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Broadband X-ray ptychography is developed based on the multi-wavelength reconstruction method and is systematically demonstrated in both simulation and experiment. In addition, a combined reconstruction approach is proposed to further increase the ptychographic imaging rate., Ptychography is a rapidly developing scanning microscopy which is able to view the internal structures of samples at a high resolution beyond the illumination size. The achieved spatial resolution is theoretically dose-limited. A broadband source can provide much higher flux compared with a monochromatic source; however, it conflicts with the necessary coherence requirements of this coherent diffraction imaging technique. In this paper, a multi-wavelength reconstruction algorithm has been developed to deal with the broad bandwidth in ptychography. Compared with the latest development of mixed-state reconstruction approach, this multi-wavelength approach is more accurate in the physical model, and also considers the spot size variation as a function of energy due to the chromatic focusing optics. Therefore, this method has been proved in both simulation and experiment to significantly improve the reconstruction when the source bandwidth, illumination size and scan step size increase. It is worth mentioning that the accurate and detailed information of the energy spectrum for the incident beam is not required in advance for the proposed method. Further, we combine multi-wavelength and mixed-state approaches to jointly solve temporal and spatial partial coherence in ptychography so that it can handle various disadvantageous experimental effects. The significant relaxation in coherence requirements by our approaches allows the use of high-flux broadband X-ray sources for high-efficient and high-resolution ptychographic imaging.

Staphylococcus aureus causes a broad spectrum of diseases in humans and animals. It is frequently associated with inflammatory skin disorders such as atopic dermatitis, where it aggravates symptoms.

Staphylococcus aureus causes a broad spectrum of diseases in humans and animals. It is frequently associated with inflammatory skin disorders such as atopic dermatitis, where it aggravates symptoms. Treatment of S. aureus-associated skin infections with antibiotics is discouraged due to their broad-range deleterious effect on healthy skin microbiota and their ability to promote the development of resistance. Thus, novel S. aureus-specific antibacterial agents are desirable. We constructed two chimeric cell wall-lytic enzymes, Staphefekt SA.100 and XZ.700, which are composed of functional domains from the bacteriophage endolysin Ply2638 and the bacteriocin lysostaphin. Both enzymes specifically killed S. aureus and were inactive against commensal skin bacteria such as Staphylococcus epidermidis, with XZ.700 proving more active than SA.100 in multiple in vitro activity assays. When surface-attached mixed staphylococcal cultures were exposed to XZ.700 in a simplified microbiome model, the enzyme selectively removed S. aureus and retained S. epidermidis. Furthermore, XZ.700 did not induce resistance in S. aureus during repeated rounds of exposure to sublethal concentrations. Finally, we demonstrated that XZ.700 formulated as a cream is effective at killing S. aureus on reconstituted human epidermis and that an XZ.700-containing gel significantly reduces bacterial numbers compared to an untreated control in a mouse model of S. aureus-induced skin infection., Antimicrobial Agents and Chemotherapy, 66 (5), ISSN:0066-4804, ISSN:1098-6596

In dairy cows, Staphylococcus aureus (S. aureus) is among the most prevalent microorganisms worldwide, causing mastitis, an inflammation of the mammary gland. Production of extracellular vesicles (EVs) is a common feature of S. aureus strains, which contributes to its pathogenesis by delivering bacterial effector molecules to host cells. In the current study, we evaluated the differences between five S. aureus mastitis isolates regarding their EV production. We found that different mastitis-related S. aureus strains differ in their behaviour of shedding EVs, with M5512VL producing the largest amount of EVs containing alpha-haemolysin, a strong cytotoxic agent. We stimulated primary cultured bovine mammary epithelial cells (pbMECs) with EVs from the S. aureus strain M5512VL. After 24 h of incubation, we observed a moderate increase in gene expression of tumour necrosis factor-alpha (TNF-α) but, surprisingly, a lack of an associated pronounced pro-inflammatory response. Our results contribute to understanding the damaging nature of S. aureus in its capacity to effectively affect mammary epithelial cells.

