Browsing by Subject "Biofilm"

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Adaptations of Prevotella bryantii B14 to short-chain fatty acids and monensin exposure
(2023) Trautmann, Andrej; Seifert, Jana
The rumen microbiome constitutes a complex ecosystem including a vast diversity of organisms that produce and consume short-chain fatty acids (SCFAs). It is of great interest to analyze these activities as they are of benefit for both, the microbiome and the host. This dissertation aims to display the proteome and metabolome of the predominant ruminal representative Prevotella bryantii B14 in presence of various SCFA and under exposure of the antibiotic monensin in pure and mixed culture (in vitro). Due to the strong contributing abundance of Prevotellaceae in the rumen microbiome, the representative P. bryantii B14 (DSM 11371) was chosen to investigate biochemical factors for the success of withstanding monensin and the impact of SCFA on their growth. The current work is composed of two effective publications. The formatting was aligned to the dissertation. The first publication, studying the supplementation of various SCFAs, showed SCFAs as growth promoting but not essential for P. bryantii B14. Pure cultures of P. bryantii B14 were grown in Hungate tubes under anaerobic conditions. Gas chromatography time of flight mass spectrometry (GC-ToF MS) was used to quantify long-chain fatty acid (LCFA) profiles of P. bryantii B14. Proteins of P. bryantii B14 were identified and quantified by using a mass spectrometry-based, label-free approach. Different growth behavior was observed depending on the supplemented SCFA. An implementation of SCFAs on LCFAs and the composition on membrane proteins became evident. Supplementing P. bryantii B14 with branched-chain fatty acids (BCFAs), in particular isovaleric acid, showed an increase of the 3-IPM pathway, which is part of the branched-chain amino acid (BCAA) metabolism. Findings point out that the structure similarity of isovaleric acid and valine is most likely enhancing the conversion of BCFA into BCAA. The required set of enzymes of the BCAA metabolism supported this perspective. The ionophore monensin has antibiotic properties which are used in cattle fattening but also for treating ketosis and acidosis in ruminants. In the second publication, P. bryantii B14 was exposed to different concentrations of monensin (0, 10, 20 and 50 uM) and to different exposure times (9, 24, 48 and 72 h) with and without monensin. Growth behavior, glucose and intracellular sodium concentration were determined. Proteins were analyzed by label-free quantification method using the same method as in the previous mentioned experiment. Fluorescence microscopy was used to observe extracellular polysaccharides (EPS) of P. bryantii B14. A progressing monensin exposure triggered disconnection between P. bryantii B14 cells to the sacrificial EPS layer by increasing its number and amount of carbohydrate active enzymes (CAZymes). Simultaneously, an increase of extracellular glucose was monitored. Reduction of intracellular sodium was likely to be performed by increasing the abundance of ion-transporters and an increased activity of Na+-translocating NADH:quinone oxidoreductase under monensin supplementation. The role of monensin supplemented Prevotella in a mixed culture of the rumen microbiome was described. Extracted rumen fluid from cows was incubated anaerobically by using the rumen simulation technique (Rusitec). Proteomics of the solid phase was applied by using a similar approach as in the previous related studies. Metabolomics of the liquid phase from the Rusitec content was performed by using 1H-nuclear magnetic resonance (NMR) spectroscopy. Further parameters such as pH, gas and methane production were monitored over time. The experiment was constituted out of three phases starting with an adaptation phase of 7 days. A subsequent treatment phase followed, where monensin was supplemented via the daily introduced total mixed ration (TMR) for further 7 days. The elution phase was the final phase when monensin supplementation was stopped and monitoring was continued for further 3 days. Metabolomics and proteomics showed that members of the genus Prevotella remained most abundant under monensin supplementation. Furthermore, shifting the ruminal metabolism to an increased production of propionate by shifting the metabolism of Prevotella sp. to an enhanced succinate production. The current work shows the impact of SCFAs on various metabolic functions of P. bryantii B14. Diverse defence mechanisms of Prevotella sp., in particular P. bryantii B14, were shown to avoid the antibiotic effects of monensin.
