Effect of dissolved oxygen and Eh and Bacteroides fragilis during continuous culture.

Bacteroides fragilis subsp. fragilis was maintained in a chemostat modified for anaerobic conditions to test the effects of dissolved oxygen and Eh on growth. Using a defined medium containing glucose and a dilution rate of 0.16 h -1, a stable population of 3 X 10(9) colony-forming units/ml was present. At this steady state, the pH was 5.6, the Eh was -50 mV, and the dissolved oxygen concentration was 0% atmospheric saturation. The Eh was then adjusted to +300 mV by adding potassium ferricyanide while oxygen was excluded; in this system there were no demonstrable changes from the steady state in viable cells, pH, glucose concentration, or volatile fatty acid production. In other experiments oxygen was introduced into the original steady state at a dissolved oxygen concentration of 10% atmospheric saturation for a period of 6 to 8 h. During O2 exposure, the viable cell count decreased at a rate comparable to the theoretical washout rate for a static bacterial culture. Similar results were obtained with a dissolved oxygen concentration of 25 and 100%. Other effects of O2 exposure included an increase in Eh from -50 to +250 mV, a decrease in glucose consumption, and a decrease in volatile fatty acid production. These results suggest that dissolved oxygen has a bacteriostatic effect on B. fragilis in continuous culture, which may be independent of changes in Eh alone.     

Anaerobe/aerobe environmental flux determines protein expression profiles of Bacteroides fragilis, a redox pathogen

The oxidation-reduction (redox) of the environment characterizes the Bacteroides fragilis pathogenic potential. Previously, using 3D confocal laser scanning microscopy, the bacteria prepared from cultures grown under oxidizing conditions (Eh(7)ca. + 100 mV) were able to penetrate into Hela cell monolayers. In contrast, when grown under reducing conditions (Eh(7)ca. - 60 mV), there were no bacteria evident within Hela cells. The influence of the anaerobe/aerobe environmental flux during the process of the anaerobe infection could be significant. In B. fragilis peritonitis, this may depend on the occurrence of aerobiosis as opposed to anaerobiosis. To this end, three clinical B. fragilis strains, two infectious and one non-infectious, were grown under oxidizing and reducing conditions; then, the outer membrane protein expressions derived from these strains were assessed, following sarcosyl extraction and SDS-PAGE. The differences between the protein profiles from these strains when cultured under oxidizing and reducing conditions were found to be statistically significant for the two infectious strains, but not for the non-infectious strain. OMP profiles under aerobic conditions compared to anaerobic conditions exhibited products with a range of apparent molecular weights suggestive of unique participation in the interaction with the host cell.     

The redox potential, rH and pH values in the gastrointestinal tract of small ruminants

Oxidation-reduction potentials (Eh), pH and rH in the gastrointestinal tract were measured in six goats and two sheep fed on ground barley and hay, with or without the addition of urea. Each ration was supplied for three weeks. The animals were slaughtered after morning feeding, the contents of relevant parts of the gastrointestinal tract were sampled, pH and Eh values were measured and rH values calculated. The range of the oxidation-reduction potential was rather extensive, from -300 to +186 mV. The variability of rH values was smaller, being between 4.6 and 12.9, except for three values. The following linear relationship holds for findings of Eh and pH: Eh (mV) = 292-69.9 pH with a correlation coefficient of r = 0.84. In those parts of the gastrointestinal tract, where the fermentation process occurs (the rumen, caecum and colon), the Eh and rH values are lower than those in the abomasum and duodenum. Urea addition has no effect on the oxidation-reduction equilibrium.     

Comparison of various atmospheric conditions for isolation and subcultivation of Mycoplasma hyorhinis from cell cultures