In dairy cows, Staphylococcus aureus (S. aureus) is among the most prevalent microorganisms worldwide, causing mastitis, an inflammation of the mammary gland. Production of extracellular vesicles (EVs) is a common feature of S. aureus strains, which contributes to its pathogenesis by delivering bacterial effector molecules to host cells. In the current study, we evaluated the differences between five S. aureus mastitis isolates regarding their EV production. We found that different mastitis-related S. aureus strains differ in their behaviour of shedding EVs, with M5512VL producing the largest amount of EVs containing alpha-haemolysin, a strong cytotoxic agent. We stimulated primary cultured bovine mammary epithelial cells (pbMECs) with EVs from the S. aureus strain M5512VL. After 24 h of incubation, we observed a moderate increase in gene expression of tumour necrosis factor-alpha (TNF-α) but, surprisingly, a lack of an associated pronounced pro-inflammatory response. Our results contribute to understanding the damaging nature of S. aureus in its capacity to effectively affect mammary epithelial cells. ISSN:2079-7737

We will present the design for the In-Situ Nanoprobe (ISN) beamline that is being developed as part of the Upgrade of the APS storage ring with an MBA magnetic lattice. The ISN will provide large working distance of 60 mm for in-situ and operando environments, and a small spot of 20 nm (25 keV) for imaging materials with small defects and functional components. To achieve both long working distance and small spot size, Kirkpatrick-Baez mirrors will be used as nanofocusing optics. The major contrast mechanisms will be XRF imaging for chemical characterization ptychography for transmission imaging with sub-10 nm resolution. Auxiliary diffraction capabilities will allow monitoring of phase change during in-situ studies. To achieve the demagnification required to achieve small spot sizes, the ISN instrument will be placed at a distance of 220 m from the x-ray source, in a satellite building outside the APS storage ring. The ISN will provide hard x-rays with photon energy between 4.8 keV and 30 keV, enabling access to the absorption edges of to most elements in the periodic system. The MBA lattice and insertion devices, coupled with the high reflectivity of the K-B mirror system, provide a very high coherent flux of above 4*1012 Ph/s at 5 keV, and 6*1012 Ph/s at 30 keV. This allows hierarchical imaging of large samples with very small spot size, as well as multidimensional imaging, such as 3D imaging and temperature change, or 2D imaging with change of several environmental parameters. The ISN will provide flow of fluids, gases, and variable temperature.

Orthopaedic device-related infection (ODRI) presents a significant challenge to the field of orthopaedic and trauma surgery. Despite extensive treatment involving surgical debridement and prolonged antibiotic therapy, outcomes remain poor. This is largely due to the unique abilities of Staphylococcus aureus, the most common causative agent of ODRI, to establish and protect itself within the host by forming biofilms on implanted devices and staphylococcal abscess communities (SACs). There is a need for novel antimicrobials that can readily target such features. Enzybiotics are a class of antimicrobial enzymes derived from bacteria and bacteriophages, which function by enzymatically degrading bacterial polymers essential to bacterial survival or biofilm formation. Here, we apply an enzybiotic-based combination regimen to a set of in vitro models as well as in a murine ODRI model to evaluate their usefulness in eradicating established S. aureus infection, compared to classical antibiotics. We show that two chimeric endolysins previously selected for their functional efficacy in human serum in combination with a polysaccharide depolymerase reduce bacterial CFU numbers 10,000-fold in a peg model and in an implant model of biofilm. The enzyme combination also completely eradicates S. aureus in a SAC in vitro model where classical antibiotics are ineffective. In an in vivo ODRI model in mice, the antibiofilm effects of this enzyme regimen are further enhanced when combined with a classical gentamicin/vancomycin treatment. In a mouse model of methicillin-resistant S. aureus (MRSA) ODRI following a fracture repair, a combined local enzybiotic/antibiotic treatment regimen showed a significant CFU reduction in the device and the surrounding soft tissue, as well as significant prevention of weight loss. These outcomes were superior to treatment with antibiotics alone. Overall, this study demonstrates that the addition of enzybiotics, which are distinguished by their extremely rapid killing efficacy and antibiofilm activities, can enhance the treatment of severe MRSA ODRI., Antibiotics, 10 (10), ISSN:2079-6382

