[Show abstract][Hide abstract] ABSTRACT: Antibiotic resistance in Gram-negative microorganisms is an increasing health care problem. The rapid detection of such resistance
is crucial for starting an early specific therapy and to enable initiation of the required hygiene measures. With continued
emphasis on reducing the cost of laboratory testing, only economical/low-cost approaches have a chance of being implemented.
During recent years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been
developed to be a standard method in microbiology laboratories for the rapid and cost-efficient identification of microorganisms.
Extending the usage of MALDI-TOF MS in the clinical microbiology laboratory to the area of resistance testing is an attractive
option. Quantitative MALDI-TOF MS using an internal standard facilitates the measurement of the quantity of peptides and small
proteins within a spectrum. These quantities correlate to the number of microorganisms and therefore to the growth of a microorganism.
The comparison of growth in the presence or absence of an antibiotic allows for analysis of the susceptibility behavior of
a strain. Here, we describe a novel method and its application in the analysis of 108 Klebsiella sp. isolates. After 1 h of incubation at a meropenem concentration of 8 μg/ml, a sensitivity of 97.3% and a specificity of
93.5% were achieved (compared to Etest results).
[Show abstract][Hide abstract] ABSTRACT: The identification of pathogens directly from blood cultures by MALDI-TOF MS can be a valuable tool to improve the treatment of patients suffering from sepsis and bacteremia. However, the increasing incidence of multi-drug-resistant Gram-negative bacteria makes it difficult to predict resistance patterns based only on pathogen identification. Most therapy regimens for sepsis caused by Gram-negative rods consist of at least one β-lactam antibiotic. Thus, it would be of great benefit to have an early marker of resistance against these drugs. In the current study, we tested 100 consecutive blood cultures containing Enterobacteriaceae for resistance against 3rd generation cephalosporins in a MALDI-TOF MS β-lactamase assay. E. coli was also tested for resistance against aminopenicillins. The results of the β-lactamase assay were compared with conventional methods. The assay permitted discrimination between E. coli strains resistant or susceptible to aminopenicillins with a sensitivity and specificity of 100%. The same was true for resistance to 3rd generation cephalosporins in Enterobacteriaceae that constitutively produced class C β-lactamases. Discrimination was more difficult in species expressing class A β-lactamases, as these enzymes can generate false positive results. Thus, sensitivity and specificity were 100% and 91.5%, respectively, for this group. The test permitted the prediction of resistance within 2.5 hours after the blood culture was flagged as positive.
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Clinical Microbiology 12/2013; DOI:10.1007/s10096-013-2031-5 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MALDI-TOF mass spectrometry profiling for microorganism detection has already been demonstrated in the 1990s, but has evolved to the first line identification method in many laboratories just during the past five years. While this application of MALDI-TOF MS has proven its broad applicability, accuracy, robustness, and cost-effectiveness it is of particular interest to expand the capabilities of the mass spectrometric platform. Resistance detection is the most desirable further application of MALDI-TOF MS in microbiology, but maybe also the most challenging. Different approaches have been published regarding diverse antibiotic drugs and distinct microorganism classes. The current review shall give an overview about the developments of the recent years and their potential to get transformed in clinical useful assays in the future.
[Show abstract][Hide abstract] ABSTRACT: Against the background of increasing numbers of resistant microorganisms the fast and cost-efficient detection of microbial resistance is an important clinical requirement for optimal therapeutic intervention. Current routine assays take at least 5 h but in most cases an overnight incubation is necessary to identify resistant isolates. The usage of MALDI-TOF MS profiling in combination with growth media containing isotopically labeled amino acids facilitates the detection of resistant microorganisms already after 3 h or less directly from the profile spectrum. Growing microorganisms incorporate isotopically labeled amino acids, increasing protein masses and thereby leading to mass shifts of their corresponding peaks in profile spectra. In the presence of antibiotics only resistant microorganisms are able to grow and to incorporate the labeled amino acids. This leads to a difference in the mass spectra of susceptible and resistant isolates allowing for their differentiation. In the presented study, we demonstrated the applicability of this novel approach for the detection of methicillin resistant Staphylococcus aureus and tested different bioinformatics approaches for automated data interpretation.
[Show abstract][Hide abstract] ABSTRACT: Nosocomial infections involving epidemic MRSA (methicillin resistant Staphylococcus aureus) strains are a serious problem in many countries. In order to analyze outbreaks, isolates have to be typed; however, most molecular methods are cost- or labor-intensive. Here we have evaluated matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) of cell extracts for molecular characterization of S. aureus strains. The peak patterns of 401 MRSA and methicillin sensitive S. aureus (MSSA), including clinical and laboratory strains, were analyzed. Database searches indicated the peptides represented by the corresponding peaks in the spectra. The identity of the peptides was confirmed by sequencing of mutants or by expression of antisense RNA fragments which resulted in the knockdown of the peptide of interest and concomitant loss of the signal or MALDI-TOF/TOF MS. It was shown that the signals derive mainly from stress proteins and ribosomal proteins. Peak shifts that differentiate the main S. aureus clonal complexes CC5, CC22, CC8, CC45, CC30 and CC1 correlate to point mutations in the respective genes. Retrospective typing of an MRSA outbreak showed that differentiation of unrelated MSSA, MRSA and borderline resistant S. aureus (BORSA) strains isolated from health care workers was possible. In conclusion, this method allows detection of epidemic lineages of S. aureus during species identification by MALDI-TOF MS analysis.
[Show abstract][Hide abstract] ABSTRACT: Carboxyterminally elongated and aminoterminally truncated Aβ peptides as well as their pyroglutamate and oxidized derivates are major constituents of human amyloid plaques. The objective of the present study was to characterize aminoterminally truncated or oxidized Aβ38, Aβ40, and Aβ42 peptide species in immunoprecipitated human cerebrospinal fluid (CSF).
We invented a novel sequential aminoterminally and carboxyterminally specific immunoprecipitation protocol and used the Aβ-SDS-PAGE/immunoblot for subsequent analysis of CSF Aβ peptide patterns.
In the present study, we identified the aminoterminally truncated Aβ peptides 2-40 and 2-42 as well as oxidized forms of Aβ1-38 and Aβ1-42 in CSF. Our protocol allowed the quantification of a pattern of Aβ peptides 1-38(ox), 2-40, and 2-42 in addition to the well known panel of Aβ 1-37, 1-38, 1-39, 1-40, 1-40(ox), and 1-42 in a group of seven patients with peripheral polyneuropathy.
In the present approach, we could broaden the range of quantifiable Aβ peptides described in previous studies (i.e., 1-37, 1-38, 1-39, 1-40, 1-40(ox), and 1-42) by Aβ 1-38(ox), 2-40, and 2-42. An exact analysis of CSF Aβ peptides regarding their carboxy- and aminoterminus as well as posttranslational modification seems promising with respect to diagnostic and pathogenic aspects.
[Show abstract][Hide abstract] ABSTRACT: Resistance against ss-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and cost efficient determination of ss-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. ss-lactam resistance is mainly based on the expression/overexpression of ss-lactamases destroying the central ss-lactam ring of these drugs by hydrolysis. Hydrolysis corresponds to a mass shift of plus 18 Da which can be easily detected by MALDI-TOF mass spectrometry. Therefore, a MALDI-TOF MS based assay was set up to investigate different enterobacteria for resistance against different ss-lactam antibiotics: Ampicillin, Piperacillin, Cefotaxime, Ceftazidime, Ertapenem, Imipenem and Meropenem. ss-lactamases are enzymes comprising a high turn over rate. Therefore, hydrolysis can already be detected by MALDI-TOF MS after a few hours of incubation of the bacteria to be tested with the respective antibiotic. The comparison of the MS-derived data with the dat
[Show abstract][Hide abstract] ABSTRACT: Resistance against β-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and cost-efficient determination of β-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. β-Lactam resistance is based mainly on the expression/overexpression of β-lactamases, which destroy the central β-lactam ring of these drugs by hydrolysis. Hydrolysis corresponds to a mass shift of +18 Da, which can be easily detected by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Therefore, a MALDI-TOF MS-based assay was set up to investigate different enterobacteria for resistance against different β-lactam antibiotics: ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem, and meropenem. β-Lactamases are enzymes that have a high turnover rate. Therefore, hydrolysis can be detected by MALDI-TOF MS already after a few hours of incubation of the bacteria to be tested with the given antibiotic. The comparison of the MS-derived data with the data from the routine procedure revealed identical classification of the bacteria according to sensitivity and resistance. The MALDI-TOF MS-based assay delivers the results on the same day. The approved routine procedures require at least an additional overnight incubation.
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Clinical Microbiology 08/2011; 31(5):767-73. DOI:10.1007/s10096-011-1373-0 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI-TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI-TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI-TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.
[Show abstract][Hide abstract] ABSTRACT: In routine clinical diagnostics, peptide biomarkers are most commonly quantified using immunological techniques but these methods often lack sensitivity and/or specificity. Hence, quantitative mass spectrometry detection is desirable as an alternative diagnostic tool. To date, quantitative mass spectrometry is mostly based on ESI-MS coupled to LC, requiring highly sophisticated instrumentation and knowledge and is time consuming and expensive. In contrast, MALDI-TOF-MS is a very simple, sensitive and rapid method for the detection of peptide biomarkers. However, the infeasibility of absolute quantification has been a tremendous handicap to the use of MS in stable clinical diagnostics. Here, we describe the development of a technical platform based on ClinProt particles and heavy-isotope internal peptide standards for the fast and reliable preparation of samples. This combines the advantages of MALDI-TOF as a read-out system with absolute quantitation of peptide biomarkers. As a proof-of-concept, this platform was successfully employed for the absolute determination of the concentration of the highly abundant serum peptide des-Ala-Fibrinopeptide A in 45 serum samples from healthy donors. Such technology essentially contributes to the development of a stable MALDI-TOF-MS-based clinical assay.
