Unlocking the microbiome.

Individual species of bacteria and yeast present in the food of wild fruit flies work together to provide the nutrients needed for larval growth.

Podcast Microbiando: produção de podcast como um projeto de extensão universitária e de divulgação científica / Microbiando Podcast: creation of a podcast as an university community project and science communication tool

A divulgação científica no Brasil ganhou notoriedade e destaque na Internet nas últimas décadas, possibilitando o surgimento de diversas iniciativas como os podcasts. Neste artigo, abordamos o fluxo de produção do Podcast Microbiando, um projeto de extensão Universitária da Universidade Federal do Rio de Janeiro na cobertura de temas da área da Microbiologia e Imunologia de forma contextualizada. Descrevemos brevemente as etapas necessárias para a produção de um episódio do Microbiando, incluindo: rotina de reuniões, produção dos roteiros, gravação dos episódios, edição, criação de artes das capas, pós-produção do episódio e divulgação. Expusemos também algumas estatísticas do Podcast Microbiando, como: quantidade de downloads, país de origem dos ouvintes, temas mais abordados e episódios mais baixados. O Podcast Microbiando é centrado na formação acadêmica dos alunos de graduação e pós-graduação que compõem o projeto de Extensão Universitária. Além disso, a equipe do Microbiando acredita que a divulgação científica é essencial na formação de cidadãos responsáveis.

Bioactive small molecules produced by the human gut microbiome modulate Vibrio cholerae sessile and planktonic lifestyles.

Humans live in symbiosis with a diverse community of microorganisms, which has evolved to carry out many specific tasks that benefit the host, including protection against invading pathogens. Within the chemical diversity of the gastrointestinal tract, small molecules likely constitute chemical cues for the communication between the microbiota and pathogens. Therefore, we sought to investigate if molecules produced by the human gut microbiota show biological activity against the human pathogen Vibrio cholerae. To probe the effects of the gut metabolome on V. cholerae, we investigated its response to small-molecule extracts from human feces, from a complex bacterial community cultivated in vitro, and from culture supernatants of Enterocloster citroniae, Bacteroides thetaiotaomicron, and Bacteroides vulgatus. Using RNA sequencing, we determined the impact of the human gut metabolome on V. cholerae global gene expression. Among the genes downregulated in the presence of the fecal extract, the most overrepresented functional category was cell motility, which accounted for 39% of repressed genes. Repression of V. cholerae motility by the fecal extract was confirmed phenotypically, and E. citroniae extracts reproduced this phenotype. A complex in vitro microbial community led to increased motility, as did extracts from B. vulgatus, a species present in this community. Accordingly, mucin penetration was also repressed by fecal and E. citroniae extracts, suggesting that the phenotypes observed may have implications for host colonization. Together with previous studies, this work shows that small molecules from the gut metabolome may have a widespread, significant impact on microbe-microbe interactions established in the gut environment.

Metabolic profiles of multidrug resistant and extensively drug resistant Mycobacterium tuberculosis unveiled by metabolomics.

Although treatable with antibiotics, tuberculosis is a leading cause of death. Mycobacterium tuberculosis antibiotic resistance is becoming increasingly common and disease control is challenging. Conventional drug susceptibility testing takes weeks to produce results, and treatment is often initiated empirically. Therefore, new methods to determine drug susceptibility profiles are urgent. Here, we used mass-spectrometry-based metabolomics to characterize the metabolic landscape of drug-susceptible (DS), multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis. Direct infusion mass spectrometry data showed that DS, MDR, and XDR strains have distinct metabolic profiles, which can be used to predict drug susceptibility and resistance. This was later confirmed by Ultra-High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry, where we found that levels of ions presumptively identified as isoleucine, proline, hercynine, betaine, and pantothenic acid varied significantly between strains with different drug susceptibility profiles. We then confirmed the identification of proline and isoleucine and determined their absolute concentrations in bacterial extracts, and found significantly higher levels of these amino acids in DS strains, as compared to drug-resistant strains (combined MDR and XDR strains). Our results advance the current understanding of the effect of drug resistance on bacterial metabolism and open avenues for the detection of drug resistance biomarkers.