As a coherent diffraction imaging technique, ptychography provides high-spatial resolution beyond Rayleigh’s criterion of the focusing optics, but it is also sensitively affected by the decoherence coming from the spatial and temporal variations in the experiment. Here we show that high-speed ptychographic data acquisition with short exposure can effectively reduce the impact from experimental variations. To reach a cumulative dose required for a given resolution, we further demonstrate that a continuous multi-pass scan via high-speed ptychography can achieve high-resolution imaging. This low-dose scan strategy is shown to be more dose-efficient, and has potential for radiation-sensitive sample studies and time-resolved imaging.

X-ray ptychography has gained tremendous success in providing quantitative high-resolution imaging for extended samples. Here we report on recent developments in ptychography imaging techniques and the improvement of reconstruction methods to increase ptychographic imaging throughput at the Advanced Photon Source.

Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells., Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.

X-ray ptychography is a rapidly developing coherent diffraction imaging technique that provides nanoscale resolution on extended field-of-view. However, the requirement of coherence and the scanning mechanism limit the throughput of ptychographic imaging. In this paper, we propose X-ray ptychography using multiple illuminations instead of single illumination in conventional ptychography. Multiple locations of the sample are simultaneously imaged by spatially separated X-ray beams, therefore, the obtained field-of-view in one scan can be enlarged by a factor equal to the number of illuminations. We have demonstrated this technique experimentally using two X-ray beams focused by a house-made Fresnel zone plate array. Two areas of the object and corresponding double illuminations were successfully reconstructed from diffraction patterns acquired in one scan, with image quality similar with those obtained by conventional single-beam ptychography in sequence. Multi-beam ptychography approach increases the imaging speed, providing an efficient way for high-resolution imaging of large extended specimens.

Listeria monocytogenes is a ubiquitous opportunistic pathogen that presents a major concern to the food industry due to its propensity to cause foodborne illness. The Listeria genus contains 15 different serovars, with most of the variance depending on the wall-associated teichoic acid glycopolymers, which confer somatic antigenicity. Strains belonging to serovars 1/2 and 4b cause the vast majority of listeriosis cases and outbreaks, meaning that regulators, as well as the food industry itself, have an interest in rapidly identifying isolates of these particular serovars in food processing environments. Current methods for phenotypic serovar differentiation are slow and lack accuracy, and the food industry could benefit from new technologies allowing serovar-specific isolation. Therefore, the novel method described here for rapid glycotype determination could present a valuable asset to detect and control this bacterium., The Gram-positive pathogen Listeria monocytogenes can be subdivided into at least 12 different serovars, based on the differential expression of a set of somatic and flagellar antigens. Of note, strains belonging to serovars 1/2a, 1/2b, and 4b cause the vast majority of foodborne listeriosis cases and outbreaks. The standard protocol for serovar determination involves an agglutination method using a set of sera containing cell surface-recognizing antibodies. However, this procedure is imperfect in both precision and practicality, due to discrepancies resulting from subjective interpretation. Furthermore, the exact antigenic epitopes remain unclear, due to the preparation of the absorbed sera and the complex nature of polyvalent antibody binding. Here, we present a novel method for quantitative somatic antigen differentiation using a set of recombinant affinity proteins (cell wall-binding domains and receptor-binding proteins) derived from a collection of Listeria bacteriophages. These proteins enable rapid, objective, and precise identification of the different teichoic acid glycopolymer structures, which represent the O-antigens, and allow a near-complete differentiation. This glycotyping approach confirmed serovar designations of over 60 previously characterized Listeria strains. Using select phage receptor-binding proteins coupled to paramagnetic beads, we also demonstrate the ability to specifically isolate serovar 1/2 or 4b cells from a mixed culture. In addition, glycotyping led to the discovery that strains designated serovar 4e actually possess an intermediate 4b-4d teichoic acid glycosylation pattern, underpinning the high discerning power and precision of this novel technique. IMPORTANCE Listeria monocytogenes is a ubiquitous opportunistic pathogen that presents a major concern to the food industry due to its propensity to cause foodborne illness. The Listeria genus contains 15 different serovars, with most of the variance depending on the wall-associated teichoic acid glycopolymers, which confer somatic antigenicity. Strains belonging to serovars 1/2 and 4b cause the vast majority of listeriosis cases and outbreaks, meaning that regulators, as well as the food industry itself, have an interest in rapidly identifying isolates of these particular serovars in food processing environments. Current methods for phenotypic serovar differentiation are slow and lack accuracy, and the food industry could benefit from new technologies allowing serovar-specific isolation. Therefore, the novel method described here for rapid glycotype determination could present a valuable asset to detect and control this bacterium.

