Sporadic type VI secretion in seventh pandemic Vibrio cholerae.

Vibrio cholerae is a pathogen that causes disease in millions of people every year by colonizing the small intestine and then secreting the potent cholera toxin. How the pathogen overcomes the colonization barrier created by the host's natural microbiota is, however, still not well understood. In this context, the type VI secretion system (T6SS) has gained considerable attention given its ability to mediate interbacterial killing. Interestingly, and in contrast to non-pandemic or environmental V. cholerae isolates, strains that are causing the ongoing cholera pandemic (7PET clade) are considered T6SS-silent under laboratory conditions. Since this idea was recently challenged, we performed a comparative in vitro study on T6SS activity using diverse strains or regulatory mutants. We show that modest T6SS activity is detectable in most of the tested strains under interbacterial competition conditions. The system's activity was also observed through immunodetection of the T6SS tube protein Hcp in culture supernatants, a phenotype that can be masked by the strains' haemagglutinin/protease. We further investigated the low T6SS activity within the bacterial populations by imaging 7PET V. cholerae at the single-cell level. The micrographs showed the production of the machinery in only a small fraction of cells within the population. This sporadic T6SS production was higher at 30 °C than at 37 °C and occurred independently of the known regulators TfoX and TfoY but was dependent on the VxrAB two-component system. Overall, our work provides new insight into the heterogeneity of T6SS production in populations of 7PET V. cholerae strains in vitro and provides a possible explanation of the system's low activity in bulk measurements.

Gut colonization by a novel Clostridium species is associated with the onset of epizootic rabbit enteropathy.

Epizootic rabbit enteropathy (ERE) represents one of the most devastating diseases affecting rabbit farms. Previous studies showing transmissibility of disease symptoms through oral inoculation of intestinal contents from sick animals suggested a bacterial infectious origin for ERE. However, no etiological agent has been identified yet. On the other hand, ERE is associated with major changes in intestinal microbial communities, pinpointing dysbiosis as an alternative cause for the disease. To better understand the role of intestinal bacteria in ERE development, we have performed a prospective longitudinal study in which intestinal samples collected from the same animals before, during and after disease onset were analyzed using high-throughput sequencing. Changes in hundreds of bacterial groups were detected after the initiation of ERE. In contrast, before ERE onset, the microbiota from rabbits that developed ERE did not differ from those that remained healthy. Notably, an expansion of a single novel Clostridium species (Clostridium cuniculi) was detected the day of ERE onset. C. cuniculi encodes several putative toxins and it is phylogenetically related to the two well-characterized pathogens C. botulinum and C. perfringens. Our results are consistent with a bacterial infectious origin of ERE and discard dysbiosis as the initial trigger of the disease. Although experimental validation is required, results derived from sequencing analysis, propose a key role of C. cuniculi in ERE initiation.

Short- and long-term effects of oral vancomycin on the human intestinal microbiota.

BACKGROUND: Oral vancomycin remains the mainstay of therapy for severe infections produced by Clostridium difficile, the most prevalent cause of healthcare-associated infectious diarrhoea in developed countries. However, its short- and long-term effects on the human intestinal microbiota remain largely unknown. METHODS: We utilized high-throughput sequencing to analyse the effects of vancomycin on the faecal human microbiota up to 22 weeks post-antibiotic cessation. The clinical relevance of the observed microbiota perturbations was studied in mice. RESULTS: During vancomycin therapy, most intestinal microbiota genera and operational taxonomic units (OTUs) were depleted in all analysed subjects, including all baseline OTUs from the phylum Bacteroidetes. This was accompanied by a vast expansion of genera associated with infections, including Klebsiella and Escherichia/Shigella. Following antibiotic cessation, marked differences in microbiota resilience were observed among subjects. While some individuals recovered a microbiota close to baseline composition, in others, up to 89% of abundant OTUs could no longer be detected. The clinical relevance of the observed microbiota changes was further demonstrated in mice, which developed analogous microbiota alterations. During vancomycin treatment, mice were highly susceptible to intestinal colonization by an antibiotic-resistant pathogen and, upon antibiotic cessation, a less-resilient microbiota allowed higher levels of pathogen colonization. CONCLUSIONS: Oral vancomycin induces drastic and consistent changes in the human intestinal microbiota. Upon vancomycin cessation, the microbiota recovery rate varied considerably among subjects, which could influence, as validated in mice, the level of susceptibility to pathogen intestinal colonization. Our results demonstrate the negative long-term effects of vancomycin, which should be considered as a fundamental aspect of the cost-benefit equation for antibiotic prescription.

Microbiome-mediated fructose depletion restricts murine gut colonization by vancomycin-resistant Enterococcus.

