Inferring early-life host and microbiome functions by mass spectrometry-based metaproteomics and metabolomics.

Humans have a long-standing coexistence with microorganisms. In particular, the microbial community that populates the human gastrointestinal tract has emerged as a critical player in governing human health and disease. DNA and RNA sequencing techniques that map taxonomical composition and genomic potential of the gut community have become invaluable for microbiome research. However, deriving a biochemical understanding of how activities of the gut microbiome shape host development and physiology requires an expanded experimental design that goes beyond these approaches. In this review, we explore advances in high-throughput techniques based on liquid chromatography-mass spectrometry. These omics methods for the identification of proteins and metabolites have enabled direct characterisation of gut microbiome functions and the crosstalk with the host. We discuss current metaproteomics and metabolomics workflows for producing functional profiles, the existing methodological challenges and limitations, and recent studies utilising these techniques with a special focus on early life gut microbiome.

Antibiofilm activity of Cutibacterium acnes cell-free conditioned media against Staphylococcus spp.

Staphylococcus spp. and Cutibacterium acnes are members of the skin microbiome but can also act as pathogens. Particularly, Staphylococcus species are known to cause medical devices-associated infections, and biofilm production is one of their main virulence factors. Biofilms allow bacteria to adhere and persist on surfaces, protecting them from antimicrobials and host defenses. Since both bacteria are found in the human skin, potentially competing for niches, we aimed to investigate if C. acnes produces molecules that affect Staphylococcus spp. biofilm formation and dispersal. Thus, we evaluated the impact of C. acnes cell-free conditioned media (CFCM) on S. aureus, S. epidermidis, S. hominis, and S. lugdunensis biofilm formation. S. lugdunensis and S. hominis biofilm formation was significantly reduced with C. acnes CFCM without impact on their planktonic growth. C. acnes CFCM also significantly disrupted S. hominis established biofilms. The active molecules against S. lugdunensis and S. hominis biofilms appeared to be distinct since initial characterization points to different sizes and sensitivity to sodium metaperiodate, although the activity is highly resistant to heat in both cases. Mass spectrometry analysis of the fractions active against S. hominis revealed several potential candidates. Investigating how species present in the same environment interact, affecting the dynamics of biofilm formation, may reveal clinically useful compounds as well as molecular aspects of interspecies interactions.

Characterization of a SPM-1 metallo-beta-lactamase-producing Pseudomonas aeruginosa by comparative genomics and phenotypic analysis.

Pseudomonas aeruginosa is one of the most common pathogens related to healthcare-associated infections. The Brazilian isolate, named CCBH4851, is a multidrug-resistant clone belonging to the sequence type 277. The antimicrobial resistance mechanisms of the CCBH4851 strain are associated with the presence of the bla[Formula: see text] gene, encoding a metallo-beta-lactamase, in combination with other exogenously acquired genes. Whole-genome sequencing studies focusing on emerging pathogens are essential to identify key features of their physiology that may lead to the identification of new targets for therapy. Using both Illumina and PacBio sequencing data, we obtained a single contig representing the CCBH4851 genome with annotated features that were consistent with data reported for the species. However, comparative analysis with other Pseudomonas aeruginosa strains revealed genomic differences regarding virulence factors and regulatory proteins. In addition, we performed phenotypic assays that revealed CCBH4851 is impaired in bacterial motilities and biofilm formation. On the other hand, CCBH4851 genome contained acquired genomic islands that carry transcriptional factors, virulence and antimicrobial resistance-related genes. Presence of single nucleotide polymorphisms in the core genome, mainly those located in resistance-associated genes, suggests that these mutations may also influence the multidrug-resistant behavior of CCBH4851. Overall, characterization of Pseudomonas aeruginosa CCBH4851 complete genome revealed the presence of features that strongly relates to the virulence and antibiotic resistance profile of this important infectious agent.

Small Molecules Produced by Commensal Staphylococcus epidermidis Disrupt Formation of Biofilms by Staphylococcus aureus.