The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus. Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections., Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureus.

Electric vehicles (EVs) can be utilized as mobile storages in a power system. The use of battery chargers can cause current harmonics in the supplied AC system. In order to analyze the impact of different EVs with regardto their number and their emission of current harmonics, a generic harmonic current model of EV types was built and implemented in the power system simulation tool PSS®NETOMAC. Based on the measurement data for different types of EVs three standardized harmonic EV models were developed and parametrized. Further, the identified harmonic models are used by the computation of load flow in a modeled, German power distribution system. As a benchmark, a case scenario was studied regarding a high market penetration of EVs in the year 2030 for Germany. The impact of the EV charging on the power distribution system was analyzed and evaluated with valid power quality standards.

X-ray ptychography, a powerful scanning lensless imaging technique, has become attractive for nondestructively imaging internal structures at nanoscale. Stage positioning overhead in conventional step-scan ptychography is one of the limiting factors on the imaging throughput. In this work, we demonstrate the use of advanced fly scan ptychography to achieve high-resolution ptychograms of modern integrated circuits on a large field-of-view at millimeter scale. By completely removing stage overheads between scan points, the imaging time for millimeter-size sample can be significantly reduced. Furthermore, we implement the orthogonal probe relaxation technique to overcome the variation of illumination across the large scan area as well as local vibrations. The capability of x-ray ptychography shown here is broadly applicable for various studies, which requires both high spatial resolution and large scan area.

Among different techniques based on x-ray nanoimaging, ptychography has become a popular tool to study specimens at nanometer-scale resolution without the need of using high-resolution optics that requires very stringent manufacturing processes. This high-resolution imaging method is compatible with other imaging modalities acquired in scanning microscopy. At the Advance Photon Source (APS), we have developed two fluorescence microscopes for simultaneous ptychography and fluorescence imaging which together provide a powerful technique to study samples in biology, environmental science, and materials science. Combined with different tilted sample projections, such correlative methods can yield high-resolution 3D structural and chemical images. More recent work has been focused on the development of a fast ptychography instrument called the Velociprobe which is built to take advantage of the over 100 times higher coherent flux provided by the coming APS upgrade source. The Velociprobe uses high-bandwidth accurate interferometry and advanced motion controls with fast continuous scanning schemes which are optimized for large-scale samples and 3D high-resolution imaging. This instrument has been demonstrated to obtain sub-10 nm resolution with different high-photon-efficient scanning schemes using fast data acquisition rate up to 3 kHz (currently limited by detector's full continuous frame rate). A ptychographic imaging rate of 100 _m2/second with a sub-20 nm spatial resolution was shown in this paper.

As a scanning version of coherent diffraction imaging (CDI), X-ray ptychography has become a popular and very successful method for high-resolution quantitative imaging of extended specimens. The requirements of mostly coherent illumination and the scanning mechanism limit the throughput of ptychographic imaging. In this paper, we will introduce the methods we use at the Advanced Photon Source (APS) to achieve highthroughput ptychography by optimizing the parameters of the illumination beam. One work we have done is increasing the illumination flux by using a double-multilayer monochromator (DMM) optics with about 0.8% bandwidth. Compared with our double-crystal monochromator (DCM) optics with 0.01% bandwidth, this DMM optics provides around 20 times more flux. A multi-wavelength reconstruction method has been implemented to deal with the consequential degraded temporal coherence from such an illumination to ensure high-quality reconstruction. In the other work, we adopt a novel use of at-top focusing optics to generate a at-top beam with the diameter of about 1.5 μm on the focal plane. The better uniformity of the probe and the large beam size allow one to significantly increase the step size in ptychography scans and thereby the imaging efficiency.

