Excision of an Unstable Pathogenicity Island in Salmonella enterica Serovar Enteritidis Is Induced during Infection of Phagocytic Cells

The availability of the complete genome sequence of several Salmonella enterica serovars has revealed the presence of unstable genetic elements in these bacteria, such as pathogenicity islands and prophages. This is the case of Salmonella enterica serovar Enteritidis (S. Enteritidis), a bacterium that causes gastroenteritis in humans and systemic infection in mice. The whole genome sequence analysis for S. Enteritidis unveiled the presence of several genetic regions that are absent in other Salmonella serovars. These regions have been denominated “regions of difference” (ROD). In this study we show that ROD21, one of such regions, behaves as an unstable pathogenicity island. We observed that ROD21 undergoes spontaneous excision by two independent recombination events, either under laboratory growth conditions or during infection of murine cells. Importantly, we also found that one type of excision occurred at higher rates when S. Enteritidis was residing inside murine phagocytic cells. These data suggest that ROD21 is an unstable pathogenicity island, whose frequency of excision depends on the environmental conditions found inside phagocytic cells

Infection of Mice by Salmonella enterica Serovar Enteritidis Involves Additional Genes That Are Absent in the Genome of Serovar Typhimurium

Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro, rfa, rfb, phoP, and phoQ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system (SEN4290 to SEN4292), the peg fimbrial operon (SEN2144A to SEN2145B), a putative pathogenicity island (SEN1970 to SEN1999), and a type VI secretion system remnant SEN1001, encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A ΔSEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro. Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro. The genes identified here may perform novel virulence functions not characterized in previous Salmonella models

Safety and immunomodulatory effects of three probiotic strains isolated from the feces of breast-fed infants in healthy adults: SETOPROB study

We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, double-blind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers’ feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects.Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3582 managed by the Fundacion General Empresa-Universidad de Granada

MASTREE+: Time-series of plant reproductive effort from six continents.

Significant gaps remain in understanding the response of plant reproduction to environmental change. This is partly because measuring reproduction in long-lived plants requires direct observation over many years and such datasets have rarely been made publicly available. Here we introduce MASTREE+, a data set that collates reproductive time-series data from across the globe and makes these data freely available to the community. MASTREE+ includes 73,828 georeferenced observations of annual reproduction (e.g. seed and fruit counts) in perennial plant populations worldwide. These observations consist of 5971 population-level time-series from 974 species in 66 countries. The mean and median time-series length is 12.4 and 10 years respectively, and the data set includes 1122 series that extend over at least two decades (≥20 years of observations). For a subset of well-studied species, MASTREE+ includes extensive replication of time-series across geographical and climatic gradients. Here we describe the open-access data set, available as a.csv file, and we introduce an associated web-based app for data exploration. MASTREE+ will provide the basis for improved understanding of the response of long-lived plant reproduction to environmental change. Additionally, MASTREE+ will enable investigation of the ecology and evolution of reproductive strategies in perennial plants, and the role of plant reproduction as a driver of ecosystem dynamics

Riociguat treatment in patients with chronic thromboembolic pulmonary hypertension: Final safety data from the EXPERT registry

Objective: The soluble guanylate cyclase stimulator riociguat is approved for the treatment of adult patients with pulmonary arterial hypertension (PAH) and inoperable or persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH) following Phase

Deletion of a prophage-like element causes attenuation of Salmonella enterica serovar Enteritidis and promotes protective immunity

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a wide host range serovar belonging to the S. enterica genus. Worldwide, it is one of the most frequent causes of food borne disease. Similar to S. Typhimurium, some virulence genes of S. Enteritidis are located in pathogenicity islands and prophages. In this study we have generated a mutant strain of S. Enteritidis lacking a prophage-like element, denominated φSE12. The resulting mutant strain was attenuated and promoted protective immunity in infected mice. Although S. Enteritidis strains lacking the complete prophage φSE12 remained capable of surviving inside phagocytic cells, they showed a significantly reduced capacity to colonize internal organs and failed to cause lethal disease in mice. Consistent with these data, infection with S. Enteritidis strains lacking prophage φSE12 promoted the production of anti-. Salmonella IgG antibodies and led to protection against a challenge with virulent strains of S. Enteritidis. The

Phosphate Favors the Biosynthesis of CdS Quantum Dots in Acidithiobacillus thiooxidans ATCC 19703 by Improving Metal Uptake and Tolerance

