Campylobacter (C.) jejuni and C. coli are common zoonotic bacteria in pigs, which typically act as asymptomatic carriers. However, the effects of Campylobacter colonisation on the porcine intestinal microbiota and metabolome remain poorly understood. This study investigated microbiome and metabolome alterations associated with co-colonisation by C. jejuni and C. coli in the different intestinal segments of pigs. Thirty-two weaned piglets were assigned to a control group and a group inoculated with C. coli ST5777/CT828 and C. jejuni ST122/CT206. Four weeks post inoculation, jejunal and caecal contents were analysed for Campylobacter counts, metabolite profiles and microbial composition. All animals remained clinically healthy. Both Campylobacter species colonised the jejunum and caecum, with higher C. coli counts in the caecum. Campylobacter-colonised pigs showed significantly altered metabolite profiles, including reduced cysteine and urea and increased glycine in the jejunum, as well as elevated 3-hydroxybutyrate levels in the caecum. In contrast, short-chain fatty acid concentrations in the caecum were unaffected by infection. Microbiota analysis revealed a significant reduction in caecal alpha diversity, whereas jejunal diversity remained unchanged. Infected pigs exhibited increased relative abundances of Lactobacillaceae and Bifidobacteriaceae and a decreased abundance of Pseudomonadota, including Enterobacteriaceae. In conclusion, Campylobacter co-colonisation induces distinct microbiome and metabolome alterations in pigs despite the absence of clinical disease. These findings highlight complex host-microbiota-pathogen interactions that may be relevant for future Campylobacter control strategies in pig production.

Abstract Vibrio natriegens is a halophilic, Gram-negative marine bacterium that is increasingly used in metabolic engineering applications due to its fast growth rate. In sparse minimal medium the organism has a doubling time of 25 minutes, which is about twice as fast as Escherichia coli under similar conditions. Given that its protein density is similarly constrained to that of E. coli , this necessitates that its metabolic enzymes are able to catalyze flux at a higher rate to sustain its metabolism. In this work, we measure the apparent turnover numbers of metabolically active enzymes in V. natriegens under a variety of growth conditions. The apparent turnover numbers of V. natriegens enzymes were measured in vivo by conducting coupled quantitative proteomics and 13 C metabolic flux analysis experiments under seven different carbon source conditions in sparse minimal medium. A high quality genome-scale metabolic model was constructed and curated using additional experimental data. This model was extended with enzyme constraints, and subsequently used to find kinetic parameters that minimize the difference between model predictions and experimental observations. This model guided data fusion approach enabled the estimation of 357 apparent turnover numbers for metabolically active enzymes in V. natriegens . Our results reveal that the metabolic enzymes of V. natriegens are in median 14-fold faster than those of E. coli under similar conditions. Moreover, we show that machine learning generated turnover number estimates substantially underestimate the kinetics of V. natriegens . Our turnover number estimates were used to parameterize multiple condition dependent enzyme constrained flux balance analysis models of V. natriegens , which improved their predictive accuracy compared to the machine learning parameterisation. The combined experimental-computational approach employed here sheds light on the mechanism V. natriegens uses to accelerate its growth. This approach can also be extended to other bacteria, increasing the availability of in vivo measured enzyme turnover numbers, and improving the predictive accuracy of enzyme constrained metabolic models of other microbes.

ObjectiveOxidative protein modifications have been linked to several diseases, but the variety and diversity of modifications are less studied.MethodsWe used the chicken egg protein ovalbumin and gas plasma technology, a potent source of various reactive species, for protein oxidation. Using high-resolution mass spectrometry and an in-house workflow, over 80 distinct oxidative protein modifications were mapped at per-amino-acid resolution. To examine how modification profiles depend on changes in reactive species types and concentrations, we generated 12 distinct argon gas plasmas by systematically varying molecular gas admixtures (water, ethanol, oxygen, and nitrogen).ResultsOptical emission spectroscopy (OES) and photometric determination of deposited long-lived species (hydrogen peroxide, nitrite, and nitrate) were applied to profile gas plasma conditions, revealing the admixture-dependent impact on the reactive oxygen/nitrogen species (ROS/RNS) fingerprint. Correlation analysis with mass spectrometry data revealed the significant involvement of atomic oxygen and hydrogen peroxide in protein oxidation. The enrichment of specific reactive species created by a defined gas plasma composition generated specific ovalbumin oxidation profiles resolved per amino acid. Feed gas-dependent oxidation hotspots, such as Trp149 for dry argon gas or Met274 for hydroxyl radical-rich humidified argon gas, were identified.DiscussionThis first-of-its-kind study reveals intricate relationships between dynamic reactive species environments and protein oxidation profiles using ovalbumin as a model system.

