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

Genetic Basis of Tetracycline Resistance in Bifidobacterium animalis subsp lactis

All strains of Bifidobacterium animalis subsp. lactis described to date show medium level resistance to tetracycline. Screening of 26 strains from a variety of sources revealed the presence of tet(W) in all isolates. A transposase gene upstream of tet(W) was found in all strains, and both genes were cotranscribed in strain IPLAIC4. Mutants with increased tetracycline resistance as well as tetracycline-sensitive mutants of IPLAIC4 were isolated and genetically characterized. The native tet(W) gene was able to restore the resistance phenotype to a mutant with an alteration in tet(W) by functional complementation, indicating that tet(W) is necessary and sufficient for the tetracycline resistance seen in B. animalis subsp. lactis

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

Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG

[Aims] The purpose of this study was to investigate the heat-shock response at molecular level in Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB-12 and their heat-tolerant derivatives and to characterize the changes that make the derivatives more robust in terms of heat stress.[Methods and results] The study strains were exposed for 2 h to a heat-shock treatment, Bif. animalis subsp. lactis BB-12 and its derivative at 50°C and the Lact. rhamnosus GG and its derivative at 60°C. Protein synthesis before and after heat shock was examined using proteomics and RT-qPCR. The analysis revealed that the regulation of seven proteins in both strain pairs was modified as a response to heat or between the original and the derivative strain. The comparison of wild-type strains and the heat-tolerant derivatives suggests that the acquisition of heat tolerance in the Bif. animalis subsp. lactis BB-12 derivative is due to a slightly increased constitutive level of chaperones, while in Lact. rhamnosus GG derivative, the main reason seems to be a higher ability to induce the production of chaperones.[Conclusions] This study revealed possible markers of heat tolerance in B. lactis and Lact. rhamnosus strains.[Significance and impact of study] This study increases our knowledge on how Lactobacillus and Bifidobacterium strains may acquire heat tolerance. These findings may be useful for improving the heat tolerance of existing probiotic strains as well as screening new heat-tolerant strains.This study was funded by TEKES (the Finnish Funding Agency for Technology and Innovation) grant number 398/31/2009.Peer Reviewe

Occurrence and diversity of CRISPR-Cas systems in the genus bifidobacterium

CRISPR-Cas systems constitute adaptive immune systems for antiviral defense in bacteria. We investigated the occurrence and diversity of CRISPR-Cas systems in 48 Bifidobacterium genomes to gain insights into the diversity and co-evolution of CRISPR-Cas systems within the genus and investigate CRISPR spacer content. We identified the elements necessary for the successful targeting and inference of foreign DNA in select Type II CRISPRCas systems, including the tracrRNA and target PAM sequence. Bifidobacterium species have a very high frequency of CRISPR-Cas occurrence (77%, 37 of 48). We found that many Bifidobacterium species have unusually large and diverse CRISPR-Cas systems that contain spacer sequences showing homology to foreign genetic elements like prophages. A large number of CRISPR spacers in bifidobacteria show perfect homology to prophage sequences harbored in the chromosomes of other species of Bifidobacterium, including some spacers that self-target the chromosome. A correlation was observed between strains that lacked CRISPR-Cas systems and the number of times prophages in that chromosome were targeted by other CRISPR spacers. The presence of prophage-targeting CRISPR spacers and prophage content may shed light on evolutionary processes and strain divergence. Finally, elements of Type II CRISPR-Cas systems, including the tracrRNA and crRNAs, set the stage for the development of genome editing and genetic engineering tools. © 2015 Briner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.This study was supported by startup funds from North Carolina State University. The authors thank GenProbio srl for financial support of the Laboratory of Probiogenomics. DvS is a member of the Alimentary pharmabiotic Centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant number SFI/12/RC/2273). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer Reviewe

Mobilidade de trabalhadores portugueses no sector da construção civil e obras públicas entre o norte de Portugal e a Galiza

European Employment ServicesUniversidade do Minho Escola de Economia e GestãoUniversidade de Vig

An in vivo permeability test protocol using iohexol to reduce and refine the use of laboratory rats in intestinal damage assessment

Assessment of intestinal damage in laboratory rats with experimentally-induced enteropathies is usually carried out by collecting and morphological interpreting tissue samples obtained surgically, endoscopically or at necropsy. Alternatively, changes in the gut mucosa may be less invasively evaluated with intestinal permeability (IP) tests. In contrast to human and veterinary patients, IP test protocols in laboratory rats have been highly variable, which may account for the limited use of this approach by investigators when evaluating intestinal damage. The objective of this study was to establish a refined IP test protocol using iohexol in rats that is able to differentiate between healthy rats and individuals with enteropathies. Iohexol was administered by oral gavage to twenty-eight Sprague-Dawley rats, before and after the induction of inflammatory bowel disease (IBD) with dextran sulphate sodium (DSS). Urine was cumulatively recovered during 24 h, and the presence of iohexol was measured by high-performance liquid chromatography with ultraviolet detection. The median percentage (and interquartile range) of administered iohexol in urine of healthy rats was 0.54% (0.3

Microbiota, Food, and Health

The effects of specific foods, such as products containing probiotics or prebiotics, on human health and the role of intestinal microbiota in this interaction have been a subject of scientific interest for several decades

Effect of an α-Tocopherol-Containing Antioxidant Parenteral Emulsion upon Gut Microbiota in Preterm Infants

Preterm babies are born with an immature intestine and are at higher risk for intestinal failure and needing for parenteral nutrition than full-term newborns. These babies show an altered intestinal microbiota, which may results of key importance for later health. However, there is a lack of information on effect of parenteral formulas upon the establishing infant microbiota. Moreover, the microbiota alterations present in preterm newborns have been related with an altered redox-potential which hinders the establishment of strict anaerobes. Thus, interventions aimed at reducing the oxidative stress in these infants, including those under parenteral nutrition, could also affect the intestinal microbiota composition. We assessed the effect upon the gut microbiota of the administration during 30 days of a lipid emulsion, containing ω3 polyunsaturated fatty acids and α-tocopherol, or a control emulsion containing soybean-bases lipids to parenterally-fed preterm newborns. In spite of its high antioxidant potential, the infants receiving the experimental fat emulsion showed a trend to increase of facultative anaerobes such as enterobacteria and specifically of Klebsiella. This study stresses the need of further work monitoring the impact of early-life parenteral nutrition on the establishment of the intestinal microbiota.This work was funded by the Luis Noé Fernández Award from Fundación Alimerka (Spain).Peer Reviewe