@phdthesis{GarciaBetancur2018,
  author    = {Garcia Betancur, Juan Carlos},
  title     = {Divergence of cell-fates in multicellular aggregates of \(Staphylococcus\) \(aureus\) defines acute and chronic infection cell types},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148059},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Staphylococcus aureus is a versatile human pathogen that normally develops acute or chronic infections. The broad range of diseases caused by this bacterium facilitates the escape from the host's immune response as well as from target-specific antimicrobial therapies. Nevertheless, the underlying cellular and molecular mechanisms that enable S. aureus to cause these disparate types of infections are largely unknown. In this work, we depicted a novel genetic program involved in the development of cell-fate decision, which promotes the differentiation of the staphylococcal cells into two genetically identical but differently heritable cell lines capable of defining the course of an infection, by simultaneously progressing to (i) a biofilm-associated chronic infection or (ii) a disperse acute bacteremia. Here, S. aureus growing in architecturally complex multicellular communities harbored different cell types that followed an exclusive developmental plan, resulting in a clonal heterogeneous population. We found that these cell types are physiologically specialized and that, this specialization impacts the collective behavior within the multicellular aggregates. Whereas one cell line that we named BRcells, promotes biofilm formation that engenders chronic infections, the second cell line, which we termed DRcells is planktonic and synthetizes virulence factors, such as toxins that can drive acute bacteremia. We identified that the positive feedback loop present in Agr quorum sensing system of S. aureus acts a bimodal switch able to antagonistically control the divergence of these two physiologically distinct, heritable cell lines. Also, we found that this bimodal switch was triggered in response to environmental signals particularly extracellular Mg2+, affecting the size of the subpopulations in specific colonization environments. Specifically, Mg2+-enriched environments enhanced the binding of this cation to the staphylococcal teichoic acids, increasing the rigidity of the cell wall and triggering a genetic program involving the alternative sigma factor σB that downregulated the Agr bimodal switch, favoring the enrichment of the BRcells type. Therefore, colonization environments with different Mg2+ content favored different outcomes in the bimodal system, defining distinct ratio in the BRcells/DRcells subpopulations and the S. aureus outcome in our in vitro model of development of multicellular aggregates and, the infection outcome in an in vivo mice infection model. In this prime human pathogen cell-fate decision-making generates a conserved pattern of heritable, physiological heterogeneity that actively contributes to determine the course of an infection through the emergence and spatio-temporal dynamics of distinct and specialized cell types. In conclusion, this work demonstrates that cell differentiation in pathogenic bacteria is a fundamental phenomenon and its understanding, is central to understand nosocomial infections and to designing new anti-infective strategies},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Audretsch2013,
  author    = {Audretsch, Christof},
  title     = {Analysing Quorum Sensing and Biofilm formation in Staphylococcus aureus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-92189},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Staphylococcus aureus (SA) causes nosocomial infections including life threatening sepsis by multi-resistant strains (MRSA). It has the ability to form biofilms to protect it from the host immune system and from anti staphylococcal drugs. Biofilm and planctonic life style is regulated by a complex Quorum-Sensing (QS) system with agr as a central regulator. To study biofilm formation and QS mechanisms in SA a Boolean network was build (94 nodes, 184 edges) including two different component systems such as agr, sae and arl. Important proteins such as Sar, Rot and SigB were included as further nodes in the model. System analysis showed there are only two stable states biofilm forming versus planctonic with clearly different subnetworks turned on. Validation according to gene expression data confirmed this. Network consistency was tested first according to previous knowledge and literature. Furthermore, the predicted node activity of different in silico knock-out strains agreed well with corresponding micro array experiments and data sets. Additional validation included the expression of further nodes (Northern blots) and biofilm production compared in different knock-out strains in biofilm adherence assays. The model faithfully reproduces the behaviour of QS signalling mutants. The integrated model allows also prediction of various other network mutations and is supported by experimental data from different strains. Furthermore, the well connected hub proteins elucidate how integration of different inputs is achieved by the QS network. For in silico as well as in vitro experiments it was found that the sae-locus is also a central modulator of biofilm production. Sae knock-out strains showed stronger biofilms. Wild type phenotype was rescued by sae complementation. To elucidate the way in which sae takes influence on biofilm formation the network was used and Venn-diagrams were made, revealing