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Discrimination of Streptococcus pneumoniae from other Streptococcus mitis group (SMG) species is still challenging but very important due to their different pathogenic potential. In this study, we aimed to develop a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based optochin susceptibility test with an objective read-out. Optimal test performance was established and evaluated by testing consecutively collected respiratory isolates. Optochin in different concentrations as a potential breakpoint concentration was added to a standardized inoculum. Droplets of 6 µL with optochin and, as growth control, without optochin were spotted onto a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard. Spectra were acquired, and results were interpreted as S. pneumoniae in the case of optochin susceptibility (no growth), or as non-S. pneumoniae in the case of optochin non-susceptibility (growth). Highest test accuracy was achieved after 20 h incubation time (95.7%). Rapid testing after 12 h incubation time (optochin breakpoint 2 µg/mL; correct classification 100%, validity 62.5%) requires improvement by optimization of assay conditions. The feasibility of the MALDI-TOF MS-based optochin susceptibility test was demonstrated in this proof-of-principle study; however, confirmation and further improvements are warranted.
In classical models of tumorigenesis, the accumulation of tumor promoting chromosomal aberrations is described as a gradual process. Next-generation sequencing-based methods have recently revealed complex patterns of chromosomal aberrations, which are beyond explanation by these classical models of karyotypic evolution of tumor genomes. Thus, the term chromothripsis has been introduced to describe a phenomenon, where temporarily and spatially confined genomic instability results in dramatic chromosomal rearrangements limited to segments of one or a few chromosomes. Simultaneously arising and misrepaired DNA double-strand breaks are also the cause of another phenomenon called chromoplexy, which is characterized by the presence of chained translocations and interlinking deletion bridges involving several chromosomes. In this study, we demonstrate the genome-wide identification of chromosomal translocations based on the analysis of translocation-associated changes in spatial proximities of chromosome territories on the example of the cutaneous T-cell lymphoma cell line Se-Ax. We have used alterations of intra- and interchromosomal interaction probabilities as detected by genome-wide chromosome conformation capture (Hi-C) to infer the presence of translocations and to fine-map their breakpoints. The outcome of this analysis was subsequently compared to datasets on DNA copy number alterations and gene expression. The presence of chained translocations within the Se-Ax genome, partly connected by intervening deletion bridges, indicates a role of chromoplexy in the etiology of this cutaneous T-cell lymphoma. Notably, translocation breakpoints were significantly overrepresented in genes, which highlight gene-associated biological processes like transcription or other gene characteristics as a possible cause of the observed complex rearrangements. Given the relevance of chromosomal aberrations for basic and translational research, genome-wide high-resolution analysis of structural chromosomal aberrations will gain increasing importance.
Matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS)-based direct-on-target microdroplet growth assay (DOT-MGA) was recently described as a novel method of phenotypic antimicrobial susceptibility testing (AST). Here, we developed the application of MALDI-TOF MS-based DOT-MGA for Gram-positive bacteria including AST from agar cultures and directly from positive blood cultures (BCs) using the detection of methicillin resistance as example. Consecutively collected, a total of 14 methicillin-resistant Staphylococcus aureus (MRSA) and 14 methicillin-susceptible S. aureus (MSSA) clinical isolates were included. Furthermore, a collection of MRSA challenge strains comprising different SCCmec types, mec genes, and spa types was tested. Blood samples were spiked with MRSA and MSSA and positive BC broth processed by three different methods: serial dilution of BC broth, lysis/centrifugation, and differential centrifugation. Processed BC broth was directly used for rapid AST using DOT-MGA. Droplets of 6 μl with and without cefoxitin at the EUCAST breakpoint concentration were spotted in triplicates onto the surface of a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard as a quality control (QC). Spectra were acquired and evaluated using MALDI Biotyper software. First, tests were considered as valid, if the growth control achieved an identification score of ≥1.7. For valid tests, same score criterion was used for resistant isolates when incubated with cefoxitin. An identification score <1.7 after incubation with cefoxitin defined susceptible isolates. On-target protein extraction using formic acid considerably improved detection of methicillin resistance in S. aureus and DOT-MGA showed feasible results for AST from agar cultures after 4 h incubation time. Comparing the different processing methods of positive BC broth, lysis/centrifugation method with a final dilution step 10–1 of the 0.5 McFarland suspension resulted in best test performance after 4 h incubation time. Overall, 96.4% test validity, 100% sensitivity, and 100% specificity were achieved for detection of methicillin resistance in clinical isolates. All strains of the MRSA challenge collection were successfully tested as methicillin-resistant. This first study on Gram-positive organisms showed feasibility and accuracy of MALDI-TOF MS-based DOT-MGA for rapid AST of S. aureus from agar cultures and directly from positive BCs.
Background: Klebsiella pneumoniae causes severe diseases including sepsis, pneumonia
and wound infections and is differentiated into hypervirulent (hvKp) and classic (cKp) pathotypes.
hvKp isolates are characterized clinically by invasive and multiple site infection and phenotypically
in particular through hypermucoviscosity and increased siderophore production, enabled by the
presence of the respective virulence genes, which are partly carried on plasmids. Methods: Here, we
analyzed two K. pneumoniae isolates of a human patient that caused severe multiple site infection.
By applying both genomic and phenotypic experiments and combining basic science with clinical
approaches, we aimed at characterizing the clinical background as well as the two isolates in-depth.
This also included bioinformatics analysis of a chromosomal virulence plasmid integration event.
