Dietary carbohydrate type and cholesterol-induced hypercholesterolemia in cynomolgus monkeys: influence of oral antibiotic.

The relationship of carbohydrate type to cholesterol-induced hypercholesterolemia and the potential role of intestinal flora in the above process were examined in 12 male cynomolgus monkeys (M. fascicularis). Semipurified diets provided two types of carbohydrates (starch or sucrose, 49% by calorie) with 0.4 mg cholesterol/kcal. Six weeks of the starch diet resulted in significantly enhanced hypercholesterolemia when compared to sucrose diet. Starch in relation to sucrose produced cholesterol enrichment of intermediate density lipoproteins and increase in low density lipoprotein particles, whereas sucrose increased high density lipoprotein constituents (phospholipids, cholesterol, and apoA-I) and triglyceride content of very low density lipoproteins. Fecal Escherichia counts were high during the starch diet as contrasted with sucrose diet, but the Escherichia, Streptococcus, and Bacteroides groups did not show differences by diet following each consecutive 4-week period of oral neomycin (107 mg/kg body wt) treatment and withdrawal. The magnitude of hypercholesterolemia during these periods also remained similar between starch and sucrose, suggesting formation of germfree metabolic characteristics. Thus, the magnitude of cholesterol-induced hypercholesterolemia can be affected by the type of carbohydrate, which may be in part determined by intestinal flora metabolism.

Sustained hypermetabolic sepsis in rats: characterization of the model.

This paper describes the development of a sustained sepsis model, using chronically catheterized conscious unrestrained rats, which simulates the progression of septicemia in man, including a sustained hypermetabolic phase. Following chronic arterial catheterization, sepsis was induced in rats by intraperitoneal administration of 0.5 ml of a pooled fecal inoculum. The pooled inoculum, which was used to ensure a uniform inoculation of microorganisms to all animals, produced a septicemia which was progressively lethal. The resultant peritonitis was characterized as polymicrobial, with gram-negative bacteria being continuously present in both peritoneal fluid and blood. Septic animals were normotensive but tachycardic, compared to time-matched controls, throughout the observation period. In contrast to the stable colonic temperature of control animals, septic rats showed a significant febrile response on the first 3 days following inoculation. The hypermetabolic response in septic rats was also manifested by a 25, 38, and 28% increase in oxygen consumption on Days 1, 2, and 3 postinoculation. Animals responded to sepsis with a fall in blood glucose (on Day 2) which remained 15--20% below control levels. Mild hyperlactacidemia (2 mM) and reduced alanine concentrations (14--33%) were also seen in the septic group on Days 2 through 5. Despite the increased lactate levels, septic animals were mildly alkalotic (pH 7.51) which probably reflected the increased (32%) respiratory rate. Light microscopic findings in the septic animals revealed a spectrum of morphologic lesions including an extensive fibrinopurulent exudate, bacterial colonies, and abscesses, which involved most of the abdominal viscera. This investigation characterizes an experimental model of sustained sepsis in rats which exhibits hemodynamics, metabolic and pathologic alterations similar to those seen in human peritonitis.

Partial reversal by sodium ascorbate of hyperoxia-induced damage to HEp-2 cell cultures.

Hyperoxia induced cellular damage was used as an experimental model system for examining the ameliorative role of antioxidants. Multiplication of HEp-2 cells in monolayer culture was inhibited after exposure to 100% O2 either hyperbarically at 3 atm absolute (atma) or normobarically at 1 atma for periods from 15 s to 4 h. The inhibition was characterized by a slower rate of replication for a period from 1 to 3 d after exposure than in unexposed cultures, and then massive cellular death. Less killing followed exposure to normobaric O2 than to hyperbaric O2, and the shorter the period of exposure to hyperoxia the less killing. Addition of 100 micrograms/ml of sodium L-ascorbate to unexposed cultures enhanced growth (cell number at 6 d) almost twofold. When added ascorbate was present only during hyperoxic exposure (but not afterward), subsequent growth in air was enhanced 1.6-fold. However, when cells were exposed without added ascorbate, there was from 2 to 12-fold greater growth in air in the presence of the added ascorbate (as compared to exposed controls). This greater growth was always only a partial reversal of the lethal effect resulting from hyperoxia. Addition of 25 micrograms/ml catalase did not affect control or exposed cultures. Addition of ascorbate plus catalase was not as effective as ascorbate alone in promoting growth; the catalase moiety antagonized some of the growth enhancing influence of ascorbate. This suggests that extracellular H2O2 was not a factor in the lethal effect resulting from hyperoxia.

Emergency autotransfusion: partial cleansing of bacteria-laden blood by cell washing.