Recently, we reported the production of Cadmium sulfide (CdS) fluorescent semiconductor nanoparticles (quantum dots, QDs) by acidophilic bacteria of the Acidithiobacillus genus. Here, we report that the addition of inorganic phosphate to Acidithiobacillus thiooxidans ATCC 19703 cultures favors the biosynthesis of CdS QDs at acidic conditions (pH 3.5). The effect of pH, phosphate and cadmium concentrations on QDs biosynthesis was studied by using Response Surface Methodology (RSM), a multivariate technique for analytical optimization scarcely used in microbiological studies to date. To address how phosphate affects intracellular biosynthesis of CdS QDs, the effect of inorganic phosphate on bacterial cadmium-uptake was evaluated. By measuring intracellular levels of cadmium we determined that phosphate influences the capacity of cells to incorporate this metal. A relation between cadmium tolerance and phosphate concentrations was also determined, suggesting that phosphate participates in the adaptation of bacteria to toxic levels of this metal. In addition, QDs-biosynthesis was also favored by the degradation of intracellular polyphosphates. Altogether, our results indicate that phosphate contributes to A. thiooxidans CdS QDs biosynthesis by influencing cadmium uptake and cadmium tolerance. These QDs may also be acting as a nucleation point for QDs formation at acidic pH. This is the first study reporting the effect of phosphates on QDs biosynthesis and describes a new cadmium-response pathway present in A. thiooxidans and most probably in other bacterial species

Spontaneous Excision of the Salmonella enterica Serovar Enteritidis-Specific Defective Prophage-Like Element φSE14 ▿ †

Salmonella enterica serovar Enteritidis has emerged as a major health problem worldwide in the last few decades. DNA loci unique to S. Enteritidis can provide markers for detection of this pathogen and may reveal pathogenic mechanisms restricted to this serovar. An in silico comparison of 16 Salmonella genomic sequences revealed the presence of an ∼12.5-kb genomic island (GEI) specific to the sequenced S. Enteritidis strain NCTC13349. The GEI is inserted at the 5′ end of gene ydaO (SEN1377), is flanked by 308-bp imperfect direct repeats (attL and attR), and includes 21 open reading frames (SEN1378 to SEN1398), encoding primarily phage-related proteins. Accordingly, this GEI has been annotated as the defective prophage SE14 in the genome of strain NCTC13349. The genetic structure and location of φSE14 are conserved in 99 of 103 wild-type strains of S. Enteritidis studied here, including reference strains NCTC13349 and LK5. Notably, an extrachromosomal circular form of φSE14 was detected in every strain carrying this island. The presence of attP sites in the circular forms detected in NCTC13349 and LK5 was confirmed. In addition, we observed spontaneous loss of a tetRA-tagged version of φSE14, leaving an empty attB site in the genome of strain NCTC13349. Collectively, these results demonstrate that φSE14 is an unstable genetic element that undergoes spontaneous excision under standard growth conditions. An internal fragment of φSE14 designated Sdf I has been used as a serovar-specific genetic marker in PCR-based detection systems and as a tool to determine S. Enteritidis levels in experimental infections. The instability of this region may require a reassessment of its suitability for such applications

Cell-Free Culture Supernatant of Bifidobacterium breve CNCM I-4035 Decreases Pro-Inflammatory Cytokines in Human Dendritic Cells Challenged with Salmonella typhi through TLR Activation

Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro-inflammatory pathways.This study was supported by Hero Spain S. A. through a number 3143 contract signed with the Fundación General Universidad de Granada Empresa and co-sponsored by a CDTI project, a public entity of the Ministry of Economy and Competitiveness of the Spanish Government

Effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 on Hepatic Steatosis in Zucker Rats

We have previously described the safety and immunomodulatory effects of Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036 in healthy volunteers. The scope of this work was to evaluate the effects of these probiotic strains on the hepatic steatosis of obese rats. We used the Zucker rat as a genetic model of obesity. Zucker-Leprfa/fa rats received one of three probiotic strains, a mixture of L. paracasei CNCM I-4034 and B. breve CNCM I-4035, or a placebo for 30 days. An additional group of Zucker-lean+/fa rats received a placebo for 30 days. No alterations in intestinal histology, in the epithelial, lamina propria, muscular layers of the ileal or colonic mucosa, or the submucosae, were observed in any of the experimental groups. Triacylglycerol content decreased in the liver of Zucker-Leprfa/fa rats that were fed L. rhamnosus, B. breve, or the mixture of B. breve and L. paracasei. Likewise, the area corresponding to neutral lipids was significantly smaller in the liver of all four groups of Zucker-Leprfa/fa rats that received probiotics than in rats fed the placebo. Zucker-Leprfa/fa rats exhibited significantly greater serum LPS levels than Zucker-lean+/fa rats upon administration of placebo for 30 days. In contrast, all four groups of obese Zucker-Leprfa/fa rats that received LAB strains exhibited serum LPS concentrations similar to those of Zucker-lean+/fa rats. Serum TNF-α levels decreased in the Zucker-Leprfa/fa rats that received B. breve, L. rhamnosus, or the mixture, whereas L. paracasei feeding decreased IL-6 levels in the serum of Zucker-Leprfa/fa rats. In conclusion, the probiotic strains reduced hepatic steatosis in part by lowering serum LPS, and had an anti-inflammatory effect in obese Zucker rats.Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3545 managed by the Fundacion General Empresa-Universidad de Granada