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- ItemAutomated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins, cephalosporins, and fluoroquinolones using specific Raman marker bands(Weinheim : Wiley-VCH-Verl., 2020) Götz, Theresa; Dahms, Marcel; Kirchhoff, Johanna; Beleites, Claudia; Glaser, Uwe; Bohnert, Jürgen A.; Pletz, Mathias W.; Popp, Jürgen; Schlattmann, Peter; Neugebauer, UteA Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemWhat the Phage: a scalable workflow for the identification and analysis of phage sequences(Oxford : Oxford University Press, 2022) Marquet, Mike; Hölzer, Martin; Pletz, Mathias W; Viehweger, Adrian; Makarewicz, Oliwia; Ehricht, Ralf; Brandt, ChristianPhages 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).
- ItemPhenotypic and Molecular Detection of Biofilm Formation in Staphylococcus aureus Isolated from Different Sources in Algeria(Basel : MDPI, 2020) Achek, Rachid; Hotzel, Helmut; Nabi, Ibrahim; Kechida, Souad; Mami, Djamila; Didouh, Nassima; Tomaso, Herbert; Neubauer, Heinrich; Ehricht, Ralf; Monecke, Stefan; El-Adawy, HosnyStaphylococcus aureus is an opportunistic bacterium causing a wide variety of diseases. Biofilm formation of Staphylococcus aureus is of primary public and animal health concern. The purposes of the present study were to investigate the ability of Staphylococcus aureus isolated from animals, humans, and food samples to form biofilms and to screen for the presence of biofilmassociated and regulatory genes. In total, 55 Staphylococcus aureus isolated from sheep mastitis cases (n = 28), humans (n = 19), and from food matrices (n = 8) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The ability of Staphylococcus aureus for slime production and biofilm formation was determined quantitatively. A DNA microarray examination was performed to detect adhesion genes (icaACD and biofilmassociated protein gene (bap)), genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), regulatory genes (accessory gene regulator (agr) and staphylococcal accessory regulator (sarA)), and the staphylococcal cassette chromosome mec elements (SCCmec). Out of 55 Staphylococcus aureus isolates, 39 (71.0%) and 23 (41.8%) were producing slime and biofilm, respectively. All Staphylococcus aureus strains isolated from food showed biofilm formation ability. 52.6% of the Staphylococcus aureus strains isolated from sheep with mastitis, and 17.9% of isolates from humans, were able to form a biofilm. Microarray analysis typed the Staphylococcus aureus into 15 clonal complexes. Among all Staphylococcus aureus isolates, four of the human isolates (21.1%) harbored the mecA gene (SCCmec type IV) typed into 2 clonal complexes (CC22-MRSA-IV and CC80-MRSA-IV) and were considered as methicillin-resistant, while two of them were slime-producing. None of the isolates from sheep with mastitis harbored the cna gene which is associated with biofilm production. The fnbB gene was found in 100%, 60% and 40% of biofilm-producing Staphylococcus aureus isolated from food, humans, and sheep with mastitis, respectively. Three agr groups were present and agr group III was predominant with 43.6%, followed by agr group I (38.2%), and agr group II (18.2%). This study revealed the capacity of Staphylococcus aureus isolates to form biofilms and highlighted the genetic background displayed by Staphylococcus aureus isolates from different sources in Algeria. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemExploring the evolution and epidemiology of European CC1-MRSA-IV: tracking a multidrug-resistant community-associated meticillin-resistant Staphylococcus aureus clone(London : Soc., 2021) Earls, Megan R.; Steinig, Eike J.; Monecke, Stefan; Samaniego Castruita, José A.; Simbeck, Alexandra; Schneider-Brachert, Wulf; Vremerǎ, Teodora; Dorneanu, Olivia S.; Loncaric, Igor; Bes, Michèle; Lacoma, Alicia; Prat Aymerich, Cristina; Wernery, Ulrich; Armengol-Porta, Marc; Blomfeldt, Anita; Duchene, Sebastian; Bartels, Mette D.; Ehricht, Ralf; Coleman, David C.This study investigated the evolution and epidemiology of the community-associated and multidrug-resistant Staphylococcus aureus clone European CC1-MRSA-IV. Whole-genome sequences were obtained for 194 European CC1-MRSA-IV isolates (189 of human and 5 of animal origin) from 12 countries, and 10 meticillin-susceptible precursors (from North-Eastern Romania; all of human origin) of the clone. Phylogenetic