The efficiency of aerobic incubation was compared with incubation under various oxygen and carbon dioxide conditions for the isolation and subcultivation of three strains of Mycoplasma hyorhinis from VERO-cell cultures and subcultivation of three laboratory strains. Under anaerobic conditions with a low oxidation-reduction potential (at or below -115 mV) as obtained in jars, with catalysts, containing mixtures of 5%-10% CO2 in H2, very poor or no growth of any of the six M. hyorhinis strains was observed. When traces of oxygen were present (that is, under conditions with higher oxidation-reduction potentials, e.g. when omitting the catalyst in the above gas mixtures or in 5% CO2 + 95% N2) isolation from cell cultures was successful in most tests, but subcultivation of these primary isolates was seldom possible under these semi-anaerobic conditions. However, in most cases these primary isolates could be subcultivated aerobically, although aerobic conditions were unsatisfactory for isolation in about half of the experiments. Isolation of M. hyorhinis was optimal in 5% O2 + 95% N2, under which condition the isolates could also always be subcultivated. Isolation failed occasionally when 5% O2 + 5% CO2 + 90% N2 was used, thus indicating that 5% CO2 was slightly inhibitory. 5% CO2 in air and 10% CO2 either in air, H2 or N2 were also inadequate for isolation from cell cultures. In contrast to the findings with these cell culture-adapted M. hyorhinis strains, the laboratory strains could be subcultivated easily under all conditions tested except those with an oxidation-reduction potential at or below -115 mV; 100% CO2 was inhibitory for all 6 strains. Our findings may partly explain why M. hyorhinis is often considered "non-cultivable" on artificial media once adapted to cell cultures. The findings emphasize the need to employ also a micro-aerophilic condition (5% O2 in 95% N2) in the examination of cell cultures for mycoplasma.     

Application of a direct fluorescence-based live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water

To evaluate the viability and survival ability of fecal Bacteroides spp. in environmental waters, a fluorescence-based live/dead staining method using ViaGram Red+ Bacterial gram stain and viability kit was combined with fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probe (referred as LDS-FISH). The proposed LDS-FISH was a direct and reliable method to detect fecal Bacteroides cells and their viability at single-cell level in complex microbial communities. The pure culture of Bacteroides fragilis and whole human feces were dispersed in aerobic drinking water and incubated at different water temperatures (4 degrees C, 13 degrees C, 18 degrees C, and 24 degrees C), and then the viability of B. fragilis and fecal Bacteroides spp. were determined by applying the LDS-FISH. The results revealed that temperature and the presence of oxygen have significant effects on the survival ability. Increasing the temperature resulted in a rapid decrease in the viability of both pure cultured B. fragilis cells and fecal Bacteroides spp. The live pure cultured B. fragilis cells could be found at the level of detection in drinking water for 48 h of incubation at 24 degrees C, whereas live fecal Bacteroides spp. could be detected for only 4 h of incubation at 24 degrees C. The proposed LDS-FISH method should provide useful quantitative information on the presence and viability of Bacteroides spp., a potential alternative fecal indicator, in environmental waters.     

The Effect of Oxidation-Reduction Potential on Spore Germination,Outgrowth, and Vegetative Growth of Clostridium tetani,Clostridium butyricum,and Bacillus subtilis

Spores of Clostridium tetani germinated in liver broth with +580 mV as the starting Eh value, and those of Clostridium butyricum germinated in liver broth with an initial Eh of +400 mV regardless of the presence or absence of a liquid paraffin covering. Spores of Bacillus subtilis germinated in liver broth with ?100 mV as the starting Eh value. Also, it was found that there are two ranges of starting Eh values for germination and vegetative growth of Cl. tetani, Cl. butyricum, and B. subtilis. In the first range these spores germinated and grew, but in the second range they only germinated and then died without outgrowth.     

Activation of a procarcinogen to a mutagen by cell-free extracts of anaerobic bacteria.

The Salmonella mutagenicity assay can be coupled to cell-free preparations derived from anaerobic bacteria (Clostridium perfringens and Bacteroides fragilis) to activate a procarcinogen to a mutagen. This activity is destroyed by heating and by digestion with pronase and it is sensitive to oxygen. These findings indicate that the Salmonella mutagenicity assay can be adapted to the study of the role of anaerobes in the activation of carcinogens.     

The response of Leuconostoc mesenteroides to low external oxidoreduction potential generated by hydrogen gas

AIMS: The physiological consequences of low external oxidoreduction potential in Leuconostoc mesenteroides were investigated. METHODS AND RESULTS: Leuconostoc mesenteroides was grown under two initial oxidoreduction potential conditions (Eh7: +200 mV and -400 mV) using nitrogen and hydrogen as reducing agents. Growth was affected by Eh7; the lag phase increased from 1 h at an initial Eh7 of +200 mV to 6 h at an initial Eh7 of -400 mV; the maximum specific growth rate at -400 mV was 68% of the one observed at +200 mV. The NADH/NAD+ ratio and (NADH + NAD+) pool were independent of the external Eh7. CONCLUSIONS: This study shows that changing the external oxidoreduction potential from +200 to -400 mV has a strong effect on the Leuc. mesenteroides physiology. The constancy of the maximum carbon and energetic fluxes (qglu, qATP) under the two Eh7 conditions accompanied by the decrease of YX/S and YATP suggested the existence of an uncoupling phenomenon, namely that some catabolized glucose and hence ATP was not associated with biomass production. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper demonstrates the usefulness of taking into account, the effect of the oxidoreduction potential on the growth of Leuc. mesenteroides in the fermentation process.     