Multidrug-resistant organisms (MDRO) are a major threat to public health. MDRO infections, including those caused by vancomycin-resistant Enterococcus (VRE), frequently begin by colonization of the intestinal tract, a crucial step that is impaired by the intestinal microbiota. However, the specific members of the microbiota that suppress MDRO colonization and the mechanisms of such protection are largely unknown. Here, using metagenomics and mouse models that mimic the patients' exposure to antibiotics, we identified commensal bacteria associated with protection against VRE colonization. We further found a consortium of five strains that was sufficient to restrict VRE gut colonization in antibiotic treated mice. Transcriptomics in combination with targeted metabolomics and in vivo assays indicated that the bacterial consortium inhibits VRE growth through nutrient depletion, specifically by reducing the levels of fructose, a carbohydrate that boosts VRE growth in vivo. Finally, in vivo RNA-seq analysis of each strain of the consortium in combination with ex vivo and in vivo assays demonstrated that a single bacterium (Olsenella sp.) could recapitulate the effect of the consortium. Our results indicate that nutrient depletion by specific commensals can reduce VRE intestinal colonization, which represents a novel non-antibiotic based strategy to prevent infections caused by this multidrug-resistant organism.

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms.

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general.

IL-17 controls central nervous system autoimmunity through the intestinal microbiome.

Interleukin-17A- (IL-17A) and IL-17F-producing CD4+ T helper cells (TH17 cells) are implicated in the development of chronic inflammatory diseases, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). TH17 cells also orchestrate leukocyte invasion of the central nervous system (CNS) and subsequent tissue damage. However, the role of IL-17A and IL-17F as effector cytokines is still confused with the encephalitogenic function of the cells that produce these cytokines, namely, TH17 cells, fueling a long-standing debate in the neuroimmunology field. Here, we demonstrated that mice deficient for IL-17A/F lose their susceptibility to EAE, which correlated with an altered composition of their gut microbiota. However, loss of IL-17A/F in TH cells did not diminish their encephalitogenic capacity. Reconstitution of a wild-type-like intestinal microbiota or reintroduction of IL-17A specifically into the gut epithelium of IL-17A/F-deficient mice reestablished their susceptibility to EAE. Thus, our data demonstrated that IL-17A and IL-17F are not encephalitogenic mediators but rather modulators of intestinal homeostasis that indirectly alter CNS-directed autoimmunity.

The Pretreatment Gut Microbiome Is Associated With Lack of Response to Methotrexate in New-Onset Rheumatoid Arthritis.

OBJECTIVE: Although oral methotrexate (MTX) remains the anchor drug for rheumatoid arthritis (RA), up to 50% of patients do not achieve a clinically adequate outcome. In addition, there is a lack of prognostic tools for treatment response prior to drug initiation. This study was undertaken to investigate whether interindividual differences in the human gut microbiome can aid in the prediction of MTX efficacy in new-onset RA. METHODS: We performed 16S ribosomal RNA gene and shotgun metagenomic sequencing on the baseline gut microbiomes of drug-naive patients with new-onset RA (n = 26). Results were validated in an additional independent cohort (n = 21). To gain insight into potential microbial mechanisms, we conducted ex vivo experiments coupled with metabolomics analysis to evaluate the association between microbiome-driven MTX depletion and clinical response. RESULTS: Our analysis revealed significant associations of the abundance of gut bacterial taxa and their genes with future clinical response (q < 0.05), including orthologs related to purine and MTX metabolism. Machine learning techniques were applied to the metagenomic data, resulting in a microbiome-based model that predicted lack of response to MTX in an independent group of patients. Finally, MTX levels remaining after ex vivo incubation with distal gut samples from pretreatment RA patients significantly correlated with the magnitude of future clinical response, suggesting a possible direct effect of the gut microbiome on MTX metabolism and treatment outcomes. CONCLUSION: Taken together, these findings are the first step toward predicting lack of response to oral MTX in patients with new-onset RA and support the value of the gut microbiome as a possible prognostic tool and as a potential target in RA therapeutics.

Roles of the intestinal microbiota in pathogen protection.

Hundreds of commensal bacterial species inhabit the gastrointestinal tract. This diverse microbial ecosystem plays a crucial role in the prevention and resolution of infectious diseases. In this review we will describe the major mechanisms by which the intestinal microbiota confers protection against infections, focusing on those caused by intestinal bacterial pathogens. These mechanisms include both non-immune- and immune-cell-mediated pathways, notably through bacterial production of inhibitory molecules and nutrient deprivation by the former and innate lymphoid cell-, myeloid cell- or lymphocyte-dependent stimulation by the latter. Finally, we will discuss novel therapeutic approaches based on commensal microbes and their products, which could potentially be used to combat infections.

Decreased bacterial diversity characterizes the altered gut microbiota in patients with psoriatic arthritis, resembling dysbiosis in inflammatory bowel disease.

