Impact of Carbohydrates on the Aggregation of Probiotic Bacteria

Probiotic bacteria can be beneficial to the gastrointestinal tract in the human body in numerous ways. Autoaggregation may play a key role in allowing probiotics to prevent pathogenic organisms from colonizing the intestinal system. Currently, scientific research does not account for the extent in which the autoaggregation capacities of probiotics may be influenced by carbohydrates. In this experiment, nine carbohydrate sources, including those with prebiotic qualities, were applied to eighteen strains of bacteria of the Lactobacillus genus. The experiment evaluated the autoaggregation abilities of the lactobacilli strains exposed to the carbohydrate treatments. Generally, no carbohydrates stimulated the autoaggregation of most strains of lactobacilli. However, experimental results confirmed the rapid autoaggregation of Lactobacillus acidophilus La-5 exposed to treatments of 2’-fucosyllactose. These experimental results are relevant in understanding how carbohydrates may indirectly impact how probiotics can prevent pathogens from colonizing the gastrointestinal tract within the human body.Ope

A keystone Methylobacterium strain in biofilm formation in drinking water

The structure of biofilms in drinking water systems is influenced by the interplay between biological and physical processes. Bacterial aggregates in bulk fluid are important in seeding biofilm formation on surfaces. In simple pure and co-cultures, certain bacteria, including Methylobacterium, are implicated in the formation of aggregates. However, it is unclear whether they help to form aggregates in complex mixed bacterial communities. Furthermore, different flow regimes could affect the formation and destination of aggregates. In this study, real drinking water mixed microbial communities were inoculated with the Methylobacterium strain DSM 18358. The propensity of Methylobacterium to promote aggregation was monitored under both stagnant and flow conditions. Under stagnant conditions, Methylobacterium enhanced bacterial aggregation even when it was inoculated in drinking water at 1% relative abundance. Laminar and turbulent flows were developed in a rotating annular reactor. Methylobacterium was found to promote a higher degree of aggregation in turbulent than laminar flow. Finally, fluorescence in situ hybridisation images revealed that Methylobacterium aggregates had distinct spatial structures under the different flow conditions. Overall, Methylobacterium was found to be a key strain in the formation of aggregates in bulk water and subsequently in the formation of biofilms on surfaces

Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium Actinomyces oris.

The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans-acting factors contributing to these processes. We report here a large-scale Tn5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis, and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn5 insertions in fimA, cafA, or srtC2, as expected; the latter were mapped to genes coding for uncharacterized proteins and various nuo genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of nuo genes and ubiE, a menaquinone C-methyltransferase-encoding gene downstream of the nuo locus, confirmed the pilus and coaggregation defects. Both nuoA and ubiE mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the ubiE mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the A. oris electron transport chain is biochemically linked to pilus assembly via oxidative protein folding.IMPORTANCE The Gram-positive actinobacterium A. oris expresses adhesive pili, or fimbriae, that are essential to biofilm formation and Actinomyces interactions with other bacteria, termed coaggregation. While the critical role of the conserved sortase machine in pilus assembly and the disulfide bond-forming catalyst MdbA in oxidative folding of pilins has been established, little is known about other trans-acting factors involved in these processes. Using a Tn5 transposon screen for mutants defective in coaggregation with Streptococcus oralis, we found that genetic disruption of the NADH dehydrogenase and menaquinone biosynthesis detrimentally alters pilus assembly. Further biochemical characterizations determined that menaquinone is important for reactivation of MdbA. This study supports the notion that the electron transport chain is biochemically linked to pilus assembly in A. oris via oxidative folding of pilin precursors

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated.IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen

Protective Yeasts Control V. anguillarum Pathogenicity and Modulate the Innate Immune Response of Challenged Zebrafish (Danio rerio) Larvae

Indexación: Web of ScienceWe investigated mechanisms involved in the protection of zebrafish (Danio rerio) larvae by two probiotic candidate yeasts, Debaryornyces hansenii 97 (Dh97) and Yarrowia Iypolitica 242 (YI242), against a Vibrio anguillarum challenge. We determined the effect of different yeast concentrations (10(4)-10(7) CFU/mL) to: (i) protect larvae from the challenge, (ii) reduce the in vivo pathogen concentration and (iii) modulate the innate immune response of the host. To evaluate the role of zebrafish microbiota in protection, the experiments were performed in conventionally raised and germ free larvae. In vitro co-aggregation assays were performed to determine a direct yeast-pathogen interaction. Results showed that both yeasts significantly increased the survival rate of conventionally raised larvae challenged with V. anguillarum. The concentration of yeasts in larvae tended to increase with yeast inoculum, which was more pronounced for Dh97. Better protection was observed with Dh97 at a concentration of 106 CFU/mL compared to 104 CFU/mL. In germ-free conditions V anguillarum reached higher concentrations in larvae and provoked significantly more mortality than in conventional conditions, revealing the protective role of the host microbiota. Interestingly, yeasts were equally (Dh97) or more effective (YI242) in protecting germ-free than conventionally-raised larvae, showing that protection can be exerted only by yeasts and is not necessarily related to modulation of the host microbiota. Although none of the yeasts co aggregated with V anguillarum, they were able to reduce its proliferation in conventionally raised larvae, reduce initial pathogen concentration in germ-free larvae and prevent the upregulation of key components of the inflammatory/anti-inflammatory response (il1b, tnfa, c3, mpx, and il10, respectively). These results show that protection by yeasts of zebrafish larvae challenged with V anguillarum relates to an in vivo anti-pathogen effect, the modulation of the innate immune system, and suggests that yeasts avoid the host-pathogen interaction through mechanisms independent of co-aggregation. This study shows, for the first time, the protective role of zebrafish microbiota against V. anguillarum infection, and reveals mechanisms involved in protection by two non-Saccharomyces yeasts against this pathogen.http://journal.frontiersin.org/article/10.3389/fcimb.2016.00127/ful