Respiratory electron transport and light-induced energy transduction in membranes from the aerobic photosynthetic bacterium Roseobacter denitrificans

Membrane fragments isolated from the aerobic phototrophic bacterium Roseobacter denitrificans were examined. Ninety-five percent of the total NADH-dependent oxidative activity was inhibited either by antimycin A or myxothiazol, two specific inhibitors of the cytochrome bc1 complex, which indicates that the respiratory electron transport chain is linear. In agreement with this finding, light-induced oxygen uptake, an electron transport activity catalyzed by the "alternative quinol oxidase pathway" in membranes of several facultative phototrophic species, was barely detectable in membranes of Rsb. denitrificans. Redox titrations at 561-575 nm, 552-540 nm, and 602-630 nm indicated the presence of three b-type cytochromes (Em,7 of +244 +/- 8, +24 +/- 3, -163 +/- 11 mV), four c-type cytochromes (Em,7 of +280 +/- 10, +210 +/- 5, +125 +/- 8, and 20 +/- 3 mV) and two a-type cytochromes (Em,7 of +335 +/- 15, +218 +/- 18 mV). The latter two a-type hemes were shown to be involved in cytochrome c oxidase activity, which was inhibited by both cyanide (I50 = 2 microM) and azide (I50 = 1 mM), while a soluble cytochrome c (c551, Em,7 = +217 +/- 2 mV) was shown to be the physiological electron carrier connecting the bc1 complex to the cytochrome c oxidase. A comparison of the ATP synthesis generated by continuous light in membranes of Rsb. denitrificans and Rhodobacter capsulatus showed that in both bacterial species photophosphorylation requires a membrane redox poise at the equilibrium (Eh > or = +80 < or = +140 mV), close to the oxidation-reduction potential of the ubiquinone pool. These data, taken together, suggest that, although the photosynthetic apparatus of Rsb. denitrificans is functionally similar to that of typical anoxygenic phototrophs, e.g. Rba. capsulatus, the in vivo requirement of a suitable redox state at the ubiquinone pool level restricts the growth capacity of Rsb. denitrificans to oxic conditions.     

Hymenolepis diminuta: effect on the oxidation-reduction potential in the mammalian gastrointestinal canal.

The oxidation-reduction potential (Eh) of the small intestine of uninfected and parasitized (ten 16-day-old Hymenolepis diminuta) rats has been determined under three different types of experimental conditions. On an ad lib. feeding regime at 10.00 hr there were highly significant (P < 0.001) differences between the intestinal Eh of uninfected and parasitized rats in every region of the small intestine. While the entire uninfected gut displayed relatively strong reducing tendencies (?28 to ?195 mV), in the parasitized gut the Eh was predominantly positive (+75 to ?76 mV) and reflected worm biomass distribution. Changes in intestinal Eh were also measured over a period of 6 hr after the feeding of a 1-g glucose meal. The Eh in uninfected animals increased from ?232 to +118 mV; in parasitized animals from ?31 to +189 mV. While the changes were greater in the former group, those of the latter reflected the changing worm biomass distribution. The most positive regional Eh value was always associated with the maximum % worm biomass distribution. Third, using surgical intestinal-loop preparations there were significantly (P < 0.05) higher positive Eh values in parasitized (+185 mV) than in uninfected (+128 mV) rats; in both animal groups the Eh of the duodenal-jejunal loops were more positive than that of the ileal loops (P < 0.05).The results are discussed in terms of the close inter-relationship between intestinal pH, Eh, the microflora and luminal pO₂ tensions.     