Genome Sequence of a Highly Virulent pvl-positive Vancomycin-intermediate-resistant Staphylococcus aureus Sequence Type 30.

BACKGROUND: Staphylococcus aureus isolates expressing the Panton-Valentine Leukocidin (PVL) have been related to a wide range of diseases. Recently, pvl-positive community-associated methicillin-resistant S. aureus belonging to USA1100 (ST30/CC30/SCCmec IV) lineage has emerged in Brazilian hospitals. OBJECTIVE: The aim of this work was to sequence the genome of a pvl-positive USA1100 Vancomycin-Intermediate-Resistant S. aureus (VISA) isolate from Rio de Janeiro, Brazil. METHODS: The 13420 genome was sequenced using the HiSeq 2500 platform. The draft genome, plasmids annotation, and genome analysis were performed using RAST. Comparison of the relative pvl gene expression of six S. aureus isolates was performed by qRT-PCR. RESULTS: The isolate presented the ϕPVL phage codifying for the H2b PVL protein isoform, and another prophage carrying a PVL variant named lukF and lukS-PV.2. The 13420 genome presented a high number of virulence determinants, such as genes codifying for serine-protease proteins, enterotoxins (egc), the immune evasion cluster (IEC), adhesion proteins, spermine/spermidine acetyltransferase gene (blt), superantigen-like proteins, as well as the ica operon. Point mutations at vraS, tcaA, and tcaB genes were detected. Moreover, the PVL mRNA relative expression of the 13420 isolate was five times higher than mRNA PVL levels of the USA300/ST8 reference strain. CONCLUSION: We described for the first time the genome sequence of a VISA isolate harboring two pvl-associated genes and other virulence factors that may improve the USA1100/ST30 lineage fitness and impact its pathogenicity and spreading at Brazilian hospitals.

The Gut Microbiome and Metabolome of Two Riparian Communities in the Amazon.

During the last decades it has become increasingly clear that the microbes that live on and in humans are critical for health. The communities they form, termed microbiomes, are involved in fundamental processes such as the maturation and constant regulation of the immune system. Additionally, they constitute a strong defense barrier to invading pathogens, and are also intricately linked to nutrition. The parameters that affect the establishment and maintenance of these microbial communities are diverse, and include the genetic background, mode of birth, nutrition, hygiene, and host lifestyle in general. Here, we describe the characterization of the gut microbiome of individuals living in the Amazon, and the comparison of these microbial communities to those found in individuals from an urban, industrialized setting. Our results showed striking differences in microbial communities from these two types of populations. Additionally, we used high-throughput metabolomics to study the chemical ecology of the gut environment and found significant metabolic changes between the two populations. Although we cannot point out a single cause for the microbial and metabolic changes observed between Amazonian and urban individuals, they are likely to include dietary differences as well as diverse patterns of environmental exposure. To our knowledge, this is the first description of gut microbial and metabolic profiles in Amazonian populations, and it provides a starting point for thorough characterizations of the impact of individual environmental conditions on the human microbiome and metabolome.

Osmotic stress induces biofilm production by Staphylococcus epidermidis isolates from neonates.

Staphylococcus epidermidis is one of the leading causes of bloodstream infections, particularly in premature neonates, and biofilm formation is a major virulence factor. We characterized biofilm formation by 50 S. epidermidis neonatal isolates under osmotic stress and evaluated the expression of biofilm-associated genes. Phenotypical analyses of biofilm production were performed in culture medium with or without addition of NaCl or glucose. In control medium (no additions), most isolates (84%) were nonproducers or weak biofilm producers. Growth in NaCl-containing medium increased the number of moderate/strong producers, and this increase was even greater in medium containing glucose. Most of the protein-enriched biofilms (60%) could be observed only during growth in glucose, whereas 50% of the polysaccharide-enriched biofilms were observed during growth in NaCl. Studies that evaluate the conditions used to characterize biofilm production are important to help us understand the dynamics of this important virulence factor in S. epidermidis and their impact on neonatal infections.

