An in vitro biological model to study the effect of Clostridium difficile toxins in real time: an opportunity to select probiotics for elders

Comunicación presentada en la 2014 ENGIHR Conference: The Gut Microbiota Throughout Life, celebrada en Karlsruhe, Alemania, del 24 al 26 de septiembre de 2014Peer Reviewe

Monitoring in real time the cytotoxic effect of Clostridium difficile upon the intestinal epithelial cell line HT29

The incidence and severity of Clostridium difficile infections (CDI) has been increased not only among hospitalized patients, but also in healthy individuals traditionally considered as low risk population. Current treatment of CDI involves the use of antibiotics to eliminate the pathogen, although recurrent relapses have also been reported. For this reason, the search of new antimicrobials is a very active area of research. The strategy to use inhibitors of toxin's activity has however been less explored in spite of being a promising option. In this regard, the lack of fast and reliable in vitro screening methods to search for novel anti-toxin drugs has hampered this approach. The aim of the current study was to develop a method to monitor in real time the cytotoxicity of C. difficile upon the human colonocyte-like HT29 line, since epithelial intestinal cells are the primary targets of the toxins. The label-free, impedance based RCTA (real time cell analyser) technology was used to follow overtime the behaviour of HT29 in response to C. difficile LMG21717 producing both A and B toxins. Results obtained showed that the selection of the medium to grow the pathogen had a great influence in obtaining toxigenic supernatants, given that some culture media avoided the release of the toxins. A cytotoxic dose- and time-dependent effect of the supernatant obtained from GAM medium upon HT29 and Caco2 cells was detected. The sigmoid-curve fit of data obtained with HT29 allowed the calculation of different toxicological parameters, such as EC50 and LOAEL values. Finally, the modification in the behaviour of HT29 reordered in the RTCA was correlated with the cell rounding effect, typically induced by these toxins, visualized by time-lapsed captures using an optical microscope. Therefore, this RTCA method developed to test cytotoxicity kinetics of C. difficile supernatants upon IEC could be a valuable in vitro model for the screening of new anti-CDI agents. © 2015 Elsevier B.V.This work was financed by the FEDER European Union funds through the projects AGL2012-33278 from the Spanish Ministry of Economy and Competitiveness (MINECO), and EQUIP11 from the “Principado de Asturias” Regional Research Plan. L. Valdés acknowledges her JAE-Pre fellowship to CSIC.Peer Reviewe

Potentially probiotic and bioprotective lactic acid bacteria starter cultures antagonise the Listeria monocytogenes adhesion to HT29 colonocyte-like cells

The capability of five lactic acid bacteria (LAB) to counteract the adhesion of Listeria monocytogenes to the epithelial intestinal cell line HT29 was studied. The highest adhesion ability to HT29 was achieved by the intestinal strain Lactobacillus rhamnosus CTC1679, followed by the meat-derived strains Lactobacillus sakei CTC494 and Enterococcus faecium CTC8005. Surprisingly, the meat strains showed significantly better adhesion to HT29 than two faecal isolates of Lactobacillus casei and even significantly higher than the reference strain L. rhamnosus GG. Additionally, the anti-listerial, bacteriocin-producer starter culture L. sakei CTC494 was able to significantly reduce the adhesion of L. monocytogenes to HT29 in experiments of exclusion, competition and inhibition. The performance was better than the faecal isolate L. rhamnosus CTC1679. Our results reinforce the fact that the ability of LAB to interact with a host epithelium model, as well as to antagonise with foodborne pathogens, is a strain-specific characteristic. Additionally, it is underlined that this trait is not dependent on the origin of the bacterium, since some food LAB behave better than intestinal ones. Therefore, the search for novel strains in food niches is a suitable approach to find those with potential health benefits. These strains are likely pre-adapted to the food environment, which would make their inclusion in the formulation of probiotic foods more feasible. © 2014 Wageningen Academic Publishers.Peer Reviewe

Degenerate PCR primers for detecting putative priming glycosyltransferase genes in Bifidobacterium strains

A new PCR-based method to detect putative exopolysaccharide (EPS) producers from the genus Bifidobacterium was developed based on the detection of two priming glycosyltransferase genes: rfbP (undecaprenyl-phosphate sugar phospho-transferase) and cpsD (galactosyl-transferase). An in silico analysis of the genomes of 28 bifidobacterial strains, belonging to 8 different species, allowed us to detect rfbP, cpsD, or both, in the large majority of the genomes. Based on DNA sequence homology studies, 24 degenerated primers were synthesised in order to select the primer pairs with the broadest capacity to detect the presence of these genes. Four primer pairs targeting internal regions of rfbP and cpsD were selected, allowing the detection of at least one of the two genes in 63 out of 99 bifidobacterial strains analysed, whereas control strains from other genera yielded negative results, suggesting that these genes are widely spread in this genus. The use of these primers is recommended to screen for the potential of Bifidobacterium strains to produce EPS. © 2015 Wageningen Academic Publishers.Peer Reviewe

