Emerging Options for the Diagnosis of Bacterial Infections and the Characterization of Antimicrobial Resistance.

Precise and rapid identification and characterization of pathogens and antimicrobial resistance patterns are critical for the adequate treatment of infections, which represent an increasing problem in intensive care medicine. The current situation remains far from satisfactory in terms of turnaround times and overall efficacy. Application of an ineffective antimicrobial agent or the unnecessary use of broad-spectrum antibiotics worsens the patient prognosis and further accelerates the generation of resistant mutants. Here, we provide an overview that includes an evaluation and comparison of existing tools used to diagnose bacterial infections, together with a consideration of the underlying molecular principles and technologies. Special emphasis is placed on emerging developments that may lead to significant improvements in point of care detection and diagnosis of multi-resistant pathogens, and new directions that may be used to guide antibiotic therapy.

Pulling the Brakes on Fast and Furious Multiple Drug-Resistant (MDR) Bacteria.

Life-threatening bacterial infections have been managed by antibiotics for years and have significantly improved the wellbeing and lifetime of humans. However, bacteria have always been one step ahead by inactivating the antimicrobial agent chemically or by producing certain enzymes. The alarming universal occurrence of multidrug-resistant (MDR) bacteria has compelled researchers to find alternative treatments for MDR infections. This is a menace where conventional chemotherapies are no longer promising, but several novel approaches could help. Our current review article discusses the novel approaches that can combat MDR bacteria: starting off with potential nanoparticles (NPs) that efficiently interact with microorganisms causing fatal changes in the morphology and structure of these cells; nanophotothermal therapy using inorganic NPs like AuNPs to destroy pathogenic bacterial cells; bacteriophage therapy against which bacteria develop less resistance; combination drugs that act on dissimilar targets in distinctive pathways; probiotics therapy by the secretion of antibacterial chemicals; blockage of quorum sensing signals stopping bacterial colonization, and vaccination against resistant bacterial strains along with virulence factors. All these techniques show us a promising future in the fight against MDR bacteria, which remains the greatest challenge in public health care.

Highly efficient ß-lactamase assay applying poly-dimethylacrylamide-based surface functionalization with ß-lactam antibiotics and ß-lactamase inhibitors.

In recent decades, the rise of ß-lactamases has substantially led to the emergence and wide spread of antibiotic resistance posing a serious global health threat. There is growing need for the development of rapid, cost-effective and user-friendly diagnostic assays for the accurate detection of ß-lactamases to optimize patient outcomes and prevent the spread of multidrug-resistances. In this article, we present a poly-dimethylacrylamide (PDMA)-based surface functionalization to immobilize ß-lactam antibiotics and ß-lactamase inhibitors of different subclasses. Immobilization was induced via UV-crosslinking through C,H-insertion reactions. The functional coatings were successfully applied in a highly efficient assay for the determination of recombinant ß-lactamases as well as ß-lactamases isolated from clinically relevant bacterial strains. Thus, this method describes an innovative approach with several significant benefits for diagnostic applications: the creation of specific detection platforms tailored for ß-lactamase activity, the development of high-throughput diagnostic assays and benefits regarding stability and shelf-life. Furthermore, this method is highly adaptable to other surfaces, antibiotics, and analytes, offering far-reaching implications for various biomedical, environmental, and antimicrobial applications.

Open-Source, Adaptable, All-in-One Smartphone-Based System for Quantitative Analysis of Point-of-Care Diagnostics.

Point-of-care (POC) diagnostics, in particular lateral flow assays (LFA), represent a great opportunity for rapid, precise, low-cost and accessible diagnosis of disease. Especially with the ongoing coronavirus disease 2019 (COVID-19) pandemic, rapid point-of-care tests are becoming everyday tools for identification and prevention. Using smartphones as biosensors can enhance POC devices as portable, low-cost platforms for healthcare and medicine, food and environmental monitoring, improving diagnosis and documentation in remote, low-resource locations. We present an open-source, all-in-one smartphone-based system for quantitative analysis of LFAs. It consists of a 3D-printed photo box, a smartphone for image acquisition, and an R Shiny software package with modular, customizable analysis workflow for image editing, analysis, data extraction, calibration and quantification of the assays. This system is less expensive than commonly used hardware and software, so it could prove very beneficial for diagnostic testing in the context of pandemics, as well as in low-resource countries.

Biosynthesis of iron oxide magnetic nanoparticles using clinically isolated Pseudomonas aeruginosa.

