Rapid detection of antibiotic resistance based on mass spectrometry and stable isotopes.

With the emergence and growing complexity of bacterial drug resistance, rapid and reliable susceptibility testing has become a topical issue. Therefore, new technologies that assist in predicting the effectiveness of empiric antibiotic therapy are of great interest. Although the use of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid detection of antibiotic resistance is an attractive option, the current methods for MALDI-TOF MS susceptibility testing are restricted to very limited conditions. Here, we describe a technique that may allow for rapid susceptibility testing to an extent that is comparable to phenotypic methods. The test was based on a stable isotope labelling by amino acids in cell culture (SILAC)-like approach. This technique was used to visualise the growth of bacteria in the presence of an antibiotic. Pseudomonas aeruginosa was chosen as the model organism, and strains were incubated in normal medium, medium supplemented with (13)C6-(15) N2-labelled lysine and medium supplemented with labelled lysine and antibiotic. Peak shifts occurring due to the incorporation of the labelled amino acids were detected by MALDI-TOF MS. Three antibiotics with different mechanisms of action, meropenem, tobramycin and ciprofloxacin, were tested. A semi-automated algorithm was created to enable rapid and unbiased data evaluation. With the proposed test, a clear distinction between resistant and susceptible isolates was possible for all three antibiotics. The application of SILAC technology for the detection of antibiotic resistance may contribute to accelerated and reliable susceptibility testing.

Rapid detection of Salmonella sp. by means of a combination of selective enrichment broth and MALDI-TOF MS.

The identification of Salmonella sp. in stool samples usually takes 2 days when employing routine procedures. Fast approaches are necessary in order to shorten the analysis time. The aim of this work was the development of a rapid procedure for the detection of Salmonella sp. from clinical stool samples. Spiked stool samples were cultured in selective selenite enrichment broth. Identifications were directly performed from the liquid broth by the MALDI Biotyper. After the evaluation of this method, the same procedure was applied to clinical samples. Coevally, the samples were streaked on Hektoen agar and single colonies were analyzed by the MALDI Biotyper. For comparison, the liquid broth was plated according to the standard laboratory procedure. A total of 4,847 samples were analyzed for Salmonella sp. In total, 108 Salmonella sp.-positive samples were identified; 66 of these were identified after the streaking of stool samples on Hektoen agar and subsequent MALDI Biotyper analysis of Salmonella sp. suspicious colonies. These and a further 34 samples were detected as Salmonella sp.-positive directly from the selenite enrichment broth on day one. Eight Salmonella sp.-positive samples were not detected before plating of the selenite broth and subsequent MALDI Biotyper analysis on day two. The combination of MALDI Biotyper analysis and selective selenite enrichment broth identification delivers positive results for the majority of the samples already after one day.

Rapid detection of antibiotic resistance by MALDI-TOF mass spectrometry using a novel direct-on-target microdroplet growth assay.

OBJECTIVES: We aimed to develop a universal phenotypic method, which allows easy and rapid antimicrobial susceptibility testing independently of underlying resistance mechanisms. METHODS: We established a novel direct-on-target microdroplet growth assay for the detection of antibiotic resistance within a few hours, which is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microorganisms were incubated with and without meropenem in nutrient broth as microdroplets directly on MALDI-TOF MS target. Subsequently, broth was separated from microbial cells by contacting the microdroplets with an absorptive material. The microorganisms grown in the presence of antibiotic were detected by MALDI-TOF MS. A total of 24 Klebsiella pneumoniae and 24 Pseudomonas aeruginosa isolates were used to assess performance for detection of meropenem resistance. The microdroplet volumes investigated were 2, 4, 6, 8 and 10 µL. RESULTS: The best performance was achieved using 6-µL microdroplets. Applying this volume, all growth controls were successfully detected (definition of valid test), and all isolates were correctly categorized as susceptible or non-susceptible after an 18-h incubation. For K. pneumoniae, rate of valid tests, sensitivity and specificity all reached 100% after a 4-h incubation of 6-µL microdroplets. Using the same microdroplet volume for P. aeruginosa, incubation for 5 h resulted in 83.3% of valid tests with 100% sensitivity and 100% specificity. CONCLUSIONS: We demonstrated easy, rapid and accurate resistance detection using carbapenem-resistant Gram-negative bacteria as an example. Our technology is suitable for automatization and expandable to further applications, e.g. simultaneous testing of multiple antibiotics as well as resistance determination directly from clinical samples.

T cell receptor specificity and mimotopes.

