77 research outputs found
Epidemiologische und diagnostische Bedeutung der genetischen Plastizität und Variabilität der β-Lactamasen
Infektionen mit multiresistenten gramnegativen Bakterien (MRGN) sind ein globales und zunehmendes Problem. Grunde hierfür liegen vor allem in den hohen Verbrauch an beta-Lactamen in ambulanten und klinischen Sektor, sowie in der Tierhaltung. Durch die plasmidale Lokalisation der Resistenzgene, die für die beta-Lactamasen kodieren, wird die schnelle Ausbreitung speziesübergreifend begünstigt. Das bring zusätzliche Herausforderungen für die Diagnostik und Therapie. Die vorliegende Arbeit befasst sich mit verschiedenen Aspekten im Kampf gegen MRGN, die im Rahmen verschiedener Förderprojekte adressiert wurden. Klinisch relevante beta-Lactamasen mit erweitertem Wirkungsspektrum (ESBL) und Carbapenemasen, die seit Mitte der 1980-er Jahre auftauchen, entstehen nicht primär durch Mutationen, sondern sind Ergebnis von Transmissionen aus dem Umweltreservoir. In einer unfangreichen Genomanalyse konnte gezeigt werden, dass dieses noch über zahlreiche unbekannate beta-Lactamasen verfügt, die ihren Weg in pathogene Bakterien nehmen können. Antrieb hierfür ist die steigende Verunreinigung der Umwelt mit Antibiotika und damit der erhöhte selektive Druck in Oberflächengewässern und Böden. Ebenfalls konnte gezeigt werden, dass Sekundärmechanismen, wie Efflux und Porinverlust bei der Ausprägung des Carbapenem-resistenten Phänotyps eine wichtige Rolle spielen, was molekulardiagnostisch nicht gut abgebildet wird aber therapeutisch durch Kombinationstherapie adressiert werden kann. Hier wäre eine mRNA-basiert Diagnostik der DNA-basierten überlegen. Die Nutzung der bakteriellem mRNA als diagnostisches Zielmaterial wurde erprobt und es konnte gezeigt werden, dass diese sogar quantitative Aussagen zu minimalen Hemmkonzentration (MHK) ermöglichen könnte. Es konnte auch durch die nachträgliche molekulare Analyse eines Carbapenemase-Kolonisierungsausbruches die Spezies-übergreifende Verbreitung eines Resistenzplasmides und des darin enthaltenden Transposons demonstriert werden
Two-Component Response Regulator DegU Controls the Expression of Bacilysin in Plant-Growth-Promoting Bacterium Bacillus amyloliquefaciens FZB42
The plant-growth-promoting-rhizobacteria Bacillus amyloliquefaciens FZB42 possess an enormous potential to synthesize a wide range of antimicrobial, antiviral and nematocidal compounds. One of them, the dipeptide antibiotic bacilysin, is synthesized by FZB42 during exponential growth. Here, we have demonstrated that bacilysin is positively regulated by the two-component response regulator DegU at the transcriptional level. In addition, ScoC (Hpr), a transition state regulator, negatively controlled expression of the bacA gene, which is the first gene within the bacilysin operon. Both DegU and ScoC were bound directly at the bacA promoter region. Furthermore, a monocistronic gene located in close vicinity of the bac operon and essential for bacilysin production, ywfH, was also regulated by DegU. Transcription of the bac operon and of the ywfH gene in B. amyloliquefaciens FZB42 was positively controlled by the DegU global regulator protein. The role of interactions within a ternary complex formed by the antagonistically acting regulators DegU and ScoC as well as the bacA promoter sequence remains to be elucidated.Peer Reviewe
What the Phage: a scalable workflow for the identification and analysis of phage sequences
Phages are among the most abundant and diverse biological entities on earth. Phage prediction from sequence data is a crucial first step to understanding their impact on the environment. A variety of bacteriophage prediction tools have been developed over the years. They differ in algorithmic approach, results, and ease of use. We, therefore, developed "What the Phage"(WtP), an easy-to-use and parallel multitool approach for phage prediction combined with an annotation and classification downstream strategy, thus supporting the user's decision-making process by summarizing the results of the different prediction tools in charts and tables. WtP is reproducible and scales to thousands of datasets through a workflow manager (Nextflow). WtP is freely available under a GPL-3.0 license (https://github.com/replikation/What_the_Phage)
Assessing genetic diversity and similarity of 435 KPC-carrying plasmids.
