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.     

Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in<Emphasis Type="Italic"> Propionibacterium freudenreichii</Emphasis>

Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.     

Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis.

The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria.     

Changes in protein synthesis and morphology during acid adaptation of Propionibacterium freudenreichii

Survival of bacteria in changing environments depends on their ability to adapt to abiotic stresses. Microorganisms used in food technology face acid stress during fermentation processes. Similarly, probiotic bacteria have to survive acid stress imposed within the stomach in order to reach the intestine and play a beneficial role. Propionibacteria are used both as cheese starters and as probiotics in human alimentation. Adaptation to low pH thus constitutes a limit to their efficacy. Acid stress adaptation in the probiotic SI41 strain of Propionibacterium freudenreichii was therefore investigated. The acid tolerance response (ATR) was evidenced in a chemically defined medium. Transient exposure to pH 5 afforded protection toward acid challenge at pH 2. Protein neosynthesis was shown to be required for optimal ATR, since chloramphenicol reduced the acquired acid tolerance. Important changes in genetic expression were observed with two-dimensional electrophoresis during adaptation. Among the up-regulated polypeptides, a biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair were identified during the early stress response, while the universal chaperonins GroEL and GroES corresponded to a later response. The beneficial effect of ATR was evident at both the physiological and morphological levels. This study constitutes a first step toward understanding the very efficient ATR described in P. freudenreichii.     

Pyrophosphate is a source of phosphoryl groups for Escherichia coli protein phosphorylation

Abstract Stress tolerance and cross-protection in Enterococcus faecalis ATCC19433 were examined after exposure to bile salts, acid or heat shock. Bile salts and heat adapted cells demonstrated induced homologous tolerance and cross-resistance. No cross-protection of heat adapted cells against acid stress is observed and pretreatment with bile salts even sensitized the cells to this challenge. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins. Some of these polypeptides are induced by more than one treatment. The greatest overlap is observed between bile salts and heat treatments. Eighteen stress proteins, including DnaK and GroEL, are common between these stresses.     

Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri

AIMS: The objective of this study was to evaluate the effect of bile salts and cholesterol in the lipid profile of Lactobacillus reuteri CRL 1098 and to determine the relationship existing between these changes: the in vitro removal of cholesterol and the tolerance of the cells to acid and cold stress. METHODS AND RESULTS: Lactobacillus reuteri CRL 1098 was grown in the following media: MRS (deMan Rogosa Sharpe; MC, control medium), MB (MC with bile salts), MCH (MC with sterile cholesterol) and MBCH (MC with bile salts and cholesterol). Fatty acids were determined by analytical gas-liquid chromatography, and phospholipids and glycolipids by colorimetric techniques. The cells from different culture media were subjected to cold and acid stress. The MB cultures displayed a decrease in phospholipids and a low ratio of saturated : unsaturated fatty acids. The presence of the unusual C18 : 0,10-OH and C18 : 0,10-oxo fatty acids was the prominent characteristic of the bile salts growing cells. The relative increase in glycolipids and the changes in the fatty acids profiles of the MB cells would be responsible for the cholesterol remotion. The changes induced by bile salts in the lipid profile did not improve the tolerance of L. reuteri CRL 1098 to freezing and acid stress. CONCLUSIONS: The changes in lipid profiles reported in this study would play a key role in the response of Lactobacilli to environmental stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides useful information about the effect of bile salts on the cell membrane of L. reuteri, a probiotic enterolactobacillus. The steady-state response of the cells subjected to bile stress seems to be the appropriate model for evaluating the bacterial behaviour in detergent-containing gastrointestinal tracts, where the bile salts stress would presumably be continuous.     

