Safety issues associated with processing soybeans in an enclosed habitat intended for long-duration space missions.

Soybeans have been selected to be grown in a habitat (BIO-Plex, Bioregenerative Planetary Life Support Systems Test Complex) designed to evaluate advanced life support systems for long-duration space missions. Soymilk and soy bread will be incorporated into this nutritious, plant-based food system. Because all consumables will be recycled and reused, food safety is a particular concern. Critical control points were identified to control microbiological hazards, particularly mycotoxins, and chemical hazards from antinutrients and volatiles emitted during processing of soymilk and soy bread. Volatile compounds, evolved during the manufacturing of soymilk and soy bread, were quantified by GC/MS to assess their impact on this closed loop system. All concentrations of volatiles evolved during soymilk production were below the 24-h Space Craft Maximum Allowable Concentration (SMAC), while acetaldehyde surpassed the SMAC criteria for soy bread. Recommendations were made for processing of soybeans in such environments to minimize risk to crew member health.

Safety analysis and hazard control during food processing and storage in the BIO-Plex Interconnecting Transfer Tunnel.

The food system, being designed for the BIO-Plex (Bioregenerative Planetary Life Support Systems Test Complex), will be a plant-based diet that requires most of the food to be grown, processed, and prepared in the BIO-Plex. Conversion of crops to edible foods will require extensive food processing within the closed environment of this habitat. Because all consumables in the BIO-Plex will be recycled and reused, food safety is a primary concern. Multifunctional equipment necessary for food processing of the baseline crops (wheat, soybeans, rice, peanuts, dried beans, potatoes, sweet potatoes, lettuce, chard, tomatoes, green onions, carrots, and radishes) was identified. Recommendations for placement of the food processing equipment in the Interconnecting Transfer Tunnel (ITT) of the BIO-Plex were made to facilitate the processing flow diagrams, increase work efficiency, and prevent cross-contamination of pathogens and antinutrients. Sanitation equipment and procedures necessary during food processing in the ITT are described.

Development of a 10-day cycle menu for Advanced Life Support.

The Advanced Life Support (ALS) program at NASA-Johnson Space Center was initiated for use in long-duration space missions. With weight and volume restrictions and prolonged periods between resupply from Earth, as much as 90% of the energy requirements must come from food grown, processed, and prepared in space. ALS involves the use of hydroponically grown crops to supply and regenerate air and food for the crew. A 10-day cycle menu has been developed consisting of items prepared from the baseline crop list: potato, sweet potato, brown rice, wheat, peanut, soybean, lettuce, tomato, carrot, chard, radish, spinach, green onion, and dry beans (pinto and lentil). Of the recipes created for the menu, resupply items contributed only 4.54% by weight and 9.18% of the total calories. The menu has been analyzed to conform to the baseline crop list and nutrient recommendations for long-duration space missions.

Food safety and sanitation during an extended mission.

In preparation for future Lunar/Mars habitats, a food system is being developed at NASA-JSC to provide Advanced Life Support for long-duration missions. As much as 90% of the food consumed on these missions is expected to be grown, processed, and prepared in space. Conversion of crops to edible foods will require extensive food processing within the closed environment of the Bioregenerative Planetary Life Support System Test Complex (BIO-Plex). Identification of hazards and critical control points associated with water recycling, biomass management, use of multifunctional equipment, and possible concentration of toxic substances in the closed system is essential for the development of safe food processing techniques and equipment. A food safety analysis, using a Hazard Analysis Critical Control Point (HACCP) approach, was conducted to identify potential hazards and critical control points during food processing of BIO-Plex-produced lettuce and wheat.

Soluble plasma antigen in experimental Salmonella typhimurium infection in mice.