Extraction of Small Molecules from Fecal Samples and Testingof Their Activity on Microbial Physiology.

The human body is colonized by vast communities of microbes, collectively known as microbiota, or microbiome. Although microbes colonize every surface of our bodies that is exposed to the external environment, the biggest collection of microbes colonizing humans and other mammals can be found in the gastrointestinal tract. Given the fact that the human gut is colonized by several hundred microbial species, our group hypothesized that the chemical diversity of this environment should be significant, and that many of the molecules present in that environment would have important signaling roles. Therefore, we devised a protocol to extract these molecules from human feces and test their signaling properties. Potentially bioactive extracts can be tested through addition to culture medium and analyses of bacterial growth and gene expression, among other properties. The protocol described herein provides an easy and rapid method for the extraction and testing of metabolites from fecal samples using Salmonella enterica as a model organism. This protocol can also be adapted to the extraction of small molecules from other matrices, such as cultured mammalian cells, tissues, body fluids, and axenic microbial cultures, and the resulting extracts can be tested against various microbial species.

Repression of Salmonella Host Cell Invasion by Aromatic Small Molecules from the Human Fecal Metabolome.

The human microbiome is a collection of microorganisms that inhabit every surface of the body that is exposed to the environment, generally coexisting peacefully with their host. These microbes have important functions, such as producing vitamins, aiding in maturation of the immune system, and protecting against pathogens. We have previously shown that a small-molecule extract from the human fecal microbiome has a strong repressive effect on Salmonella enterica serovar Typhimurium host cell invasion by modulating the expression of genes involved in this process. Here, we describe the characterization of this biological activity. Using a series of purification methods, we obtained fractions with biological activity and characterized them by mass spectrometry. These experiments revealed an abundance of aromatic compounds in the bioactive fraction. Selected compounds were obtained from commercial sources and tested with respect to their ability to repress the expression of hilA, the gene encoding the master regulator of invasion genes in Salmonella We found that the aromatic compound 3,4-dimethylbenzoic acid acts as a strong inhibitor of hilA expression and of invasion of cultured host cells by Salmonella Future studies should reveal the molecular details of this phenomenon, such as the signaling cascades involved in sensing this bioactive molecule.IMPORTANCE Microbes constantly sense and adapt to their environment. Often, this is achieved through the production and sensing of small extracellular molecules. The human body is colonized by complex communities of microbes, and, given their biological and chemical diversity, these ecosystems represent a platform where the production and sensing of molecules occur. In previous work, we showed that small molecules produced by microbes from the human gut can significantly impair the virulence of the enteric pathogen Salmonella enterica Here, we describe a specific compound from the human gut that produces this same effect. The results from this work not only shed light on an important biological phenomenon occurring in our bodies but also may represent an opportunity to develop drugs that can target these small-molecule interactions to protect us from enteric infections and other diseases.

A Highly Effective Component Vaccine against Nontyphoidal Salmonella enterica Infections.