Brandt C, Viehweger A, Singh A, et al. Assessing genetic diversity and similarity of 435 KPC-carrying plasmids. Scientific reports. 2019;9(1): 11223.The global spread and diversification of multidrug-resistant Gram-negative (MRGN) bacteria poses major challenges to healthcare. In particular, carbapenem-resistant Klebsiella pneumoniae strains have been frequently identified in infections and hospital-wide outbreaks. The most frequently underlying resistance gene (blaKPC) has been spreading over the last decade in the health care setting. blaKPC seems to have rapidly diversified and has been found in various species and on different plasmid types. To review the progress and dynamics of this diversification, all currently available KPC plasmids in the NCBI database were analysed in this work. Plasmids were grouped into 257 different representative KPC plasmids, of which 79.4% could be clearly assigned to incompatibility (Inc) group or groups. In almost half of all representative plasmids, the KPC gene is located on Tn4401 variants, emphasizing the importance of this transposon type for the transmission of KPC genes to other plasmids. The transposons also seem to be responsible for the occurrence of altered or uncommon fused plasmid types probably due to incomplete transposition. Moreover, many KPC plasmids contain genes that encode proteins promoting recombinant processes and mutagenesis; in consequence accelerating the diversification of KPC genes and other colocalized resistance genes
Biocide Susceptibility and Antimicrobial Resistance of Escherichia coli Isolated from Swine Feces, Pork Meat and Humans in Germany
Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum β-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials
Label-free multimodal imaging of infected Galleria mellonella larvae
Non-linear imaging modalities have enabled us to obtain unique morpho-chemical insights into the tissue architecture of various biological model organisms in a label-free manner. However, these imaging techniques have so far not been applied to analyze the Galleria mellonella infection model. This study utilizes for the first time the strength of multimodal imaging techniques to explore infection-related changes in the Galleria mellonella larvae due to massive E. faecalis bacterial infection. Multimodal imaging techniques such as fluorescent lifetime imaging (FLIM), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second harmonic generation (SHG) were implemented in conjunction with histological HE images to analyze infection-associated tissue damage. The changes in the larvae in response to the infection, such as melanization, vacuolization, nodule formation, and hemocyte infiltration as a defense mechanism of insects against microbial pathogens, were visualized after Enterococcus faecalis was administered. Furthermore, multimodal imaging served for the analysis of implant-associated biofilm infections by visualizing biofilm adherence on medical stainless steel and ePTFE implants within the larvae. Our results suggest that infection-related changes as well as the integrity of the tissue of G. mellonella larvae can be studied with high morphological and chemical contrast in a label-free manner
Regulation der Phytaseexpression in Bacillus amyloliquefaciens
Viele Bacillus Stämme sekretieren Phytasen, diese katalysieren die Dephosphorylierung von myo-Inositolhexakisphosphat (phytate). Das monocystronische phyC Gen aus dem im Boden lebenden Bacillus amyloliquefaciens FZB45 konnte als Teil des, durch Phosphatmangel induzierbaren, PhoPR-Regulons identifiziert werden. Der Transkriptionsstart des SigmaA-abhängigen Promotors wurde 27 bp upstream von Translationsstart bestimmt. Der Promotor weist jedoch eine ungewöhnliche Struktur auf, da die –35 und die –10 Region durch ein 21 bp Fenster voneinander getrennt sind. Die in vitro Transkriptionsanalyse zeigte, dass PhoP-P für die Initiation der phyC-Transkription notwendig ist. Die PhoP-Bindungsstellen der meisten B. subtilis Promotoren, die durch PhoP aktiviert werden, besitzen mindestens vier TTAACA-ähnliche Sequenzmotive, welche durch 5-6 bp- Intervalle getrennt sind. Die upstream Region von phyC aus B. amyloliquefaciens FZB45 weicht von dieser Struktur ab. Hier konnten nur zwei solcher Motive, die für die Bindung eines PhoP-Dimeres zuständig sind und die Positionen -47 und -35 überlagern, identifiziert werden. Ein weiteres Motiv befindet sich zwischen -13 und –8 und überlappt um eine Base mit der –10 Region. Die Funktionen der drei Bindungsstellen konnten durch DNaseI-Footprinting ermittelt werden. Ein PhoP-Dimer besetzt die –35 Region und dirigiert dabei die RNA-Polymerase wahrscheinlich in Richtung –10 Region. Durch die Bindung von PhoP an die PhoP-Erkennungsstelle bei –10 wird die Transkription gehemmt. Diese PhoP-vermittelte duale Kontrolle des phyC-Promotors scheint bis lang einzigartig für Pho-Regulongene zu sein. Der globale Regulator AbrB konnte als weiterer Repressor des phyC identifiziert werden. Die Bindungsstelle befinden sich upstream von –147 und downsteam von +29, müssen aber in weiteren Experimenten genauer spezifiziert werden. Diese Arbeit stellt als erste die Modelle der phyC-Regulation durch PhoP und AbrB vor.Several Bacillus strains secrete phytase, an enzyme catalysing dephosphorylation of myo-inositol hexakisphosphate (phytate). The monocistronic phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 was identified as a member of the phosphate-starvation inducible PhoPR regulon. The transcriptional start was determined downstream of a sigmaA-like promoter region located 27 bp upstream of the translation start codon. Inspection of the phyC promoter sequence revealed an unusual structure, since the -35 and -10 region are separated by a window of 21 bp. In vitro transcription analysis established that PhoP-P is necessary to initiate the transcription from phyC promoter. PhoP binding boxes occurring in most B. subtilis promoters activated by PhoP consist of at least four TTAACA-like sequences repeated at intervals of 5-6 bps. The upstream region of the B. amyloliquefaciens FZB45 phyC gene deviates from this general architecture in that there is only one appropriate binding site for the dimeric PhoP protein, which consists of two motives centered at -47 and -35 and separated by five base pairs. A single PhoP binding site is located at -13 to -8, nearly matching the -10 consensus. Functionality of the three PhoP binding boxes was demonstrated by DNaseI footprinting suggesting that a pair of dimeric PhoP molecules cover the -35 region directing the RNA-polymerase to the –10 region. Binding of PhoP at a single PhoP binding site covering the -10 consensus repress the transcription. It seems to be a unique feature of the phyC promoter structure and has not been reported for any other member of the PhoPR regulon, previously. Furthermore an inhibitory effect via AbrB, a global regulator, could be verified. The binding regions could be determine upstream of –147 and downstream of +29, but have to be specify in further experiments. This work presents for the first time the models for the regulation of phyC in Bacillus via PhoP and AbrB
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