Immune modulating capability of two exopolysaccharide-producing bifidobacterium strains in a wistar rat model

Fermented dairy products are the usual carriers for the delivery of probiotics to humans, Bifidobacterium and Lactobacillus being the most frequently used bacteria. In this work, the strains Bifidobacterium animalis subsp. lactis IPLA R1 and Bifidobacterium longum IPLA E44 were tested for their capability to modulate immune response and the insulin-dependent glucose homeostasis using male Wistar rats fed with a standard diet. Three intervention groups were fed daily for 24 days with 10% skimmed milk, or with 109 cfu of the corresponding strain suspended in the same vehicle. A significant increase of the suppressor-regulatory TGF-β cytokine occurred with both strains in comparison with a control (no intervention) group of rats; the highest levels were reached in rats fed IPLA R1. This strain presented an immune protective profile, as it was able to reduce the production of the proinflammatory IL-6. Moreover, phosphorylated Akt kinase decreased in gastroctemius muscle of rats fed the strain IPLA R1, without affecting the glucose, insulin, and HOMA index in blood, or levels of Glut-4 located in the membrane of muscle and adipose tissue cells. Therefore, the strain B. animalis subsp. lactis IPLA R1 is a probiotic candidate to be tested in mild grade inflammation animal models. © 2014 Nuria Salazar et al.This work was financed by the Spanish Ministry of Economy and Competitiveness (MINECO) and the FEDER European Union funds through the projects AGL2010-16525 and AGL2012-33278. The authors acknowledge Dr. Baltasar Mayo (IPLA-CSIC) for kindly supplying the strain IPLA E44.Peer Reviewe

Capability of exopolysaccharide-producing Lactobacillus paraplantarum BGCG11 and its non-producing isogenic strain NB1, to counteract the effect of enteropathogens upon the epithelial cell line HT29-MTX

The putative protective role of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11, and its non-EPS-producing isogenic strain NB1, was tested upon HT29-MTX monolayers challenged with seven opportunistic pathogens. The probiotic strain Lactobacillus rhamnosus LMG18243 (GG) was used as a reference bacterium. Tested lactobacilli were able to efficiently reduce the attachment to HT29-MTX of most pathogens. Lb. paraplantarum NB1 and Lb. rhamnosus GG were more efficient reducing the adhesion of Clostridium difficile or Yersinia enterocolitica than Lb. paraplantarum BGCG11, while stain BGCG11 reduced, to a greater extent, the adhesion of Escherichia coli and Listeria monocytogenes. The detachment and cell lysis of HT29-MTX monolayers in the presence of pathogens alone and co-incubated with lactobacilli or purified EPS was followed. L. monocytogenes induced the strongest cell detachment among the seven tested pathogens and this effect was prevented by addition of purified EPS-CG11. The results suggest that this EPS could be an effective macromolecule in protection of HT29-MTX cells from the pathogen-induced lysis. Regarding innate intestinal barrier, the presence of C. difficile induced the highest IL-8 production in HT29-MTX cells and this capability was reinforced by the co-incubation with Lb. paraplantarum NB1 and Lb. rhamnosus GG. However, the increase in IL-8 production was not noticed when C difficile was co-incubated with EPS-producing Lb. paraplantarum BGCG11 strain or its purified EPS-CG11 polymer, thus indicating that the polymer could hinder the contact of bacteria with the intestinal epithelium. The measurement of mucus secreted by HT29-MTX and the expression of mud, muc2, muc3B and muc5AC genes in the presence of pathogens and lactobacilli suggested that all lactobacilli strains are weak "co-adjuvants" helping some pathogens to slightly increase the secretion of mucus by HT29-MTX, while purified EPS-CG11 did not induce mucus secretion. Taking altogether, Lb. paraplantarum BGCG11 could act towards the reinforcement of the innate mucosal barrier through the synthesis of a physical-protective EPS layer which could make difficult the contact of the pathogens with the epithelial cells.This is the peer reviewed version of the paper: Zivkovic, M., Hidalgo-Cantabrana, C., Kojic, M., Gueimonde, M., Golic, N., & Ruas-Madiedo, P. (2015). Capability of exopolysaccharide-producing Lactobacillus paraplantarum BGCG11 and its non-producing isogenic strain NB1, to counteract the effect of enteropathogens upon the epithelial cell line HT29-MTX. Food Research International, 74, 199–207.[ https://doi.org/10.1016/j.foodres.2015.05.012]Published version : [https://imagine.imgge.bg.ac.rs/handle/123456789/794

