Nondigestible Oligosaccharides Enhance Bacterial Colonization Resistance against Clostridium difficile In Vitro

Clostridium difficile is the principal etiologic agent of pseudomembranous colitis and is a major cause of nosocomial antibiotic-associated diarrhea. A limited degree of success in controlling C. difficile infection has been achieved by using probiotics; however, prebiotics can also be used to change bacterial community structure and metabolism in the large gut, although the effects of these carbohydrates on suppression of clostridial pathogens have not been well characterized. The aims of this study were to investigate the bifidogenicity of three nondigestible oligosaccharide (NDO) preparations in normal and antibiotic-treated fecal microbiotas in vitro and their abilities to increase barrier resistance against colonization by C. difficile by using cultural and molecular techniques. Fecal cultures from three healthy volunteers were challenged with a toxigenic strain of C. difficile, and molecular probes were used to monitor growth of the pathogen, together with growth of bifidobacterial and bacteroides populations, over a time course. Evidence of colonization resistance was assessed by determining viable bacterial counts, short-chain fatty acid formation, and cytotoxic activity. Chemostat studies were then performed to determine whether there was a direct correlation between bifidobacteria and C. difficile suppression. NDO were shown to stimulate bifidobacterial growth, and there were concomitant reductions in C. difficile populations. However, in the presence of clindamycin, activity against bifidobacteria was augmented in the presence of NDO, resulting in a further loss of colonization resistance. In the absence of clindamycin, NDO enhanced colonization resistance against C. difficile, although this could not be attributed to bifidobacterium-induced inhibitory phenomena.     

Inhibition of Clostridium difficile growth and adhesion to enterocytes by Bifidobacterium supernatants

The antimicrobial and anti-adhesive effects of extracellular factors from 27 strains of bifidobacteria isolated from healthy infants were tested against two reference strains of Clostridium difficile (ATCC 9689 and ATCC 43593). All bifidobacterial supernatants at pHs between 5.0 and 4.1 were able to produce strain-dependent growth inhibition of clostridia in the agar-diffusion assay. Six strains of Bifidobacterium produced during growth extracellular factors able to antagonize the adhesion of C. difficile ATCC 9689 and ATCC 43593 to cultured human enterocytes (Caco-2/TC7). Factors responsible for the anti-adhesive effect were thermolabile, active at neutral pH and unaffected by proteolytic cleavage (proteinase K and chymotrypsin). Results of the present paper show the potential of selected bifidobacteria to antagonize key mechanisms involved in the virulence of C. difficile.     

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.     

Molecular analysis of the composition of the bifidobacterial and lactobacillus microflora of humans.

The bifidobacterial and lactobacillus populations of fecal samples collected from two human subjects during a 12-month period were studied. The total numbers of bifidobacteria were stable throughout the study period in both subjects, but lactobacillus numbers were less constant. Analysis of the composition of the bifidobacterial populations by using ribotyping or pulsed-field gel electrophoresis to differentiate between bacterial strains demonstrated major differences between the subjects. Subject 1 harbored five strains of bifidobacteria throughout the 12-month period, and one strain was numerically predominant. In contrast, subject 2 harbored a more complex bifidobacterial population (five to six strains per sample) whose composition fluctuated throughout the 12 months. One lactobacillus strain was numerically predominant throughout the study in both subjects. Strains of bifidobacteria and lactobacilli common to both subjects were not detected.     

