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Background: Methanogenic archaea represent a less investigated and likely underestimated part of the intestinal tract microbiome in swine.
Aims/Methods: This study aims to elucidate the archaeome structure and function in the porcine intestinal tract of healthy and H1N1 infected swine. We performed multi-omics analysis consisting of 16S rRNA gene profiling, metatranscriptomics and metaproteomics.
Results and discussion: We observed a significant increase from 0.48 to 4.50% of archaea in the intestinal tract microbiome along the ileum and colon, dominated by genera Methanobrevibacter and Methanosphaera. Furthermore, in feces of naïve and H1N1 infected swine, we observed significant but minor differences in the occurrence of archaeal phylotypes over the course of an infection experiment. Metatranscriptomic analysis of archaeal mRNAs revealed the major methanogenesis pathways of Methanobrevibacter and Methanosphaera to be hydrogenotrophic and methyl-reducing, respectively. Metaproteomics of archaeal peptides indicated some effects of the H1N1 infection on central metabolism of the gut archaea.
Conclusions/Take home message: Finally, this study provides the first multi-omics analysis and high-resolution insights into the structure and function of the porcine intestinal tract archaeome during a non-lethal Influenza A virus infection of the respiratory tract, demonstrating significant alterations in archaeal community composition and central metabolic functions.
Introduction: The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients.
Methods and results: In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio.
Conclusion: Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.
Infective/bacterial endocarditis is a rare but life-threatening disease with a hospital mortality rate of 22.7% and a 1-year mortality rate of 40%. Therefore, continued research efforts to develop efficient anti-infective implant materials are of the utmost importance. Equally important is the development of test systems that allow the performance of new materials to be comprehensively evaluated. In this study, a novel antibacterial coating based on dalbavancin was tested in comparison to rifampicin/minocycline, and the suitability of a recently developed mouse tail vein model for testing the implant coatings was validated. Small polymeric stent grafts coated with a poly-L-lactic acid (PLLA) layer and incorporated antibiotics were colonized with Staphylococcus (S.) aureus before implantation into the tail vein of mice. The main assessment criteria were the hematogenous spread of the bacteria and the local tissue reaction to the contaminated implant. For this purpose, colony-forming units (CFU) in the blood, spleen and kidneys were determined. Tail cross sections were prepared for histological analysis, and plasma cytokine levels and expression values of inflammation-associated genes were examined. Both antibiotic coatings performed excellently, preventing the onset of infection. The present study expands the range of available methods for testing the anti-infectivity of cardiovascular implants, and the spectrum of agents for effective surface coating.
The anaerobic, gastrointestinal pathogen Clostridioides difficile can cause severe forms of enterocolitis which is mainly mediated by the toxins it produces. The RNA polymerase inhibitor Fidaxomicin is the current gold standard for the therapy of C. difficile infections due to several beneficial features including its ability to suppress toxin synthesis in C. difficile. In contrast to the Rifamycins, Fidaxomicin binds to the RNA polymerase switch region, which is also the binding site for Myxopyronin B. Here, serial broth dilution assays were performed to test the susceptibility of C. difficile and other anaerobes to Myxopyronin B, proving that the natural product is considerably active against C. difficile and that there is no cross-resistance between Fidaxomicin and Myxopyronin B in a Fidaxomicin-resistant C. difficile strain. Moreover, mass spectrometry analysis indicated that Myxopyronin B is able to suppress early phase toxin synthesis in C. difficile to the same degree as Fidaxomicin. Conclusively, Myxopyronin B is proposed as a new lead structure for the design of novel antibiotics for the therapy of C. difficile infections.
Influenza A Virus (IAV) infection followed by bacterial pneumonia often leads to hospitalization and death in individuals from high risk groups. Following infection, IAV triggers the process of viral RNA replication which in turn disrupts healthy gut microbial community, while the gut microbiota plays an instrumental role in protecting the host by evolving colonization resistance. Although the underlying mechanisms of IAV infection have been unraveled, the underlying complex mechanisms evolved by gut microbiota in order to induce host immune response following IAV infection remain evasive. In this work, we developed a novel Maximal-Clique based Community Detection algorithm for Weighted undirected Networks (MCCD-WN) and compared its performance with other existing algorithms using three sets of benchmark networks. Moreover, we applied our algorithm to gut microbiome data derived from fecal samples of both healthy and IAV-infected pigs over a sequence of time-points. The results we obtained from the real-life IAV dataset unveil the role of the microbial families Ruminococcaceae, Lachnospiraceae, Spirochaetaceae and Prevotellaceae in the gut microbiome of the IAV-infected cohort. Furthermore, the additional integration of metaproteomic data enabled not only the identification of microbial biomarkers, but also the elucidation of their functional roles in protecting the host following IAV infection. Our network analysis reveals a fast recovery of the infected cohort after the second IAV infection and provides insights into crucial roles of Desulfovibrionaceae and Lactobacillaceae families in combating Influenza A Virus infection. Source code of the community detection algorithm can be downloaded from https://github.com/AniBhar84/MCCD-WN.
