Effect of Various Concentrations of Brilliant Green and Bile Salts on Salmonellae and Other Microorganisms

A study of the inhibitory effect of 24 different combinations of brilliant green and bile salts concentrations was conducted, using seven species of microorganisms capable of fermenting mannitol. The inhibitory effect of brilliant green decreased as the bile salts concentration was increased. Staphylococcus aureus and Proteus rettgeri were inhibited by all test media. Escherichia coli was inhibited in all but two combinations of brilliant green and bile salts. Aerobacter aerogenes generally followed a pattern of growth similar to that of three species of salmonellae. Three of the 24 combinations of brilliant green and bile salts showed little or no inhibition of salmonellae but did inhibit the other organisms studied.     

Involvement of Trihydroxyconjugated Bile Salts in Cholesterol Assimilation by Bifidobacteria

To determine the conditions of cholesterol assimilation, various strains of Bifidobacterium species were cultured in the presence of cholesterol and bile salts. During culturing, Bifidobacterium breve ATCC 15700 assimilates cholesterol in the presence of oxgall at pH values lower than 6. This strain was selected to study the influence of conjugated (taurocholic acid) and deconjugated (cholic acid) bile salts on cholesterol assimilation. B. breve ATCC 15700 assimilated cholesterol (up to 51%) when cultures were undertaken in the presence of taurocholic acid, whereas less than 13% of the initial amount of cholesterol was measured in the cells in the presence of cholic acid. Cultured in the presence of six individual di- or trihydroxyconjugated bile salts, bifidobacteria strains assimilated cholesterol. This assimilation appeared to be more important in the presence of trihydroxyconjugated bile salts (tauro- and glycocholic acids). It is concluded that trihydroxyconjugated bile salts are involved in the assimilation of cholesterol by bifidobacteria. Received: 20 June 1996 / Accepted: 19 July 1996     

Bile Salt Hydrolase Activity and Resistance to Toxicity of Conjugated Bile Salts Are Unrelated Properties in Lactobacilli

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.     

Bifidobacteria Strain Behavior Toward Cholesterol: Coprecipitation with Bile Salts and Assimilation

Resting cells and growing cells of bifidobacteria strains exhibited an ability to remove cholesterol in the presence of bile salts. In resting cell assays, the removed cholesterol was precipitated in the presence of cholic acid at pH values lower than 5.4. However, this precipitated cholesterol was redissolved when the pellets were washed with phosphate buffer, pH 7, and no cholesterol was found in the cells. It appears that this precipitation is a transient phenomenon. In the case of growing cells, the removed cholesterol was partially recovered when cells were washed with phosphate buffer, pH 7, while the remaining cholesterol was extracted from the cells. Cultured in the presence of radiolabeled free or esterified cholesterol, bifidobacteria strains were able to assimilate esterified cholesterol. It is concluded that the removal of cholesterol from the growth medium by bifidobacteria strains is due to both bacterial assimilation and precipitation of cholesterol. Received: 8 February 1996/Accepted: 11 March 1996     

Metabolism of Bile Salts in Mice Influences Spore Germination in Clostridium difficile

Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.     

Bile Salts Modulate the Mucin-Activated Type VI Secretion System of Pandemic Vibrio cholerae

The causative agent of cholera, Vibrio cholerae, regulates its diverse virulence factors to thrive in the human small intestine and environmental reservoirs. Among this pathogen’s arsenal of virulence factors is the tightly regulated type VI secretion system (T6SS). This system acts as an inverted bacteriophage to inject toxins into competing bacteria and eukaryotic phagocytes. V. cholerae strains responsible for the current 7^th pandemic activate their T6SS within the host. We established that T6SS-mediated competition occurs upon T6SS activation in the infant mouse, and that this system is functional under anaerobic conditions. When investigating the intestinal host factors mucins (a glycoprotein component of mucus) and bile for potential regulatory roles in controlling the T6SS, we discovered that once mucins activate the T6SS, bile acids can further modulate T6SS activity. Microbiota modify bile acids to inhibit T6SS-mediated killing of commensal bacteria. This interplay is a novel interaction between commensal bacteria, host factors, and the V. cholerae T6SS, showing an active host role in infection.     

