@phdthesis{Balasubramanian2018,
  author    = {Balasubramanian, Srikkanth},
  title     = {Novel anti-infectives against pathogenic bacteria},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163882},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities. Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria. However, not much is known about their anti-infective or anti-virulence potential against human pathogens. This Ph.D. project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics. This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics. The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes. Diarrheal infections pose a huge burden in several developing and developed countries. Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated. Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis. Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications. Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed. In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit. It was found that treatment with the extracts did not lead to significant reduction in Stx production. However, strepthonium A isolated from the culture of Streptomyces sp. SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 μM concentration) in EHEC strain EDL933 without affecting the bacterial growth. The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments. This demonstrated the possible application of strepthonium A in restraining EHEC infections. VI In the second study, the effect of marine sponge-associated actinomycetes on biofilm formation of staphylococci was assessed. Medical devices such as contact lenses, metallic implants, catheters, pacemakers etc. are ideal ecological niches for formation of bacterial biofilms, which thereby lead to device-related infections. Bacteria in biofilms are multiple fold more tolerant to the host immune responses and conventional antibiotics, and hence are hard-to-treat. Here, the anti-biofilm potential of an organic extract derived from liquid fermentation of Streptomyces sp. SBT343 (previously cultivated from the Mediterranean sponge Petrosia ficiformis) was reported. Results obtained in vitro demonstrated its anti-biofilm (against staphylococci) and non-toxic nature (against mouse macrophage (J774.1), fibroblast (NIH/3T3) and human corneal epithelial cell lines). Interestingly, SBT343 extract could inhibit staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces without affecting the bacterial growth. High Resolution Fourier Transform Mass Spectrometry (HR-MS) analysis indicated the complexity and the chemical diversity of components present in the extract. Preliminary physio-chemical characterization unmasked the heat stable and non-proteinaceous nature of the active component(s) in the extract. Finally, fractionation experiments revealed that the biological activity was due to synergistic effects of multiple components present in the extract. In the third study, anti-biofilm screening of 50 organic extracts generated from solid and liquid fermentation of 25 different previously characterized sponge-derived actinomycetes was carried out. This led to identification of the anti-biofilm organic extract derived from the solid culture of Streptomyces sp. SBT348 (previously cultivated from the Mediterranean sponge Petrosia ficiformis). Bioassay-guided fractionation was employed to identify the active fraction Fr 7 in the SBT348 crude extract. Further purification with semi-preparative HPLC led to isolation of the bioactive SKC1, SKC2, SKC3, SKC4 and SKC5 sub-fractions. The most active sub-fraction SKC3 was found to be a pure compound having BIC90 and MIC values of 3.95 μg/ml and 31.25 μg/ml against S. epidermidis RP62A. SKC3 had no apparent toxicity in vitro on cell lines and in vivo on the greater wax moth Galleria melonella larvae. SKC3 was stable to heat and enzymatic treatments indicating its non-proteinaceous nature. HR-MS analysis revealed the mass of SKC3 to be 1258.3 Da. Structure elucidation of SKC3 with the aid of 1D and 2D-NMR data is currently under investigation. Further, to obtain insights into the mode of action of SKC3 on S. epidermidis RP62A, RNA sequencing was done. Transcriptome data revealed that SKC3 was recognized by RP62A at 20 min and SKC3 negatively interfered with the central metabolism of staphylococci at 3 h. Taken VII together, these findings suggest that SKC3 could be a lead structure for development of new anti-staphylococcal drugs. Overall, the results obtained from this work underscore the anti-infective attributes of actinomycetes consortia associated with marine sponges, and their applications in natural product drug discovery programs.},
  subject      = {Marine sponges},
  language  = {en}
}
@phdthesis{Soundararajan2020,
  author    = {Soundararajan, Manonmani},
  title     = {Investigations into the mechanisms behind the antagonistic effects and phage resistance of probiotic Escherichia coli strain Nissle 1917},