Magnetotactic bacteria are microscale complex natural systems that synthesize magnetic nanoparticles through biologically controlled mineralization. Nanoparticles produced by this process are biocompatible due to the presence of surrounding membranes. The mechanism controlling synthesis is cost-effective and is executed by complex genomes (operons). The results are monodispersed magnetic nanoparticles displaying advantages over polydispersed ones synthesized by physical and chemical methods. In this work, we isolated Pseudomonas aeruginosa from clinical samples and demonstrated its ability to biosynthesize magnetic nanoparticles. P. aeruginosa was thrived in a carbon-minimal medium supplemented with iron at low pH. The cells aligned parallel to a magnetic field, confirming their magnetic properties. The magnetic nanoparticles were extracted, purified, and characterized using electron microscopy, magnetometry, dynamic light scattering, and X-ray diffraction. This work represents the first isolation of a magnetotactic bacterium from clinical samples. The aerobic nature of these bacteria allows them to be easily cultured under laboratory conditions, unlike their well-known microaerophilic counterparts. The biosynthesized magnetic nanoparticles can be used in many applications, including magnetic resonance imaging, diagnostics, and therapeutics (i.e., magnetic hyperthermia).

Methacryloyl-GlcNAc Derivatives Copolymerized with Dimethacrylamide as a Novel Antibacterial and Biocompatible Coating.

Improving medical implants with functional polymer coatings is an effective way to further improve the level of medical care. Antibacterial and biofilm-preventing properties are particularly desirable in the area of wound healing, since there is a generally high risk of infection, often with a chronic course in the case of biofilm formation. To prevent this we here report a polymeric design of polymer-bound N-acetyl-glucosamine-oligoethylene glycol residues that mimic a cationic, antibacterial, and biocompatible chitosan surface. The combination of easy to use, crosslinkable, thin, potentially 3D-printable polymethacrylate layering with antibacterial and biocompatible functional components will be particularly advantageous in the medical field to support a wide range of implants as well as wound dressings. Different polymers containing a N-acetylglucosamine-methacryloyl residue with oligoethylene glycol linkers and a methacryloyl benzophenone crosslinker were synthesized by free radical polymerization. The functional monomers and corresponding polymers were characterized by 1H, 13C NMR, and infrared (IR) spectroscopy. The polymers showed no cytotoxic or antiadhesive effects on fibroblasts as demonstrated by extract and direct contact cell culture methods. Biofilm formation was reduced by up to 70% and antibacterial growth by 1.2 log, particularly for the 5% GlcNAc-4EG polymer, as observed for Escherichia coli and Staphylococcus aureus as clinically relevant Gram-negative and Gram-positive model pathogens.

Vertical Scanning Interferometry for Label-Free Detection of Peptide-Antibody Interactions.

Peptide microarrays are a fast-developing field enabling the mapping of linear epitopes in the immune response to vaccinations or diseases and high throughput studying of protein-protein interactions. In this respect, a rapid label-free measurement of protein layer topographies in the array format is of great interest but is also a great challenge due to the extremely low aspect ratios of the peptide spots. We have demonstrated the potential of vertical scanning interferometry (VSI) for a detailed morphological analysis of peptide arrays and binding antibodies. The VSI technique is shown to scan an array area of 5.1 square millimeters within 3⁻4 min at a resolution of 1.4 µm lateral and 0.1 nm vertical in the full automation mode. Topographies obtained by VSI do match the one obtained by AFM measurements, demonstrating the accuracy of the technique. A detailed topology of peptide-antibody layers on single spots was measured. Two different measurement regions are distinguished according to the antibody concentration. In the case of weakly diluted serum, the thickness of the antibody layer is independent of the serum dilution and corresponds to the physical thickness of the accumulated antibody layer. In strongly diluted serum, the thickness measured via VSI is linearly proportional to the serum dilution.

Identification of a Tetanus Toxin Specific Epitope in Single Amino Acid Resolution.

Vaccinations are among the most potent tools to fight infectious diseases. However, cross-reactions are an ongoing problem and there is an urgent need to fully understand the mechanisms of the immune response. For the development of a methodological workflow, the linear epitopes in the immune response to the tetanus toxin is investigated in sera of 19 vaccinated Europeans applying epitope mapping with peptide arrays. The most prominent epitope, appearing in nine different sera (923 IHLVNNESSEVIVHK937 ), is investigated in a substitution analysis to identify the amino acids that are crucial for the binding of the corresponding antibody species - the antibody fingerprint. The antibody fingerprints of different individuals are compared and found to be strongly conserved (929 ExxEVIVxK937 ), which is astonishing considering the randomness of their development. Additionally, the corresponding antibody species is isolated from one serum with batch chromatography using the amino acid sequence of the identified epitope and the tetanus specificity of the isolated antibody is verified by ELISA. Studying antibody fingerprints with peptide arrays should be transferable to any kind of humoral immune response toward protein antigens. Furthermore, antibody fingerprints have shown to be highly disease-specific and, therefore, can be employed as reliable biomarkers enabling the study of cross-reacting antigens.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays.

Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.

Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays.

The antibody species that patrol in a patient's blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding "their" antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases.