The influence of polyunsaturated fatty acids on probiotic growth and adhesion

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.     

Adaptation of adhesion test using Caco-2 cells for anaerobic bacterium<Emphasis Type="Italic">Pseudobutyrivibrio xylanivorans</Emphasis>, a probiotic candidate

Pseudobutyrivibrio xylanivorans strain Mz5(T), an anaerobic bacterium (originating from the rumen of a Holstein-Friesian cow), has some attributes that make it a possible probiotic strain (very active hydrolases, bacteriocin and conjugated linoleic acid production). For the estimation of its adhesion ability, the adhesion test on Caco-2 cells was introduced and adapted. The adhesion was performed in an anaerobic glove box in standard 24-well plates at neutral pH for 30 min. The best method for separation of the adhered bacteria from Caco-2 cells appeared to be homogenization with an automatic pipette. The number of adhered bacteria was too small to be determined microscopically, so a new approach, i.e. detection of the apparent lag phase in liquid growth medium was tested. Under the selected assay conditions 1.04 bacterial cells from the late exponential phase adhered to one Caco-2 cell, which confirms the adhesion capability of P. xylanivorans Mz5(T). The adapted adhesion test using Caco-2 cells is suitable for estimation of adhesion capability of anaerobic bacteria.     

Comparison of in vitro models to study bacterial adhesion to the intestinal epithelium

Aims:  To evaluate the adhesion ability of intestinal bacteria to different in vitro models of intestinal epithelia, and to estimate the suitability of these models and the type of interactions involved.
Methods and results:  The adhesion of probiotic ( Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp . lactis Bb12), commensal ( B. animalis IATA-A2 and B. bifidum IATA-ES2) and potentially pathogenic bacteria ( E. coli and L. monocytogenes ) was determined. The adhesion models used were polycarbonate-well plates, with or without mucin, and different configurations of Caco-2 and/or HT29-MTX cell cultures. All bacteria adhered to wells without mucin (2·6–27·3%), the values being highly variable depending on the bacterial strain. Adhesion percentages of potentially probiotic bacteria to Caco-2 cultures were remarkably lower ( P  <   0·05) than those to mucin, and more similar to those of pathogenic strains. The lowest adhesion of different bacterial strains was detected on HT29-MTX (0·5–2·3%) cultures and Caco-2/HT29-MTX (0·6–3·2%) cocultures, while these values were increased in Caco-2 cultures plus mucin.
Conclusions:  The results suggested that bacterial strains exhibit different capacities to adhere to cellular components and several types of mucin present in different models, showing preferences for intestinal MUC2.
Significance and impact of the study:  The use of Caco-2 cells monolayer plus mucin (type II) better approaches the physiological characteristics of in vivo situation, providing a reliable and suitable in vitro model to evaluate bacterial adhesion.     

应用实时荧光定量PCR方法定量检测双歧杆菌对Caco-2细胞的黏附

【目的】采用实时荧光定量PCR的方法定量分析黏附于Caco-2细胞的双歧杆菌,并建立一种快速有效分离黏附于细胞的细菌的方法。【方法】采用Triton X-100溶液处理黏附于Caco-2细胞上的菌体,确定获得最佳分离效果的处理时间;建立实时荧光定量PCR定量检测双歧杆菌的方法,获得标准曲线,进行特异性、灵敏度、重复性评价;应用建立的方法分析11株双歧杆菌对Caco-2细胞的黏附能力。【结果】Triton X-100处理黏附于Caco-2细胞的双歧杆菌的最佳作用时间为10 min。实时荧光定量PCR定量检测双歧杆菌的方法重复性好、特异性强、灵敏度高;起始模板浓度范围在104?108 CFU/mL之间具有良好的线形关系,相关系数>99%,在该浓度范围线性方程为：y=?3.345 2x+37.637 0。应用建立的方法定量分析双歧杆菌的黏附能力,与直接镜检法相比差异不显著(P>0.05),检测时间由48 h缩短至4 h。【结论】Triton X-100分离处理结合实时荧光定量PCR方法是一种快速、有效的检测双歧杆菌对Caco-2细胞黏附能力的方法。     

A fluorescence-based method for the detection of adhesive properties of lactic acid bacteria to Caco-2 cells

AIMS: The ability of probiotic micro-organisms to adhere to the intestinal surface is regarded as a substantial advantage in terms of bacteria persistence in the gastrointestinal tract. The aim of the present study was the development of a method based on fluorescent staining of bacteria and subsequent spectrofluorimetric detection to quantify the adhesion of several strains of Lactobacillus and Bifidobacterium to Caco-2 cells. METHODS AND RESULTS: Lactic acid bacteria strains were subjected to fluorescent staining using the viable probe carboxyfluorescein diacetate and subsequently incubated on Caco-2 monolayers. The adhesion of the micro-organisms was determined by spectrofluorimetry following the lysis of the attached bacterial cells and expressed as adhesion percentage. The values obtained for the micro-organisms tested ranged from 4% for Bifidobacterium infantis Bi1 to 10% for a Bifidobacterium mixture containing three different strains. CONCLUSIONS: In the present study we successfully applied fluorescent labelling and fluorimetric detection to investigate the adhesive properties of some Lactobacillus and Bifidobacterium strains and a Bifidobacterium mixture to Caco-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The results proved that fluorescent labelling is suitable for adhesion studies and provides a reliable and safer alternative to radioactive labelling.     

Bacterial monocultures, propionate, butyrate and H2O2 modulate the expression, secretion and structure of the fasting-induced adipose factor in gut epithelial cell lines

Previous research showed that an intestinal microbial community represses the fasting-induced adipose factor (FIAF) in the gut epithelium, thereby increasing fat storage in the host. This study was designed to investigate the overall effect of different bacterial species and metabolites on FIAF in intestinal (Caco-2, HT-29 and HCT-116) and hepatic (HepG2) cancer cell lines. First, we showed that FIAF was present in different isoforms, and secreted as N-glycosylated proteins, exclusively at the basal side of the cell monolayer. Second, co-incubation of cell lines with bacterial monocultures and metabolites altered both FIAF production and isoform appearance. Propionate and/or butyrate treatment increased FIAF expression and cleavage in all tested cell lines. In contrast, different bacteria induced cell line-specific FIAF modulation. Clostridium perfringens induced FIAF isoform changes in Caco-2 cells. Enterococcus faecalis and Bacteroides thetaiotaomicron treatment resulted in cell line-specific FIAF increases, whereas Escherichia coli significantly decreased FIAF expression in HCT-116 cells. Treatment with H(2) O(2) and peroxide-producing E. faecalis strains induced FIAF isoform changes in Caco-2 cells. Since bacteria and bacterial metabolites alter both FIAF production and isoform appearance, further investigation may reveal an important role for bacteria in FIAF-regulated physiological processes, such as cell differentiation and fat metabolism.     

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination.     

Quantitative approach in the study of adhesion of lactic acid bacteria to intestinal cells and their competition with enterobacteria

To describe the phenomena of bacterial adhesion to intestinal cells and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of Lactobacillus sp. and Escherichia coli cells that can adhere to Caco-2 cells and intestinal mucus; they also characterize the affinity of the bacteria to Caco-2 cells and intestinal and fecal mucus and the theoretical adhesion ratio of two bacteria present in a mixed suspension. The competition for adhesion between Lactobacillus rhamnosus GG and E. coli TG1 appeared to follow the proposed kinetics, whereas the competition between Lactobacillus casei Shirota and E. coli TG1 may involve multiple adhesion sites or a soluble factor in the culture medium of the former. The displacement of the adhered Lactobacillus by E. coli TG1 seemed to be a rapid process, whereas the displacement of E. coli TG1 by the Lactobacillus took more than an hour.     

Assessing the effect of different ultrasonic frequencies on bacterial viability using flow cytometry

Aims: This research investigated the effect of sonication at frequencies of 20, 40 and 580 kHz and approximately the same acoustic intensity on the viability and declumping of two micro‐organisms (Escherichia coli and Klebsiella pneumonia). Methods and Results: Two analytical methods were employed; viable plate counts (CFU ml^?1) and flow cytometry to identify and quantify both live/viable and dead bacteria in the bulk liquid. Flow cytometry results for E. coli and Kl. pneumonia indicated a high sensitivity to 20 and 40 kHz frequency with a continuous decrease in the viable cells and an increase in dead cells during experiments. In contrast, results using the higher frequency of 580 kHz indicate predominantly deagglomeration of bacterial clumps rather than cell membrane disruption (Joyce et al. 2003). Results indicate a good correlation between flow cytometry and viable plate count methodology. Conclusions: Sonication has two different effects on bacteria (i) inactivation and (ii) declumping; however, the scale of these effects is dependent on intensity and frequency. Flow cytometry provides a method to distinguish between and quantify the effects through the observation of two subpopulations: (i) live/viable and (ii) dead bacterial cells. Significance and Impact of the study: Treatment using power ultrasound has been shown to have a significant impact on microbial activity. This is the first time a study has compared the influence of a range of different frequencies, but at similar power settings on the survival of bacteria in phosphate buffer saline (PBS). This work is of importance for applications where ultrasound has been considered for use in industry as a means of disinfection including the treatment and pretreatment of water and also for the sterilization of liquid foods.     

A Quantitative Real-time PCR Assay for Quantification of Viable Listeria Monocytogenes Cells After Bacteriocin Injury in Food-First Insights

Quantitative real-time PCR may be a rapid and automated procedure for detection of bacterial pathogens from food samples. Nevertheless, when testing the effects of antimicrobials on the viability of bacterial pathogens in foods, we found that DNA from dead cells interfered greatly in the detection of viable Listeria monocytogenes after treatment with the broad-spectrum bacteriocin enterocin AS-48. To overcome this problem, a quantitative real-time PCR (qRT-PCR) assay based on bacterial mRNA was adapted to quantify viable L. monocytogenes in food after bacteriocin treatments. The procedure allowed a better and faster estimation of viable cells compared to PALCAM viable cell counts when the threshold level was 2 log units/g of food, while PALCAM viable count allowed detection of one log unit/g. This procedure may be useful to verify the efficacy of bacteriocins against L. monocytogenes in foods.     

Adhesion of lactic acid bacteria to caco-2 cells and their effect on cytokine secretion

Cytokines secreted by human enterocytes play a critical role in mucosal and systemic immunity. Intestinal microorganisms can influence this secretion. In the present study, 30 strains of lactic acid bacteria were characterized for their adhesion to Caco-2 cells and their potential to stimulate proinflammatory cytokine secretion by this cell line. The bacteria adhered in a strain-dependent manner to Caco-2 cells. Contact with lactobacilli did not result in the production of IL-6 or IL-8. A slight IL-6 and IL-8 production by a Caco-2 cell was detected after exposure to 8 of the tested Bifidobacterium strains. No correlation was found between adhesion and cytokine induction among the bacteria tested. This indicates that lactic acid bacteria, even those with strong adhesive properties, are not very likely to trigger an inflammatory response in human enterocytes.     

Quantification of bacterial internalization by host cells using a beta-lactamase reporter strain: Neisseria gonorrhoeae invasion into cervical epithelial cells requires bacterial viability

Neisseria gonorrhoeae can invade into cervical epithelial cells to overcome this host defense barrier. We developed a beta-lactamase reporter system that allowed us to quantify at the single cell level if a host cell internalized a viable or nonviable microorganism. We autodisplayed beta-lactamase on the surface of FA1090 [FA1090Phi(bla-iga')] and demonstrated by confocal fluorescence microscopy and flow cytometry that FA1090Phi(bla-iga') cleaved the beta-lactamase substrate CCF2-AM loaded into host cells only when gonococci were internalized by these host cells. While FA1090Phi(bla-iga') adhered to almost all ME180 cells, viable N. gonorrhoeae were internalized by only a subset of cells during infection. Nonviable gonococci adhered to, but were not internalized by ME180 cells, and failed to recruit F-actin to sites of adherent bacteria. Overall, we show that epithelial cell invasion is a dynamic process that requires viable N. gonorrhoeae. We demonstrate the advantages of the beta-lactamase reporter system over the gentamicin protection assay in quantifying bacterial invasion. The reporter system that we have developed can be adapted to studying the internalization of any bacterial species into any host cell.     

