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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.
Coagulase-negative staphylococci (CoNS) are common opportunistic pathogens, but also ubiquitous human and animal commensals. Infection-associated CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin- and multidrug-resistant isolates. Less is known about AMR patterns of CoNS colonizing the general population. Here we report on AMR in commensal CoNS recovered from 117 non-hospitalized volunteers in a region of Germany with a high livestock density. Among the 69 individuals colonized with CoNS, 29 had reported contacts to either companion or farm animals. CoNS were selectively cultivated from nasal swabs, followed by species definition by 16S rDNA sequencing and routine antibiotic susceptibility testing. Isolates displaying phenotypic AMR were further tested by PCR for presence of selected AMR genes. A total of 127 CoNS were isolated and Staphylococcus epidermidis (75%) was the most common CoNS species identified. Nine isolates (7%) were methicillin-resistant (MR) and carried the mecA gene, with seven individuals (10%) being colonized with at least one MR-CoNS isolate. While resistance against gentamicin, phenicols and spectinomycin was rare, high resistance rates were found against tetracycline (39%), erythromycin (33%) and fusidic acid (24%). In the majority of isolates, phenotypic resistance could be associated with corresponding AMR gene detection. Multidrug-resistance (MDR) was observed in 23% (29/127) of the isolates, with 33% (23/69) of the individuals being colonized with MDR-CoNS. The combined data suggest that MR- and MDR-CoNS are present in the community, with previous animal contact not significantly influencing the risk of becoming colonized with such isolates.
Antibiotic resistance is increasing worldwide making it necessary to search for alternative antimicrobials. Sodium bituminosulfonate is a long-known substance, whose antimicrobial inhibitory activity has recently been re-evaluated. However, to the best of our knowledge, the bactericidal mode of action of this substance has not been systematically characterized. The aim of this study was to investigate the in vitro bactericidal activity of sodium bituminosulfonate by determining the minimal bactericidal concentrations (MBC), as well as the rapidity of bactericidal effect by time-kill curves. Clinical isolates of methicillin-susceptible (MSSA, n = 20) and methicillin-resistant (mecA/mecC-MRSA, n = 20) Staphylococcus aureus were used to determine MBC by a broth microdilution method. Sodium bituminosulfonate (Ichthyol® light) was tested in double-dilution concentration steps ranging from 0.03 g/L to 256 g/L. For time-kill analysis, two reference and two clinical S. aureus strains were tested with different concentrations of sodium bituminosulfonate (1× minimal inhibitory concentration (MIC), 2× MIC, 4× MIC, 16× MIC and 256× MIC). For MSSA isolates, MBC50, MBC90 and the MBC range were 0.5 g/L, 1.0 g/L and 0.125–1.0 g/L; (MBC/MIC ratio)50, (MBC/MIC ratio)90 and the range of the MBC/MIC ratio were 4, 4 and 1–8, respectively. Among MRSA isolates, MBC50, MBC90 and the MBC range amounted to 0.5 g/L, 1.0 g/L and 0.06–1.0 g/L; (MBC/MIC ratio)50, (MBC/MIC ratio)90 and the range of the MBC/MIC ratio were 2, 4 and 1–8, respectively. Time-kill kinetics revealed a bactericidal effect after 30 min for sodium bituminosulfonate concentrations of 16× MIC and 256× MIC. The bactericidal activity against MSSA and MRSA was demonstrated for sodium bituminosulfonate. The killing was very rapid with the initial population reduced by 99.9% after only short incubation with concentrations of 16× MIC and higher.
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.
Knowledge on differences in the severity and symptoms of infections with the SARS-CoV-2 Omicron variants BA.2 (Pango lineage B.1.529.2) and BA.5 (Pango lineage B.1.529.5) is still scarce. We investigated epidemiological data available from the public health authorities in Mecklenburg-Western Pomerania, Northeast Germany, between April and July 2022 retrospectively. Comparative analyses revealed significant differences between recorded symptoms of BA.2 and BA.5 infected individuals and found strong correlations of associations between symptoms. In particular, the symptoms ‘chills or sweating’, ‘freeze’ and ‘runny nose’ were more frequently reported in BA.2 infections. In contrast, ‘other clinical symptoms’ appeared more frequently in Omicron infections with BA.5. However, the results obtained in this study provide no evidence that BA.5 has a higher pathogenicity or causes a more severe course of infection than BA.2. To our knowledge, this is the first report on clinical differences between the current Omicron variants BA.2 and BA.5 using public health data. Our study highlights the value of timely investigations of data collected by public health authorities to gather detailed information on the clinical presentation of different SARS-CoV-2 subvariants at an early stage.