Capability of exopolysaccharide-producing Lactobacillus paraplantarum BGCG11 and its non-producing isogenic strain NB1, to counteract the effect of enteropathogens upon the epithelial cell line HT29-MTX

The putative protective role of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11, and its non-EPS-producing isogenic strain NB1, was tested upon HT29-MTX monolayers challenged with seven opportunistic pathogens. The probiotic strain Lactobacillus rhamnosus LMG18243 (GG) was used as a reference bacterium. Tested lactobacilli were able to efficiently reduce the attachment to HT29-MTX of most pathogens. Lb. paraplantarum NB1 and Lb. rhamnosus GG were more efficient reducing the adhesion of Clostridium difficile or Yersinia enterocolitica than Lb. paraplantarum BGCG11, while strain BGCG11 reduced, to a greater extent, the adhesion of Escherichia coli and Listeria monocytogenes. The detachment and cell lysis of HT29-MTX monolayers in the presence of pathogens alone and co-incubated with lactobacilli or purified EPS was followed. L. monocytogenes induced the strongest cell detachment among the seven tested pathogens and this effect was prevented by addition of purified EPS-CG11. The results suggest that this EPS could be an effective macromolecule in protection of HT29-MTX cells from the pathogen-induced lysis. Regarding innate intestinal barrier, the presence of C. difficile induced the highest IL-8 production in HT29-MTX cells and this capability was reinforced by the co-incubation with Lb. paraplantarum NB1 and Lb. rhamnosus GG. However, the increase in IL-8 production was not noticed when C. difficile was co-incubated with EPS-producing Lb. paraplantarum BGCG11 strain or its purified EPS-CG11 polymer, thus indicating that the polymer could hinder the contact of bacteria with the intestinal epithelium. The measurement of mucus secreted by HT29-MTX and the expression of muc1, muc2, muc3B and muc5AC genes in the presence of pathogens and lactobacilli suggested that all lactobacilli strains are weak >co-adjuvants> helping some pathogens to slightly increase the secretion of mucus by HT29-MTX, while purified EPS-CG11 did not induce mucus secretion. Taking altogether, Lb. paraplantarum BGCG11 could act towards the reinforcement of the innate mucosal barrier through the synthesis of a physical-protective EPS layer which could make difficult the contact of the pathogens with the epithelial cells. © 2015 Elsevier Ltd.This work was financed by the Spanish Ministry of Economy and Competiveness (MINECO) and FEDER funds (European Union) through the project AGL2012-33278, as well as by the Ministry of Education, Science and Technological Development of the Republic of Serbia grant No. 173019. The bilateral collaboration project AIB2010SE-00386 between Spain and the Republic of Serbia allowed the mobility of personnel between both institutions. C. Hidalgo-Cantabrana acknowledges his FPI pre-doctoral fellowship to MINECO (BES-2010-038270).Peer Reviewe

Gene Replacement and Fluorescent Labeling to Study the Functional Role of Exopolysaccharides in Bifidobacterium animalis subsp. lactis

An extracellular layer of exopolysaccharides (EPS) covers the surface of some Bifidobacterium animalis subsp. lactis strains, which could be of relevance for its probiotic performance. In order to understand the functional characteristics of B. animalis subsp. lactis, two isogenic strains that differ in their EPS-producing phenotype, due to a single mutation in the gene Balat_1410, were studied. By means of a double crossover recombination strategy, successfully used for the first time in bifidobacteria, Balat_1410 in the type strain B. animalis subsp. lactis DSM10140 was replaced by a mutated gene containing a non-synonymous mutation previously associated with the appearance of a mucoid-ropy phenotype. Nuclear magnetic resonance and SEC-MALS analyses showed that the novel strain harboring the mutation acquired a ropy phenotype, due to the production of a high molecular weight (HMW)-EPS that is not produced in the wild-type strain. Fluorescence labeling of both strains with two fluorescent proteins, m-Cherry and Green Fluorescent Protein, was achieved by expressing the corresponding genes under the control of a native selected promoter (the elongation factor Tu promoter). Remarkably, qualitative and quantitative fluorescence analyses demonstrated that the ropy strain displays a lower capability to adhere to human intestinal epithelial cells. In addition, the presence of the HMW-EPS reduced the capability of the producing strain to form biofilms upon three different abiotic surfaces. This work also highlights the fact that different EPS confer variable functional characteristics to the bifidobacterial surface, which may be relevant for the performance of B. animalis subsp. lactis as a probiotic. The construction of molecular tools allowing the functional characterization of surface structures in next generation probiotics is still a challenging issue that deserves further attention, given the relevant role that such molecules must play in the interaction with the host.España, MINECO AGL2015-64901-R. NC-

