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- ItemCharacterization and prediction of the mechanism of action of antibiotics through NMR metabolomics(London : BioMed Central, 2016) Hoerr, Verena; Duggan, Gavin E.; Zbytnuik, Lori; Poon, Karen K.H.; Große, Christina; Neugebauer, Ute; Methling, Karen; Löffler, Bettina; Vogel, Hans J.Background: The emergence of antibiotic resistant pathogenic bacteria has reduced our ability to combat infectious diseases. At the same time the numbers of new antibiotics reaching the market have decreased. This situation has created an urgent need to discover novel antibiotic scaffolds. Recently, the application of pattern recognition techniques to identify molecular fingerprints in ‘omics’ studies, has emerged as an important tool in biomedical research and laboratory medicine to identify pathogens, to monitor therapeutic treatments or to develop drugs with improved metabolic stability, toxicological profile and efficacy. Here, we hypothesize that a combination of metabolic intracellular fingerprints and extracellular footprints would provide a more comprehensive picture about the mechanism of action of novel antibiotics in drug discovery programs. Results: In an attempt to integrate the metabolomics approach as a classification tool in the drug discovery processes, we have used quantitative 1H NMR spectroscopy to study the metabolic response of Escherichia coli cultures to different antibiotics. Within the frame of our study the effects of five different and well-known antibiotic classes on the bacterial metabolome were investigated both by intracellular fingerprint and extracellular footprint analysis. The metabolic fingerprints and footprints of bacterial cultures were affected in a distinct manner and provided complementary information regarding intracellular and extracellular targets such as protein synthesis, DNA and cell wall. While cell cultures affected by antibiotics that act on intracellular targets showed class-specific fingerprints, the metabolic footprints differed significantly only when antibiotics that target the cell wall were applied. In addition, using a training set of E. coli fingerprints extracted after treatment with different antibiotic classes, the mode of action of streptomycin, tetracycline and carbenicillin could be correctly predicted. Conclusion: The metabolic profiles of E. coli treated with antibiotics with intracellular and extracellular targets could be separated in fingerprint and footprint analysis, respectively and provided complementary information. Based on the specific fingerprints obtained for different classes of antibiotics, the mode of action of several antibiotics could be predicted. The same classification approach should be applicable to studies of other pathogenic bacteria.
- ItemCorrelation of crystal violet biofilm test results of Staphylococcus aureus clinical isolates with Raman spectroscopic read-out(Chichester [u.a.] : Wiley, 2021) Ebert, Christina; Tuchscherr, Lorena; Unger, Nancy; Pöllath, Christine; Gladigau, Frederike; Popp, Jürgen; Löffler, Bettina; Neugebauer, UteBiofilm-related infections occur quite frequently in hospital settings and require rapid diagnostic identification as they are recalcitrant to antibiotic therapy and make special treatment necessary. One of the standard microbiological in vitro tests is the crystal violet test. It indirectly determines the amount of biofilm by measuring the optical density (OD) of the crystal violet-stained biofilm matrix and cells. However, this test is quite time-consuming, as it requires bacterial cultivation up to several days. In this study, we correlate fast Raman spectroscopic read-out of clinical Staphylococcus aureus isolates from 47 patients with different disease background with their biofilm-forming characteristics. Included were low (OD < 10), medium (OD ≥ 10 and ≤20), and high (OD > 20) biofilm performers as determined by the crystal violet test. Raman spectroscopic analysis of the bacteria revealed most spectral differences between high and low biofilm performers in the fingerprint region between 750 and 1150 cm−1. Using partial least square regression (PLSR) analysis on the Raman spectra involving the three categories of biofilm formation, it was possible to obtain a slight linear correlation of the Raman spectra with the biofilm OD values. The PLSR loading coefficient highlighted spectral differences between high and low biofilm performers for Raman bands that represent nucleic acids, carbohydrates, and proteins. Our results point to a possible application of Raman spectroscopy as a fast prediction tool for biofilm formation of bacterial strains directly after isolation from the infected patient. This could help clinicians make timely and adapted therapeutic decision in future.
- ItemThe Staphylococcus aureus extracellular matrix protein (Emp) has a fibrous structure and binds to different extracellular matrices(Berlin : Nature Pulishing, 2017) Geraci, Jennifer; Neubauer, Svetlana; Pöllath, Christine; Hansen, Uwe; Rizzo, Fabio; Krafft, Christoph; Westermann, Martin; Hussain, Muzaffar; Peters, Georg; Pletz, Mathias W.; Löffler, Bettina; Makarewicz, Oliwia; Tuchscherr, LorenaThe extracellular matrix protein Emp of Staphylococcus aureus is a secreted adhesin that mediates interactions between the bacterial surface and extracellular host structures. However, its structure and role in staphylococcal pathogenesis remain unknown. Using multidisciplinary approaches, including circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, transmission electron (TEM) and immunogold transmission electron microscopy, functional ELISA assays and in silico techniques, we characterized the Emp protein. We demonstrated that Emp and its truncated forms bind to suprastructures in human skin, cartilage or bone, among which binding activity seems to be higher for skin compounds. The binding domain is located in the C-terminal part of the protein. CD spectroscopy revealed high contents of β-sheets (39.58%) and natively disordered structures (41.2%), and TEM suggested a fibrous structure consisting of Emp polymers. The N-terminus seems to be essential for polymerization. Due to the uncommonly high histidine content, we suggest that Emp represents a novel type of histidine-rich protein sharing structural similarities to leucine-rich repeats proteins as predicted by the I-TASSER algorithm. These new findings suggest a role of Emp in infections of deeper tissue and open new possibilities for the development of novel therapeutic strategies.
- ItemClinical S. aureus Isolates Vary in Their Virulence to Promote Adaptation to the Host(Basel : MDPI, 2019) Tuchscherr, Lorena; Pöllath, Christine; Siegmund, Anke; Deinhardt-Emmer, Stefanie; Hoerr, Verena; Svensson, Carl-Magnus; Figge, Marc Thilo; Monecke, Stefan; Löffler, BettinaStaphylococcus aureus colonizes epithelial surfaces, but it can also cause severe infections. The aim of this work was to investigate whether bacterial virulence correlates with defined types of tissue infections. For this, we collected 10–12 clinical S. aureus strains each from nasal colonization, and from patients with endoprosthesis infection, hematogenous osteomyelitis, and sepsis. All strains were characterized by genotypic analysis, and by the expression of virulence factors. The host–pathogen interaction was studied through several functional assays in osteoblast cultures. Additionally, selected strains were tested in a murine sepsis/osteomyelitis model. We did not find characteristic bacterial features for the defined infection types; rather, a wide range in all strain collections regarding cytotoxicity and invasiveness was observed. Interestingly, all strains were able to persist and to form small colony variants (SCVs). However, the low-cytotoxicity strains survived in higher numbers, and were less efficiently cleared by the host than the highly cytotoxic strains. In summary, our results indicate that not only destructive, but also low-cytotoxicity strains are able to induce infections. The low-cytotoxicity strains can successfully survive, and are less efficiently cleared from the host than the highly cytotoxic strains, which represent a source for chronic infections. The understanding of this interplay/evolution between the host and the pathogen during infection, with specific attention towards low-cytotoxicity isolates, will help to optimize treatment strategies for invasive and therapy-refractory infection courses.