Intraoperative autotransfusions for selected patients can ease demands on blood banks. In 33 cases (trauma, elective vascular, and cardiac procedures), we saved 80.7 L of blood with a cell washer blood recovery system (Cell Saver). In eight trauma cases, including three with intestinal perforation, cell washing salvaged 31.5 L of shed blood as a lifesaving emergency procedure. This stimulated interest in defining the limits of the instrumentation in cleansing blood of bacteria. In 18 in vitro experiments, discarded banked blood was inoculated with Escherichia coli to simulate light, moderate, or heavy bacterial contamination. Volumes of blood (500 ml) containing a total of 6.3 X 10(5), 4.8 X 10(7), or 3.2 X 10(10) bacteria were processed. The original blood, filtered blood, plasma supernate, effluent wash solution, and final washed packed red cells were cultured quantitatively in each experiment. The mean total number of E. coli retained in the final washed packed red cells was 1.4 X 10(5), 6.3 X 10(6), or 1.6 X 10(9) bacteria, or 23%, 13%, and 5% of each original inoculum. Additional washing with 10 L of saline did not remove significantly more bacteria (p greater than 0.20). Although emergency autotransfusion of blood contaminated with intestinal contents was lifesaving, we recommend caution since these results show that cell washing does not remove all bacteria. Further laboratory and clinical studies are needed to determine the levels of bacterial contamination of autotransfused blood that can be tolerated, and to determine adjunctive means of rendering contaminated or potentially contaminated blood safe for autotransfusion.

Sepsis-induced depression of phagocytosis in experimental canine peritonitis.

Experimental canine peritonitis was produced in 14 dogs by appendiceal ligation. Phagocytic activity in blood and peritoneal fluid was examined during the peritonitis and following surgical intervention. In nine dogs, the gangrenous appendix was resected after 43 hours, the peritoneal cavity was irrigated, and fibrinous exudate debrided. The remaining five dogs were not tested. Leukocytes in blood and peritoneal fluid were counted preligation and at 24, 43, 46, and 55 hours postligation. Polymorphonuclear leukocytes (PMNs) predominated. Phagocytosis and killing of Candida tropicalis by blood and peritoneal PMNs were assayed by a coverslip method. As sepsis progressed, phagocytosis by blood PMNs declined and at 43 hours was 60 per cent of the preoperative level. After resection of the gangrenous appendix, phagocytic activity returned to 90 per cent of preoperative levels at 12 hours postresection. Peritoneal PMNs exhibited a similar, but more depressed pattern of phagocytic activity. Phagocytosis in five sham-operated dogs was unchanged. Thus, phagocytosis by blood and peritoneal PMNs was depressed in peritonitis and was restored following surgical treatment.

Intraperitoneal povidone-iodine in experimental canine and murine peritonitis.

In dogs with appendicitis-peritonitis, intraperitoneal povidone-iodine caused death more rapidly than the instillation of saline solution. The bacterial content of canine peritoneal fluid increased with time. Although fewer bacteria were found in fluid from povidone-iodine-treated dogs, the differences were not statistically significant. Qualitative chemical analysis of peritoneal fluid revealed iodide, but not free iodine, 15 to 30 minutes after instillation of povidone-iodine. Iodine was present in the peritoneum at 2 hours but not at 3 or 6 hours. The antibacterial effect of povidone-iodine was demonstrated in mice challenged intraperitoneally with lethal doses of Escherichia coli. Povidone-iodine diminished mortality when injected immediately (p less than 0.005) but not when given 1 to 3 hours later. Immediate injection of povidone-iodine into mice lowered the number of E. coli by 3 logs. Injection of povidone-iodine 3 hours after bacterial challenge lowered the number of E. coli by only 1/3 log. This lesser bactericidal effect in mice is attributed to greater dispersal and sequestration of bacteria throughout the peritoneal cavity with time and with inactivation of povidone-iodine by reduction to iodide in vivo. In dogs with appendicitis-peritonitis, the more rapid death after treatment with povidone-iodine was not associated with differences in peritoneal microflora but with peritoneal absorption of excessive amounts of iodide.

Comparison between moist swab and tissue biopsy methods for quantitation of bacteria in experimental incisional wounds.

The relationship between tissue-associated and wound fluid-associated levels of bacteria in closed, healing experimental incisional wounds seeded with Escherichia coli was examined in 200 rats. Tissue specimens and moist swab specimens were taken simultaneously and cultured by a single plate serial dilution method. Colony counts of approximately 10(5) bacteria/gm of tissue were found to be equivalent to colony counts of 10(3) bacteria/ml of specimen obtained on a moist swab. Moist swab sampling of an incisional wound therefore offers a direct and simple method of ascertaining infection (defined by others as greater than 10(5) bacteria/gm of tissue). Moist swab sampling obviates the necessity of surgical manipulation of the patient and subsequent weighing and grinding of tissue in the laboratory.