analysis was performed using a maximum-likelihood approach, a time-measured phylogeny was reconstructed using Bayesian analysis, and in silico microarray genotyping was performed to identify resistance, virulence-associated and SCCmec (staphylococcal cassette chromosome mec) genes. Isolates were typically sequence type 1 (190/204) and spa type t127 (183/204). Bayesian analysis indicated that European CC1-MRSA-IV emerged in approximately 1995 before undergoing rapid expansion in the late 1990s and 2000s, while spreading throughout Europe and into the Middle East. Phylogenetic analysis revealed an unstructured meticillin-resistant S. aureus (MRSA) population, lacking significant geographical or temporal clusters. The MRSA were genotypically multidrug-resistant, consistently encoded seh, and intermittently (34/194) encoded an undisrupted hlb gene with concomitant absence of the lysogenic phage-encoded genes sak and scn. All MRSA also harboured a characteristic ~5350 nt insertion in SCCmec adjacent to orfX. Detailed demographic data from Denmark showed that there, the clone is typically (25/35) found in the community, and often (10/35) among individuals with links to South-Eastern Europe. This study elucidated the evolution and epidemiology of European CC1-MRSA-IV, which emerged from a meticillin-susceptible lineage prevalent in North-Eastern Romania before disseminating rapidly throughout Europe.
- ItemImaging the invisible—Bioorthogonal Raman probes for imaging of cells and tissues(Weinheim [u.a.] : Wiley-VCH, 2020) Azemtsop Matanfack, Georgette; Rüger, Jan; Stiebing, Clara; Schmitt, Michael; Popp, JürgenA revolutionary avenue for vibrational imaging with super-multiplexing capability can be seen in the recent development of Raman-active bioortogonal tags or labels. These tags and isotopic labels represent groups of chemically inert and small modifications, which can be introduced to any biomolecule of interest and then supplied to single cells or entire organisms. Recent developments in the field of spontaneous Raman spectroscopy and stimulated Raman spectroscopy in combination with targeted imaging of biomolecules within living systems are the main focus of this review. After having introduced common strategies for bioorthogonal labeling, we present applications thereof for profiling of resistance patterns in bacterial cells, investigations of pharmaceutical drug-cell interactions in eukaryotic cells and cancer diagnosis in whole tissue samples. Ultimately, this approach proves to be a flexible and robust tool for in vivo imaging on several length scales and provides comparable information as fluorescence-based imaging without the need of bulky fluorescent tags. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemCharacterization of Staphylococci and Streptococci Isolated from Milk of Bovides with Mastitis in Egypt(Basel : MDPI, 2020) Ahmed, Wedad; Neubauer, Heinrich; Tomaso, Herbert; El Hofy, Fatma Ibrahim; Monecke, Stefan; Abdeltawab, Ashraf Awad; Hotzel, HelmutThe aim of this study was to characterize staphylococci and streptococci in milk from Egyptian bovides. In total, 50 milk samples were collected from localities in the Nile Delta region of Egypt. Isolates were cultivated, identified using matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistanceassociated genes. Thirty-eight Staphylococcus isolates and six Streptococcus isolates could be cultivated. Staphylococcus aureus isolates revealed a high resistance rate to penicillin, ampicillin, clindamycin, and erythromycin. The mecA gene defining methicillin-resistant Staphylococcus aureus, erm(C) and aac-aphD genes was found in 87.5% of each. Coagulase-negative staphylococci showed a high prevalence of mecA, blaZ and tetK genes. Other resistance-associated genes were found. All Streptococcus dysgalactiae isolates carried blaZ, erm(A), erm(B), erm(C) and lnuA genes, while Streptococcus suis harbored erm(C), aphA-3, tetL and tetM genes, additionally. In Streptococcus gallolyticus, most of these genes were found. The Streptococcus agalactiae isolate harbored blaZ, erm(B), erm(C), lnuA, tetK, tetL and tetM genes. Streptococcus agalactiae isolate was analyzed by DNA microarray analysis. It was determined as sequence type 14, belonging to clonal complex 19 and represented capsule type VI. Pilus and cell wall protein genes, pavA, cadD and emrB/qacA genes were identified by microarray analysis. © 2020 by the authors.