抗生素在体内外对厌氧菌和艰难梭菌细胞毒素生成的作用

氯林霉素、灭滴灵和甲砜霉素对大多数肠道厌氧菌的生长具抑制作用。氯林霉素还会破坏肠道菌群平衡,使原来受抑制的艰难梭菌得以定植,并在艰难梭菌浓度达10~8/g盲肠内含物时,检测到艰难梭菌细胞毒素。培养基中亚抑菌浓度的氯林霉素和灭滴灵会推迟艰难梭菌细胞霉素的生成。灭滴灵还可保护无菌小鼠及受氯林霉素处理的悉生小鼠免遭艰难梭菌细胞毒素的致死作用,从而证实了灭滴灵在伪膜性结肠炎临床治疗中的可用性。     

Evaluation of the effect of medicines on biological properties of Clostridium difficile

The presence of the persistence factors (anti-lysozyme and anti-complement activity) in the vegetative forms of C. difficile was experimentally proved. The effect of different medicines (vitamins B1, B6 and C, prebiotic inulin, probiotics Bifidumbacterin and Enterol) on the persistence factors of C. difficile and microbial resistance to vancomycin, thienam, lincomycin, clindamycin was evaluated. The anti-lysozyme and anti-complement activity of C. difficile was found to decrease under the influence of vitamins B1, B6, C, inulin, exometabolites of bifidobacteria. Under the impact of the preparations used in this study changes in the sensitivity of C. difficile to antibiotics of the lincoamide, carbapenem, glycopeptide groups were found to occur. The data obtained reveal one of the possible mechanisms of the corrective action of the medicines under study on the intestinal microbiocenosis in patients with antibiotic-associated colitis.     

Polydextrose, Lactitol, and Fructo-Oligosaccharide Fermentation by Colonic Bacteria in a Three-Stage Continuous Culture System

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.     

Analysis of fecal populations of bifidobacteria and lactobacilli and investigation of the immunological responses of their human hosts to the predominant strains.

The bifidobacterial and lactobacillus populations of fecal samples collected from 10 human subjects were studied. The numbers of bifidobacteria were similar in the fecal samples of all of the subjects, but lactobacillus numbers varied, even between samples collected from the same individual. Analysis of the composition of the bacterial populations by ribotyping and pulsed-field gel electrophoresis to differentiate between strains showed that, at least for the numerically predominant strains, each subject harbored a unique collection of bifidobacteria and lactobacilli. Predominant bifidobacterial and lactobacillus strains detected in the feces of each subject were used in immunological assays (lymphocyte transformation, serum antibody titers) to determine the influence of the bacteria on the immune system of their host. Immunoglobulin G antibodies reactive with lactobacilli were detected at high concentrations; antibodies reactive with bifidobacteria were present at lower concentrations. The antibodies appeared to be genus specific rather than strain specific. The results of the study emphasized the complexity of the relationship that exists between the intestinal microflora and the human host.     

Increased resistance of mice to Salmonella enterica serovar Typhimurium infection by synbiotic administration of Bifidobacteria and transgalactosylated oligosaccharides

AIMS: The anti-infectious activity of Bifidobacteria in combination with transgalactosylated oligosaccharides (TOS) against Salmonella enterica serovar Typhimurium LT-2 in an opportunistic antibiotic-induced murine infection model in mice was examined. METHODS AND RESULTS: B. breve (strain Yakult) with natural resistance to streptomycin sulphate (SM, MIC: > 4 mg ml(-1)), when given daily at a dose of 108 cfu/mouse orally under SM treatment was constantly excreted at 10(10) cfu g(-1) faeces so long as SM was administered, even at 2 weeks after discontinuing administration of B. breve. Explosive intestinal growth and subsequent extra-intestinal translocation of orally infected LT-2 under SM treatment were inhibited by B. breve colonization, and this anti-infectious activity was strengthened by synbiotic administration of TOS with B. breve. Comparison of anti-Salmonella activity among several Bifidobacterium strains with natural resistance to SM revealed that strains such as B. bifidum ATCC 15696 and B. catenulatum ATCC 27539T conferred no activity, even when they reached high population levels similar those of effective strains such as strain Yakult and B. pseudocatenulatum DSM 20439. Both the increase in the concentration of organic acids and the lowered pH in the intestine due to bifidobacterial colonization correlated with the anti-infectious activity. Moreover, the crude cecal extract of B. breve-colonized mice exerted growth-inhibitory activity against LT-2 in vitro, whereas that of the ineffective B. bifidum-colonized cecum showed much lower activity. CONCLUSIONS: Intestinal colonization by bifidobacteria given exogenously together with TOS during antibiotic treatment prevents the antibiotic-induced disruption of colonization resistance to oral infection with S. enterica serovar Typhimurium, and the metabolic activity needed to produce organic acids and lower the intestinal pH is important in the anti-infectious activity of synbiotics against enteric infection with Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that certain bifidobacteria together with prebiotics may be used for the prophylaxis against opportunistic intestinal infections with antibiotic-resistant pathogens.     