The anaerobic pathogen Clostridioides difficile is perfectly equipped to survive and persist inside the mammalian intestine. When facing unfavorable conditions C. difficile is able to form highly resistant endospores. Likewise, biofilms are currently discussed as form of persistence. Here a comprehensive proteomics approach was applied to investigate the molecular processes of C. difficile strain 630Δerm underlying biofilm formation. The comparison of the proteome from two different forms of biofilm-like growth, namely aggregate biofilms and colonies on agar plates, revealed major differences in the formation of cell surface proteins, as well as enzymes of its energy and stress metabolism. For instance, while the obtained data suggest that aggregate biofilm cells express both flagella, type IV pili and enzymes required for biosynthesis of cell-surface polysaccharides, the S-layer protein SlpA and most cell wall proteins (CWPs) encoded adjacent to SlpA were detected in significantly lower amounts in aggregate biofilm cells than in colony biofilms. Moreover, the obtained data suggested that aggregate biofilm cells are rather actively growing cells while colony biofilm cells most likely severely suffer from a lack of reductive equivalents what requires induction of the Wood-Ljungdahl pathway and C. difficile’s V-type ATPase to maintain cell homeostasis. In agreement with this, aggregate biofilm cells, in contrast to colony biofilm cells, neither induced toxin nor spore production. Finally, the data revealed that the sigma factor SigL/RpoN and its dependent regulators are noticeably induced in aggregate biofilms suggesting an important role of SigL/RpoN in aggregate biofilm formation.
Lichens represent self-supporting symbioses, which occur in a wide range of terrestrial habitats and which contribute significantly to mineral cycling and energy flow at a global scale. Lichens usually grow much slower than higher plants. Nevertheless, lichens can contribute substantially to biomass production. This review focuses on the lichen symbiosis in general and especially on the model species Lobaria pulmonaria L. Hoffm., which is a large foliose lichen that occurs worldwide on tree trunks in undisturbed forests with long ecological continuity. In comparison to many other lichens, L. pulmonaria is less tolerant to desiccation and highly sensitive to air pollution. The name-giving mycobiont (belonging to the Ascomycota), provides a protective layer covering a layer of the green-algal photobiont (Dictyochloropsis reticulata) and interspersed cyanobacterial cell clusters (Nostoc spec.). Recently performed metaproteome analyses confirm the partition of functions in lichen partnerships. The ample functional diversity of the mycobiont contrasts the predominant function of the photobiont in production (and secretion) of energy-rich carbohydrates, and the cyanobiont’s contribution by nitrogen fixation. In addition, high throughput and state-of-the-art metagenomics and community fingerprinting, metatranscriptomics, and MS-based metaproteomics identify the bacterial community present on L. pulmonaria as a surprisingly abundant and structurally integrated element of the lichen symbiosis. Comparative metaproteome analyses of lichens from different sampling sites suggest the presence of a relatively stable core microbiome and a sampling site-specific portion of the microbiome. Moreover, these studies indicate how the microbiota may contribute to the symbiotic system, to improve its health, growth and fitness.
Background: Plasma-generated compounds (PGCs) such as plasma-processed air (PPA) or plasma-treated water (PTW) offer an increasingly important alternative for the control of microorganisms in hard-to-reach areas found in several industrial applications including the food industry. To this end, we studied the antimicrobial capacity of PTW on the vitality and biofilm formation of Listeria monocytogenes, a common foodborne pathogen.
Results: Using a microwave plasma (MidiPLexc), 10 ml of deionized water was treated for 100, 300, and 900 s (pre-treatment time), after which the bacterial biofilm was exposed to the PTW for 1, 3, and 5 min (post-treatment time) for each pre-treatment time, separately. Colony-forming units (CFU) were significantly reduced by 4.7 log10 ± 0.29 log10, as well as the metabolic activity decreased by 47.9 ± 9.47% and the cell vitality by 69.5 ± 2.1%, compared to the control biofilms. LIVE/DEAD staining and fluorescence microscopy showed a positive correlation between treatment and incubation times, as well as reduction in vitality. Atomic force microscopy (AFM) indicated changes in the structure quality of the bacterial biofilm.
Conclusion: These results indicate a promising antimicrobial impact of plasma-treated water on Listeria monocytogenes, which may lead to more targeted applications of plasma decontamination in the food industry in the future.
An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
(2021)
Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.
The human pathogen Clostridioides difficile has evolved into the leading cause of nosocomial diarrhea. The bacterium is capable of spore formation, which even allows survival of antibiotic treatment. Although C. difficile features an anaerobic lifestyle, we determined a remarkably high oxygen tolerance of the laboratory reference strain 630Δerm. A mutation of a single nucleotide (single nucleotide polymorphism [SNP]) in the DNA sequence (A to G) of the gene encoding the regulatory protein PerR results in an amino acid substitution (Thr to Ala) in one of the helices of the helix-turn-helix DNA binding domain of this transcriptional repressor in C. difficile 630Δerm. PerR is a sensor protein for hydrogen peroxide and controls the expression of genes involved in the oxidative stress response. We show that PerR of C. difficile 630Δerm has lost its ability to bind the promoter region of PerR-controlled genes. This results in a constitutive derepression of genes encoding oxidative stress proteins such as a rubrerythrin (rbr1) whose mRNA abundance under anaerobic conditions was increased by a factor of about 7 compared to its parental strain C. difficile 630. Rubrerythrin repression in strain 630Δerm could be restored by the introduction of PerR from strain 630. The permanent oxidative stress response of C. difficile 630Δerm observed here should be considered in physiological and pathophysiological investigations based on this widely used model strain.
IMPORTANCE The intestinal pathogen Clostridioides difficile is one of the major challenges in medical facilities nowadays. In order to better combat the bacterium, detailed knowledge of its physiology is mandatory. C. difficile strain 630Δerm was generated in a laboratory from the patient-isolated strain C. difficile 630 and represents a reference strain for many researchers in the field, serving as the basis for the construction of insertional gene knockout mutants. In our work, we demonstrate that this strain is characterized by an uncontrolled oxidative stress response as a result of a single-base-pair substitution in the sequence of a transcriptional regulator. C. difficile researchers working with model strain 630Δerm should be aware of this permanent stress response.