UNLABELLED: Nontyphoidal Salmonella enterica (NTS) infections are a major burden to global public health, as they lead to diseases ranging from gastroenteritis to systemic infections and there is currently no vaccine available. Here, we describe a highly effective component vaccine against S. enterica serovar Typhimurium in both gastroenteritis and systemic murine infection models. We devised an approach to generate supernatants of S. enterica serovar Typhimurium, an organism that is highly abundant in virulence factors. Immunization of mice with this supernatant resulted in dramatic protection against a challenge with serovar Typhimurium, showing increased survival in the systemic model and decreased intestinal pathology in the gastrointestinal model. Protection correlated with specific IgA and IgG levels in the serum and specific secretory IgA levels in the feces of immunized mice. Initial characterization of the protective antigens in the bacterial culture supernatants revealed a subset of antigens that exhibited remarkable stability, a highly desirable characteristic of an effective vaccine to be used under suboptimal environmental conditions in developing countries. We were able to purify a subset of the peptides present in the supernatants and show their potential for immunization of mice against serovar Typhimurium resulting in a decreased level of colonization. This component vaccine shows promise with regard to protecting against NTS, and further work should significantly help to establish vaccines against these prevalent infections. IMPORTANCE: Salmonella enterica infections other than typhoid and paratyphoid fever are a major global health burden, as they cause high morbidity and mortality worldwide. Strategies that prevent Salmonella-related diseases are greatly needed, and there is a significant push for the development of vaccines against nontyphoidal Salmonella enterica serovars. In this work, we describe an S. Typhimurium supernatant-derived vaccine that is effective in reducing bacterial colonization in mouse models of gastroenteritis as well as invasive disease. This is a component vaccine that shows high stability to heat, a feature that is important for use under suboptimal conditions, such as those found in sub-Saharan Africa.

Antivirulence activity of the human gut metabolome.

The mammalian gut contains a complex assembly of commensal microbes termed microbiota. Although much has been learned about the role of these microbes in health, the mechanisms underlying these functions are ill defined. We have recently shown that the mammalian gut contains thousands of small molecules, most of which are currently unidentified. Therefore, we hypothesized that these molecules function as chemical cues used by hosts and microbes during their interactions in health and disease. Thus, a search was initiated to identify molecules produced by the microbiota that are sensed by pathogens. We found that a secreted molecule produced by clostridia acts as a strong repressor of Salmonella virulence, obliterating expression of the Salmonella pathogenicity island 1 as well as host cell invasion. It has been known for decades that the microbiota protects its hosts from invading pathogens, and these data suggest that chemical sensing may be involved in this phenomenon. Further investigations should reveal the exact biological role of this molecule as well as its therapeutic potential. Importance: Microbes can communicate through the production and sensing of small molecules. Within the complex ecosystem formed by commensal microbes living in and on the human body, it is likely that these molecular messages are used extensively during the interactions between different microbial species as well as with host cells. Deciphering such a molecular dialect will be fundamental to our understanding of host-microbe interactions in health and disease and may prove useful for the design of new therapeutic strategies that target these mechanisms of communication.

Autophagy facilitates Salmonella replication in HeLa cells.

Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. IMPORTANCE As a host defense system, autophagy is known to target a population of Salmonella for degradation and hence restricting Salmonella replication. In contrast to this concept, a recent report showed that knockdown of Rab1, a GTPase required for autophagy of Salmonella, decreases Salmonella replication in HeLa cells. Here, we have reexamined the fate of Salmonella targeted by autophagy by various cell biology-based assays. We found that the association of autophagy components with cytosolic Salmonella increases shortly after initiation of intracellular bacterial replication. Furthermore, through a live-cell imaging method, a subset of cytosolic Salmonella was found to be extensively associated with autophagy components p62 and/or LC3, and they replicated quickly. Most importantly, depletion of autophagy components significantly reduced the replication of cytosolic Salmonella in HeLa cells. Hence, in contrast to previous reports, we propose that autophagy facilitates Salmonella replication in the cytosol of HeLa cells.

Neutrophil elastase alters the murine gut microbiota resulting in enhanced Salmonella colonization.

The intestinal microbiota has been found to play a central role in the colonization of Salmonella enterica serovar Typhimurium in the gastrointestinal tract. In this study, we present a novel process through which Salmonella benefit from inflammatory induced changes in the microbiota in order to facilitate disease. We show that Salmonella infection in mice causes recruitment of neutrophils to the gut lumen, resulting in significant changes in the composition of the intestinal microbiota. This occurs through the production of the enzyme elastase by neutrophils. Administration of recombinant neutrophil elastase to infected animals under conditions that do not elicit neutrophil recruitment caused shifts in microbiota composition that favored Salmonella colonization, while inhibition of neutrophil elastase reduced colonization. This study reveals a new relationship between the microbiota and the host during infection.