Despite the extensive research that has been conducted into the metabolism of Gram-positive bacteria, relatively little is known about the adaptations of Streptococcus dysgalactiae subsp. equisimilis (SDSE) during biofilm maturation. This study uses targeted metabolomics and MALDI MS imaging to investigate time-dependent changes in the metabolome composition. Key metabolites of energy metabolism were quantified in planktonic and biofilm bacteria. A distinct decline in the abundance of metabolites from all analyzed metabolic pathways was observed in sessile SDSE. Furthermore, we observed a broad decrease of nutrient utilization in biofilm-associated bacteria. Additionally, we examined the impact of deleting streptokinase on SDSE metabolism. Especially, concentrations of glycolysis intermediates and adenosine phosphates exhibited significant alterations when comparing the S118 strain with its mutant. The absence of the ska gene led to drastic changes in both energy and structural metabolism. These findings provide new insight into the metabolic adaptations of SDSE and represent an initial step toward understanding its role in pathogenesis.

As one of the most-consumed drugs worldwide, ibuprofen (IBU) reaches the environment in considerable amounts as environmental pollutant, necessitating studies of its biotransformation as potential removal mechanism. Here, we screened bacteria with known capabilities to degrade aromatic environmental pollutants, belonging to the genera Bacillus, Priestia (formerly also Bacillus), Paenibacillus, Mycobacterium, and Cupriavidus, for their ability to transform ibuprofen. We identified seven transformation products, namely 2-hydroxyibuprofen, carboxyibuprofen, ibuprofen pyranoside, 2-hydroxyibuprofen pyranoside, 4-carboxy-α-methylbenzene-acetic acid, 1-[4-(2-hydroxy-2-methylpropyl)phenyl]ethanone, and 2-hydroxyibuprofenmethyl ester. Based on our screening results, we focused on ibuprofen biotransformation by Priestia megaterium SBUG 518, to identify structures of transformation products, and to shed light on the drug’s impact on bacterial physiology. Biotransformation reactions by P. megaterium SBUG 518 involved (A) the hydroxylation of the isobutyl side chain at two positions, and (B) conjugate formation via esterification with a sugar molecule of the carboxylic group of ibuprofen and an ibuprofen hydroxylation product. Glycosylation seems to be a detoxification process, since the ibuprofen conjugate (ibuprofen pyranoside) was considerably less toxic than the parent compound to P. megaterium SBUG 518. Based on proteome profile changes and inhibition assays, cytochrome P450 systems appear to be central for ibuprofen transformation in P. megaterium SBUG 518. The toxic effect of ibuprofen appears to be caused by interference of the drug with different physiological pathways, especially sporulation.

Aster tataricus is a plant used in Traditional Chinese Medicine. From its roots, we isolated four endophytic fungi strains. After mass spectrometry analysis and subsequent molecular networking and dereplication, one of the strain’s extracts showed a cluster of yet undescribed natural products. Additionally, the extract was found to be lethal for the nematode Caenorhabditis elegans and cytotoxic against eukaryotic cell lines. The fungal strain was characterized by morphological and molecular studies, allowing its description as a new species in the genus Tengochaeta (Chaetomiaceae), Tengochaeta bulbillosa. After cultivation and extraction of the strain, the major secondary metabolites were isolated. Structure elucidation based on nuclear magnetic resonance spectroscopy and high-resolution tandem mass spectrometry revealed these compounds to be five new azaphilones. Additionally, the localization of these azaphilones in the host plant was studied by mass spectrometry imaging of different plant tissues, revealing that they were mainly localized in the aerial parts of the plant. The main compound, bulbillosin A, was evaluated for its activity against sixty cancer cell lines, revealing a differential cytotoxicity profile.

