Direct detection of human Staphylococcus aureus carriage in the nose using the Lightcycler Staphylococcus kit

The Lightcycler Staphylococcus kit is a diagnostic tool for direct real-time detection of Staphylococcus aureus in clinical materials. We show here that detection of S. aureus nasal carriage using this test is hampered by competition of DNA from coagulase-negative staphylococci. However the test is well suited for species identification after culture and the identification of high-load S. aureus carriers.     

Cloning of the ribokinase gene of Staphylococcus hyicus subsp. hyicus in Staphylococcus carnosus

Summary A gene library with DNA of Staphylococcus hyicus subsp. hyicus was established in S. carnosus by using the plasmid vector pCT20. Two clones of S. carnosus were isolated which were able to ferment d-ribose. The two hybrid plasmids (pRib 1) and (pRib 2) were isolated and characterized. They contained inserted DNA fragments of S. hyicus subsp. hyicus with sizes of 10.2 and 8.2 kb, respectively. d-Ribose uptake and enzyme activities were studied. All strains tested [S. hyicus subsp. hyicus, S. carnosus (wild type) and the two S. carnosus clones] possessed an inducible uptake system for d-ribose. S. hyicus subsp. hyicus possessed in addition enzyme activities of d-ribokinase and d-ribose-5-P isomerase. None of these enzyme activities could be detected in S. carnosus (wildtype). Only in the S. carnosus clones containing (pRib 1) or (pRib 2) could a d-ribokinase activity be demonstrated, indicating that the gene for d-ribokinase of S. hyicus subsp. hyicus was cloned in S. carnosus.Abbreviations bp base pairs - C-TLC cellulose-thin layer chromoatography - kb kilo base pairs - pR_ib 1 and 2 ribokinase activity conferring hybridplasmids - MBq megabequerel - wt wild type     

Profiling the surfacome of Staphylococcus aureus

Staphylococcus aureus is a widespread opportunistic pathogen that can cause a wide variety of life‐threatening diseases. Especially for the colonization of human tissues and the development of invasiveness, surface‐exposed proteins are of major importance. In the present studies, we optimized a proteolytic shaving approach to identify those surface‐exposed protein domains – the surfacome – of S. aureus that are accessible to extracellular bio‐macromolecules, for example in the host milieu. Subsequently, this approach was applied to define the surfacomes of four strains with different genetic backgrounds. This resulted in the identification of 96 different proteins. Surprisingly, the overlap between the surfacomes of the four different strains was below 10% and each strain displayed its own characteristic set of surface‐exposed proteins. The data were also evaluated at the peptide level and here we observed a similar phenomenon. From 190 unique peptides only five were commonly found in the four strains. Besides well known cell wall proteins, we also identified some essential proteins, several yet uncharacterized exported proteins and predicted intracellular proteins. These results show for the first time that the cell surface of different S. aureus strains is not only highly variable, but also that the displayed proteins are very heterogeneous.     

Range of the antigenic specificity of peptidoglycan in Staphylococcus aureus compared to other representatives of the Staphylococcus genus

In investigations based on the use of a highly sensitive test system permitting the detection of normal human antibodies to S. aureus peptidoglycan, the antigenic relationships between the peptidoglycans of S. aureus and other representatives of the genus Staphylococcus have been studied. Among other staphylococcal species, S. simulans, S. xylosus, S. hyicus, S. cohnii, S. hyicus s. s. chromogenes have been found to possess peptidoglycans most closely related to S. aureus peptidoglycans, while S. warneri and S. epidermidis peptidoglycans have proved to be least closely related to it.     

Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibro cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.     

Staphylococcus and the healing power of pus

Staphylococcus aureus infections induce formation of neutrophil-rich abscesses filled with debris from dead phagocytes. Corbin and colleagues report that this pus has antimicrobial powers through the activity of calprotectin. Calprotectin, a member of the S100 family of proinflammatory proteins, acts through chelation of manganese. As manganese is an essential cofactor for several enzymes in S. aureus, this impacts bacterial growth and the bacterium's ability to withstand oxidative stress.     

Detecting the Enterotoxigenicity of Staphylococcus aureus Strains

An optimal sensitivity plate method for examining large number of staphylococcal strains for production of the known enterotoxins (A-E) is presented. Small volumes of relatively concentrated enterotoxin are produced by the semi-solid agar, cellophane-over-agar, or sac culture techniques. Detection of the enterotoxin in the supernatant fluid is accomplished with the optimal sensitivity plate method. In this method small plastic petri dishes (50 mm) were used for a modified Ouchterlony of high sensitivity.