Monitoring in real time the formation and removal of biofilms from clinical related pathogens using an impedance-based technology

Bacteria found in diverse ecosystems grow in a community of aggregated cells that favors their survival and colonization. Different extracellular polymeric substances are used to entrap this multispecies community forming a biofilm, which can be associated to biotic and abiotic surfaces. This widespread and successful way of bacterial life, however, can lead to negative effects for human activity since many pathogen and spoiling bacteria form biofilms which are not easy to eradicate. Therefore, the search for novel anti-biofilm bio-active molecules is a very active research area for which simple, reliable, and fast screening methods are demanded. In this work we have successfully validated an impedance-based method, initially developed for the study of adherent eukaryotic cells, to monitor the formation of singlespecies biofilms of three model bacteria in real time. The xCelligence real time cell analyzer (RTCA) equipment uses specific microtiter E-plates coated with gold-microelectrodes that detect the attachment of adherent cells, thus modifying the impedance signal. In the current study, this technology allowed the distinction between biofilm-producers and non-producers of Staphylococcus aureus and Staphylococcus epidermidis, as well as the formation of Streptococcus mutans biofilms only when sucrose was present in the culture medium. Besides, different impedance values permitted discrimination among the biofilm-producing strains tested regardless of the nature of the polymeric biofilm matrix. Finally, we have continuously monitored the inhibition of staphylococcal biofilm formation by the bacteriophage phi-IPLA7 and the bacteriophage-encoded endolysin LysH5, as well as the removal of a preformed biofilm by this last antimicrobial treatment. Results observed with the impedance-based method showed high correlation with those obtained with standard approaches, such as crystal violet staining and bacteria enumeration, as well as with those obtained upon other abiotic surfaces (polystyrene and stainless steel). Therefore, this RTCA technology opens new opportunities in the biofilm research arena and its application could be further explored for other bacterial genera as well as for different bio-active molecules.This work was financed by the FEDER European Union funds through the projects AGL2015-64901-R and AGL201565673-R from the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad MINECO) and through the grants EQUIP11 and GRUPIN14-139 (Program of Science, Technology and Innovation, Principado de Asturias). DG and CH-C hold FPI-scholarships of MINECO.Peer Reviewe

Red Wine Consumption Is Associated with Fecal Microbiota and Malondialdehyde in a Human Population

[Objectives] Red wine intake has been associated with a lower risk of cardiovascular disease; its polyphenol content is the primary cause of antioxidant and anti-inflammatory properties attributed to this beverage. However, the way in which these activities are exerted is not yet clear, although some authors have proposed that intestinal microbiota could be implicated.[Methods] The association between red wine intake, inflammation, and oxidative stress parameters and fecal microbial populations has been explored in 38 adult volunteers. Food intake was recorded by means of an annual food frequency questionnaire (FFQ). Energy, cholesterol, and ethanol intake were analyzed using the nutrient Food Composition Tables developed by Centro de Enseñanza Superior de Nutrición y Dietética (CESNID) and polyphenol intake was obtained from the Phenol-Explorer Database. Fecal levels of Akkermansia, Bacteroides, Bifidobacterium, Blautia coccoides group, Clostridium leptum group, Lactobacillus group, and Faecalibacterium prausnitzii were determined by quantitative polymerase chain reaction. Serum concentrations of C-reactive protein (CRP), malondialdehyde (MDA), total antioxidant capacity (TAC), cholesterol, triglycerides and glucose were analyzed by standard methods.[Results] Subjects with regular consumption of red wine (mean = 100 ml/day) had lower serum concentrations of MDA and lower fecal levels of B. coccoides, C. leptum, Bifidobacterium, and Lactobacillus. A positive association between MDA levels and B. coccoides and Lactobacillus was also found.[Conclusion] Regular consumption of red wine appears to be associated with a reduced serum lipoperoxidation in which the intestinal microbiota may be involved.This work was funded by Biopolis SL within the framework of the e-CENIT Project SENIFOOD from the Spanish Ministry of Science and Innovation.Peer Reviewe