Browsing by Subject "Kot"

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    Ecological studies of the Lactobacillus biota in the human digestive tract and adaptation of intestinal lactobacilli to the sourdough ecosystem
    (2005) Dal Bello, Fabio; Hertel, Christian
    Among the bacteria inhabiting the human gut, lactobacilli have received considerable attention, due to their putative health promoting effects (Reid, 1999; Vaughan et al., 1999). Cultivation of lactobacilli is considered to be reliable and numerous studies using plating on selective media have been performed to investigate these bacteria in intestinal ecosystems (Tannock, 1995; Reuter, 2001). Recently, the application of PCR-DGGE in combination with primers specific for lactic acid bacteria (LAB) detected species which are not considered to be intestinal inhabitants but food-associated, such as Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides and Pediococcus pentosaceus (Walter et al., 2001; Heilig et al., 2002). Remarkably, these species could not be recovered by traditional bacteriological culture on Rogosa SL agar (Walter et al., 2001). In Chapter III, different cultivation media, as well as new incubation conditions were applied to overcome these difficulties. Human faecal samples were plated on selective and non-selective media and incubated under standard condition (37°C, anaerobiosis) for faecal LAB as well as alternative condition (30°C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected stronger by the incubation condition than by the used medium. It was observed that food-associated LAB such as L. sakei and Lc. mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition on Rogosa SL agar showed that L. sakei is one of the predominant food-associated LAB species in faecal samples, reaching counts of up to 106 CFU per gram faeces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in faeces. Examination of the faecal Lactobacillus populations over longer periods has revealed marked variation in the complexity and stability of these populations among human subjects (Vanhoutte et al., 2004, Walter et al., 2001). Ecological studies indicate that most Lactobacillus species found in the human gastrointestinal tract (GIT) are likely to be transient (allochthonous), originating from either the oral cavity or food (reviewed in Bibiloni et al., 2004). In order to investigate if oral lactobacilli constitute a part of the faecal Lactobacillus biota, the Lactobacillus biota of saliva and faeces of three human subjects were investigated and compared at two time-points in a three months interval (Chapter IV). The species composition of the Lactobacillus biota of human saliva and faeces was found to be subject-specific and fluctuated to some degree, but the species Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus vaginalis were detected at both time-points in saliva and faecal samples of individual subjects. RAPD-PCR analysis indicated that several strains of these species were present both in the oral cavity and in the faecal samples of the same subject. Oral isolates of the species L. gasseri and L. vaginalis showing identical RAPD types were found to persist over time, suggesting that these species are autochthonous to the oral cavity. The results of Chapter IV, together with recently published data (reviewed in Bibiloni et al., 2004), give strong evidence that some lactobacilli found in human faeces are allochthonous to the intestine and originate from the oral cavity. Lactobacilli have been detected in diverse environments and have been the subject of considerable research due to their commercial use in the food industry (reviewed in Hammes and Hertel, 2003). Several Lactobacillus species are commonly detected in both fermented food and the human GIT, but the genetic background for this ecological versatility is poorly understood. Lactobacillus reuteri is a dominant member of the microbiota of type II sourdough fermentations (Meroth et al., 2003) and is considered one of the truly autochthonous Lactobacillus species in humans (Reuter, 2001). The in vivo expression technology (IVET) developed by Walter et al. (2003) was used to identify genes (so-called ivi genes) of the sourdough isolate L. reuteri LTH5531 that show elevated levels of expression during growth of this organism in a type II sourdough fermentation (Chapter V) and during passage through the GIT of mice (Chapter VI). Thirty-eight induced fusions were found to be highly expressed during the sourdough fermentation (Chapter V), and 29 genes could be identified on the basis of the available sequence information. Four genes encoded stress-related functions (e.g. acid and general stress response) reflecting the harsh conditions prevailing during sourdough fermentation. Further eight genes were involved in acquisition and synthesis of amino acids and nucleotides, indicating their limited availability in sourdough. The remaining genes were either part of functionally unrelated pathways or encoded hypothetical proteins. The identification of a putative proteinase and a component of the arginine deiminase pathway are of technological interest, as they are potentially involved in the formation of aroma precursors. Remarkably, IVET with the genomic library that was successfully used in the sourdough study (Chapter V) did not detect ivi promoters when LTH5531 inhabited the GIT of mice (Chapter VI). With IVET, active promoters are selected by expression of an "essential growth factor" (in our system the erythromycin resistance mediated by ErmGT) that allows the organism to colonize and/ or grow in the ecosystem (Rainey, 1999, Walter et al., 2003). Expression of ivi promoters in particular ecosystems must therefore be permanent and strong in order to allow comparable growth rates of ivi clones and clones bearing constitutive promoters, especially in the GIT, where inactive bacteria are washed out. The findings of Chapter V and VI indicate that L. reuteri LTH5531 does not possess strongly expressed "GIT inducible" genes, while possessing 38 ones specifically induced in sourdough. Ivi genes are more likely to contribute to the ecological performance of an organism in a specific environment than genes expressed equally in a broad range of habitats (Rainey, 1999, Gal et al, 2003, Walter et al., 2005). Therefore, traits encoded by ivi genes are likely to be adaptive and the extent of their expression would be shaped by natural selection to improve ecological fitness. The presence of thirty-eight "sourdough specific" ivi fusions in L. reuteri LTH5531 probably reflects the long term adaptation of LTH5531 to the sourdough environment, just as ivi genes detected in strain 100-23 reflect adaptation of this GIT isolate to the rodent GIT (Walter et al., 2003). Indeed, LTH5531 was isolated from an experimental sourdough that had been inoculated with an industrial starter. This industrial starter has been propagated over several years, giving the organisms present sufficient time to adapt. In accordance with this, by using RAPD-PCR, Meroth et al. (2003) showed that strain LTH5531 was present in a commercial type II sourdough starter collected 10 years prior isolation of LTH5531, thus indicating that this strain has adapted to the sourdough environment for at least 10 years. The results of Chapter V clearly demonstrated that knowledge of gene expression and metabolic activities of bacteria during food fermentations can be obtained by applying IVET. The results collected provide an important molecular basis on which improved starter strains can be developed for industrial exploitation. Moreover, the results of Chapter VI show the importance of working with highly adapted, autochthonous strains in studies of microbial ecology in order to reveal the adaptive interactions responsible for the ecological success of these bacteria in their natural environment or during food fermentations.