In Vitro Production of Clostridium perfringens Enterotoxin and Its Detection by Reversed Passive Hemagglutination

The reversed passive hemagglutination (RPHA) test yielded a positive reaction in 2 h with as little as 0.5 ng of purified Clostridium perfringens enterotoxin (CPE) per ml as well as with cultures of some C. perfringens grown in Duncan-Strong (DS) medium. This method is the most sensitive, the simplest, and the fastest among all reported. The time course of CPE production of Clostridium perfringens NCTC 8798 in DS was investigated by RPHA. CPE in culture was detectable at 4 h, increased gradually, reached a maximum at 12 to 14 h, and remained at a high level of 20 mug/ml through 48 h of incubation. CPE synthesized within cells is released easily by sonic disruption of young cultures and by aging the cultures 20 h or more. Heat shock of the cell inoculum was essential for CPE production by C. perfringens in DS.     

Effect of redox potential on the growth of Yarrowia lipolytica and the biosynthesis and activity of heterologous hydroperoxide lyase

The aim of this study was to investigate the effect of redox potential (Eh) on the growth of the yeast Yarrowia lipolytica in both oxidizing (Eh = +350 mV) and reducing (Eh = −150 mV) media and its effect on the expression and activity of hydroperoxide lyase (HPL). HPL activity was assayed in media with Eh values ranging from −250 to +720 mV. In order to change the Eh value of the media, reducing agents including dithiotreitol (1 g/L) and hydrogen (4%) as well as oxidants such as potassium ferricyanide (1 g/L) and oxygen (100%), were used. The experimental findings showed that oxidizing conditions, with Eh of +350 mV, were favorable for the growth of the yeast, whereas reducing conditions, with Eh values of −150 mV, resulted in a higher expression of HPL. In addition, the results showed that the enzymatic activity of the purified HPL was enhanced in the presence of 0.5 mM dithiotreitol but decreased with 1 mM potassium ferricyanide and bubbling O₂. However, HPL activity increased 1.5 times in the presence of 4% hydrogen with an Eh value of −170 mV.     

Inhibition of Clostridium perfringens sporulation by Bacteroides fragilis and short-chain fatty acids

The effect a common fecal organism, Bacteroides fragilis, has on the sporulation of Clostridium perfringens, an organism linked to some cases of antibiotic associated diarrhea, was examined. Established B. fragilis cultures significantly decreased the number of heat resistant spores formed by C. perfringens ATCC 12915 and increased the number of vegetative cells. To determine if short-chain fatty acids (SCFA), fermentation products of B. fragilis, inhibited sporulation, the SCFA were added to sporulation broth. Sporulation decreased in the presence of acetate, isobutyrate, isovalerate, and succinate. Vegetative cell number for C. perfringens decreased in the cultures with isobutyrate. Propionate did not affect sporulation or vegetative cell number. The data support the hypothesis that the decrease in short-chain fatty acid concentration following antibiotic therapy predisposes patients to diarrheas caused by C. perfringens.     

Convenient Non-Chromatographic Assays for the Microbial Deconjugation and 7α-OH Bioconversion of Taurocholate

We described two convenient assay methods to estimate bile acid deconjugation and bile acid bioconversion at the 7alpha-OH position by individual microorganisms grown in media containing taurocholic acid. The methods are based on (i) a selective chemical assay for taurine conjugates previously described and (ii) the use of a cell-free preparation of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli to directly quantify 7alpha-OH groups. These non-chromatographic approaches have been applied to the study of three model strains of intestinal organisms, E. coli, Bacteroides fragilis, and Clostridium perfringens, grown in standard media in the presence of purified tritiated taurocholate. Assay results were confirmed by thin-layer chromatography solvent systems designed to separate conjugated from unconjugated bile acid and unmodified cholic acid nucleus from 7alpha-OH bioconversion product(s) (primarily 3alpha, 12alpha dihydroxy, 7-keto-cholanoic acid). In addition, 7alpha-hydroxysteroid dehydrogenase activity was demonstrated in cell-free extracts of all three organisms. Of the three organisms, only C. perfringens was demonstrated to (i) deconjugate taurocholic acid, (ii) contain 3alpha-hydroxysteroid dehydrogenase activity, (iii) convert cholic acid into at least five labeled metabolites visible on thin-layer chromatography, and (iv) catalyze significant tritium exchange with water in the medium.     

Response of Clostridium perfringens and its L form to bacteriocins of C. perfringens

Clostridium perfringens strain No. 28 and its penicillin-induced stable L form were treated with 10 different bacteriocins of C. perfringens. Viable count and labelled amino acid incorporation experiments revealed that the L form was sensitive to two and possibly three bacteriocins to which the bacillus was not, while both forms were commonly sensitive to two other bacteriocins and resistant to five others. Adsorption of bacteriocin, immunity factors, or perhaps uptake of bacteriocin might be proposed to explain the responses of these organisms to bacteriocins.     