Bile salts and glycine as cogerminants for Clostridium difficile spores

Spore formation by Clostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile-associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile. A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.     

Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods

Aims:  To detect and enumerate bifidobacteria in faeces with a new quantitative multiplex real-time PCR (qPCR) method and to compare the results obtained with fluorescence in situ hybridization (FISH) methods.
Methods and Results:  A multiplex qPCR assay was developed, which enabled the enumeration of Bifidobacterium spp. by targeting the bifidobacterial xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene ( xfp ) and total bacteria using universal Eub-primers targeting 16S rRNA gene from the domain bacteria. The qPCR assay showed high sensitivity and specificity and a low detection limit of about 2·5 × 10³ bifidobacterial cells per gram of faeces. The qPCR results were compared with FISH combined with microscopy or flow cytometry (FCM). No statistical differences among bifidobacterial counts averages measured in adult faeces with the three methods were observed. Total bacterial count averages were higher with the FISH method coupled with microscopic analyses compared to FISH with FCM, whereas total cell numbers estimated by qPCR were intermediate between the two FISH methods.
Conclusions:  The new qPCR assay was shown to be sensitive, rapid and accurate for enumerating bifidobacteria in faeces.
Significance and Impact of the Study:  This method is a valuable alternative for other molecular methods for detecting faecal bifidobacteria, especially when their counts are below the detection limit of the FISH methods.     

Susceptibility of bifidobacteria to lysozyme as a possible selection criterion for probiotic bifidobacterial strains

Resistance or susceptibility of bifidobacteria to lysozyme and growth of bifidobacteria in human milk were tested. Susceptible bifidobacterial strains stopped their growth almost immediately after the addition of lysozyme (400 μg/ml), moderately susceptible strains exhibited reduced growth rate, and growth curves of resistant strains were not affected. Strains of human origin were more resistant to lysozyme than animal strains. While strains of B. bifidum grew well in human milk samples, the growth B. animalis strains was inhibited after inoculation to human milk. The resistance to lysozyme seems to be a promising criterion for the selection of new probiotic bifidobacterial strains.     

Insights into physiological and genetic mupirocin susceptibility in bifidobacteria

Mupirocin is an antibiotic commonly used in selective media for the isolation of bifidobacteria. However, little is known about the genetic traits responsible for bifidobacterial resistance to mupirocin. Our investigation demonstrates that all of the bifidobacteria tested exhibit a phenotype of generally high resistance to this antibiotic. The genotypic reason for bifidobacterial mupirocin resistance was further characterized by sequencing of the isoleucyl-tRNA synthetase gene (ileS) coupled with three-dimensional modeling of the encoded protein and cloning of the ileS gene of Bifidobacterium bifidum PRL2010 in a mupirocin-sensitive Escherichia coli strain. These analyses revealed key amino acid residues of the IleS protein that apparently are crucial for conferring a mupirocin resistance phenotype to bifidobacteria.     