nodes regulated by sae and changed in biofilms. In these Venn-diagrams nucleases and extracellular proteins were found to be promising nodes. The network revealed DNAse to be of great importance. Therefore qualitatively the DNAse amount, produced by different SA mutants was measured, it was tried to dissolve biofilms with according amounts of DNAse and the concentration of nucleic acids, proteins and polysaccharides were measured in biofilms of different SA mutants. With its thorough validation the network model provides a powerful tool to study QS and biofilm formation in SA, including successful predictions for different knock-out mutant behaviour, QS signalling and biofilm formation. This includes implications for the behaviour of MRSA strains and mutants. Key regulatory mutation combinations (agr-, sae-, sae-/agr-, sigB+, sigB+/sae-) were directly tested in the model but also in experiments. High connectivity was a good guide to identify master regulators, whose detailed behaviour was studied both in vitro and in the model. Together, both lines of evidence support in particular a refined regulatory role for sae and agr with involvement in biofilm repression and/or SA dissemination. With examination of the composition of different mutant biofilms as well as with the examination of the reaction cascade that connects sae to the biofilm forming ability of SA and also by postulating that nucleases might play an important role in that, first steps were taken in proving and explaining regulatory links leading from sae to biofilms. Furthermore differences in biofilms of different mutant SA strains were found leading us in perspective towards a new understanding of biofilms including knowledge how to better regulate, fight and use its different properties.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Bauchart2010,
  author    = {Bauchart, Philippe Michel Paul},
  title     = {Evaluation of the Zoonotic Risk of Escherichia coli Strains involved in Extraintestinal Infections of Humans and Animals. Characterization of New Virulences Factors in ExPEC},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48848},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Avian pathogenic Escherichia coli (APEC) represent a subset of the so-called extraintestinal pathogenic Escherichia coli (ExPEC) pathotype that can cause various extraintestinal infections in humans and animals. APEC are the causative agent of localized colibacillosis or systemic infection in poultry. In this latter case, the syndrome starts as an infection of the upper respiratory tract and develops into a systemic infection. Generally, ExPEC are characterized by a broad variety of virulence-associated factors that may contribute to pathogenesis. Major virulence factors, however, that clearly define this pathotype, have not been identified. Instead, virulence-associated genes of ExPEC and thus also of APEC could be used in a mix-and-match-fashion. Both pathotypes could not be clearly distinguished by molecular epidemiology, and this suggested a hypothetical zoonotic risk caused by APEC. Accordingly, the main scientific question of this study was to characterize common traits as well as differences of APEC and human ExPEC variants that could either support the possible zoonotic risk posed by these pathogenic E. coli strains or indicate factors involved in host specificity. Comparative genomic analysis of selected APEC and human ExPEC isolates of the same serotype indicated that these variants could not be clearly distinguished on the basis of (i) general phenotypes, (ii) phylogeny, (iii) the presence of typical ExPEC virulence genes, and (iv) the presence of pathoadaptive mutations. Allelic variations in genes coding for adhesins such as MatB and CsgA or their regulators MatA and CsgD have been observed, but further studies are required to analyze their impact on pathogenicity. On this background, the second part of this thesis focused on the analysis of differences between human ExPEC and APEC isolates at the gene expression level. The analysis of gene expression of APEC and human ExPEC under growth conditions that mimick their hosts should answer the question whether these bacterial variants may express factors required for their host-specificity. The transcriptomes of APEC strain BEN374 and human ExPEC isolate IHE3034 were compared to decipher whether there was a specific or common behavior of APEC and human ExPEC, in response to the different body temperatures of man (37°C) or poultry (41°C). Only a few genes were induced at 41 °C in each strain relative to growth at 37 °C. The group of down-regulated genes in both strains was markedly bigger and mainly included motility and chemotaxis genes. The results obtained from the transcriptome, genomic as well as phenotypic comparison of human ExPEC and APEC, supports the idea of a potential zoonotic risk of APEC and certain human ExPEC variants. In the third part of the thesis, the focus was set on the characterization of Mat fimbriae, and their potential role during ExPEC infection. Comparison of the mat gene cluster in K-12 strain MG1655 and O18:K1 isolate IHE3034 led to the discovery of differences in (i) DNA sequence, (ii) the presence of transcriptional start and transcription factor binding sites as well as (iii) the structure of the matA upstream region that account for the different regulation of Mat fimbriae expression in these strains. A negative role of the H-NS protein on Mat fimbriae expression was also proven at 20 °C and 37 °C by real-time PCR. A major role of this