Gut microbiota disturbance during antibiotic therapy: a multi-omic approach.

OBJECTIVE: Antibiotic (AB) usage strongly affects microbial intestinal metabolism and thereby impacts human health. Understanding this process and the underlying mechanisms remains a major research goal. Accordingly, we conducted the first comparative omic investigation of gut microbial communities in faecal samples taken at multiple time points from an individual subjected to ß-lactam therapy. METHODS: The total (16S rDNA) and active (16S rRNA) microbiota, metagenome, metatranscriptome (mRNAs), metametabolome (high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry) and metaproteome (ultra high performing liquid chromatography coupled to an Orbitrap MS(2) instrument [UPLC-LTQ Orbitrap-MS/MS]) of a patient undergoing AB therapy for 14 days were evaluated. RESULTS: Apparently oscillatory population dynamics were observed, with an early reduction in Gram-negative organisms (day 6) and an overall collapse in diversity and possible further colonisation by 'presumptive' naturally resistant bacteria (day 11), followed by the re-growth of Gram-positive species (day 14). During this process, the maximum imbalance in the active microbial fraction occurred later (day 14) than the greatest change in the total microbial fraction, which reached a minimum biodiversity and richness on day 11; additionally, major metabolic changes occurred at day 6. Gut bacteria respond to ABs early by activating systems to avoid the antimicrobial effects of the drugs, while 'presumptively' attenuating their overall energetic metabolic status and the capacity to transport and metabolise bile acid, cholesterol, hormones and vitamins; host-microbial interactions significantly improved after treatment cessation. CONCLUSIONS: This proof-of-concept study provides an extensive description of gut microbiota responses to follow-up ß-lactam therapy. The results demonstrate that ABs targeting specific pathogenic infections and diseases may alter gut microbial ecology and interactions with host metabolism at a much higher level than previously assumed.

Industrial biotechnology of Pseudomonas putida and related species.

Since their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.

The logicome of environmental bacteria: merging catabolic and regulatory events with Boolean formalisms.

The regulatory and metabolic networks that rule biodegradation of pollutants by environmental bacteria are wired to the rest of the cellular physiology through both transcriptional factors and intermediary signal molecules. In this review, we examine some formalisms for describing catalytic/regulatory circuits of this sort and advocate the adoption of Boolean logic for combining transcriptional and enzymatic occurrences in the same biological system. As an example, we show how known regulatory and metabolic actions that bring about biodegradation of m-xylene by Pseudomonas putida mt-2 can be represented as clusters of binary operations and then reconstructed as a digital network. Despite the many simplifications, Boolean tools still capture the gross behaviour of the system even in the absence of kinetic constants determined experimentally. On this basis, we argue that still with a limited volume of data binary formalisms allow us to penetrate the raison d'être of extant regulatory and metabolic architectures.

Strategies for structuring interdisciplinary education in Systems Biology: an European perspective.

Systems Biology is an approach to biology and medicine that has the potential to lead to a better understanding of how biological properties emerge from the interaction of genes, proteins, molecules, cells and organisms. The approach aims at elucidating how these interactions govern biological function by employing experimental data, mathematical models and computational simulations. As Systems Biology is inherently multidisciplinary, education within this field meets numerous hurdles including departmental barriers, availability of all required expertise locally, appropriate teaching material and example curricula. As university education at the Bachelor's level is traditionally built upon disciplinary degrees, we believe that the most effective way to implement education in Systems Biology would be at the Master's level, as it offers a more flexible framework. Our team of experts and active performers of Systems Biology education suggest here (i) a definition of the skills that students should acquire within a Master's programme in Systems Biology, (ii) a possible basic educational curriculum with flexibility to adjust to different application areas and local research strengths, (iii) a description of possible career paths for students who undergo such an education, (iv) conditions that should improve the recruitment of students to such programmes and (v) mechanisms for collaboration and excellence spreading among education professionals. With the growing interest of industry in applying Systems Biology approaches in their fields, a concerted action between academia and industry is needed to build this expertise. Here we present a reflection of the European situation and expertise, where most of the challenges we discuss are universal, anticipating that our suggestions will be useful internationally. We believe that one of the overriding goals of any Systems Biology education should be a student's ability to phrase and communicate research questions in such a manner that they can be solved by the integration of experiments and modelling, as well as to communicate and collaborate productively across different experimental and theoretical disciplines in research and development.

From the environment to the host: re-wiring of the transcriptome of Pseudomonas aeruginosa from 22°C to 37°C.

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe²âº extracellular concentration at 37°C and a peak in Fe³âº extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature.

Effects of ß-lactam antibiotics and fluoroquinolones on human gut microbiota in relation to Clostridium difficile associated diarrhea.

Clostridium difficile infections are an emerging health problem in the modern hospital environment. Severe alterations of the gut microbiome with loss of resistance to colonization against C. difficile are thought to be the major trigger, but there is no clear concept of how C. difficile infection evolves and which microbiological factors are involved. We sequenced 16S rRNA amplicons generated from DNA and RNA/cDNA of fecal samples from three groups of individuals by FLX technology: (i) healthy controls (no antibiotic therapy); (ii) individuals receiving antibiotic therapy (Ampicillin/Sulbactam, cephalosporins, and fluoroquinolones with subsequent development of C. difficile infection or (iii) individuals receiving antibiotic therapy without C. difficile infection. We compared the effects of the three different antibiotic classes on the intestinal microbiome and the effects of alterations of the gut microbiome on C. difficile infection at the DNA (total microbiota) and rRNA (potentially active) levels. A comparison of antibiotic classes showed significant differences at DNA level, but not at RNA level. Among individuals that developed or did not develop a C. difficile infection under antibiotics we found no significant differences. We identified single species that were up- or down regulated in individuals receiving antibiotics who developed the infection compared to non-infected individuals. We found no significant differences in the global composition of the transcriptionally active gut microbiome associated with C. difficile infections. We suggest that up- and down regulation of specific bacterial species may be involved in colonization resistance against C. difficile providing a potential therapeutic approach through specific manipulation of the intestinal microbiome.

Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida.

BACKGROUND: Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterial phosphotransferase system (PTS) together with the PEP/pyruvate node in central metabolism represents a signaling unit that acts as a sensory element and measures the activity of the central metabolism. Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru) and a second branch (PTSNtr), which communicate with each other by phosphate exchange. Recent experimental results showed a cross talk between the two branches. However, the functional role of the crosstalk remains open. RESULTS: A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one of the PTS proteins, for different environmental conditions and different strain variants. Additionally, data from flux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based on the calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructose could be determined. CONCLUSION: Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major part of the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a new role of the second branch of the PTS and will serve as a basis for further experimental studies.

A gene cluster involved in degradation of substituted salicylates via ortho cleavage in Pseudomonas sp. strain MT1 encodes enzymes specifically adapted for transformation of 4-methylcatechol and 3-methylmuconate.

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.