Regulation of cellular functions by an exogenous and non-invasive approach has the means of revolutionizing the field of tissue engineering. In this direction, use of electric fields has garnered significant interest due to its positive influence on cell adhesion, proliferation, and differentiation. Recently, this has been achieved by placing electrodes in direct contact with cells, or through a non-contact approach by inducing deformation of piezoelectric membranes. In this work, we have developed 3D magnetoelectric inverse opal scaffolds that can generate localized electric fields upon the application of magnetic fields. These scaffolds were composed of biodegradable poly(l-lactic acid), and cobalt ferrite@bismuth ferrite magnetoelectric nanoparticles and were designed to mimic the natural micro-environment of cancellous bone by endowing them with piezoelectric properties and porosity. The effect of magnetic field induced electric stimulation on the proliferation of human-derived MG63 osteoblast cells, a model for primary osteoblast cells, was investigated on 2D membranes and 3D scaffolds by applying a magnetic field of 13 mT at 1.1 kHz. During this study, a 134% increase in cell proliferation was achieved on stimulated 3D scaffolds in comparison to non-stimulated ones, and in case of 2D membranes, we have obtained an increase of 43% of stimulated 2D membranes in comparison to non-stimulated ones. These findings showcase the importance of designing scaffolds with 3D characteristics that provide a suitable micro-environment for host cells. The results obtained from this work further demonstrate the beneficial influence that the magnetoelectric effect has on regulating cellular functions and draws light on the possibility of exploiting this effect for tissue engineering and regenerative medicine in the future.

Many cosmological and astrophysical observations indicate the existence of Dark Matter (DM) in the Universe with an about five times higher contribution to the energy content of the Universe than baryonic matter. The search for DM in the mono-Higgs (bb) channel in proton-proton collisions at a center-of-mass energy of 13 TeV in the mono-Higgs (bb) channel is presented, where the Higgs boson, decaying into a bottom (b) quark pair, is produced in association with large missing transverse energy, which is due to the pair produced DM particles. Datasets corresponding to integrated luminosities of 36.1/fb and 79.8/fb recorded with the ATLAS detector at the Large Hadron Collider (LHC) have been analysed. Eine Vielzahl kosmologischer und astrophysikalischer Beobachtungen weist auf die Existenz Dunkler Materie (DM) mit etwa fünfach höherem Beitrag zum Energieinhalt des Universums als baryonische Materie. Es wird über die Suche nach DM im Mono-Higgs-Kanal in Proton-Proton-Kollisionen bei einer Schwerpunktsenergie 13 TeV im Mono-Higgs (bb)-Kanal berichtet, wobei das in ein bottom (b)-Quarkpaar zerfallende Higgs-Boson zusammen mit hoher fehlender Transversalenergie produziert wird, die von der Paarproduktion der Teilchen der Dunklen Materie herrührt. Es wurden Datensätze entsprechend integrierter Luminositäten von 36.1/fb und 79.8/fb analysiert, die mit dem ATLAS Detektor am Large Hadron Collider (LHC) aufgezeichnet wurden.