fimbrial adhesin was demonstrated for biofilm formation, but a significant role of Mat fimbriae for APEC in vivo virulence could not yet be determined. Interestingly, the absence of either a functional matA gene or that of the structural genes matBCDEF independently resulted in upregulation of motility in E. coli strains MG1655 and IHE3034 by a so far unknown mechanism. In conclusion, the results of this thesis indicate a considerable overlap between human and animal ExPEC strains in terms of genome content and phenotypes. It becomes more and more apparent that the presence of a common set of virulence-associated genes among ExPEC strains as well as similar virulence gene expression patterns and phylogenetic backgrounds indicate a significant zoonotic risk of avian-derived E. coli isolates. In addition, new virulence factors identified in human ExPEC may also play a role in the pathogenesis of avian ExPEC.},
  subject      = {Escherichia coli},
  language  = {en}
}
@phdthesis{Rachid2000,
  author    = {Rachid, Shwan},
  title     = {Molecular investigation of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1882},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2000},
  abstract  = {Biofilm production is an important step in the pathogenesis of S. epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin (PIA). The PIA represents a sugar polymer consisting of ß-1,6 linked N-acetyl glucosaminoglycans and mediates the intercellular adherence of the bacteria to each other and the accumulation of a multilayered biofilm. Epidemiological and experimental studies strongly suggest that PIA-production and subsequently biofilm formation contributes significantly to the virulence of specific S. epidermidis strains. This work aimed on the investigation of external factors regulating the ica expression in S. epidermidis. For this purpose, a reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from E. coli was constructed and integrated into the chromosome of an ica positive S. epidermidis clinical isolate. The reporter gene fusion was used to investigate the influence of external factors and of sub-MICs of different antibiotics on the ica expression. It was shown that the S. epidermidis biofilm formation is growth phase dependent with a maximum expression in the late logarithmic and early stationary growth phase. The optimal expression was recorded at 42 °C at a neutral pH ranging from 7.0 to 7.5. The glucose content of the medium was found to be essential for biofilm formation, since concentrations of 1.5 to 2 per cent glucose induced the ica expression. In addition, external stress factors as high osmolarity (mediated by 3 to 5 per cent sodium chloride), and sub-lethal concentrations of detergents, ethanol, hydrogene peroxide, and urea significantly enhanced the biofilm production. Subinhibitory concentrations of tetracyline, the semisynthetic streptogramin quinupristin/dalfopristin and the streptogramin growth promoter virginiamycin were found to enhance the ica expression 8 to 11-fold, respectively, whereas penicillin, oxacillin, gentamicin, clindamycin, vancomycin, teicoplanin, ofloxacin, and chloramphenicol had no effects. A weak induction was recorded for sub-MICs of erythromycin. Both quinupristin/ dalfopristin and tetracyline exhibited a strong postexposure effect on the S. epidermidis ica expression, respectively, even when the substances were immediately removed from the growth medium. The results were confirmed by Northern blot analysis of the ica transcription and quantitative analysis of biofilm formation in a colorimetric assay. Expression of the icaprom::lacZ reporter gene plasmid in Bacillus subtilis and S. epidermidis revealed that the ica induction by sub-MICs of streptogramins and tetracycline might depend on unidentified regulatory elements which are specific for the staphylococcal cell. In contrast, the activation by external stress signals seems to be mediated by factors which are present both in Staphylococci and in Bacillus subtilis. Construction and analysis of an agr-mutant in a biofilm-forming S. epidermidis strain excluded the possibility that the Agr-quorum-sensing system significantly contributes to the ica expression in the stationary growth phase. However, clear evidence was provided that in S. aureus the ica transcription depends on the expression of the alternative transcription factor sigmaB, which represents a global regulator of the stress response in S. aureus as well as in B. subtilis. For this purpose, a sigB knockout mutant had been constructed in a biofilm-forming S. aureus. This mutant showed a markedly decrease of the ica transcription and biofilm-production, whereas a complement strain carrying the sigB gene on an expression vector completely restored the biofilm-forming phenotype of the S. aureus wild type. Southern blot analysis indicated that the the sigB gene is also present in S. epidermidis and Northern analyses of the sigB and the ica transcription revealed that both genes are activated under identical conditions (i. e. in the stationary growth phase and by external stress factors) suggesting a similar regulatory pathway as in S. aureus. However, since neither in S. aureus nor in S. epidermidis the ica promoter has obvious similiarities to known SigB-dependent promotoer sequences it is tempting to speculate that the ica activation is not directely mediated by SigB, but might be indirectely controlled by other SigB-dependent regulatory elements which remain to be elucidated.},
  subject      = {Staphylococcus epidermidis},
  language  = {en}
}