In Vivo Relationship between Intestinal Bifidobacteria Overgrowth and Bacteroides fragilis Repression Induced by Consumption of Bifidobacterial Cell-free Whey

Cell-free whey from a selected strain, Bifidobacterium breve C50, induced an increase in bifidobacteria associated with a Bacteroides fragilis reduction in the gut of conventional mice and humans. The purpose of our study was to investigate the mechanism of B. fragilis repression. C50 cell-free whey was given for 15 days to conventional or ex-germ-free mice mono-associated to the strain B. fragilis CFPL 358. Conventional and ex-germ-free control mice received whey which was incapable of promoting intestinal bifidobacteria and of reducing B. fragilis. Bacterial counting was carried out in the ileum, caecum and colon of both mouse models. The C50 cell-free whey induced a significant increase in endogenous bifidobacteria in the ileum of conventional mice, whereas B. fragilis was below detectable levels throughout the intestine. In ex-germ-free mice mono-associated with B. fragilis, the strain was seen to be at a high level through the whole intestine and no significant difference in counts was observed according to the whey administered to animals. The data indicated that a prerequisite for C50 cell-free whey repressive activity against B. fragilis is colonization of the mouse gut with complex bacterial microflora. With the exception of the distal ileum, the bifidobacterial overgrowth did not, however, support B. fragilis reduction. It is likely that in the caecum and colon some other bacteria participated in the process.     

Does the intestinal bifidobacterial colonisation affect bacterial translocation?

The aim of this work was to investigate the possible role of the intestinal anaerobic flora (especially bifidobacteria) in regulating bacterial translocation (BT) which can be defined as the passage of intestinal microbes through the mucosa to internal organs. Default in BT regulation concurs with pathogenesis of sepsis in various human conditions, such as acute pancreatitis, cirrhosis, necrotising enterocolitis or multiple organ failure. The intestinal flora was studied in human flora associated mice (HF mice) and BT was quantified in Peyer's patches (PP), blood, spleen, liver and lungs. HF mice displayed a heterogenic intestinal colonisation with bifidobacteria. High colonisation of both caecum and colon by bifidobacteria led to a poorer bacterial contamination of blood, liver and lungs. Moreover, ileal, caecal and colonic bifidobacterial counts negatively correlated with the bacterial dissemination (number of contaminated organs per mouse). In contrast, Bacteroides fragilis group counts positively correlated with bacteraemia, lungs contamination or bacterial dissemination. Additionally, clostridia localised in the colon affected bacterial uptake by PP and lungs contamination as indicated by positive correlations between bacterial populations in these respective locations. These results indicate that bifidobacteria, when established in high counts, reduced BT to liver, blood and lungs, whereas B. fragilis group favoured the bacterial passage. Clostridia established in the distal ileum also seemed to favour BT to lungs. The manipulation of the bacterial flora to optimise the regulatory effect on BT should therefore focus on the selective promotion of bifidobacteria and avoid an increase in potentially detrimental populations such as B. fragilis group and clostridia.     

Effect of various antibiotics on gastrointestinal colonization and dissemination by Candida albicans

Mice were treated orally with various antibiotics to determine which members of the indigenous intestinal microflora normally suppress Candida albicans colonization and dissemination from the gastrointestinal (GI) tract. The mice were given penicillin, clindamycin, vancomycin, erythromycin, or gentamicin for 3 days, and then challenged orally with C. albicans. Penicillin, clindamycin, and vancomycin, but not gentamicin or erythromycin, decreased the total anaerobic bacterial populations in the animals ceca, and increased the enteric bacilli population levels. All three of the former antibiotics allowed C. albicans to proliferate in the gut and, subsequently, disseminate from the GI tract to visceral organs. The ability of C. albicans to associate with intestinal mucosal surfaces was also tested. It was found that antibiotics which reduced anaerobic population levels, but not enteric bacilli or aerobes, also predisposed animals to mucosal association by C. albicans. It is suggested that the strictly anaerobic bacterial populations which predominate in the gut ecosystem are responsible for the inhibition of C. albicans adhesion, colonization and dissemination from the GI tract.     