The successful sustainable cultivation of the well-known medicinal plant sundew on rewetted peatlands not only leads to the preservation of natural populations, but also provides a basis for the sustainable pharmaceutical use of the plant. The bioactive compounds of sundew, flavonoids and naphthoquinones, show biofilm-inhibiting properties against multidrug-resistant, ESBL-producing E. coli strains and open up new therapeutic possibilities. This study investigates the molecular mechanisms of these compounds in biofilm inhibition through proteomic analyses. Specific fractions of flavonoids and naphthoquinones, as well as individual substances like 7-methyljuglone and 2″-O-galloylhyperoside, are analyzed. Results show that naphthoquinones appear to act via central regulatory proteins such as OmpR and alter the stress response while flavonoids likely affect biofilm formation by creating an iron-poor environment through iron complexation and additionally influence polyamine balance, reducing intracellular spermidine levels. Further investigations including assays for iron complexation and analysis of polyamines confirmed the proteomic data. Safety evaluations through cytotoxicity tests in 3D cell cultures and the Galleria mellonella in vivo model confirm the safety of the extracts used. These findings highlight sundew as a promising candidate for new phytopharmaceuticals.

Biocatalysis provides an attractive approach to facilitate synthetic reactions in aqueous media. Motivated by the discovery of promiscuous aminolysis activity of esterases, we exploited the esterase from Pyrobaculum calidifontis VA1 (PestE) for the synthesis of carbamates from different aliphatic, aromatic, and arylaliphatic amines and a set of carbonates such as dimethyl-, dibenzyl-, or diallyl carbonate. Thus, aniline and benzylamine derivatives, aliphatic and even secondary amines could be efficiently converted into the corresponding benzyloxycarbonyl (Cbz)- or allyloxycarbonyl (Alloc)-protected products in bulk water, with (isolated) yields of up to 99 %.

Biofilms of pathogenic bacteria are responsible for persistent infections in humans, therefore investigations of biofilm formation and treatment strategies are required. The gram-negative enterobacterium Escherichia (E.) coli is the most common pathogen causing chronic or recurring urinary tract infections. Metabolomics approaches targeted the bacterium to investigate specific metabolic patterns of biofilms and regulatory influences on biofilm formation. In this study, we aimed to investigate the metabolome of biofilms formed by the multidrug-resistant extended-spectrum beta-lactamase-producing (ESBL) E. coli PBIO729. For this purpose, a protocol for fast sampling of the macrocolony biofilms and efficient extraction of metabolites was optimized. Validation of an LC-MS/MS method confirmed its usability for the analysis of nucleotides and other phosphorylated metabolites. A GC-MS approach was used to monitor nutrient uptake from the medium in addition to the analysis of amino acid content and metabolites of glycolysis and TCA cycle in E. coli biofilms.

Introduction There are only few studies comparing differences in the outcome of primary versus secondary gliosarcoma. This study aimed to review the outcome and survival of patients with primary or secondary gliosarcoma following surgical resection and adjuvant treatment. The data were also matched with data of patients with primary and secondary glioblastoma (GBM).Patients and methods Treatment histories of 10 patients with primary gliosarcoma and 10 patients with secondary gliosarcoma were analysed and compared. Additionally, data of 20 patients with primary and 20 patients with secondary GBM were analysed and compared. All patients underwent surgical resection of the tumour in our department. Follow-up data, progression-free survival (PFS), and median overall survival (mOS) were evaluated.Results The median PFS in patients with primary gliosarcoma was significantly higher than in patients with secondary gliosarcoma (p = 0.037). The 6-month PFS rates were 80.0% in patients with primary and 30.0% in patients with secondary gliosarcoma. Upon recurrence, five patients with primary gliosarcoma and four patients with secondary gliosarcoma underwent repeat surgical resection. The mOS of patients with primary gliosarcoma was significantly higher than that of patients with secondary gliosarcoma (p = 0.031). The percentage of patients surviving at 1-year/2-year follow-up in primary gliosarcoma was 70%/20%, while it was only 10%/10% in secondary gliosarcoma. When PFS and mOS of primary gliosarcoma was compared to primary GBM, there were no statistically differences (p = 0.509; p = 0.435). The PFS and mOS of secondary gliosarcoma and secondary GBM were also comparable (p = 0.290 and p = 0.390).Conclusion Patients with primary gliosarcoma have a higher PFS and mOS compared to those with secondary gliosarcoma. In the case of tumour recurrence, patients with secondary gliosarcoma harbour an unfavourable prognosis with limited further options. The outcome of patients with primary or secondary gliosarcoma is comparable to that of patients with primary or secondary GBM.

