The influence of microbiota in human health is well-known. Imbalances in microbiome structure have been linked to several diseases. Modulation of microbiota composition through probiotic therapy is an attempt to harness the beneficial effects of commensal microbiota. Although, there is wide knowledge of the responses induced by gut microbiota, the microbial factors that mediate these effects are not well-known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a secretion mechanism of microbial factors, which have an important role in intercellular communication. Here, we investigated whether OMVs from the probiotic Escherichia coli strain Nissle 1917 (EcN) or the commensal E. coli strain ECOR12 trigger immune responses in various cellular models: (i) peripheral blood mononuclear cells (PBMCs) as a model of intestinal barrier disruption, (ii) apical stimulation of Caco-2/PMBCs co-culture as a model of intact intestinal mucosa, and (iii) colonic mucosa explants as an ex vivo model. Stimulations with bacterial lysates were also performed. Whereas, both OMVs and lysates activated expression and secretion of several cytokines and chemokines in PBMCs, only OMVs induced basolateral secretion and mRNA upregulation of these mediators in the co-culture model. We provide evidence that OMVs are internalized in polarized Caco-2 cells. The activated epithelial cells elicit a response in the underlying immunocompetent cells. The OMVs effects were corroborated in the ex vivo model. This experimental study shows that OMVs are an effective strategy used by beneficial gut bacteria to communicate with and modulate host responses, activating signaling events through the intestinal epithelial barrier.

BackgroundEscherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells.ResultsHere we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections.ConclusionsSat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0591-5) contains supplementary material, which is available to authorized users.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response.

Abiotrophia and Granulicatella species are fastidious organisms, representing around 1%–3% of infective endocarditis (IE). Little is known about the optimal antibiotic treatment of these species, and daptomycin has been suggested as a therapeutic option. We describe the antimicrobial profile in Abiotrophia and Granulicatella IE isolates, investigate high-level daptomycin resistance (HLDR) development and evaluate daptomycin activity in combination therapy. In vitro studies with 16 IE strains (6 A. defectiva , 9 G. adiacens and 1 G. elegans ) were performed using microdilution to determine minimal inhibitory concentration (MIC) and time-kill methodology to evaluate combination therapy. Daptomycin non-susceptibility (DNS; MIC≥ 2 mg/L) and HLDR (MIC≥256 mg/L) were based on existing Clinical and Laboratory Standards (CLSI) breakpoints for viridans streptococci. All isolates were susceptible to vancomycin: G. adiacens was more susceptible to penicillin and ampicillin than A. defectiva (22% vs. 0%, and 67% vs. 33%) but less susceptible to ceftriaxone and daptomycin (56% vs. 83%, and 11% vs. 50%). HLDR developed in both A. defectiva (33%) and G. adiacens (78%) after 24h exposure to daptomycin. Combination therapy did not prevent the development of daptomycin resistance with ampicillin (2/3 strains), gentamicin (2/3 strains), ceftriaxone (2/3 strains) or ceftaroline (2/3 strains). Once developed, HLDR was stable for a prolonged time (>3 weeks) in G. adiacens , whereas in A. defectiva the HLDR it reversed to baseline MIC at day 10. This study is first to demonstrate rapid HLDR development in Abiotrophia and Granulicatella species in vitro . Resistance was stable, and most combination therapies did not prevent it.