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    The intestinal microbiome and metabolome of dairy cows under challenging conditions
    (2022) Tröscher-Mußotter, Johanna; Seifert, Jana
    The modern dairy cow is confronted with a multitude of stressors throughout live. Especially calving, transition, and microbial infections are strong challenges that can have long-lasting impacts on the cow’s health and performance. Yet, individuals can differ in their response towards these challenges, raising the question which characteristics in the dairy cow contribute to a more or less robust animal. Apart from genetics, the gut microbiome and the entailed metabolome is assumed to play an important role in buffering or promoting host stress. This is also due to the fact that the gut microbiome is strongly involved in the hosts energy metabolism and immune system. As dairy cows often show performance impairments during high energy demanding periods, it could be suggested that improving energy metabolism in these specific phases might reduce the negative phenotypic outcomes. This was tested using dietary L-carnitine, a metabolite inevitably necessary for energy metabolism. However, no supplement effects on the intestinal microbiome or metabolome have been found in the present work. Supplementation was continued throughout the complete trial. Calving functioned as an individual stimulus, and an intra-venous LPS injection induced a standardized inflammatory challenge, as a specific amount of LPS per kg of bodyweight was applied per cow. Supplemented animals were compared to a control group. In total, the animals were studied across 168 days and sampled extensively at several sites. The focus of this thesis was to analyze the bacterial consortia and metabolites of both, host and bacteria, in rumen, duodenum, and feces throughout the given period. This was to elucidate the metabolic reactions and bacterial shifts during the mentioned challenging periods and their response to the L-carnitine supplementation. First, the ruminal and duodenal fluid microbiome of eight double cannulated animals during the two respective challenges was analysed. Before calving and feed change, rumen and duodenal fluid bacterial consortia were significantly different, thereafter very alike. Strong microbial community shifts were observed throughout the complete trial irrespectively of the matrix. Both matrices varied in their metabolite patterns indicating functional variation among sites. Also, a strong increase of Bifidobacterium at three days after calving was observed in almost all animals pointing towards a strong biological purpose. This needs to be investigated in upcoming studies. The study could show increasing ketogenic activities in the animals after calving and proposes a possible protective host-microbial interaction, against a ruminal collapse induced by LPS challenge, here described as "microbial airbag". The second part included fecal samples of the same animals, which were analyzed for their bacterial consortia and targeted metabolites. Different dynamics and diversities of microbial communities amongst the individuals were observed, according to which animals could be grouped into three microbiome clusters. These showed in part fundamentally different metabolic, health, and performance parameters, indicating strong host-microbiome-metabolite interactions. The study demonstrated that microbiome clustering may contribute to identifying different metabo- and production types. Again, the study observed a strong increase of Bifidobacterium at three days after calving and even during the LPS challenge supporting the findings of the former study. This strengthens the hypothesis that also for the cow Bifidobacterium may have protective effects, as this genus is largely involved in health promoting activities. The power of this project lies in the massive sampling of different body sites in dairy cows across a very long period of time and finally, merging of the collected data. This, however, requires high computational efforts as numerous time points, matrices, animals, measurements, treatments, feeding regimen, and challenges resulted into a large bandwidth of parameters and metadata. Yet, it bears the potential to better elucidate and understand actions and reactions of the host, its microbiome and metabolism, as well as organ-axes in dairy cows and thereby gaining a more holistic picture of these complex animals. The aim of analyzing the host, its microbiome and metabolome throughout challenging periods resulted into the following main findings. Time, calving, and feed change remarkably change the microbial communities and to a lesser extent the metabolomes in all three matrices. Rumen and proximal duodenal fluid samples significantly differ in their metabolomes but not in their microbiome. In all matrices, an increase of Bifidobacterium is seen within three days after calving, which has to be further researched. Across the herd, three distinct microbiome clusters are found, which significantly differ in their production and health parameters.