The microbiota influences host health through several mechanisms, including protecting it from pathogen colonization. Staphylococcus epidermidis is one of the most frequently found species in the skin microbiota, and its presence can limit the development of pathogens such as Staphylococcus aureusS. aureus causes diverse types of infections ranging from skin abscesses to bloodstream infections. Given the increasing prevalence of S. aureus drug-resistant strains, it is imperative to search for new strategies for treatment and prevention. Thus, we investigated the activity of molecules produced by a commensal S. epidermidis isolate against S. aureus biofilms. We showed that molecules present in S. epidermidis cell-free conditioned media (CFCM) caused a significant reduction in biofilm formation in most S. aureus clinical isolates, including all 4 agr types and agr-defective strains, without any impact on growth. S. epidermidis molecules also disrupted established S. aureus biofilms and reduced the antibiotic concentration required to eliminate them. Preliminary characterization of the active compound showed that its activity is resistant to heat, protease inhibitors, trypsin, proteinase K, and sodium periodate treatments, suggesting that it is not proteinaceous. RNA sequencing revealed that S. epidermidis-secreted molecules modulate the expression of hundreds of S. aureus genes, some of which are associated with biofilm production. Biofilm formation is one of the main virulence factors of S. aureus and has been associated with chronic infections and antimicrobial resistance. Therefore, molecules that can counteract this virulence factor may be promising alternatives as novel therapeutic agents to control S. aureus infections.IMPORTANCES. aureus is a leading agent of infections worldwide, and its main virulence characteristic is the ability to produce biofilms on surfaces such as medical devices. Biofilms are known to confer increased resistance to antimicrobials and to the host immune responses, requiring aggressive antibiotic treatment and removal of the infected surface. Here, we investigated a new source of antibiofilm compounds, the skin microbiome. Specifically, we found that a commensal strain of S. epidermidis produces molecules with antibiofilm activity, leading to a significant decrease of S. aureus biofilm formation and to a reduction of previously established biofilms. The molecules potentiated the activity of antibiotics and affected the expression of hundreds of S. aureus genes, including those associated with biofilm formation. Our research highlights the search for compounds that can aid us in the fight against S. aureus infections.

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.

Impact of violacein from Chromobacterium violaceum on the mammalian gut microbiome.

Violacein is a violet pigment produced by Chromobacterium violaceum that possesses several functions such as antibacterial, antiviral, antifungal, and antioxidant activities. The search for potential compounds and therapies that may interfere with and modulate the gut microbial consortia without causing severe damage and increased resistance is important for the treatment of inflammatory, allergic, and metabolic diseases. The aim of the present work was to evaluate the ability of violacein to change microbial patterns in the mammalian gut by favoring certain groups over the others in order to be used as a therapy for diseases associated with changes in the intestinal microflora. To do this, we used male Wistar rats, and administered violacein orally, in low (50 µg/ml) and high (500 µg/ml) doses for a month. Initially, the changes in the microbial diversity were observed by DGGE analyses that showed that the violacein significantly affects the gut microbiota of the rats. Pyrosequencing of 16S rDNA was then employed using a 454 GS Titanium platform, and the results demonstrated that higher taxonomic richness was observed with the low violacein treatment group, followed by the control group and high violacein treatment group. Modulation of the microbiota at the class level was observed in the low violacein dose, where Bacilli and Clostridia (Firmicutes) were found as dominant. For the high violacein dose, Bacilli followed by Clostridia and Actinobacteria were present as the major components. Further analyses are crucial for a better understanding of how violacein affects the gut microbiome and whether this change would be beneficial to the host, providing a framework for the development of alternative treatment strategies for intestinal diseases using this compound.

Bacteroides species produce Vibrio harveyi autoinducer 2-related molecules.

Quorum sensing is a density-dependent gene regulation mechanism that has been described in many bacterial species in the last decades. Bacteria that use quorum sensing as part of their gene regulation circuits produce molecules called autoinducers that accumulate in the environment and activate target genes in a quorum-dependent way. Some specific clues led us to hypothesize that Bacteroides species can produce autoinducers and possess a quorum sensing system. First, Bacteroides are anaerobic bacteria that are frequently involved in polymicrobial infections. These infections often involve Pseudomonas aeruginosa and Staphylococcus aureus, two of the best understood examples of bacteria that employ quorum sensing systems as part of their pathogenesis. Also, studies have detected the presence of a quorum sensing gene involved in the production of autoinducers in Porphyromonas gingivalis, a species closely related to the Bacteroides genus. These and other evidences prompted us to investigate if Bacteroides strains could produce autoinducer molecules that could be detected by a Vibrio harveyi reporter system. In this paper, we show that supernatants of B. fragilis, B. vulgatus and B. distasonis strains are able to stimulate the V. harveyi quorum sensing system 2. Also, we were able to demonstrate that the stimulation detected is due to the production of autoinducer molecules and not the growth of reporter strains after addition of supernatant. Moreover, the phenomenon observed does not seem to represent the degradation of repressors possibly present in the culture medium used. We could also amplify bands from some of the strains tested using primers designed to the luxS gene of Escherichia coli. Altogether, our results show that B. fragilis, B. vulgatus and B. distasonis (but possibly some other species) can produce V. harveyi autoinducer 2-related molecules. However, the role of such molecules in the biology of these organisms remains unknown.