Bacteria are an integral part of human life on earth and surround us from the beginning to the end of our existence. In this thesis, we present two projects that permit better understanding and control of two major pathogens from the bacterial order Bacillales: Staphylococcus aureus and Listeria monocytogenes. Both bacteria are Gram-positive and foodborne pathogens, have various virulence factors and can invade human cells. S. aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistant strains. In addition, intracellular localization of S. aureus plays a key role in recurrent infections, as intracellular pathogens are protected from antibiotics and immune responses. Bacteriophage encoded endolysins and other peptidoglycan hydrolases (PGHs) are alternative therapeutics, which are currently under clinical investigation. They offer several advantages such as effective and specific killing by means of an active mechanism, which eliminates metabolically inactive and drug resistant S. aureus. However, PGHs are limited in their ability to target intracellular bacteria and in Manuscript 1 of this thesis, we describe the preclinical development of PGHs for effective targeting of intracellular S. aureus. To this end, we screened 322 PGHs for staphylolytic activity simulating intracellular and other relevant physiological conditions. The most active constructs were extensively characterized in vitro and six enzymes were fused to selected cell penetrating peptides (CPPs) to enhance cellular uptake, as demonstrated by time-resolved fluorescence of Europium labeled enzymes. Cellular infection models with different S. aureus strains (including methicillin resistant S. aureus) were employed in combination with fluorescence microscopy to identify the most suitable combinations of PGHs and CPPs. We demonstrate killing of intracellular S. aureus by up to 4.5 log units. PGHs fused to the CCP "TAT", yielded the best results, and efficacy could be further enhanced by combining synergistically active PGHs. Finally, synergistically active PGH TAT cocktails and their individual components with and without TAT were evaluated in a murine abscess model. The synergistic PGH-TAT cocktail showed significantly improved killing compared to all other tested treatments, with a total S. aureus reduction of 2.2 log units. The PGH TAT cocktail also significantly enhanced killing of intracellular Staphylococci, additionally abscess size and body weight loss were most reduced in animals treated with this cocktail. Altogether, the superior performance of the PGH TAT cocktail is based on the careful selection of appropriate enzymes by activity screening, the synergistic action of its components and their increased penetration due to CPP fusion. In conclusion, CPP fused PGHs represent promising novel protein therapeutics against intracellular, drug tolerant and drug resistant S. aureus. In the future, their safety should be assessed and a clinical trial should be designed for the treatment of diseases with reported intracellular localization of S. aureus, such as atopic dermatitis. Manuscript 2 focuses on Listeria monocytogenes, a bacterium, which can be ubiquitous found in the environment, particularly in soil. It is a major concern in the food industry, as it frequently causes listeriosis outbreaks due to contamination of fresh produce, dairy products and pre cut meat or fish. Listeriosis can lead to abortion and infected individuals have high mortality rates of 20-30%. Listeria monocytogenes can be divided into 12 different serovars, which diverge in their wall-teichoic acid glycosylation. The current standard procedure for serovar determination is based on an agglutination method using antisera. The differential binding patterns of those antisera to the somatic and flagellar antigens of Listeria allow distinction of different serovars. Only strains designated as serovars 1/2a, 1/2b and 4b are regarded as highly virulent and are responsible for 98% of all listeriosis cases. Unfortunately, serotyping is imperfect in both, precision and practicality, due to wide discrepancies in the subjective interpretation of results. Additionally, the exact antigen epitopes remain unclear. We used structural predictions to find proteins derived from bacteriophage endolysins and receptor binding proteins to develop of a novel method for somatic antigen differentiation. The six selected proteins recognize differentially glycosylated wall teichoic acids on the Listeria surface. Their fusion to green fluorescent protein permits quantification of surface glycosylation, which is directly linked to different Listeria serovars. This objective glycotyping approach enables clear, non-biased determination of all somatic antigen structures with the ability to quantify the relative amount of surface receptor present on each strain. As such, the developed glycotyping system facilitated near complete and quantitative serovar differentiation of over 60 Listeria strains from a cohort of previously characterized Listeria. In addition, we used the selected proteins to functionalize paramagnetic beads. These functionalized beads can now specifically isolate strains belonging to the pathogenic serovars 1/2 and 4b from a mixture of Listeria strains. Furthermore our serovar-specific profiling method led to the discovery that strains designated as SV 4e possess an intermediate 4b-4d glycosylation phenotype, revealing the high discriminative power of this novel technique. In the future, our system may prove valuable for enrichment from foods, prior to detection with highly sensitive systems, such as novel luciferase reporter phages constructed in our laboratory. In summary, in this work we use bacteriophage proteins to meet the demand for novel diagnostic tools and ways to combat drug-resistant bacterial pathogens.