Stability of human fecal flora.

Recent experimental studies show that neither increased dietary fiber nor its absence alters the numbers and major groups of fecal bacteria. Although the total number of bacteria per gram of feces remains constant, the daily fecal mass doubles with added dietary fiber and is halved in its absence. Thus, the total output of fecal bacteria is related to dietary fiber.

Quantitative cidal activity of hyperbaric oxygen for opportunistic yeast pathogens.

Cidal activities of 24-h exposures to 100% O2 and 95% O2 + 5% CO2 were assayed at 1 and 3 ATA. Studied were 21 yeasts isolated from humans: Candida albicans (8 strains), C. tropicalis (3 strains), C. krusei (3 strains), C. parapsilosis (2 strains), C. guilliermondii (2 strains), and one strain each of C. pseudotropicalis, C. stellatoidea, and Torulopsis sp. Generally, these were extremely sensitive to hyperbaric oxygen, although species and strain differences were observed. Indices of kill from 80-100 (total kill) characterized 17 of the 21 yeasts (81%). Hyperoxia (O2 +/- CO2 at 1 ATA) was not lethal. Deprivation of CO2 as a consequence of hyperbaric exposure to 100% O2 enhanced cidal activity for only 2 of 21 yeasts, whereas hyperbaric exposure to the mixture enhanced activity against four yeasts. Cidal activities were not significantly different for the remaining 15 yeasts. This response to deprivation of CO2 is different from that of bacteria, and manifests fundamental differences between procaryotic and eucaryotic cells.

Microbial flora of the diseased stomach at resection.

The relation of microbial flora in stomach contents and stomach wall was examined in paired specimens from 10 surgical patients and in specimens of wall from 10 additional patients. The flora of both contents and wall were similar. Paired specimens from five patients contained the same kinds of bacteria. Paired specimens from these patients were sterile. Contents from two patients contained bacteria, but the wall was sterile. Microorganisms isolated were streptococci, lactobacilli, bifidobacteria, bacteroides, staphylococci yeast, and coliforms. Bacterial counts ranged from 0 to 10(7.5) per ml or g in both contents and tissue. Survey of the literature shows that most specimens from patients with gastric ulcers or gastric malignancies are positive for bacteria, while only about 60% of specimens from patients with duodenal ulcers are positive.

Evaluation of wound irrigation by pulsatile jet and conventional methods.

Irrigation of wounds to remove bacteria and foreign material is an essential of wound management along with debridement. The effectiveness of saline lavage by high pressure (50 psi) pulsatile jet irrigation has been compared with conventional gravity flow and bulb syringe procedures. Experimental paravertebral incisional surface wounds in 234 randomized rats were either clean or traumatized and soiled. Wounds in 200 of the rats were seeded with E. coli (log 8.80). Swab specimens of each wound were taken at incision, after seeding, after irrigation, and at three, seven, and ten days after closure. Eulates of more than 1600 specimens were cultured. No anaerobes were found. Irrigation diminished bacterial counts in all wounds, but only pulsatile jet irrigation brought about significant (P less than 0.05) reduction of bacteria in each type of wound. After three days E. coli was significantly diminished in all wounds, regardless of irrigation or none, owing to host defense mechanisms. Nevertheless, clean contaminated wounds were infected at three days but not at seven days after lavage, while traumatized wounds remained infected at ten days except for those initially irrigated by pulsatile jet. Thus, pulsatile jet irrigation removed bacterial from experimental wounds more efficiently than conventional procedures.

Intracolonic tensions of oxygen and carbon dioxide in germfree, conventional, and gnotobiotic rats.

The relation of intracolonic gaseous tension to fecal microflora was investigated by mass spectrometric measurements of intracolonic O2 and CO2 in unanesthetized germfree, conventional, and gnotobiotic rats; ip measurements were obtained in rats whose colons became perforated accidentally; fecal bacterial flora and cecal size were also determined. Gnotobiotes were monoassociated with Escherichia coli, Bacteroides fragilis and Staphylococcus epidermidis, and were diassociated with E. coli plus B. fragilis and with Bacillus macerans plus an aerobic diphtheroid. Mean intracolonic Po2 in conventional rats (11.1 mmHg) was significantly lower than in germfree rats (12.8 mmHg); mean intracolonic Pco2 in conventionals (83.4 mmHg) was greater than in germfree rats (54.6 mmHg). Differences of Po2 values among all rats were slight. However, intracolonic Pco2 values were directly related to CO2 production by the normal intestinal flora, and were thus significantly lower in both germfree and gnotobiotic rats. Intraperitoneal tensions were independent of the status of rats, and the mean ip Po2 and Pco2 (38.9 and 49.1 mmHg, respectively) agreed with values in the literature. The enlarged cecum, characteristic of germfree rats, was also present in gnotobiotic rats. Counts of each bacterial species in feces of monoassociated rats were 10(8) to 10(10)/g; counts were progressively smaller in feces of diassociated rats and conventional rats (10(8) to 10(9)/g and 10(5) to 10(8)/g, respectively). Intracolonic gaseous tensions of CO2 clearly reflected the presence of a normal flora inconventional rats, and were inversely proportional to cecal size.