Effect of production conditions on the stability of a human bifidobacterial species Bifidobacterium longum in yogurt

Aims:  Human bifidobacteria are more sensitive to external environmental factors than animal bifidobacteria, and it is difficult to ensure their stable survival in yogurt. The purpose of this investigation was to observe the survival of human bifidobacteria in yogurts produced under various production conditions.
Methods:  Frozen or lyophilized bifidobacteria starters containing Bifidobacterium longum BB536 originally isolated from an infant, and commercial lyophilized yogurt starters were used for yogurt preparation. After producing yogurts under various conditions, the survival of bifidobacteria in these yogurts over various storage periods was observed.
Results:  Although there were some differences in bifidobacterial survival in yogurt between various production conditions, more than 1·0 × 10⁷ CFU g⁻¹ of Bif. longum survived in yogurt after 35 days' storage at 5°C. Lower fermentation temperature (37°C) and inclusion of Lactococcus lactis in the starter significantly ( P  < 0·05) improved survival of Bif. longum in the yogurt.
Conclusion:  In this investigation, the human bifidobacterial strain Bif. longum survived adequately in yogurt, although the fermentation temperature and starter composition affect bifidobacterial survival.
Significance and Impact of the Study:  This investigation indicates that stable probiotic yogurt using human bifidobacteria can be produced by choosing optimal production conditions.     

新生儿肠道菌群的动态观察及临床意义

本文以厌氧菌双歧杆菌和需氧菌大肠杆菌为代表对29例健康足月新生儿(阴道分娩儿13例、剖腹产分娩儿16倒)生后1周内肠道细菌的定植,进行了动态观察。结果表明:两组新生儿首先肠道定植细菌是大肠杆菌,后为双歧杆菌,而且阴道分娩儿肠道优势菌双歧杆菌定植及达优势化时间均早于剖腹产分娩儿。提示,分娩方式也是影响肠道细菌定植的因素之一,为临床治疗和护理提供了参考。     

Bovine lactoferrin region responsible for binding to bifidobacterial cell surface proteins

Bovine lactoferrin promotes bifidobacterial growth. Its binding to bifidobacteria is thought to be responsible for such action. After separating the bovine lactoferrin half molecule and extraction of surface proteins from bifidobacteria, binding profiles were observed by immunoblotting. No binding appeared when lactoferrin C-lobe was reacted with the cell surface proteins on a polyvinylidene difluoride membrane. Conversely, a 50-kDa band appeared when the surface proteins were reacted with either intact or nicked bovine lactoferrin. This result strongly suggests that the binding region could be lactoferrin N-lobe. Interestingly, despite the absence of binding, C-lobe enhanced bifidobacterial growth.     

Influence of oral doxycycline therapy on the diversity and antibiotic susceptibility of human intestinal bifidobacterial population

Aim:  To evaluate the influence of doxycycline therapy on the composition and antibiotic susceptibility of intestinal bifidobacteria.
Methods and Results:  Faecal samples were collected from nine subjects receiving doxycycline therapy and ten control subjects, and analysed for bifidobacteria by culturing and PCR–DGGE (denaturing gradient gel electrophoresis). A marked decrease in the diversity (average number of amplicons detected by PCR–DGGE 0·8 in the antibiotic vs 4·3 in the control group) of Bifidobacterium populations was observed during doxycycline therapy. The proportion of a tetracycline-resistant bifidobacterial population was higher in the antibiotic group than in the control group (83% vs 26%). Based on the tet gene PCR, resistance could be associated with the presence of tet (W). In two subjects, strains representing highly similar genetic fingerprints but different tetracycline susceptibilities were detected. A mutation causing lack of functionality in the tet (W) was observed in one of the susceptible strains.
Conclusions:  Doxycycline therapy had a drastic effect on the diversity and tetracycline susceptibility of intestinal Bifidobacterium populations.
Significance and Impact of the Study:  The use of broad-spectrum antibiotics increased the pool of tetracycline-resistant commensal bacteria in the intestine. The detection of resistance genes alone is not sufficient for the evaluation of bacterial antibiotic resistance.