To detect and characterize Salmonella antigen in blood, outbred CF-1 female mice were inoculated intraperitoneally with S. typhimurium LT-2 and blood was assayed by ELISA for Salmonella common structural antigen. Plasma antigen was detectable early in the course of infection and increased in quantity later in the course of illness when animals showed high grade bacteremia and high counts of splenic bacteria. Antigen was associated with a cell-free plasma fraction of blood, passed through filters with cut-offs of 0.2 mu and molecular mass of 1000 kDa, and was enhanced in detectability after heating to 100 degrees C for 15 min. Antigen was concentrated by diluting plasma 1:4 in 0.1 M EDTA, heating to 100 degrees C, and concentrating the supernate with an ultrafiltration membrane with a molecular mass cut-off of 15 kDa. By gel filtration, antigen was associated with a peak at about molecular mass 300 kDa in heated plasma and a peak at about 380 kDa in unheated plasma. These results indicate that murine typhoid infection results in circulating soluble plasma antigen, which is heat-stable with a molecular mass of approximately 300 kDa.

The anaerobic microflora of the human body.

An overview is presented of the kinds of anaerobic bacteria that inhibit the surfaces of the human body. The anaerobic floras of the skin, oral cavity, alimentary tract, and genitourinary tract are described. The activities of these organisms that impact on the human host and their interactions are discussed. Emphasis is placed on the protective roles of the floras of the various bodily surfaces in preventing infections. Identified mechanisms of protection on the skin and in the colon and the vagina are explained.

Influence of infant diets on the ecology of the intestinal tract of human flora-associated mice.

The effect of diet on intestinal ecology was studied in germ-free mice that were inoculated orogastrically with predominant intestinal flora components isolated from the feces of breast-fed human infants. The flora components colonized the intestines of mice and persisted at fixed population levels. Groups of flora- associated mice were fed either human milk, bovine milk, whey-dominant formula, or formula modifications exclusively for 2 weeks, and then examined for changes in small intestinal and cecal flora composition, cecal pH, and resistance to intestinal colonization with Salmonella typhimurium. Dietary variations influenced the composition of the flora to a moderate degree but the differences were generally not statistically significant. However, the addition of bovine lactoferrin to the whey-dominant formula resulted in significantly greater counts of Bifidobacterium, Bacteroides, Enterococcus and total aerobes in the small intestine when compared with mice fed unsupplemented formula. Bifidobacterium was present in large numbers in both the ceca and small intestines of mice fed the lactoferrin-supplemented formula. Despite similarities in intestinal flora patterns among mice fed the various diets, human milk consumption resulted in a lower pH of cecal contents and a greater resistance to colonization by Salmonella typhimurium after orogastric challenge than the consumption of the other diets.

Colonization of mice by Campylobacter jejuni.

Both streptomycin-treated and untreated Swiss white mice were irregularly colonized when challenged orogastrically with between 1 and 10(11) viable organisms of either of two strains of Campylobacter jejuni. The organisms were occasionally recovered from portions of the intestinal tracts of these animals in numbers ranging from 10(1) to 10(3)/g when the challenge doses were 10(10) or more. When germfree mice were challenged with 10(8) organisms of either strain, the entire intestinal tracts of all the animals were colonized with C. jejuni in numbers ranging from 10(4) to 10(9)/g. The ceca were most heavily colonized. Both strains of C. jejuni multiplied anaerobically in brucella broth, except when the broth contained 60.80 mu eq of total volatile fatty acids (VFA) per ml at pH 6.75, simulating conditions in the ceca of untreated mice, or when it contained 21.63 mu eq/ml at pH 7.04, simulating conditions in the ceca of streptomycin-treated mice. Active multiplication occurred, however, in brucella broth without VFA at pH 7.02 that was incubated microaerobically, simulating conditions in the ceca of germfree mice. The results suggest that VFA operating under anaerobic conditions present in the intestinal tract of both streptomycin-treated and untreated conventional mice interfere with the multiplication of C. jejuni. The organisms actively multiply, on the other hand, in the absence of VFA at the higher oxidation-reduction potential of the intestinal tract of germfree mice.

Effect of streptomycin administration on association of enteric pathogens with cecal tissue of mice.

The effect of streptomycin on the ability of Shigella sonnei 3SR and enterotoxigenic Escherichia coli 2SR to associate with cecal tissue of mice in vivo and in vitro was examined. After orogastric challenge, both pathogens associated in significantly greater numbers (P less than or equal to 0.05) with the cecal tissue of streptomycin-treated mice than with the tissue of untreated mice. The population levels of the pathogens were also significantly greater (P less than or equal to 0.05) in the cecal contents of streptomycin-treated mice than in untreated mice. When excised cecal tissues from the two groups of mice were exposed to the pathogens in vitro, the extent of the association of the pathogens was markedly greater with tissues from streptomycin-treated mice than with tissues from untreated mice. There was also a positive correlation between the numbers of the pathogens in the suspending fluid and the extent of the tissue associations. The population size of fusiform organisms, which are the major components of the mucus layer of the ceca of mice, was reduced 100-fold by streptomycin administration. This was determined by microscopic count. Sections of cecal tissue obtained from the mice and stained with hematoxylin and eosin demonstrated that streptomycin administration greatly decreased the number of fusiform bacteria present in the mucosal microbial layer. We speculate that the partial elimination of fusiform organisms from this layer by streptomycin administration provides available association sites for pathogens so that they can successfully colonize the mouse cecum.

Impact of LY146032 on Streptococcus (Enterococcus) faecalis translocation in mice.

The susceptibility of Swiss White mice to colonization with Streptococcus (Enterococcus) faecalis was greatly increased when the animals were given 5 mg of streptomycin sulfate per ml in their drinking water. One week after initiation of streptomycin treatment, the mice were challenged orogastrically with graded doses of streptomycin-resistant S. faecalis. The number of S. faecalis cells required to implant the intestinal tract of 50% of untreated mice was 2.9 X 10(9), but was only 4.8 X 10(3) for streptomycin-treated animals. When both groups of mice were challenged orogastrically with 4.6 X 10(6) viable S. faecalis cells, the cecum and small intestine of 100% of the streptomycin-treated animals, but only 10% of the untreated animals, were colonized with the organism. Similarly, translocation of S. faecalis to extraintestinal sites occurred in a majority of streptomycin-treated mice, but in only a small number of untreated mice. Subcutaneous administration of the experimental antibiotic LY146032 (Eli Lilly & Co., Indianapolis, Ind.) to streptomycin-treated mice concomitant with orogastric challenge with 5.5 X 10(5) viable S. faecalis cells resulted in a significant decrease in the incidence of intestinal colonization by the organism, a significant reduction in S. faecalis populations, and the absence of the organism in the liver, spleen, and heart. However, once intestinal colonization had occurred and extraintestinal infections were established, LY146032 did not significantly reduce S. faecalis populations or ameliorate the infections. We conclude that LY146032 effectively prevents translocation of S. faecalis from the intestinal tract of mice but does not resolve established extraintestinal infections.

Factors responsible for increased susceptibility of mice to intestinal colonization after treatment with streptomycin.

Streptomycin sulfate (5 mg/ml) was added to the drinking water of Swiss white mice. After treatment for 1 week, the mice were challenged orogastrically with 10(8) Pseudomonas aeruginosa cells. The organism failed to multiply in the intestinal tract of either treated or untreated animals, but could be recovered from contents and tissues after 48 h. In a previous study, Salmonella typhimurium was shown to multiply in the intestines of streptomycin-treated but not untreated mice when 10(3) organisms were used as inoculum. Streptomycin administration had little effect on Eh, protein or carbohydrate concentrations of cecal contents, or intestinal motility. However, it caused a statistically significant increase in water content and pH of contents and a decrease in the concentrations of acetic, propionic, butyric, and valeric acids. S. typhimurium multiplied in pooled cecal contents obtained from both streptomycin-treated and untreated animals, but its multiplication rate and total populations were significantly greater in contents from treated animals. P. aeruginosa did not multiply in contents from either treated or untreated mice. Similar results were obtained when the organisms were inoculated into nutrient broth adjusted to simulate the pH levels and volatile fatty acid (VFA) concentrations in cecal contents of treated and untreated mice. The addition of brain heart infusion broth to cecal contents from untreated animals, in concentrations that support multiplication of S. typhimurium and P. aeruginosa, did not reverse inhibition. The addition of VFA to cecal contents from treated animals to equal the concentration in cecal contents from untreated animals caused inhibition of a magnitude observed in cecal contents from untreated animals. The results indicate that VFA operating at the pH level of cecal contents of conventional mice inhibit the multiplication of both S. typhimurium and P. aeruginosa and restrict colonization of the intestine by these organisms. The decrease in VFA concentrations that occurs as a result of streptomycin administration adequately explains the increased susceptibility of treated mice to colonization with S. typhimurium. It does not explain the increased susceptibility of treated mice to P. aeruginosa colonization, however.

Effect of streptomycin administration on colonization resistance to Salmonella typhimurium in mice.

The addition of 5 mg of streptomycin sulfate per ml to the drinking water of Swiss white mice resulted in a 100,000-fold reduction in the 50% implantation dose of streptomycin-resistant Salmonella typhimurium for the animals. When streptomycin-treated and untreated mice were challenged orogastrically with 10(3) viable S. typhimurium organisms, 100% of the treated and none of the untreated mice excreted the pathogen in their feces. Similarly, translocation of S. typhimurium from the intestinal tract to the liver, spleen, and mesentery occurred in 10 of 10 treated mice but in none of the untreated mice 7 days after challenge with 10(3) CFU. Studies of colonization dynamics showed that S. typhimurium was present at high population levels in the intestines of streptomycin-treated mice and in detectable levels in the liver, spleen, and mesentery within 72 h after challenge with 10(3), 10(5), or 10(8) organisms. In untreated mice challenged with either 10(3) or 10(5) S. typhimurium organisms, the organisms were isolated from ileal and cecal tissues but not from ileal or cecal contents or from extraintestinal tissue 72 h after challenge. When untreated mice were challenged with 10(8) organisms, however, S. typhimurium was present in all organs and in intestinal contents. Streptomycin treatment, therefore, facilitated colonization and development of streptomycin-resistant S. typhimurium populations in intestines of mice and the subsequent translocation of the organisms from the intestinal tract to other tissues.

Protective role of intestinal flora against infection with Pseudomonas aeruginosa in mice: influence of antibiotics on colonization resistance.

Swiss white mice were given ampicillin, clindamycin, kanamycin, metronidazole, or streptomycin in drinking water for a period of 3 weeks. One week after the initiation of antibiotic administration, the treated mice and untreated control mice were challenged orally with approximately 10(8) viable, streptomycin-resistant (SR) Pseudomonas aeruginosa isolates. All five of the antibiotics decreased the resistance of the mice to intestinal colonization with SR P. aeruginosa, as reflected by an increased fecal carriage of the organism and an increase in population levels of SR P. aeruginosa in feces as compared with untreated controls. Metronidazole was least effective in this regard. The antibiotics lowered the dose of SR P. aeruginosa that resulted in implantation in 50% of the mice ID50 to various degrees. Administration of streptomycin, the most effective antibiotic, caused a 10,000-fold decrease in ID50 as compared with untreated controls. Oral inoculation of approximately 10(8) organisms of SR P. aeruginosa resulted in translocation of the organism to the mesenteric lymph nodes, spleens, or livers of 13 or 17 streptomycin-treated mice, 1 of 20 clindamycin-treated mice, and 1 of 14 metronidazole-treated mice. Translocation was not observed, however, in ampicillin- or kanamycin-treated animals. Antibiotic activity was detected in the cecal contents of streptomycin-, kanamycin, and clindamycin-treated mice but not in the cecal contents of ampicillin- or metronidazole-treated animals.

