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Comparison of Different Label-Free Raman Spectroscopy Approaches for the Discrimination of Clinical MRSA and MSSA Isolates
Methicillin-resistant Staphylococcus aureus (MRSA) is classified as one of the priority pathogens that threaten human health. Resistance detection with conventional microbiological methods takes several days, forcing physicians to administer empirical antimicrobial treatment that is not always appropriate. A need exists for a rapid, accurate, and cost-effective method that allows targeted antimicrobial therapy in limited time. In this pilot study, we investigate the efficacy of three different label-free Raman spectroscopic approaches to differentiate methicillin-resistant and -susceptible clinical isolates of S. aureus (MSSA). Single-cell analysis using 532 nm excitation was shown to be the most suitable approach since it captures information on the overall biochemical composition of the bacteria, predicting 87.5% of the strains correctly. UV resonance Raman microspectroscopy provided a balanced accuracy of 62.5% and was not sensitive enough in discriminating MRSA from MSSA. Excitation of 785 nm directly on the petri dish provided a balanced accuracy of 87.5%. However, the difference between the strains was derived from the dominant staphyloxanthin bands in the MRSA, a cell component not associated with the presence of methicillin resistance. This is the first step toward the development of label-free Raman spectroscopy for the discrimination of MRSA and MSSA using single-cell analysis with 532 nm excitation. IMPORTANCE Label-free Raman spectra capture the high chemical complexity of bacterial cells. Many different Raman approaches have been developed using different excitation wavelength and cell analysis methods. This study highlights the major importance of selecting the most suitable Raman approach, capable of providing spectral features that can be associated with the cell mechanism under investigation. It is shown that the approach of choice for differentiating MRSA from MSSA should be single-cell analysis with 532 nm excitation since it captures the difference in the overall biochemical composition. These results should be taken into consideration in future studies aiming for the development of label-free Raman spectroscopy as a clinical analytical tool for antimicrobial resistance determination.
Link to glow - iEDDA conjugation of a Ruthenium(II) tetrazine complex leading to dihydropyrazine and pyrazine complexes with improved 1O2 formation ability
The synthesis and photophysical properties of the Ru-polypyridyl type complex [(tbbpy)2Ru(bptz)]2+ (Ru-bptz, tbbpy: 4,4’-di-tert-butyl-2,2’-bipyridine, bptz: 2,6-dipyrido-1,2,4,5-tetrazine), and the complexes [(tbbpy)2Ru(L)]2+ formed by inverse electron demand Diels Alder reaction (iEDDA) of Ru-bptz with with alkenes and alkynes, where L is 3,6-dipyrido-2,5-dihydropyridazine (bpdhpn) or 3,6-dipyrido-pyridazine (bppn) are described. A combination of steady-state and time-resolved spectroscopy complemented by the computation of state-specific absorption properties by means of time-dependent density functional theory reveals that the intense visible absorption band stems from Ru → tbbpy and Ru → L metal-to-ligand charge-transfer (MLCT) excitations. The studies show that lowest-lying L-centered MLCT states (3MLCTL) show comparably low emission quantum yields (3–9%) and lifetimes (90–150 ns). This correlates with the singlet oxygen generation ability, following the trend: Ru-bppn > Ru-bpdhpn > Ru-bptz.
Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8
Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.
ConsensusPrime—A Bioinformatic Pipeline for Ideal Consensus Primer Design
Background: High-quality oligonucleotides for molecular amplification and detection procedures of diverse target sequences depend on sequence homology. Processing input sequences and identifying homogeneous regions in alignments can be carried out by hand only if they are small and contain sequences of high similarity. Finding the best regions for large and inhomogeneous alignments needs to be automated. Results: The ConsensusPrime pipeline was developed to sort out redundant and technical interfering data in multiple sequence alignments and detect the most homologous regions from multiple sequences. It automates the prediction of optimal consensus primers for molecular analytical and sequence-based procedures/assays. Conclusion: ConsensusPrime is a fast and easy-to-use pipeline for predicting optimal consensus primers that is executable on local systems without depending on external resources and web services. An implementation in a Docker image ensures platform-independent executability and installability despite the combination of multiple programs. The source code and installation instructions are publicly available on GitHub.
Characterization of PVL-Positive MRSA Isolates in Northern Bavaria, Germany over an Eight-Year Period
Purpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton–Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period. Methods: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa-typing. Results: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites. Conclusions: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.
Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques
Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton–Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.
Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics
Burkholderia 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.
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).
Revealing the Chemical Composition of Birch Pollen Grains by Raman Spectroscopic Imaging
The investigation of the biochemical composition of pollen grains is of the utmost interest for several environmental aspects, such as their allergenic potential and their changes in growth conditions due to climatic factors. In order to fully understand the composition of pollen grains, not only is an in-depth analysis of their molecular components necessary but also spatial information of, e.g., the thickness of the outer shell, should be recorded. However, there is a lack of studies using molecular imaging methods for a spatially resolved biochemical composition on a single-grain level. In this study, Raman spectroscopy was implemented as an analytical tool to investigate birch pollen by imaging single pollen grains and analyzing their spectral profiles. The imaging modality allowed us to reveal the layered structure of pollen grains based on the biochemical information of the recorded Raman spectra. Seven different birch pollen species collected at two different locations in Germany were investigated and compared. Using chemometric algorithms such as hierarchical cluster analysis and multiple-curve resolution, several components of the grain wall, such as sporopollenin, as well as the inner core presenting high starch concentrations, were identified and quantified. Differences in the concentrations of, e.g., sporopollenin, lipids and proteins in the pollen species at the two different collection sites were found, and are discussed in connection with germination and other growth processes.
The Other Dimension—Tuning Hole Extraction via Nanorod Width
Solar-to-hydrogen generation is a promising approach to generate clean and renewable fuel. Nanohybrid structures such as CdSe@CdS-Pt nanorods were found favorable for this task (attaining 100% photon-to-hydrogen production efficiency); yet the rods cannot support overall water splitting. The key limitation seems to be the rate of hole extraction from the semiconductor, jeopardizing both activity and stability. It is suggested that hole extraction might be improved via tuning the rod’s dimensions, specifically the width of the CdS shell around the CdSe seed in which the holes reside. In this contribution, we successfully attain atomic-scale control over the width of CdSe@CdS nanorods, which enables us to verify this hypothesis and explore the intricate influence of shell diameter over hole quenching and photocatalytic activity towards H2 production. A non-monotonic effect of the rod’s diameter is revealed, and the underlying mechanism for this observation is discussed, alongside implications towards the future design of nanoscale photocatalysts.