  doi       = {10.25972/OPUS-21525},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-215256},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Gastrointestinal infections account for high morbidity and mortality in humans every year across the globe. The increasing emergence of antibiotic resistance among the gastrointestinal pathogens and the induction of virulence factors by antibiotics makes it highly risky to only depend on antibiotic therapy for intestinal infections. Most of these infections are associated with an imbalance in the gut microbial population whereas the restoration of the balance with probiotic supplements can result in an improvement of the health condition. Probiotics are therefore considered as successful support in the treatment of gastrointestinal infections. E. coli Nissle 1917 (EcN) is the active component of the probiotic medication Mutaflor® and has been used in the treatment of various gastrointestinal disorders for more than 100 years. Several studies have reported antagonistic effects of EcN against enterohemorrhagic E. coli (EHEC) in vitro and in vivo. However, detailed investigations on the probiotic mechanisms and safety aspects of EcN are a pre-requisite, for administering EcN to treat EHEC infected patients or to use EcN as a prophylactic for the patient's close contacts. In this regard, the first part of the study aimed to understand the nature and behaviour of EcN in the presence of pathogenic or non-pathogenic E. coli strains. Transcriptomic analysis was deployed to this end. We investigated the changes in EcN's transcriptome after different time points of coculture with the EHEC strain EDL933 or the K-12 strain MG1655. The transcriptome data reported a strain-specific response in EcN at all the investigated time points (3 h, 5 h, 7 h and 8 h) of coincubation. The alterations in gene regulation of EcN were highly pronounced in initial timepoints (3 h and 5 h) of coincubation with EDL933, which gradually decreased over time. In the presence of MG1655, the alterations were strongly differentially regulated only at later time points (7 h and 8 h). The unique transcriptional response of EcN towards two different E. coli strains, that are genetically more than 98 \% identical, was startling. 12 More importantly, this can be considered as a beneficial trait of EcN over a chemical-pharmaceutical preparation like an antibiotic that might act identically on all target cells. Bacteriophages are one of the most abundant members of gut microbiota. On the one hand, the infection of a probiotic strain by a lysogenic phage could transfer genetic material coding for pathogenic factors or antibiotic resistance into an otherwise beneficial probiotic bacterium and thereby converting it into a virulent pathogenic bacterium. On the other hand, infection by a lytic phage could result in bacterial lysis and prevent the bacterium from exerting its probiotic effect. Thus, in order to successfully establish and colonise the gut, it is crucial for any probiotic to be resistant against phage infections. To address this, in the second part of the study, we investigated the phage resistance of EcN towards the lysogenic lambda and the lytic T4 phage. EcN showed complete resistance against tested phages and was also able to inactivate these phages upon coincubation. In the case of lambda phages, the resistance was attributed to the presence of a lambdoid prophage (prophage 3) in the genome of EcN. In addition, the overexpression of one of the early genes of EcN's prophage 3 (i.e. phage repressor gene pr) in the phage sensitive MG1655 conferred partial protection against lambda phage infection. Moreover, the inactivation was mediated by binding of lambda phages to its receptor LamB. Experiments with lytic T4 phages revealed that the EcN's K5 polysaccharide capsule was crucial for its T4 phage resistance, while its lipopolysaccharide (LPS) inactivated the T4 phages. Apart from protecting itself, EcN displayed even a protective role for the tested K-12 strains, by interfering with the lysogeny and lysis by these phages. In summary, this work highlights two novel positive traits of the probiotic strain EcN: i) the strain-specific response that was evident from the global transcriptome analysis of EcN when incubated with other E. coli strains, and ii) lytic and lysogenic phage resistance. Both these traits are additional safety aspects for a well-characterised probiotic strain and encourage its application in therapeutics.},
  language  = {en}
}
@phdthesis{Rund2014,
  author    = {Rund, Stefan A.},
