. Pigs fed the C-NP diet also showed significantly increased number of anaerobic bacteria (P < 0.05), increased concentrations of organic acids, and reduced pH in the stomach. In addition, pigs fed the C-NP diet showed increased in vitro death rate of S. enterica serovar Typhimurium DT12 in content from the stomach (P < 0.001). Pigs fed the C-NP diet had a significantly higher concentration of undissociated lactic acid in gastric content than pigs fed the other diets (P < 0.001). A strong correlation between the concentration of undissociated lactic acid and the death rate of S. enterica serovar Typhimurium DT12 was found. In the distal small intestine, cecum, and midcolon, significantly lower numbers of coliform bacteria were observed in pigs fed the coarse diets than in pigs fed the fine diets (P < 0.01). Pigs fed the C-NP diet showed the lowest number of coliform bacteria in these segments of the gastrointestinal tract. Pigs fed the coarse diets showed increased concentration of butyric acid in the cecum (P < 0.05) and colon (P < 0.10) compared with pigs fed the fine diets. It was concluded that feeding a coarsely ground meal feed to pigs changes the physicochemical and microbial properties of content in the stomach, which decreases the survival of Salmonella during passage through the stomach. In this way the stomach acts as a barrier preventing harmful bacteria from entering and proliferating in the lower part of the gastrointestinal tract.     

Establishment of stable cell lines producing anti-Pseudomonas aeruginosa monoclonal antibodies and their protective effects for the infection in mice.

Human-human hybridomas producing monoclonal antibodies (MoAbs) specific for five major serotypes of Pseudomonas aeruginosa were developed by fusing P. aeruginosa primed and Epstein-Barr virus-transformed cells with human myeloma P109 cells using polyethyleneglycol. The MoAbs which were produced by the hybridomas were protective against lethal intraperitoneal (i.p.) challenge of P. aeruginosa (10 LD50) in mice. The 50% effective dose (ED50) values of MoAbs ranged from 0.5 to 10.2 micrograms/mouse and were 26 to 240 times more protective than a commercial human IgG preparation. MoAb administration to mice promoted bacterial clearance in peritoneal cavity, and prevented bacterial invasion into blood in the way of increasing both the number of bacteria trapped by a macrophage and the ratio of macrophages that trapped bacteria. MoAbs also showed protective effects against lethal infection of P. aeruginosa in the mice which were decreased in polymorphonuclear cells (PMN) by cyclophosphamide (CY). All MoAbs showed serotype-specific binding to the clinical isolates of P. aeruginosa as well as to the immunized strains. The hybridoma cell lines maintained their capacity to produce MoAb continuously for more than 12 months and produced 10 to 60 micrograms MoAbs per 10(6) cells in 24 hr. It is practicable to use these cell lines for large-scale production of anti-P. aeruginosa MoAbs and such MoAbs must be useful for the therapeutics of patients with P. aeruginosa infection.     

Synergistic Antibacterial and Anti-Inflammatory Activity of Temporin A and Modified Temporin B In Vivo

Temporins are antimicrobial peptides secreted by the granular glands of the European red frog (Rana temporaria). They are 10–14 amino acid long polypeptides active prevalently against gram positive bacteria. This study shows that a synthetic temporin B analogue (TB-YK), acquires the capacity to act in synergism with temporin A and to exert antimicrobial and anti-inflammatory activity in vivo against gram positive and gram negative bacteria. Administration of 3.4 mg/Kg of temporin A (TA)+1.6 mg/Kg TB-YK, given to individual mice concurrently with a lethal dose of bacteria (gram positive or negative), rescued 100% of the animals. More importantly, the same doses of temporins, administered one week after experimental infection with a sub lethal dose of bacteria, sterilized 100% of the animals within 3–6 days. Also, it is described an animal model based on the use of sub lethal doses of bacteria, which closely mimics bacterial infection in humans. The model offers the possibility to test in a preclinical setting the true potential of TA and TB-YK in combination as antimicrobial and anti-inflammatory agents.     

