Refine
Document Type
- Article (4)
Language
- English (4)
Has Fulltext
- yes (4)
Is part of the Bibliography
- no (4)
Keywords
- - (3)
- coagulase-negative staphylococci (2)
- Germany (1)
- MALDI-TOF (1)
- MALDI-TOF MS (1)
- MRSA (1)
- MRSA - Methicillin-resistant (1)
- One Health (1)
- adhesion (1)
- antimicrobial resistance (1)
Publisher
- Frontiers Media S.A. (4) (remove)
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.
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.
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.