Quantitative Approach in the Study of Adhesion of Lactic Acid Bacteria to Intestinal Cells and Their Competition with Enterobacteria

To describe the phenomena of bacterial adhesion to intestinal cells and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of Lactobacillus sp. and Escherichia coli cells that can adhere to Caco-2 cells and intestinal mucus; they also characterize the affinity of the bacteria to Caco-2 cells and intestinal and fecal mucus and the theoretical adhesion ratio of two bacteria present in a mixed suspension. The competition for adhesion between Lactobacillus rhamnosus GG and E. coli TG1 appeared to follow the proposed kinetics, whereas the competition between Lactobacillus casei Shirota and E. coli TG1 may involve multiple adhesion sites or a soluble factor in the culture medium of the former. The displacement of the adhered Lactobacillus by E. coli TG1 seemed to be a rapid process, whereas the displacement of E. coli TG1 by the Lactobacillus took more than an hour.     

Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.     

Kefiran protects Caco-2 cells from cytopathic effects induced by <Emphasis Type="Italic">Bacillus cereus</Emphasis> infection

The aim of this work was to evaluate the ability of kefiran to antagonize cytopathic effects triggered by Bacillus cereus strain B10502 on cultured human enterocytes (Caco-2 cells). Cell damage was evaluated by F-actin labelling, scanning electron microscopy and determination of ratios of necrotic and detached cells. To assess the interaction between kefiran and bacteria or eukaryotic cells, flow cytometric analysis was conducted with FITC-labelled kefiran. Kefiran significantly protected infected cells from cytopathic effects induced by B. cereus such as cell necrosis, F-actin disorganisation and microvilli effacement, although presence of kefiran did not modify the adhesion of microorganisms to cultured human enterocytes. Results could be ascribed to the ability of kefiran to interact with both bacteria and eukaryotic cells thus antagonizing interactions necessary for maximal biological effects. Our findings encourage further research on the use of bacterial exopolysaccharides to antagonize virulence factors associated to direct bacteria–cell interactions.     

蒙脱石对细菌黏附Caco-2细胞的影响

采用Caco-2细胞培养模型,观察两歧双歧杆菌、嗜酸乳杆菌、嗜水气单胞菌、副溶血弧菌、大肠杆菌、鼠伤寒沙门菌的黏附率,并在培养液中加入蒙脱石,计算蒙脱石对细菌黏附的阻断率,探讨蒙脱石对上述细菌黏附作用的影响。结果表明:所试菌与Caco-2细胞均有不同程度的黏附作用;蒙脱石对细菌黏附Caco-2细胞均有不同程度的阻断作用,对病原菌黏附Caco-2细胞的阻断作用要明显大于其对益生菌的阻断效果,其中对大肠杆菌、鼠伤寒沙门菌、嗜水气单胞菌、副溶血弧菌黏附的阻断率分别为54.22%、48.41%、60.53%、50.64%,而对两歧双歧杆菌、嗜酸乳杆菌黏附的阻断率分别为25.64%和21.49%。结果提示蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位。     

Study of the Adhesion of <Emphasis Type="Italic">Bifidobacterium bifidum</Emphasis> MIMBb75 to Human Intestinal Cell Lines

The aim of this study was to investigate the adhesive phenotype of the human intestinal isolate Bifidobacterium bifidum MIMBb75 to human colon carcinoma cell lines. We have previously shown that the adhesion of this strain to Caco-2 cells is mediated by an abundant surface lipoprotein named BopA. In this study, we found that this strain adheres to Caco-2 and HT-29 cells, and that its adhesion strongly depends on the environmental conditions, including the presence of sugars and bile salts and the pH. Considerably more adhesion to a Caco-2 monolayer occurred in the presence of fucose and mannose and less when MIMBb75 grew in Oxgall bile salts compared to standard environmental conditions. In particular, growth in Oxgall bile salts reduced the adhesion ability of MIMBb75 and modified the SDS-PAGE profile of the cell wall associated proteins of the strain. The pH markedly affected both adhesion to Caco-2 and bacterial autoaggregation. Finally, experiments with sodium metaperiodate suggested that not only proteinaceous determinants are involved in the adhesion process of B. bifidum. In conclusion, it seems that the colonization strategy of this bacterium can be influenced by factors varying along the gastrointestinal tract, such as the presence of specific sugars and bile salts and the pH, possibly limiting the adhesion of B. bifidum to only restricted distal sites of the gut.