Extracellular Deoxyribonuclease Production by Anaerobic Bacteria

The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common. All strains of Fusobacterium, including eight species, as well as Bacteroides fragilis and B. melaninogenicus, displayed enzyme activity. Whereas the gram-positive bacteria were generally less productive, all strains of Clostridium perfringens, Peptostreptococcus intermedius, and P. anaerobius specifically produced deoxyribonuclease. The test is taxonomically valuable, particularly in the characterization of gram-positive cocci, since a deoxyribonuclease-producing coccus indicates P. intermedius or P. anaerobius. Additionally, possession of the enzyme may prove to be a useful correlate of the potential pathogenicity of anaerobes.     

Benzylpenicillin-induced filament formation of Clostridium perfringens

Growth of Clostridium perfringens with low concentrations of benzylpenicillin inhibited septum formation and division of the organisms. This resulted in continued growth of the organisms as aseptate filaments. The effect was reversed on removal of the antibiotic. The composition of walls isolated from organisms grown with the antibiotic was similar to that of walls from untreated bacteria. In addition, both contained non-N-acetylated glucosamine residues in their peptidoglycan. No differences were detected in the degree of cross-linkage of peptidoglycan. Clostridium perfringens contains six membrane-associated penicillin-binding proteins (PBPs) which have different affinities for [3H]benzylpenicillin. Concentrations of the antibiotic which were sufficient to cause filamentation of apparently all organisms in a culture caused almost complete saturation of PBPs 3, 4, 5 and 6. At these concentrations there was no measurable interaction with PBPs 1 and 2. Thus interaction of the antibiotic with the lower molecular weight PBPs is correlated with the inhibition of septum formation in C. perfringens.     

Phosphatase Activity of Anaerobic Organisms

Anaerobic organisms were tested for phosphatase activity in different pH ranges. Several groups of organisms displayed characteristic patterns. Bacteroides fragilis, B. melaninogenicus, and B. ruminicola produced phosphatase with strongest activity at pH 8.6. Fusobacterium mortiferum was the only species of this genus to show strong hydrolysis. The enzyme was active in both acid and alkaline ranges. The activity of gram-positive organisms was variable, the most active groups being Clostridium perfringens, Peptostreptococcus intermedius, P. micros, and Peptococcus constellatus. The incorporation of phosphatase activity into the identification scheme of anaerobes seems feasible. There was a correlation of hydrolysis with several important pathogens.     

Factors involved in the transformation of previously non-transformable Clostridium perfringens type B

Abstract The pre-shock incubation of cells plus DNA and the methylation state of plasmid DNA were found to play a role in the electroporation-based transformation of Clostridium perfringens 3626B. Following pre-shock incubation, the highest number of C. perfringens 3626B transformants was obtained when plasmid pGK201 was both dam⁺ dcm⁺ modified, while no transformants were obtained when pGK201 was not methylated or only dcm methylated. This is consistent with the observation that plasmid pGK201 was protected against digestion by C. perfringens 3626B cell-associated nucleases for up to 3 min when methylated by both methylases. C. perfringens 3626B was successfully transformed only within a narrow cell recovery rate window. The erm AM gene associated with pGK201 and pAK102 was found to integrate into the chromosome of C. perfringens strains 13A and 3626B.     

The size pH, and redox potential of the cecum in mice associated with various microbial floras.

Cecal size and in situ redox potential and pH of cecal contents were determined in conventionally reared mice and mice reared under a variety of gnotobiotic conditions: germfree, monoassociated with a cecal Clostridium sp., hexaflora-associated and thermoduric polyflora-associated. The mean Eh was approximately +200 mV in germfree and -200 mV in conventional mice. The Eh was close to zero in the monoassociated mice, thus occupying a position intermediate between the germfree and conventional mice. The potentials observed in the hexaflora and the thermoduric flora groups were indistinguishable from those of conventional animals. The degree of normalization was more advanced with respect to the redox potential than to the cecal size in the various gnotobiotic groups. In the thermoduric polyflora-associated group, normalization was observed in both cecal size and redox potential. This demonstrates that normalization can be accomplished with a relatively simplified microflora, at least with regard to the parameters studied.