Degeneracy rather than unique ligand specificity seems to guide T cell functions. This view has evolved from analyses of T cell development and responses in vivo, as well as studies with synthetic molecular libraries in vitro, and has opened new prospects both for understanding T cell biology and for applied immunology.

Altered allogeneic response in neonatally anti-MHC class I-treated mice.

Neonatal treatment of C3H mice (H-2k) with anti-Kk monoclonal antibodies results in altered cytotoxic responses against allogeneic targets. After 2-3 weeks of antibody treatment, no difference in the number of CD4+8- or CD4-8+ T cells was observed between the antibody- and saline-treated mice. However, antibody-treated mice had a significantly reduced cytotoxic response against various allogeneic major histocompatibility complex (MHC) class I-expressing targets. The strongest reduction was observed in very young mice (up to 2 weeks of age). As the mice got older, the allo MHC-specific responses reached control levels. No significant changes in T-cell receptor (TCR)-V-region usage was observed even in young antibody-treated mice. The results suggest that the reduction in the number of positively selecting elements reduces alloreactivity and most likely also the diversity of TCR-repertoire. However, the reduced alloresponsiveness was not restricted to either allogeneic K- or D-encoded molecules, suggesting that self MHC-D-region encoded molecules can mediate positive selection of T cells able to react against both K and D region-encoded allogeneic MHC class I molecules.

Electrophoresis-related protein modification: alkylation of carboxy residues revealed by mass spectrometry.

In recent years, the combination of gel electrophoresis and mass spectrometry has developed into one of the most powerful approaches for the analysis of proteins. However, a number of gel electrophoresis-induced protein modifications have been described. Cysteine is the most endangered amino acid readily reacting with mercaptoethanol or free acrylamide. In the course of studies on glucan phosphorylases (E.C.2.4.1.1) from white potato (Solanum tuberosum L.) and the T cell receptor, we noticed that proteolytic peptides from these proteins can undergo an unexpected modification, giving rise to a mass increment of 14 Da. By post-source decay (PSD) analysis the modification was identified as methylation of the glutamic acid side chain carboxyl group. The methylation takes place during Coomassie blue staining of proteins if both trichloroacetic acid and methanol are present in the staining solution. Replacement of methanol by ethanol under otherwise unchanged conditions results in ethylation of the peptides. The in vitro alkylation was further studied by using synthetic peptides which contain, at different positions: glutamic acid, aspartic acid or the corresponding amides. The kinetic analysis of the observed reactions revealed that glutamic acid is preferentially methylated. The three other amino acid residues can be methylated but with a velocity at least one order of magnitude lower. Although these modifications complicate the interpretation of the spectra, they provide valuable structural information.

Combined laser tweezers and dielectric field cage for the analysis of receptor-ligand interactions on single cells.

A new technique based on the combination of optical and chip-based dielectrophoretical trapping was developed and employed to manipulate cells and beads with micrometer precision. The beads were trapped with optical tweezers (OT) and brought into contact for defined times with cells held in the dielectrophoretic field cage (DFC). The well-defined ligand-receptor system biotin-streptavidin was used to study the multiple interaction between biotinylated live cells and streptavidin-coated beads. The biotin density on the cell surface was varied down to a few single bonds (3 +/- 2 bonds/microm2) to control the valency of the binding. The quantitative relationship between the contact area, ligand density and its diffusion rate in the outer membrane of the cell could be demonstrated. The increase of the strength of the cell-bead adhesion was strictly dependent on the increase of individual bond numbers in the contact area. This is in part due to accumulation of ligands (D approxiamtely (0.5 +/- 0.1) 10(-8) cm2/s) in the contact area as seen by confocal laser scanning microscopy. Individual receptor-ligand rupture forces were evaluated and are compatible with values obtained by biomembrane force probe techniques. To summarize, the combination leads to a new powerful microsystem for cell handling and pN-force measurements on the single-cell level.

Analysis of a naturally occurring HLA class I-restricted viral epitope.

A previously described nonapeptide sequence motif for antigens recognized by T cells in the context of the human major histocompatibility complex (MHC) molecule HLA-A2.1 was used to identify the natural epitope of influenza A virus matrix protein. We show here that the peptide with the sequence GILGFVFTL is the synthetic analogue of the natural epitope by demonstrating the presence of the corresponding peptide on MHC molecules of virus-infected cells. The role of the hydrophobic anchor amino acids in positions 2 and 9, which constitute the epitope motif, was investigated with synthetic variants of the epitope and cytotoxic T lymphocytes as indicator cells. The crucial role of the side chains of amino acids in those positions was evidence by their influence on the efficiency of T-cell stimulation.