Repression of Salmonella enterica phoP expression by small molecules from physiological bile.

Infection with Salmonella enterica serovar Typhi in humans causes the life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate. Additionally, we showed that Salmonella Typhimurium can use bile phospholipids to grow at high rates. These abilities are likely important for quick colonization of the gallbladder during typhoid fever and further pathogen dissemination through fecal shedding. To further characterize the interactions between Salmonella and the gallbladder environment, we compared the transcriptomes of Salmonella cultures grown in LB broth or physiological murine bile. Our data showed that many genes involved in bacterial central metabolism are affected by bile, with the citric acid cycle being repressed and alternative respiratory systems being activated. Additionally, our study revealed a new aspect of Salmonella interactions with bile through the identification of the global regulator phoP as a bile-responsive gene. Repression of phoP expression could also be achieved using physiological, but not commercial, bovine bile. The biological activity does not involve PhoPQ sensing of a bile component and is not caused by bile acids, the most abundant organic components of bile. Bioactivity-guided purification allowed the identification of a subset of small molecules from bile that can elicit full activity; however, a single compound with phoP inhibitory activity could not be isolated, suggesting that multiple molecules may act in synergy to achieve this effect. Due to the critical role of phoP in Salmonella virulence, further studies in this area will likely reveal aspects of the interaction between Salmonella and bile that are relevant to disease.

Output targets and transcriptional regulation by a cyclic dimeric GMP-responsive circuit in the Vibrio parahaemolyticus Scr network.

The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ΔscrABC and wild-type strains revealed expression differences with respect to ∼100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit.

Metabolomics reveals phospholipids as important nutrient sources during Salmonella growth in bile in vitro and in vivo.

During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to occur successfully. This requires the bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to the pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Additionally, Salmonella can successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. To gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit.

The intestinal microbiota plays a role in Salmonella-induced colitis independent of pathogen colonization.

The intestinal microbiota is composed of hundreds of species of bacteria, fungi and protozoa and is critical for numerous biological processes, such as nutrient acquisition, vitamin production, and colonization resistance against bacterial pathogens. We studied the role of the intestinal microbiota on host resistance to Salmonella enterica serovar Typhimurium-induced colitis. Using multiple antibiotic treatments in 129S1/SvImJ mice, we showed that disruption of the intestinal microbiota alters host susceptibility to infection. Although all antibiotic treatments caused similar increases in pathogen colonization, the development of enterocolitis was seen only when streptomycin or vancomycin was used; no significant pathology was observed with the use of metronidazole. Interestingly, metronidazole-treated and infected C57BL/6 mice developed severe pathology. We hypothesized that the intestinal microbiota confers resistance to infectious colitis without affecting the ability of S. Typhimurium to colonize the intestine. Indeed, different antibiotic treatments caused distinct shifts in the intestinal microbiota prior to infection. Through fluorescence in situ hybridization, terminal restriction fragment length polymorphism, and real-time PCR, we showed that there is a strong correlation between the intestinal microbiota composition before infection and susceptibility to Salmonella-induced colitis. Members of the Bacteroidetes phylum were present at significantly higher levels in mice resistant to colitis. Further analysis revealed that Porphyromonadaceae levels were also increased in these mice. Conversely, there was a positive correlation between the abundance of Lactobacillus sp. and predisposition to colitis. Our data suggests that different members of the microbiota might be associated with S. Typhimurium colonization and colitis. Dissecting the mechanisms involved in resistance to infection and inflammation will be critical for the development of therapeutic and preventative measures against enteric pathogens.

Bringing Koch's postulates to the table in IBD.

In this issue of Cell Host & Microbe, Bloom and colleagues elegantly show that commensal Bacteroides species fulfill Koch's postulates for inflammatory bowel disease in a host-genotype-specific way. This study showcases the use of a non-germ-free mouse model to identify specific members of the microbiota involved in disease development.