Acinetobacter calcoaceticus plays a bridging function in drinking water biofilms

Intergeneric coaggregation of six drinking water autochthonous heterotrophic bacteria isolated from a model laboratory system were tested for their ability to coaggregate by a visual assay and by two microscopic techniques (epifluorescence and scanning electron microscopies). One isolate, identified as Acinetobacter calcoacticus, was found not only to autoaggregate, but also to coaggregate with four of the five other isolates (Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.) to different degrees as assessed by the visual assay, highlighting a possible bridging function in a biofilm consortium. In its absence, no coaggregation was found. Microscopic observations revealed a higher degree of interaction for all the aggregates than did the visual assay. Heat and protease reversed autoaggregation and coaggregation, suggesting that interactions were lectin-saccharide mediated. The increase/decrease in the level of extracellular proteins and polysaccharides produced during intergeneric bacteria association was not correlated with coaggregation occurrence, but probably with coaggregation strength. The bridging function of A. calcoaceticus was evidenced by multispecies biofilm studies through a strain exclusion process.Este trabalho investiga a co-agregação intergenérica de seis bactérias heterotróficas autóctones de água potável isoladas de um sistema laboratorial modelo, testando assim a sua capacidade de co-agregação através do ensaio visual e de duas técnicas microscópicas (microscopia de epifluorescência e microscopia electrónica de varrimento). Para o isolado identificado como Acinetobacter calcoaceticus, foi detectado através do ensaio visual que não só auto-agrega, mas também co-agrega, a diferentes intensidades, com quatro dos outros cinco isolados (Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp.), realçando a possível função de ligação em biofilmes multi-espécie. Na sua ausência não foi detectada co-agregação. As observações microscópicas revelaram um maior grau de interacção para todos os agregados do que a detectada pelo ensaio visual. O tratamento com calor e protease reverteram a auto-agregação e a co-agregação, sugerindo que as interacções são mediadas por lectinas-açúcares. O aumento/diminuição no nível de proteínas e polissacarídeos extracelulares produzidos durante os fenómenos de co-agregação não estão relacionados com a sua ocorrência, mas provavelmente com a sua força de interacção. A função de ligação da A. calcoaceticus nos consórcios microbianos foi evidenciada pela formação de biofilmes multi-espécie, através de um processo de exclusão bacteriana.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/31661/2006, FRH/BPD/20582/200

Prevotella intermedia and Prevotella nigrescens serotypes, ribotypes and binding characteristics

type strains and 62 clinical isolates of Prevotella intermedia and Prevotella nigrescens were typed with the use of genomic DNA fingerprints and rRNA gene probes. The strains were further serotyped with monoclonal antibodies and characterized with SDS-PAGE, enzymatic activities, hemolysis and hemagglutination and coaggregation with Streptococcus and Actinomyces spp. P. intermedia and P. nigrescens were found to have distinct ribotype patterns which correspond to previously defined serotypes I and II/III, respectively. No clear phenotypic difference related to hemolysis, hemagglutination and coaggregation with Streptococcus and Actinomyces species, or expression of aminopeptides and lipase was found between P. intermedia and P. nigrescen

Escherichia coli and Enterococcus faecalis are able to incorporate and enhance a pre-formed Gardnerella vaginalis biofilm

Gardnerella vaginalis is the most frequent microorganism found in bacterial vaginosis (BV), while Escherichia coli and Enterococcus faecalis are amongst the most frequent pathogens found in urinary tract infections (UTIs). This study aimed to evaluate possible interactions between UTIs pathogens and G. vaginalis using an in vitro dual-species biofilm model. Our results showed that dual-species biofilms reached significantly higher bacterial concentration than mono-species biofilms. Moreover, visualization of dual-populations species in the biofilms, using the epifluorescence microscopy, revealed that all of the urogenital pathogens co-existed with G. vaginalis. In conclusion, our work demonstrates that uropathogens can incorporate into mature BV biofilms.This work was supported by Portuguese National Funds (FCT) under the Strategic Project of UID/BIO/04469/2013 unit and co-funded by the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). JC and DM acknowledge the financial support of individual grants SFRH/BD/93963/2013 and SFRH/BD/87569/2012, respectively. NC is an Investigador FCT