Results: Our genomic analysis revealed that the two isolates were clonal and belonged to sequence
type 420, which is not only the first description of this K. pneumoniae subtype in Germany but also
suggests belonging to the hvKp pathotype. The latter was supported by the clinical appearance and
our phenotypic findings revealing increased siderophore production and hypermucoviscosity similar
to an archetypical, hypervirulent K. pneumoniae strain. In addition, our in-depth bioinformatics
analysis suggested the insertion of a hypervirulence plasmid in the bacterial chromosome, mediated
by a new IS5 family sub-group IS903 insertion sequence designated ISKpn74. Conclusion: Our study
contributes not only to the understanding of hvKp and the association between hypervirulence and
clinical outcomes but reveals the chromosomal integration of a virulence plasmid, which might lead
to tremendous public health implications.
Investigation of In-Vitro Adaptation toward Sodium Bituminosulfonate in Staphylococcus aureus
(2020)
Hair follicles constitute important drug delivery targets for skin antisepsis since they contain ≈25% of the skin microbiome. Nanoparticles are known to penetrate deeply into hair follicles. By massaging the skin, the follicular penetration process is enhanced based on a ratchet effect. Subsequently, an intrafollicular drug release can be initiated by various trigger mechanisms. Here, we present novel ultraviolet A (UVA)-responsive nanocapsules (NCs) with a size between 400 and 600 nm containing hydroxyethyl starch (HES) functionalized by an o-nitrobenzyl linker. A phase transfer into phosphate-buffered saline (PBS) and ethanol was carried out, during which an aggregation of the particles was observed by means of dynamic light scattering (DLS). The highest stabilization for the target medium ethanol as well as UVA-dependent release of ethanol from the HES-NCs was achieved by adding 0.1% betaine monohydrate. Furthermore, sufficient cytocompatibility of the HES-NCs was demonstrated. On ex vivo porcine ear skin, a strong UVA-induced release of the model drug sulforhodamine 101 (SR101) could be demonstrated after application of the NCs in cyclohexane using laser scanning microscopy. In a final experiment, a microbial reduction comparable to that of an ethanol control was demonstrated on ex vivo porcine ear skin using a novel UVA-LED lamp for triggering the release of ethanol from HES-NCs. Our study provides first indications that an advanced skin antisepsis based on the eradication of intrafollicular microorganisms could be achieved by the topical application of UVA-responsive NCs.
Previous research identified veterinary clinics as hotspots with respect to accumulation and spread of multidrug resistant extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (EC). Therefore, promoting the prudent use of antibiotics to decrease selective pressure in that particular clinical environment is preferable to enhance biosecurity for animal patients and hospital staff. Accordingly, this study comparatively investigated the impact of two distinct perioperative antibiotic prophylaxis (PAP) regimens (short-term versus prolonged) on ESBL-EC carriage of horses subjected to colic surgery. While all horses received a combination of penicillin/gentamicin (P/G) as PAP, they were assigned to either the “single-shot group” (SSG) or the conventional “5-day group” (5DG). Fecal samples collected on arrival (t0), on the 3rd (t1) and on the 10th day after surgery (t2) were screened for ESBL-EC. All isolates were further investigated using whole genome sequences. In total, 81 of 98 horses met the inclusion criteria for this study. ESBL-EC identified in samples available at t0, t1 and t2 were 4.8% (SSG) and 9.7% (5DG), 37% (SSG) and 47.2% (5DG) as well as 55.6% (SSG) and 56.8% (5DG), respectively. Regardless of the P/G PAP regimen, horses were 9.12 times (95% CI 2.79–29.7) more likely to carry ESBL-EC at t1 compared to t0 (p < 0.001) and 15.64 times (95% CI 4.57–53.55) more likely to carry ESBL-EC at t2 compared to t0 (p < 0.001). ESBL-EC belonging to sequence type (ST) 10, ST86, ST641, and ST410 were the most prevalent lineages, with blaCTX–M–1 (60%) being the dominant ESBL gene. A close spatio-temporal relationship between isolates sharing a particular ST was revealed by genome analysis, strongly indicating local spread. Consequently, hospitalization itself has a strong impact on ESBL-EC isolation rates in horses, possibly masking differences between distinct PAP regimens. The results of this study reveal accumulation and spread of multi-drug resistant ESBL-EC among horses subjected to colic surgery with different P/G PAP regimens, challenging the local hygiene management system and work-place safety of veterinary staff. Moreover, the predominance of particular ESBL-EC lineages in clinics providing health care for horses needs further investigation.
Drug alternatives to combat methicillin-resistant Staphylococcus aureus (MRSA) in human and animal healthcare are urgently needed. Recently, the recombinant bacteriophage endolysins, PRF-119 and its successor substance HY-133, have proven to be highly active against various S. aureus clonal lineages and to exhibit a very rapid bactericidal effect when standard methods for susceptibility testing are applied. Along with subsequent growth curve experiments, a re-growth phenomenon was observed in vitro necessitating its clarification for the assessment of the agent’s stability and activity as well as for methodological aspects of endolysin testing in general. Distinct in vitro parameters were comparatively examined applying also scanning electron microscopy, fluorescence assays and SDS-PAGE analysis. The shape and material of the culture vessels as well as the shaking conditions were identified as factors influencing the in vitro stability and activity of HY-133. The highest function maintenance was observed in plain centrifuge tubes. Based on this, the conditions and parameters of assays for testing the antimicrobial activities of phage endolysins were determined and adjusted. In particular, shear forces should be kept to a minimum. Our results form the basis for both future test standardization and re-growth-independent experiments as prerequisites for exact determination of the antimicrobial activities of engineered endolysins.