- ItemDeep learning a boon for biophotonics(Weinheim : Wiley-VCH-Verl., 2020) Pradhan, Pranita; Guo, Shuxia; Ryabchykov, Oleg; Popp, Juergen; Bocklitz, Thomas W.This review covers original articles using deep learning in the biophotonic field published in the last years. In these years deep learning, which is a subset of machine learning mostly based on artificial neural network geometries, was applied to a number of biophotonic tasks and has achieved state-of-the-art performances. Therefore, deep learning in the biophotonic field is rapidly growing and it will be utilized in the next years to obtain real-time biophotonic decision-making systems and to analyze biophotonic data in general. In this contribution, we discuss the possibilities of deep learning in the biophotonic field including image classification, segmentation, registration, pseudostaining and resolution enhancement. Additionally, we discuss the potential use of deep learning for spectroscopic data including spectral data preprocessing and spectral classification. We conclude this review by addressing the potential applications and challenges of using deep learning for biophotonic data. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemBladder tissue characterization using probe-based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction(Weinheim : Wiley-VCH-Verl., 2020) Cordero, Eliana; Rüger, Jan; Marti, Dominik; Mondol, Abdullah S.; Hasselager, Thomas; Mogensen, Karin; Hermann, Gregers G.; Popp, Jürgen; Schie, Iwan W.Existing approaches for early-stage bladder tumor diagnosis largely depend on invasive and time-consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real-time tumor diagnosis can enable immediate laser-based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real-time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe-based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low- and high-grade tumor. © 2019 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemCharacterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt(Basel : MDPI, 2021) Ahmed, Wedad; Neubauer, Heinrich; Tomaso, Herbert; El Hofy, Fatma Ibrahim; Monecke, Stefan; Abd El-Tawab, Ashraf Awad; Hotzel, HelmutThis study aimed to investigate the prevalence and antimicrobial resistance of enterococci- and ESBL-producing E. coli isolated from milk of bovine mastitis cases in Egypt. Fifty milk samples of dairy animals were collected from localities in the Nile Delta region of Egypt. Isolates were identified using MALDI-TOF MS, and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Seventeen Enterococcus isolates and eight coliform isolates could be cultivated. Vancomycin resistance rate was high in Ent. faecalis. The VITEK 2 system confirmed all E. coli isolates as ESBL-producing. All Ent. faecalis isolates harbored erm(B), tetL and aac-aphD genes. The vanA gene was detected in Ent. faecalis isolate, vanB was found in other Enterococcus, while one isolate of E. casseliflavus exhibited the vanA gene. E. coli isolates exhibited high prevalence of erm(B) and tetL. E. coli isolates were analyzed by DNA microarray analysis. Four isolates were determined by O-serotyping as O8 (n = 1), O86 (n = 2) and O157 (n = 1). H-serotyping resulted in H11, H12, H21 (two isolates each) and one was of H16 type. Different virulence-associated genes were detected in E. coli isolates including lpfA, astA, celB, cmahemL, intI1 and intI2, and the iroN gene was identified by DNA microarray analysis.
- ItemProtein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics(Washington, DC : American Society for Microbiology, 2022) Wagner, Gabriel E.; Berner, Andreas; Lipp, Michaela; Kohler, Christian; Assig, Karoline; Lichtenegger, Sabine; Saqib, Muhammad; Müller, Elke; Trinh, Trung T.; Gad, Anne-Marie; Söffing, Hans Hermann; Ehricht, Ralf; Laroucau, Karine; Steinmetz, IvoBurkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.