The coagulase-negative staphylococcal (CoNS) species Staphylococcus lugdunensis is unique in causing serious infections in humans that resemble those of Staphylococcus aureus rather than those of other CoNS species. The colonization and invasion of host tissue presupposes the presence of adherence factors, but only a few proteins mediating adhesion of S. lugdunensis to biotic surfaces are known yet. Here, we report on the functionality of the S. lugdunensis enolase (SlEno), which performs two distinct roles, first, as the metabolic enzyme of the glycolysis, and second, as an adherence factor to the extracellular matrix (ECM) of cells. Phylogenetic analyses of the SlEno confirmed their high conservation to enolases of other species and revealed a closer relationship to Staphylococcus epidermidis than to S. aureus. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry and Western blot experiments, we identified SlEno to be located in the cytoplasm as well as on the cell surface of S. lugdunensis. Recombinantly generated and surface-associated SlEno showed the usual enolase activity by catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate but, in addition, also displayed strong binding to immobilized laminin, fibronectin, fibrinogen, and collagen type IV in a dose-dependent manner. We also showed a strong binding of SlEno to plasminogen (Plg) and observed a tissue plasminogen activator (tPA)-dependent conversion of Plg to plasmin (Pln) whereby the Plg activation significantly increased in the presence of SlEno. This interaction might be dependent on lysines of the SlEno protein as binding to Plg was inhibited by ε-aminocaproic acid. Furthermore, the enhanced activation of the Plg/Pln system by SlEno enabled S. lugdunensis to migrate through a fibrin matrix. This migration was about 10-fold higher than without exogenously added SlEno. Finally, we observed a significantly higher clearance of S. lugdunensis by freshly prepared granulocytes and in the presence of anti-SlEno antibodies. In conclusion, these data demonstrate for the first time a moonlighting function of the S. lugdunensis enolase, which is an underrated virulence factor for colonization and invasion of tissues. Hence, SlEno might be a potential vaccine candidate to prevent severe infections caused by this pathogen.
Counting of microbial colonies is a common technique employed in research and diagnostics. To simplify this tedious and time-consuming process, automated systems have been proposed. This study aimed to elucidate the reliability of automated colony counting. We evaluated a commercially available instrument (UVP ColonyDoc-It Imaging Station) in regard to its accuracy and potential time savings. Suspensions of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus faecium, and Candida albicans (n = 20 each) were adjusted to achieve growth of approximately 1,000, 100, 10, and 1 colony per plate, respectively, after overnight incubation on different solid media. Compared with manual counting, each plate was automatically counted by the UVP ColonyDoc-It with and without visual adjustment on a computer display. For all bacterial species and concentrations automatically counted without visual correction, an overall mean difference from manual counts of 59.7%, a proportion of isolates with overestimation/underestimation of colony numbers of 29%/45%, respectively, and only a moderate relationship (R2 = 0.77) with the manual counting were shown. Applying visual correction, the overall mean difference from manual counts was 1.8%, the proportion of isolates with overestimation/underestimation of colony numbers amounted to 2%/42%, respectively, and a strong relationship (R2 = 0.99) with the manual counting was observed. The mean time needed for manual counting compared with automated counting without and with visual correction was 70 s, 30 s, and 104 s, respectively, for bacterial colonies through all concentrations tested. Generally, similar performance regarding accuracy and counting time was observed with C. albicans. In conclusion, fully automatic counting showed low accuracy, especially for plates with very high or very low colony numbers. After visual correction of the automatically generated results, the concordance with manual counts was high; however, there was no advantage in reading time.
IMPORTANCE Colony counting is a widely utilized technique in the field of microbiology. The accuracy and convenience of automated colony counters are essential for research and diagnostics. However, there is only sparse evidence on performance and usefulness of such instruments. This study examined the current state of reliability and practicality of the automated colony counting with an advanced modern system. For this, we thoroughly evaluated a commercially available instrument in terms of its accuracy and counting time required. Our findings indicate that fully automatic counting resulted in low accuracy, particularly for plates with very high or very low colony numbers. Visual correction of the automated results on a computer screen improved concordance with manual counts, but there was no benefit in counting time.
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.