Susceptibility and Adaptive Response to Bile Salts in Propionibacterium freudenreichii: Physiological and Proteomic Analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

A New Type of Liquid Silymarin Proliposome Containing Bile Salts: Its Preparation and Improved Hepatoprotective Effects

Silymarin, a known extract, is used in the treatment of liver diseases with various origins, but its current administration form cannot target the liver because of its poor oral bioavailability. A new type of oral silymarin proliposome aimed at improving silymarin’s poor bioavailability and hepatoprotective effects, is introduced in this work. Silymarin-loaded liquid proliposome were prepared using a simple dissolving process. The morphology, particle size, zeta potential, and entrapment efficiency of the silymarin liposomes were analysed. The everted gut sac transport model was used to measure the intestinal transport of liposomes. The liposomal hepatoprotective activity was evaluated in three types of experimental hepatitis animal models. After staining with haematoxylin and eosin, the livers were microscopically examined to analyse any pathological changes. The prepared silymarin proliposome formed silymarin liposomes with a multilayer liposome structure and improved intestinal transport. In an injured liver, the silymarin liposomes produced a stronger hepatoprotective effect through a significant decrease in both the aminotransferase and MDA levels and a significant increase in the SOD and GSH-PX levels compared to orally administered silymarin tablets. This effect was also confirmed histopathologically. In a word, incorporation of silymarin into a liposomal carrier system increased intestinal absorption and showed better hepatoprotective effects compared to silymarin tablets.     

The Effects of Serum Albumin and Related Amino Acids on Pancreatic Lipase and Bile Salts Inhibited Microbial Lipases

The activities of microbial lipases were inhibited by bile salts in a non-emulsifying assay system. To protect lipase activities from inactivation, the effects of proteins and amino acids were investigated. Bovine serum albumin (BSA) and α-lactalbumin (α-LA) stored the bile salts inhibited microbial lipases. Among N-end amino groups contained in BSA, L-histidine restored the activities of the bile salts inhibited microbial lipases. On the other hand, pancreatic lipase activity was stimulated by not only BSA, but L-histidine and L-aspartic acid as N-end amino groups of BSA and additionally accelerated it in combination with bile salts.     

Effect of Counterions on Physicochemical Properties of Prazosin Salts

This study evaluated the effect of counterions on the physicochemical properties of prazosin salts. Salt forms of prazosin, namely, mesylate, besylate, tosylate, camsylate, oxalate, and maleate, were prepared and compared with the marketed anhydrous and polyhydrate forms of prazosin hydrochloride. Physicochemical characterization was performed in the order of crystallinity, hygroscopicity, solubility, and stability to select the optimal salt(s). Permeability study in Caco-2 cell lines and in vivo bioavailability study in rat model were investigated to ascertain their biopharmaceutical advantage. All salt forms were crystalline, nonhygroscopic (except the anhydrous hydrochloride salt), and had solubility in the range of 0.2 to 1.6 mg/ml. All salts were physically and chemically stable at 40°C/75% relative humidity, but degraded in UV-visible light, except the anhydrous hydrochloride salt. Prazosin mesylate was selected as the optimal salt, as it possessed higher solubility, permeability, and bioavailability, compared to the commercial hydrochloride salts. Hydrochloride salt is reported to have poor bioavailability that is partially attributed to its low solubility and extensive common-ion effect in the gastric region. Factors like hydrophilicity of the counterion, hydration state of the salt, and melting point of the salt contribute to the physicochemical properties of the salts. This study has implications in the selection of an optimal salt form for prazosin, which is suitable for further development.     

Characterization of Cholylglycine Hydrolase from a Bile-Adapted Strain of Xanthomonas maltophilia and Its Application for Quantitative Hydrolysis of Conjugated Bile Salts

Purified bile salt hydrolase from bile-adapted Xanthomonas maltophilia displays Michaelis-Menten kinetics on cholylglycine and cholyltaurine and hydrolyzes bile salts also in crude bovine bile. The protein is a dimer and is resistant to proteinases and to heating at 55 to 60°C for up to 60 min, in agreement with calorimetric data.     

Long-Term Survival of Salmonella enterica Serovar Typhimurium Reveals an Infectious State That Is Underrepresented on Laboratory Media Containing Bile Salts