Background Streptococcus dysgalactiae subspecies equisimilis (SDSE) is increasingly recognized as an emerging cause of invasive diseases including necrotizing soft tissue infections (NSTIs). In contrast to the closely related Streptococcus pyogenes, SDSE infections mainly affect older and comorbid patients. Biofilm formation has been demonstrated in soft tissue biopsies of S. pyogenes NSTI cases. Results Here, we show that bacterial aggregations indicative of biofilms are also present in SDSE NSTI. Although streptokinase (Ska) activity and biofilm formation did not correlate in a diverse set of clinical SDSE isolates, addition of exogenous Ska at an early time point prevented biofilm formation for selected strains. Deletion of ska in SDSE S118 strain resulted in increased biofilm forming capacity. Ska-deficient mutant strain was characterized by a higher metabolic activity and consequent metabolome profiling of biofilms identified higher deposition of a wide range of metabolites as compared to the wild-type. Conclusions Our results argue that Ska suppresses biofilm formation in SDSE independent of its original plasminogen converting activity. However, the impact of biofilms and its consequences for patient outcomes in streptococcal NSTIs remain to be elucidated.

Cold physical plasma shows promising preclinical results as an anticancer strategy. The technology generates a variety of reactive oxygen species (ROS) mediating gas plasma-induced effects in cells and tissues. On the cellular level, ROS can trigger oxidative stress-related responses. On the biomolecular level, ROS can introduce oxidative modifications, potentially leading to functional alterations. To better understand plasma treatment in oncology therapies, we treated tyrosine kinase inhibitors (TKIs) with plasma to investigate the efficacy upon oxidation as well as plasma pre-treated A549 lung cancer cells before TKI treatment to investigate combination effects. Specifically, a library of 37 compounds was exposed to the atmospheric pressure argon plasma jet kINPen before being added to the cells. Most gas plasma-treated TKIs showed a significant decline in anticancer efficacy. The experimental compound NVP-AEW541 showed elevated tumor-toxic effects after exposure to gas plasma. In A549 cells pre-treated with gas plasma, all TKIs but one showed additive toxicity. In summary, this first study on gas plasma treatment of TKIs and lung cancer cells in combination treatments revealed that direct gas plasma TKI treatment decreased the activity of most but not all compounds investigated, while gas plasma pre-treated cells mostly showed additive toxicity in response to TKI exposure.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia worldwide. A key pathogenic mechanism that exacerbates severity of disease is the disruption of the alveolar-capillary barrier. However, the specific virulence mechanisms responsible for this in the human lung are not yet fully understood. In this study, we infected living human lung tissue with Strep. pneumoniae and observed a significant degradation of the central junctional proteins occludin and vascular endothelial cadherin, indicating barrier disruption. Surprisingly, neither pneumolysin, bacterial hydrogen peroxide nor pro-inflammatory activation were sufficient to cause this junctional degradation. Instead, pneumococcal infection led to a significant decrease of pH (similar to 6), resulting in the acidification of the alveolar microenvironment, which was linked to junctional degradation. Stabilising the pH at physiological levels during infection reversed this effect, even in a therapeutic-like approach. Further analysis of bacterial metabolites and RNA sequencing revealed that sugar consumption and subsequent lactate production were the major factors contributing to bacterially induced alveolar acidification, which also hindered the release of critical immune factors. Our findings highlight bacterial metabolite-induced acidification as an independent virulence mechanism for barrier disruption and inflammatory dysregulation in pneumonia. Thus, our data suggest that strictly monitoring and buffering alveolar pH during infections caused by fermentative bacteria could serve as an adjunctive therapeutic strategy for sustaining barrier integrity and immune response.