  title     = {Interferenz des probiotischen Escherichia coli Stammes Nissle 1917 mit Adh{\"a}sion, Replikation und Shiga Toxin Produktion von EHEC St{\"a}mmen in vitro},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-104837},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {E. coli Nissle 1917 (EcN) z{\"a}hlt durch seine fast hundertj{\"a}hrige Nutzung als Arzneimittel und aufgrund der weitreichenden Forschung w{\"a}hrend der letzten Jahrzehnte mittlerweile zu einem der am besten untersuchten Probiotika. EcN wird als Medikament zur Remissionserhaltung von Patienten mit Kolitis, bei chronischer Verstopfung und bei Durchfall von Kleinkindern eingesetzt. Der enteroaggregative - h{\"a}morrhagische - E. coli (EAHEC) mit dem Serotyp O104:H4 war 2011 in Deutschland f{\"u}r den bisher gr{\"o}ßten EHEC-Ausbruch seit Beginn der Aufzeichnungen verantwortlich. Es fehlt bis zum heutigen Tage immer noch an effektiven M{\"o}glichkeiten einer Infektionsprophylaxe oder einer Behandlung der Erkrankung. Ein alternatives Therapeutikum wird daher dringend ben{\"o}tigt. In dieser Arbeit wurden die antagonistischen Effekte von EcN auf pathogene E. coli St{\"a}mme wie dem EHEC Stamm EDL933 oder klinischen EAHEC O104:H4 Isolaten untersucht. Es wurden die Auswirkungen von EcN auf die Adh{\"a}sion an humane Epithelzellen, das Wachstum und die Shiga Toxin Produktion der pathogenen St{\"a}mme untersucht. Zus{\"a}tzlich wurde die Resistenz von EcN gegen{\"u}ber Shiga Toxin Phagen nachgewiesen. Zun{\"a}chst wurde die Adh{\"a}sionseffizienz der verschiedenen E. coli St{\"a}mme bestimmt. Der am schlechtesten an die humanen Epithelzelllinien Caco-2 und LS-174T adh{\"a}rierende Stamm war EcN. Dies ist insofern {\"u}berraschend, da von Probiotika erwartet wird, besser als Pathogene an Epithelzellen zu adh{\"a}rieren. Dem ungeachtet konnte jedoch gezeigt werden, dass EcN die Adh{\"a}sion von zwei EAHEC O104:H4 Isolaten, des nahe verwandten enteroaggregativen E. coli (EAEC) Stammes 55989 und des enteroh{\"a}morrhagischen (EHEC) E. coli Stammes O157:H7 EDL933 an beide Zelllinen hemmt. Die von EcN produzierten Mikrozine M und H47 konnten hier f{\"u}r einen Teil des beobachteten anti-adh{\"a}siven Effektes von EcN auf die pathogenen E. coli St{\"a}mme verantwortlich gemacht werden. Die Mikrozine wurden hier als einzige Substanz, die das Wachstum der pathogenen E. coli St{\"a}mme beeinflusst, identifiziert. Einer der wichtigsten Virulenzfaktoren von EAHEC und EHEC St{\"a}mmen ist das Shiga Toxin. In dieser Arbeit konnte gezeigt werden, dass EcN die Shiga Toxin Produktion der am h{\"a}ufigsten auftretenden EHEC St{\"a}mme (´Big Five´: O157:H7, O26:H11, O103:H2, O111:H-, O145:H25) und der klinischen Isolate von EAHEC O104:H4 im Zellkulturmedium DMEM hemmt. Auff{\"a}llig war, dass die Stx1 Produktion von EHEC O103:H2 und O111:H- nicht nur von EcN, sondern auch von E. coli K-12 Stamm MG1655, gehemmt wurde, im Gegensatz zur EcN-spezifischen Blockierung der Stx2-Produktion in den Serotypen O104:H4, O26:H11, O145:H25. Die Reduktion der Stx-Produktion in EAHEC O104:H4 TY3730 und TY3456, sowie EHEC O26:H11 war zum Teil von der Mikrozinproduktion abh{\"a}ngig. Diese hatte jedoch keinen Einfluss auf die Stx-Produktion in EHEC O157:H7 EDL933 und EHEC O145:H25. Bei Verwendung von LB-Medium zeigte sich im Gegensatz zum DMEM-Medium keine Mikrozin-Abh{\"a}ngigkeit der Toxinproduktion bei den EAHEC Isolaten TY3730 und TY3456. Die Toxinproduktion von EHEC EDL933 wurde ebenfalls nicht durch die Deletion der Mikrozin-Gene in EcN beeinflusst. Studien der Toxinproduktion in SCEM-Medium zeigten ebenfalls eine EcN-Dosisabh{\"a}ngige Reduktion der Stx-Produktion in Co-Kultur. Um den Mechanismus der Hemmung der Stx-Produktion zu untersuchen, wurden Versuche mit der EcN-Mutante EcN::luxS durchgef{\"u}hrt. Diese Deletion des AI-2 ´Quorum sensing´ Molek{\"u}ls in EcN hatte allerdings keinen Einfluss auf die Hemmung der Stx-Produktion. Der Einsatz von Acetat f{\"u}hrte, im Gegensatz zu publizierten Ergebnissen, nicht zu einer Reduktion der Stx-Produktion. Auch eine Beeinflussung der Lyse der EHEC-Bakterien, oder der Verminderung der Sekretion von Shiga Toxin durch EcN, konnte widerlegt werden. Zur Untersuchung der Stx-Expression wurde ein Assay mit einem biolumineszenten C-P (Chromosom-Plasmid) Reporter System etabliert. Damit konnte die Shiga Toxin Expression im Stammhintergrund EHEC EDL933 in Echtzeit untersucht werden. Hier wurde wiederum eine Reduktion der Shiga Toxin Expression in Co-Kultur mit EcN erfolgreich nachgewiesen. In weiteren Versuchen konnte gezeigt werden, dass EcN nicht nur die Shiga Toxin Produktion von nicht-induzierten EAHEC Bakterien, sondern auch in mit Mitomycin C induzierten Bakterien hemmt. Als wichtiger Sicherheitsaspekt einer Behandlung mit EcN wurde die Resistenz von EcN gegen{\"u}ber Shiga Toxin Phagen untersucht. Die Infektion der Bakterien wurde hierbei mit stx-spezifischer PCR, Phagen-Plaque-Assay, Stx-ELISA und K+-Efflux Assay untersucht. Es konnte durch diese verschiedenen Methoden erfolgreich gezeigt werden, dass EcN nicht durch Shiga Toxin Phagen infiziert wird. Als m{\"o}glicher Resistenzmechanismus kommt hier eine Mutation vom Phagenrezeptor LamB in Frage, was jedoch noch best{\"a}tigt werden muss. Zusammenfassend wurden in dieser Arbeit wichtige antagonistische Effekte von EcN auf pathogene E. coli St{\"a}mme untersucht, die als Grundlage von neuen und dringend ben{\"o}tigten Behandlungen von EHEC-Infektionen dienen k{\"o}nnen.},
  subject      = {EHEC},
  language  = {de}
}