Ligionella pneumophila: Avirulent to virulent conversion through passage in cultured human embryonic lung fibroblasts

Legionella pneumophila isolated in guinea pigs from human lung tissues was highly virulent as determined by its infectivity and lethality in guinea pigs. Repeated passages of the bacteria on agar media resulted in the loss of virulence in guinea pigs. Virulence, however, was restored by cultivating the avirulent bacteria in cell cultures of human embryonic lung fibroblasts. Death of the host animals was the result of infection; no lethal toxin was detected in the cultural filtrate. These findings indicate that the virulent form ofL. pneumophilia is capable of surviving inside the host cells either through its endogenous resistance to environmental factors within the host cells or by host cell selection. Intracellular multiplication of the virulent bacteria followed by destruction of host cells appears to be an important pathogenic mechanism of Legionnaires' disease.     

Suppression of lordosis in guinea pigs by ethamoxy-triphetol (MER-25) given at long intervals (34-46 hr) after estradiol benzoate treatment

Ovariectomized guinea pigs were given estradiol benzoate (EB) followed 40 hr later by progesterone (P). Behavioral testing commenced 1 hr after P injection and continued at hourly intervals for 8 hr. This treatment activated lordosis in almost 100% of animals. Administration of the antiestrogen MER-25 (75 mg/kg body wt per injection) between 2 hr before and 6 hr after EB treatment did not cause a significant decline in proportion of animals displaying lordosis, but did cause a decrease in length of time the lordosis position was held (maximum lordosis, sec). In contrast, $\text{13}\text{14}$ animals given MER-25 at 2 hr before and 2 hr after P and $\text{8}\text{10}$ animals given MER-25 simultaneously with and 2 hr after P, failed to show lordosis. Administration of supplementary EB at around the time of P injection, partially alleviated these behavior-blocking effects of MER-25. When MER-25 was given 2–6 hr after administration of P there was a significant decrease in duration of heat (hr). These results suggest that in addition to its early “triggering” effects, estrogen has important “maintenance” effects which determine the character of heat in guinea pigs. Continued presence of estrogen in the nervous system may be a requirement for the facilitatory actions of P on sexual behavior in guinea pigs, but such a requirement may not exist in other rodents such as rats.     

Effects of age and starvation on the gastrointestinal microflora and the heat resistance of fecal bacteria in rats.

The microflora of the gastrointestinal tract in rats 1 day to 100 weeks old, of the cecal contents and wall in starved rats, and of heat-treated feces of normal rats was determined by cultural examination. Streptococci, staphylococci, lactobacilli, actinobacilli, and coliforms colonized the tract during the 1st week of life. Bacteroidaceae, veillonellae, catenabacteria (composed of eubacteria and anaerobic lactobacilli), clostridia, bifidobacteria, anaerobic gram-positive cocci, fusiform bacteria, curved rods, and spirochetes appeared when the rats were 2 to 4 weeks old. Yeasts were slower in colonizing the tract than any other organism. Dramatic changes occurred in the microflora of rats 2 to 4 weeks of age. There was a time lag between the changes in enterococcal and coliform populations. The enterococcal population was depressed over a period from 2 to 6 weeks of age. Bifidobacteria showed a larger population at 4 to 9 weeks than at any other age. The microflora of the stomach was the same as that of the small intestine, with some exceptions. It differed markedly from that of the cecum. The ratio of total aerobic count to anaerobic count gradually increased in the stomach, but decreased in the cecum, with advance in age. The microorganisms distributed in the tract could be divided roughly into 3 types. The population of each organism, except spirochetes, in the cecal wall was approximately 1/1,000 of that in the cecal contents. One of the 2 types of spirochetes was found only in the cecal wall and in a high incidence, forming a large population. In rats starved for 48 hr, coliforms, Proteus spp., anaerobic gram-positive cocci, Clostridia, and some bacteroidaceae showed an increase in population in the cecum, but lactobacilli, veillonellae, and spirochetes decreased. The major organisms cultured from the heat-treated feces were fusiform and curved bacteria, some members of Bacillus, minor anaerobic cocci, and straight rods.