ABSTRACT The conserved protein WhiA is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motif and is a transcription regulator of the key cell division gene ftsZ in actinomycetes. In Bacillus subtilis, the absence of WhiA influences both cell division and chromosome segregation; however, the protein does not regulate any gene involved in these processes. In this study, we addressed three alternative mechanisms by which WhiA might exert its activity in B. subtilis and examined whether WhiA influences either (i) central carbon metabolism, (ii) fatty acid composition of the cell membrane, or (iii) chromosome organization. Mutations in glycolytic enzymes have been shown to influence both cell division and DNA replication. To measure the effect of WhiA on carbon metabolism, we tested different carbon sources and measured exometabolome fluxes. This revealed that the absence of WhiA does not affect glycolysis but does influence the pool of branched-chain fatty acid precursors. Due to the effect of WhiA on chromosome segregation, we examine chromosome organization in a ∆whiA mutant using chromosome conformation capture (Hi-C) analysis. This revealed a local reduction in short-range chromosome interactions. Together, these findings provide new avenues for future research into how this protein works in the non-actinomycete firmicutes. IMPORTANCE WhiA is a conserved DNA-binding protein that influences cell division in many Gram-positive bacteria and, in B. subtilis, also chromosome segregation. How WhiA works in Bacillus subtilis is unknown. Here, we tested three hypothetical mechanisms using metabolomics, fatty acid analysis, and chromosome confirmation capture experiments. This revealed that WhiA does not influence cell division and chromosome segregation by modulating either central carbon metabolism or fatty acid composition. However, the inactivation of WhiA reduces short-range chromosome interactions. These findings provide new avenues to study the molecular mechanism of WhiA in the future.

Background Klebsiella pneumoniae , which is frequently associated with hospital- and community-acquired infections, contains multidrug-resistant (MDR), hypervirulent (hv), non-MDR/non-hv as well as convergent representatives. It is known that mostly international high-risk clonal lineages including sequence types (ST) 11, 147, 258, and 307 drive their global spread. ST395, which was first reported in the context of a carbapenemase-associated outbreak in France in 2010, is a less well-characterized, yet emerging clonal lineage. Methods We computationally analyzed a large collection of K. pneumoniae ST395 genomes ( n = 297) both sequenced in this study and reported previously. By applying multiple bioinformatics tools, we investigated the core-genome phylogeny and evolution of ST395 as well as distribution of accessory genome elements associated with antibiotic resistance and virulence features. Results Clustering of the core-SNP alignment revealed four major clades with eight smaller subclades. The subclades likely evolved through large chromosomal recombination, which involved different K. pneumoniae donors and affected, inter alia , capsule and lipopolysaccharide antigen biosynthesis regions. Most genomes contained acquired resistance genes to extended-spectrum cephalosporins, carbapenems, and other antibiotic classes carried by multiple plasmid types, and many were positive for hypervirulence markers, including the siderophore aerobactin. The detection of “hybrid” resistance and virulence plasmids suggests the occurrence of the convergent ST395 pathotype. Conclusions To the best of our knowledge, this is the first study that investigated a large international collection of K. pneumoniae ST395 genomes and elucidated phylogenetics and detailed genomic characteristics of this emerging high-risk clonal lineage.

BACKGROUND:Reactive oxygen species (ROS) are implicated in cancer therapy and as drivers of microenvironmental tumour cell adaptations. Medical gas plasma is a multi-ROS generating technology that has been shown effective for palliative tumour control in head and neck cancer (HNC) patients before tumour cells adapted to the oxidative stress and growth regressed fatally.METHODS:In a bedside-to-bench approach, we sought to explore the oxidative stress adaptation in two human squamous cell carcinoma cell lines. Gas plasma was utilised as a putative therapeutic agent and chronic oxidative stress inducer.RESULTS:Cellular responses of single and multiple treated cells were compared regarding sensitivity, cellular senescence, redox state and cytokine release. Whole transcriptome analysis revealed a strong correlation of cancer cell adaption with increased interleukin 1 receptor type 2 (IL1R2) expression. Using magnetic resonance imaging, tumour growth and gas plasma treatment responses of wild-type (WT) and repeatedly exposed (RE) A431 cells were further investigated in a xenograft model in vivo. RE cells generated significantly smaller tumours with suppressed inflammatory secretion profiles and increased epidermal growth factor receptor (EGFR) activity showing significantly lower gas plasma sensitivity until day 8.CONCLUSIONS:Clinically, combination treatments together with cetuximab, an EGFR inhibitor, may overcome acquired oxidative stress resistance in HNC.

Additional file 1: Table S1. Characteristics of the isolates from the ST395 dataset. Table S2. Comparison of cgMLST profiles of isolates representing ST395 subclades. Table S3. Location of carbapenemase genes.

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.