Inhibition of pathogenic enteric bacteria by hyperbaric oxygen: enhanced antibacterial activity in the absence of carbon dioxide.

The antibacterial effects of 24-h exposures to high-pressure oxygen in relation to environmental CO(2) were studied at 3 atm absolute (ata) and at 1 ata. Eight gram-negative, aerobic and facultatively aerobic, pathogenic enteric bacteria (Salmonella typhosa, Salmonella paratyphi, Salmonella schottmuelleri, Shigella dysenteriae, Shigella flexneri, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli) were exposed as shallow-broth cultures and agar surface cultures. Although broths supplemented with 0.2% glucose permitted some growth of Salmonella typhosa, Salmonella schottmuelleri, Shigella dysenteriae, and Shigella flexneri during exposure to high-pressure oxygen in the presence of CO(2), the other species grew only after the exposure, indicating a bacteriostatic effect. Both bacteriostatic and bactericidal effects were demonstrated on the surface of Trypticase soy agar, where killing of Salmonellea typhosa, Proteus vulgaris, and Pseudomonas aeruginosa was significantly greater after exposure to pure O(2) at 3 ata than at 1 ata. At 3 ata, significantly more killing occurred upon exposure of all species (except Shigella dysenteriae and S. flexneri) on an agar surface to 100% O(2) as compared with exposure to a mixture of 95% O(2) + 5% CO(2). Thus, deprivation of CO(2) during exposure to pure O(2) enhanced the bactericidal effect of high-pressure oxygen.

Stability of normal human fecal flora during a chemically defined, low residue liquid diet.

Ten adult male volunteers (medical students) subsisted for seven days upon a chemically defined, low residue liquid diet, and consumed 1200-1800 calories per day. All stools were collected; three were cultured within the hour-a prediet stool, one collected on the seventh day, and a postdiet stool. Specimens were diluted anaerobically, and anaerobic cultures were streaked upon plates of prereduced agar media and incubated in Brewer jars. During the low residue diet, total fecal mass was relatively small and each subject passed only two or three stools. The mean reduction in daily fecal output was 70%. Mean counts of total aerobes were 10-7/gm throughout the study, and mean counts of total anaerobes were 10-10/gm. There was no overgrowth by opportunistic bacteria or fungi. The low residue food did not alter fecal flora; there was neither disappearance nor reduction of any bacterial group.

Development of tetracycline-resistant Staphylococcus aureus in gnotobiotic mice.

Germ-free mice were colonized with a pigmented, tetracycline-sensitive strain of Staphylococcus aureus and maintained in flexible plastic isolators. Treatment of the gnotobiotic mice with oral tetracycline (20 mg/ml) resulted in the development of staphylococci resistant to tetracycline (5 mug/ml or higher). Resistant staphylococci did not appear in feces until several days after exposure of mice to the antibiotic and persisted for as long as specimens were collected (64 days subsequently). Resistance developed after a single exposure of gnotobiotes to antibiotic. Resistant staphylococci were present in the intestinal tracts of mice at counts of 10(3) per g of contents, whereas sensitive organisms coexisted at counts of 10(5) to 10(11) per g. Resistant staphylococci were isolated only from treated mice and not from untreated mice in adjacent cages. Initial colonization of germ-free mice with sensitive staphylococci interfered with subsequent colonization by resistant staphylococci and provided an example of bacterial interference. Resistance to tetracycline was not associated with resistance to chloramphenicol, penicillin, ampicillin, erythromycin, streptomycin, or kanamycin. Hydrolysis of gelatin was the only biochemical characteristic in which isolates varied but was not correlated with resistance to tetracycline or pigmentation of colonies. A nonpigmented, gray variant of S. aureus appeared in all specimens after colonization with the original, pigmented strain. Only the nonpigmented strain was obtained from gnotobiotes colonized with the nonpigmented variant. Contact between bacteria and antibiotic in the intestinal tract of gnotobiotes was considered to be essential for the development of tetracycline-resistant staphylococci.