Cells in desiccated Salmonella enterica serovar Typhimurium rdar (red, dry, and rough) morphotype colonies were examined for culturability and infectivity after 30 months. Culturability decreased only 10-fold; however, cells were underrepresented on Salmonella selective media containing bile salts. These cells were mildly attenuated compared to the infectivity of freshly grown cells but still able to cause systemic infections in mice.Salmonella enterica serovar Typhimurium (hereafter referred to as S. Typhimurium) is a gram-negative enteric pathogen of humans and other mammalian species. S. Typhimurium causes self-limiting gastroenteritis in humans and typhoid-like fever in mice (4). When propagated on the surface of nutrient-limited laboratory media, Salmonella cells form patterned colonies (1) involving the production of an extracellular matrix comprised of thin aggregative fimbriae (Tafi or curli) and cellulose (16, 21) and other polysaccharides. This rdar morphotype is characterized by the formation of red, dry, and rough colonies on solid agar medium containing Congo red as an indicator dye (8). Cells within rdar colonies are resistant to desiccation and commonly used disinfectants (14, 18), and the propensity to form the rdar morphotype is conserved throughout the salmonellae (7, 19). These findings, coupled with the observations that neither Tafi nor cellulose is required for virulence (13, 17), suggest that the rdar phenotype may contribute to the environmental survival of Salmonella (17).Previous experiments by our group established that approximately 10% (∼10⁸) of bacteria within a rdar morphotype colony were able to survive a 9-month period of starvation and desiccation (18). In this study, we examined survival after a longer time period of 30 months. S. Typhimurium ATCC 14028 cells were grown at 28°C for 4 days on tryptone agar, at which time colonies were detached from the agar surface and stored at room temperature in sterile, 24-well plates, as previously described (18). The results were compared to those for planktonic S. Typhimurium cells grown overnight in 1% tryptone and washed with distilled water prior to storage on plastic. Surprisingly, the number of viable cells recovered from rdar colonies after 30 months was similar to that obtained at the 9-month time point (Fig. ​(Fig.1).1). In contrast, planktonic S. Typhimurium cells exhibited a decrease in viability of 4 orders of magnitude within 14 days. However, if planktonic cells were stored in water or isotonic saline, the viability decreased by only 2 orders of magnitude after 84 days (Fig. ​(Fig.1).1). This finding indicated that desiccation rather than starvation was the primary cause of bacterial death under these conditions. The survival after 30 months of isogenic ΔcsgD mutant cells, which are deficient in extracellular matrix production (8, 18), was reduced 25-fold compared to that of wild-type cells (data not shown), confirming that cellulose and Tafi polymers have an important role in long-term survival. Together, these results indicated that extreme longevity of cells within rdar colonies is a morphotype-specific phenomenon rather than a general property of S. Typhimurium ATCC 14028.Open in a separate windowFIG. 1.Long-term survival of S. Typhimurium cells in rdar morphotype colonies or from planktonic culture under conditions of desiccation and nutrient absence. Approximately 10⁹ CFU of planktonic cells (▴) or cells within rdar colonies (•) were placed on sterile plastic, or planktonic cells were stored in sterile water (▪). At time points shown, cells were rehydrated, homogenized, and serially diluted before being grown on LB medium to enumerate the CFU. Data points represent averages and error bars show the standard deviations of the results from at least three biological samples.Under most laboratory conditions, S. Typhimurium is resistant to bile concentrations exceeding 60% (wt/vol) (3, 5, 6, 15); therefore, many selective media used in microbiological laboratories for culturing Salmonella contain bile salts. During storage on plastic with no nutrients or water, cells within rdar colonies became sensitive to sodium cholate and sodium deoxycholate (bile salts) over time (Table ​(Table1).1). As expected, freshly grown cells (exponential or stationary phase) did not display bile sensitivity (data not shown). The culturability of cells in 30-month-old colonies on media containing bile salts was significantly reduced compared to that of cells from 2-day-old or 2-week-old rdar colonies or colonies that were lyophilized for 1 week (Table ​(Table1).1). In contrast, culturability was not reduced to the same extent when aged cells were cultured on media that did not contain bile salts (brilliant green agar or XLD and LSA without bile salts [see Table ​Table11 for media]). Within diagnostic laboratories, bacterial isolates in environmental samples are commonly incubated in recovery broth prior to enumeration on selective medium (2). When cells derived from 30-month-old rdar colonies were incubated overnight in buffered peptone water, they recovered their resistance to bile salts (data not shown).

TABLE 1.

Relative recovery of S. Typhimurium ATCC 14028 on common Salmonella selective media

Effect of Bile and Desoxycholate on Gram-Negative Anaerobic Bacteria

The bile tests for characterizing gram-negative anaerobic bacilli were reevaluated in prereduced anaerobically sterilized peptone-yeast-glucose broth, in thioglycollate broth, and on blood agar plates. Blood agar plates were unsatisfactory. The combination of 20% bile with 0.1% desoxycholate inhibited Fusobacterium, Bacteroides melaninogenicus, and B. oralis and sometimes Sphaerophorus necrophorus, but not B. fragilis or other Sphaerophorus species studied. Ten per cent bile with 0.05% desoxycholate was less satisfactory. There was no significant difference between fresh and commercial powdered bile. Desoxycholate (0.1% in thioglycollate broth) inhibited B. fragilis, Fusobacterium, B. melaninogenicus, B. oralis, and S. necrophorus, but not S. varius or S. mortiferus/S. ridiculosus. The bile and desoxycholate tests are simple to perform and helpful for characterization and classification of gram-negative anaerobic bacilli.