The effect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92

Bile tolerance is an important criterion in the selection of microbial strains for probiotic use. The survival and morphological changes of a potential probiotic strain, Lactobacillus acidophilus M92, in the presence of bile salts were examined. Lactobacillus acidophilus M92 has shown a satisfactory degree of tolerance against oxgall and individual bile salts tested, especially to taurocholate. The higher resistance of L. acidophilus M92 against taurine-conjugated bile salts relative to deconjugated and glycine-conjugated bile salts was attributed to its reaction to the stronger acidity of the former. Furthermore, bile salt hydrolase (BSH) was active when L. acidophilus M92 was grown in the presence of sodium taurocholate. The rate of BSH activity was highest at the exponential growth phase. It was hypothesised that BSH activity may be important for the bile salt resistance of this strain. The colonial and cellular morphology may also be a valuable parameter in the selection of bile salt-resistant Lactobacillus strains for probiotic use. Smooth (S) and rough (R) colonies, appeared in the original L. acidophilus M92 bacterial culture and demonstrated a different degree of bile tolerance. Rough colonies were more sensitive to bile salts than smooth ones. The R colony cells assumed a round form, probably induced by gaps in the cell wall caused by the cytotoxicity of glycodeoxycholate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acid-bile, antibiotic resistance and inhibitory properties of propionibacteria isolated from Turkish traditional home-made cheeses

In this study, a total of 29 Propionibacterium spp. were isolated from traditional home-made Turkish cheese samples. As a result of the identification, isolates were identified as Propionibacterium freudenreichii subsp. freudenreichii (15 strains), Propionibacterium jensenii (12), and Propionibacterium thoenii (2). All isolates and 5 reference strains were examined for their abilities to survive at pH 2.0, 3.0, 4.0, 5.0 and in the presence of 0.06, 0.15 and 0.30% bile salts, their influence on the growth of food-borne and spoilage bacteria, as well as their sensitivity against 11 selected antibiotics. Only seven propionibacteria strains survived in both the acidic and bile salt environments. Propionibacterium spp. strains strongly inhibited growth of the Escherichia coli ATCC 11229 and Shigella sonnei Mu:57 strains (91%). All propionibacteria strains were sensitive to a majority of the antibiotics used in the investigations. Overall, dairy propionibacteria showed high antibacterial activity, resistance to pH 4.0, 5.0, high resistance to bile salts and will provide an alternative source to Lactobacillus and Bifidobacterium as probiotic culture.     

Method for direct selection of potentially probiotic Bifidobacterium strains from human feces based on their acid-adaptation ability

A method for direct selection of acid-resistant Bifidobacterium strains was developed by prolonged exposure of human feces to homologous lethal stress conditions. The recovered strains were intrinsically resistant to acidic gastric conditions (pH 2.0) and also showed good tolerance to high concentrations of bile salts and NaCl. Bifidobacterium breve and Bifidobacterium adolescentis were, respectively, the infant- and adult-type bifidobacterial species showing the highest ability to develop an acid-tolerant phenotype. Therefore, this procedure could be applicable to the direct selection of Bifidobacterium strains with improved stability in adverse environments and, probably, contribute to expand the spectra of probiotic species of human origin currently marketed.     

Anticodon nuclease encoding virus-like elements in yeast

The aim of this study was to apply flow cytometric (FCM) analysis to assess the use of sucrose and lecithin vesicles for the protection of probiotic lactic acid bacteria in response to the challenge of gastric acidity and bile salts. FCM analysis in combination with fluorescent probes carboxyfluorescein (cF) and propidium iodide was used to reveal the physiological heterogeneity in the stressed bacteria population. Three subpopulations (intact, stressed, and damaged) were differentiated by FCM in all six examined strains. Significant changes were observed in the presence of the selected protectants. The addition of 20?mM sucrose in the simulated gastric fluid substantially increased the number of intact cells over 20 folds and reduced the damaged subpopulation by half. The presence of 2?% (w/v) lecithin vesicles was shown to protect 50?% more intact cells from the challenge of bile salts. The improved survival as evaluated by FCM analysis was further assessed for the proliferation capacity by sorting a number of cells from each subpopulation on nutrient agar plate. The result confirmed conformity between the proliferation-based cultivability and the probe-indicated viability in the samples of the intact and the damaged subpopulations. However, it also revealed the complexities of the stressed (injured) subpopulation. In conclusion, FCM analysis confirmed that the selected protectants could improve the survival of the probiotic strains in the simulated GI environments. The FCM analysis also proved to be a useful analytical tool for the probiotics research.     

Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis.

Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 degrees C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.     

Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics

The identification of cell determinants involved in probiotic features is a challenge in current probiotic research. In this work, markers of bile tolerance in Lactobacillus casei were investigated using comparative proteomics. Six L. casei strains were classified on the basis of their ability to grow in the presence of bile salts in vitro. Constitutive differences between whole cell proteomes of the most tolerant strain (L. casei Rosell-215), the most sensitive one (L. casei ATCC 334), and a moderately tolerant strain (L. casei DN-114 001) were investigated. The ascertained subproteome was further studied for the six strains in both standard and bile stressing conditions. Focus was on proteins whose expression levels were correlated with observed levels of bile tolerance in vitro, particularly those previously reported to be involved in the bile tolerance process of lactobacilli. Analysis revealed that 12 proteins involved in membrane modification (NagA, NagB, and RmlC), cell protection and detoxification (ClpL and OpuA), as well as central metabolism (Eno, GndA, Pgm, Pta, Pyk, Rp1l, and ThRS) were likely to be key determinants of bile tolerance in L. casei and may serve as potential biomarkers for phenotyping or screening purposes. The approach used enabled the correlation of expression levels of particular proteins with a specific probiotic trait.     

Adaptation and response of Bifidobacterium animalis subsp. lactis to bile: a proteomic and physiological approach

Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.     

Different probiotic properties for Lactobacillus fermentum strains isolated from swine and poultry

Systematic procedures were used to evaluate the probiotic properties of Lactobacillus fermentum (L. fermentum) strains isolated from swine and poultry. The major properties included their capabilities to adhere to the intestinal epithelium of swine and poultry, the inhibition on pathogenic bacteria, and their tolerance to the gastric juice and bile salts. Results showed that L. fermentum strains from poultry digestive tract showed better adherence to the swine intestine and chicken crop epithelial cells as compared to those strains from the swine origin. In addition, six strains from poultry and one strain from swine showed adhesion specificity to their own intestinal epithelium. Four poultry isolates and one swine isolate were able to adhere to the epithelial cells from both swine and chicken. For gastric juice and bile tolerance, most of the strains isolated from swine or poultry were acid tolerant but less strains were bile intolerant. The spent culture supernatant (SCS) of these L. fermentum strains showed antagonistic effect against the indicator bacteria, such as Escherichia coli, Salmonella spp., Shigella sonnei and some enterotoxigenic Staphylococcus aureus. From the above studies, some L. fermentum strains isolated from poultry were found to have the probiotic properties required for use in animal feed supplement. This study suggested that poultry digestive tract may serve as potential source for the isolation of probiotic lactic acid bacteria.     