Vergleichende Transkriptomanalyse und funktionelle Untersuchungen von enterohämorrhagischen Escherichia coli nach Kultivierung in Pflanzenmedium
(2020) Bufe, Thorsten; Schmidt, Herbert
Enterohemorrhagic Escherichia coli (EHEC) are human pathogens which are able to cause severe gastrointestinal diseases in humans. The gastrointestinal tract of cattle is considered as the main reservoir for EHEC and contaminated raw meat represents the primary source of infection. Yet there have been increasing reports over the last few decades of EHEC infections that were linked to the consumption of raw vegetables. Today it is generally accepted that EHEC bacteria are able to use plants as their secondary hosts, thus favouring the transmission to humans. To improve the understanding of this pathogen-plant interaction fundamental knowledge about the pathogens’ molecular adaptions towards plant material is urgently required. In the cope of this study the adaption of different EHEC strains towards components of the plant was examined. Therefore O157:H7 strain Sakai, O104:H4 strain C227-11phicu and O157:H strain 3072/96 were chosen as surrogates. In growth experiments performed with an artificial lettuce medium it could be shown that components of the lettuce were sufficient for the proliferation of the three strains. RNA-sequencing was performed to study the differential gene expression of the three strains after the growth in lettuce medium compared to the growth in M9 minimal medium. In order to compare genes according to standardized gene denotations, the differential gene expression analysis was performed on the basis of a shared genome including the genomes of the three pathogenic strains as well as the genome of Escherichia coli strain K-12 substrain MG1655. Analogous to the successful growth in presence of components of the plant an upregulation of genes involved in carbohydrate and peptide metabolism throughout all three strains was observed. Especially genes involved in the catabolism of lactose (lacZ), ribose (rbsAC) and xylose (xylF) were found to be uniformly upregulated. The greatest differences among the strains accounted for the regulation of motility and chemotaxis genes. O104:H4 strain C227-11phicu showed a strong upregulation of all three classes of the flagellar hierarchy (class I, II and III) in presence of plant derived compounds. These included genes involved in the establishment of the basal body hook structure (fli, flg), the synthesis of the flagellar filament (fliC), and the chemotaxis-system (che, tap, tar). In contrast, O157:H7 strain Sakai only featured upregulation of class I and class II genes. According to the transcriptional data both of these strains also showed increased swimming and swarming behaviour on motility plates in presence of lettuce extract. Solely O157:H- strain 3072/96, which is non-motile due to a deletion in the flhC gene, showed an upregulation of virulence factors encoded on the LEE pathogenicity island, including genes involved in the establishment of the T3SS (esc) and T3SS secreted effectors (esp). Interestingly, it was shown for O157:H- strain 3072/96 to have a powerful capacity to form biofilms in M9 minimal medium. Furthermore it was proven that the complementation of an intact flhC gene restored motility in O157:H- strain 3072/96. In this regard it could be shown that the deletion in flhC was not the mere reason for the augmented biofilm formation capacity. In addition to the biofilm formation, the strains’ potential to adhere to HT-29 cells was examined. Here a significantly increased adherence potential for O157:H- strain 3072/96 with respect to the motile strains could be observed, the lowest adherence potential was determined for O157:H7 strain Sakai. The results presented in this study clearly indicate that the different EHEC strains are capable to adapt towards the nutrient availability provided by their plantal host. It can be assumed that flagella and the chemotaxis system play a fundamental role in the finding and exploitation of the plant. Furthermore curli structures might play a crucial role in the initial adherence and the subsequent establishment of a biofilm on plant tissues. Presumably, besides the typical plant associated outbreak strain O157:H7 strain Sakai, there are further strains capable of utilizing their genetic repertoire in order to adapt towards the atypical environmental conditions within this niche. The findings of this study suggest that the strains, besides sharing multiple coinciding mechanisms, are able to adapt in a strain specific manner and use different strategies in coping with plants as their secondary hosts.