Hydrolytic enzymes of anaerobic bacteria isolated from human infections.

Thirty-three strains of anaerobic bacteria isolated from human clinical specimens were examined for the presence of heparinase, hyaluronidase, chondroitin sulfatase, gelatinase, collagenase, fibrinolysin, lecithinase, and lipase activities. Pronounced heparinase activity was limited to species of the genus Bacteroides. A number of species of the genera Bacteroides and Clostridium produced hyaluronidase and chondroitin sulfatase. Gelatinase, collagenase, and fibrinolysin activities were encountered in isolates of the genera Bacteroides, Clostridium, and Peptostreptococcus. All strains capable of degrading collagen also hydrolyzed other protein substrates. Lipolytic activity was minimal among these anaerobic bacteria. No specific hydrolytic activity was consistently associated with the isolates.

Chemotaxis by lipids isolated from Bacteroides fragilis.

Living, heat or formalin killed Bacteroides fragilis and a crude preparation of their cell walls were examined by the Boyden technique for chemotactic activity upon guinea pig peritoneal exudate cells. Their relative chemotactic activity ranged from 3.0 to 5.2 compared to an average value of 6.4 for the positive control, an endotoxic culture filtrate of Escherichia coli. A culture filtrate of B. fragilis and an index of 3.7. Miocrogram quantities of cytoplasmic preparations obtained by ammonium sulphate precipitation had chemotactic indices ranging from 2.8 to 6.4, the highest value being displayed by the precipitate formed between 50 and 75% saturation with ammonium sulphate. This fraction retained leucotactic activity after exposure to strong acid and heat. The leucotactic potency of these fractions did not correlate directly with their protein content. Further precipitation of the most active fraction with 80% ethanol revealed that there was little chemotactic activity attributable to polysaccharides. Gas liquid chromatography of a chloroform-methanol extract of the cells which had a chemotactic index of 6.1 revealed the presence of more than thirty fatty acids ranging in carbon length from C8 to C25. These results suggest a role of lipids as initiators of the leucotactic response associated with infections caused by B. fragilis.

The intestinal flora and infant botulism.

The intestinal flora of experimental animals interferes with infection by species of Salmonella and Shigella. Protection against infection with these organisms appears to be related to high concentrations of volatile acids, low pH, and low oxidation-reduction potential of the intestinal contents of animals with an intact flora. There are no data to show that the flora influences colonization of the intestine with clostridial species, but indirect evidence suggests that the intestinal flora may be involved in this process. The impact of the intestinal flora on the ecology of the large intestine may be the most important determinant of resistance to infant botulism.

Antibodies detectable by counterimmunoelectrophoresis against Bacteroides antigens in serum of patients with inflammatory bowel disease.

Heat-extracted antigens from seven species of Bacteroides were used in passive hemagglutination and counterimmunoelectrophoretic tests. Sera from 87 normal persons (group I) and 15 patients with ulcerative colitis (group II) were of low and equal reactivity in passive hemagglutination tests; all positive tests were eliminated by 2-mercaptoethanol reduction of the sera. When these same sera were tested by counterimmunoelectrophoresis with six of the Bacteroides antigens, no significant difference in the percentage of positive reactions was noted. However, using the chi-square test, the seventh antigen, prepared from Bacteroides vulgatus, successfully distinguished the two populations at the 0.025 level. Counterimmunoelectrophoretic tests with the B. vulgatus antigen also provided a means to separate the patients in group II with active disease from those in remission at a P value of 0.01. All the sera from 12 patients with defined Crohn's disease activity indexes reacted with the B. vulgatus antigen in counterimmunoelectrophoretic tests. Reduction and alkylation of patient sera with 2-mercaptoethanol and iodoacetamide removed detectable antibody in 78% of the samples, which suggested a dominant role of immunoglobulin M in the response to Bacteroides antigens.