Adaptation and preadaptation of Salmonella enterica to Bile

Bile possesses antibacterial activity because bile salts disrupt membranes, denature proteins, and damage DNA. This study describes mechanisms employed by the bacterium Salmonella enterica to survive bile. Sublethal concentrations of the bile salt sodium deoxycholate (DOC) adapt Salmonella to survive lethal concentrations of bile. Adaptation seems to be associated to multiple changes in gene expression, which include upregulation of the RpoS-dependent general stress response and other stress responses. The crucial role of the general stress response in adaptation to bile is supported by the observation that RpoS(-) mutants are bile-sensitive. While adaptation to bile involves a response by the bacterial population, individual cells can become bile-resistant without adaptation: plating of a non-adapted S. enterica culture on medium containing a lethal concentration of bile yields bile-resistant colonies at frequencies between 10(-6) and 10(-7) per cell and generation. Fluctuation analysis indicates that such colonies derive from bile-resistant cells present in the previous culture. A fraction of such isolates are stable, indicating that bile resistance can be acquired by mutation. Full genome sequencing of bile-resistant mutants shows that alteration of the lipopolysaccharide transport machinery is a frequent cause of mutational bile resistance. However, selection on lethal concentrations of bile also provides bile-resistant isolates that are not mutants. We propose that such isolates derive from rare cells whose physiological state permitted survival upon encountering bile. This view is supported by single cell analysis of gene expression using a microscope fluidic system: batch cultures of Salmonella contain cells that activate stress response genes in the absence of DOC. This phenomenon underscores the existence of phenotypic heterogeneity in clonal populations of bacteria and may illustrate the adaptive value of gene expression fluctuations.     

Importance of microbial defence systems to bile salts and mechanisms of serum cholesterol reduction

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.     

Enhancing the stress responses of probiotics for a lifestyle from gut to product and back again

Before a probiotic bacterium can even begin to fulfill its biological role, it must survive a battery of environmental stresses imposed during food processing and passage through the gastrointestinal tract (GIT). Food processing stresses include extremes in temperature, as well as osmotic, oxidative and food matrix stresses. Passage through the GIT is a hazardous journey for any bacteria with deleterious lows in pH encountered in the stomach to the detergent-like properties of bile in the duodenum. However, bacteria are equipped with an array of defense mechanisms to counteract intracellular damage or to enhance the robustness of the cell to withstand lethal external environments. Understanding these mechanisms in probiotic bacteria and indeed other bacterial groups has resulted in the development of a molecular toolbox to augment the technological and gastrointestinal performance of probiotics. This has been greatly aided by studies which examine the global cellular responses to stress highlighting distinct regulatory networks and which also identify novel mechanisms used by cells to cope with hazardous environments. This review highlights the latest studies which have exploited the bacterial stress response with a view to producing next-generation probiotic cultures and highlights the significance of studies which view the global bacterial stress response from an integrative systems biology perspective.     

Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport

The role of the hepatocyte microtubular system in the transport and excretion of bile salts and biliary lipid has not been defined. In this study the effects of microtubule inhibition on biliary excretion of micelle- and non-micelle-forming bile salts and associated lipid were examined in rats. Low-dose colchicine pretreatment had no effect on the baseline excretion of biliary bile salts and phospholipid in animals studied 1 hr after surgery (basal animals), but slightly retarded the excretion of tracer [14C]taurocholate relative to that of lumicolchicine-pretreated (control) rats. However, colchicine pretreatment resulted in a marked reduction in the excretion of 2 mumol/100 g doses of a series of four micelle-forming bile salts of differing hydrophilicity, but had no significant effect on the excretion of the non-micelle-forming bile salt, taurodehydrocholate. Continuous infusion of 0.2 mumol of taurocholate/(100 g.min) following 24 hr of biliary drainage (depleted/reinfused animals) resulted in physiologic bile flow with biliary excretion rates of bile salts, phospholipid, and cholesterol that were markedly inhibited (mean 33, 39, and 42%, respectively) by colchicine or vinblastine pretreatment. Excretion of tracer [14C]taurocholate also was markedly delayed by colchicine in these bile salt-depleted/reinfused animals. In contrast, colchicine did not inhibit bile salt excretion in response to reinfusion of taurodehydrocholate. Thus, under basal conditions, the microtubular system appears to play a minor role in hepatic transport and excretion of bile salts and biliary lipid. However, biliary excretion of micelle-forming bile salts and associated phospholipid and cholesterol becomes increasingly dependent on microtubular integrity as the transcellular flux and biliary excretion of bile salts increases, in both bile salt-depleted and basal animals. We postulate that cotransport of micelle-forming bile salts and lipids destined for biliary excretion, via an intracellular vesicular pathway, forms the basis for this microtubule dependence.