OBJECTIVE: To characterize the diversity and taxonomic relative abundance of the gut microbiota in patients with never-treated, recent-onset psoriatic arthritis (PsA). METHODS: High-throughput 16S ribosomal RNA pyrosequencing was utilized to compare the community composition of gut microbiota in patients with PsA (n = 16), patients with psoriasis of the skin (n = 15), and healthy, matched control subjects (n = 17). Samples were further assessed for the presence and levels of fecal and serum secretory IgA (sIgA), proinflammatory proteins, and fatty acids. RESULTS: The gut microbiota observed in patients with PsA and patients with skin psoriasis was less diverse when compared to that in healthy controls. This could be attributed to the reduced presence of several taxa. Samples from both patient groups showed a relative decrease in abundance of Coprococcus species, while samples from PsA patients were also characterized by a significant reduction in Akkermansia, Ruminococcus, and Pseudobutyrivibrio. Supernatants of fecal samples from PsA patients revealed an increase in sIgA levels and decrease in RANKL levels. Analysis of fatty acids revealed low fecal quantities of hexanoate and heptanoate in both patients with PsA and patients with psoriasis. CONCLUSION: Patients with PsA and patients with skin psoriasis had a lower relative abundance of multiple intestinal bacteria. Although some genera were concomitantly decreased in both conditions, PsA samples had a lower abundance of reportedly beneficial taxa. This gut microbiota profile in PsA was similar to that previously described in patients with inflammatory bowel disease and was associated with changes in specific inflammatory proteins unique to this group, and distinct from that in patients with skin psoriasis and healthy controls. Thus, the role of the gut microbiome in the continuum of psoriasis-PsA pathogenesis and the associated immune response merits further study.

[Molecular epidemiology studies on the immigrant population in Spain]. / Estudios de epidemiología molecular en población inmigrante en España.

BACKGROUND: Molecular epidemiology is a new scientific discipline which allows to integrate information on the genetic variation of infectious pathogens with their diffusion in a population and its subgroups including, for instance, resistance mutations to antibiotics and antiretrovirals. We present the results of an analysis of scientific publications that analyze the health status of the immigrant population in Spain from a molecular epidemiology perspective. METHODS: We reviewed original articles published in 1998-2014 with the keywords "molecular epidemiology", "molecular typing", "sequencing", "immigrant", and "Spain". RESULTS: From a total of 267 articles identified initially, only 50 passed through the established filters. Most of them (36) analyzed infections by Mycobacterium tuberculosis (3) and HIV (3), followed at a large distance by Staphylococcus aureus and hepatitis B virus. The main goal of these works was the typing of the pathogen and to determine the frequency of resistance mutations. CONCLUSION: Is difficult to generalize the conclusions from the analyzed articles because most of them have a purely descriptive and quite restricted scope, considering the type and size of the samples studied. Several studies are focused on the most likely origin for the strains or variants of the pathogen but others also reveal transmissions from the local to the immigrant populations.

Estudios de epidemiología molecular en población inmigrante en España / Molecular epidemiology studies on the immigrant population in Spain

Fundamentos: La epidemiología molecular es una nueva disciplina que permite la integración de la información sobre la variabilidad genética de patógenos infecciosos con su difusión en la población y subgrupos de la misma incluyendo, por ejemplo, las mutaciones de resistencia a antibióticos y antivirales. El objetivo es conocer qué posibles diferencias existe en las características genéticas de los agentes infecciosos que afectan a las poblaciones inmigrante y autóctoctona en España.. Métodos: Se revisaron artículos originales publicados entre 1998- 2013, con las palabras clave "epidemiología molecular", "tipado molecular", "secuenciación", "inmigrante", "España". Resultados: De un total de 267 artículos identificados inicialmente, 50 pasaron los diferentes filtros establecidos. De ellos, 36 analizan las infecciones por Mycobacterium tuberculosis y VIH, seguidos de los que analizan infecciones por Staphylococcus aureus (3) y el Virus de la Hepatitis B (3). Conclusiones: Los objetivos principales de estos trabajos fueron el tipado del patógeno y la determinación de la frecuencia de mutaciones de resistencia. Los estudios más frecuentes correspondieron a cohortes retrospectivas, seguidos por los estudios ecológicos y los ensayos clínicos. En general los estudios son descriptivos y su ámbito por el tipo y tamaño de muestra es bastante restringido. En varios se determina que las cepas o variantes del patógeno encontradas en inmigrantes tienen su origen más probable en sus países de origen, si bien otros también ponen de manifiesto la transmisión desde la población autóctona a la inmigrante (AU)

Background: Molecular epidemiology is a new scientific discipline which allows to integrate information on the genetic variation of infectious pathogens with their diffusion in a population and its subgroups including, for instance, resistance mutations to antibiotics and antiretrovirals. We present the results of an analysis of scientific publications that analyze the health status of the immigrant population in Spain from a molecular epidemiology perspective. Methods:We reviewed original articles published in 1998-2014 with he keywords "molecular epidemiology", "molecular typing", "sequencing", "immigrant", and "Spain". Results: Froma total of 267 articles identified initially, only 50 passed through the established filters. Most of them (36) analyzed infections by Mycobacterium tuberculosis (3) and HIV (3), followed at a large distance by Staphylococcus aureus and hepatitis B virus. The main goal of these works was the typing of the pathogen and to determine the frequency of resistance mutations. Conclusion: Is difficult to generalize the conclusions from the analyzed articles because most of them have a purely descriptive and quite restricted scope, considering the type and size of the samples studied. Several studies are focused on the most likely origin for the strains or variants of the pathogen but others also reveal transmissions from the local to the immigrant populations (AU)