[Show abstract][Hide abstract] ABSTRACT: Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively.
Journal of biomolecular techniques: JBT 10/2007; 18(4):252-8.
[Show abstract][Hide abstract] ABSTRACT: 2,5-Dihydroxybenzoic acid (DHB) is the matrix of choice for carbohydrate and glycopeptide analysis, but due to the inhomogeneous surface morphology of samples prepared with DHB, it is typically incompatible with automated measurements.
[Show abstract][Hide abstract] ABSTRACT: Proteomic analyses typically produce massive amounts of mass spectrometric data, which are analyzed in an automated way by database search engines for retrieval of peptide sequences and subsequent inference on the corresponding protein sequences. However, this process turned out to be error prone, producing false positives and multiple hits for the same proteins for various reasons.
[Show abstract][Hide abstract] ABSTRACT: Concanavalin A, boronic acid and Wheat germ agglutinin functionalized magnetic micro-particles were developed to enrich glycosylated peptides and proteins. The bead functionalities were validated according to their specificity by analyses of model proteins. Validated beads were employed for the enrichment of glycosylated human serum proteins. Eluted glycoproteins were digested by trypsin and the resulting peptides were purified by magnetic MB-HIC C8 beads. Each fraction was analyzed by MALDI-TOF MS and single peaks were subjected to MALDI-TOF/TOF MS with the objective to identify the respective proteins by database search. Search results revealed overlapping profiles of known serum glycoproteins.
Journal of Chromatography B 09/2006; 840(1):29-36. DOI:10.1016/j.jchromb.2006.06.028 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As the differential diagnosis of dementias based on established clinical criteria is often difficult, biomarkers for applicable diagnostic testing are currently under intensive investigation. Amyloid plaques deposited in the brain of patients suffering from Alzheimer's disease, dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD) mainly consist of carboxy-terminally elongated forms of amyloid-beta (Abeta) peptides, such as Abeta1-42. Absolute Abeta1-42 levels in CSF have shown diagnostic value for the diagnosis of Alzheimer's disease, but the discrimination among Alzheimer's disease, DLB and PDD was poor. A recently established quantitative urea-based Abeta-sodium-dodecylsulphate-polyacrylamide-gel-electrophoresis with Western immunoblot (Abeta-SDS-PAGE/immunoblot) revealed a highly conserved Abeta peptide pattern of the carboxy-terminally truncated Abeta peptides 1-37, 1-38, 1-39 in addition to 1-40 and 1-42 in human CSF. We used the Abeta-SDS-PAGE/immunoblot to investigate the CSF of 23 patients with Alzheimer's disease, 21 with DLB, 21 with PDD and 23 non-demented disease controls (NDC) for disease-specific alterations of the Abeta peptide patterns in its absolute and relative quantities. The diagnostic groups were matched for age and severity of dementia. The present study is the first attempt to evaluate the meaning of Abeta peptide patterns in CSF for differential diagnosis of the three neurodegenerative diseases--Alzheimer's disease, DLB and PDD. The Abeta peptide patterns displayed disease-specific variations and the ratio of the differentially altered Abeta1-42 to the Abeta1-37 levels subsequently discriminated all diagnostic groups from each other at a highly significant level, except DLB from PDD. Additionally, a novel peptide with Abeta-like immunoreactivity was observed constantly in the CSF of all 88 investigated patients. The pronounced percentage increase of this peptide in DLB allowed a highly significant discrimination from PDD. Using a cut-off point of 0.954%, this marker yielded a diagnostic sensitivity and specificity of 81 and 71%, respectively. From several lines of indication, we consider this peptide to represent an oxidized alpha-helical form of Abeta1-40 (Abeta1-40*). The increased abundance of Abeta1-40* probably reflects a disease-specific alteration of the Abeta1-40 metabolism in DLB. We conclude that Abeta peptide patterns reflect disease-specific pathophysiological pathways of different dementia syndromes as distinct neurochemical phenotypes. Although Abeta peptide patterns failed to fulfil the requirements for a sole biomarker, their combined evaluation with other biomarkers is promising in neurochemical dementia diagnosis. It is noteworthy that DLB and PDD exhibit distinct clinical temporal courses, despite their similar neuropathological appearance. Their distinct molecular phenotypes support the view of different pathophysiological pathways for each of these neurodegenerative diseases.
[Show abstract][Hide abstract] ABSTRACT: 2,5-Dihydroxyacetophenone (DHAP) is presented as a matrix which enables highly sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides, proteins and glycoproteins on AnchorChip targets. Depending on the protein, lower fmol amounts can be detected due to the increased homogeneity and concentration of the crystallization of the analyte/matrix mixture on the anchors. Best results could be generated in the mass range of 8-100 kDa. All sample/matrix preparation steps starting from mixing of DHAP matrix solution with sample solution to the transfer of the mixture to the MALDI-TOF target can be performed manually or automatically allowing low- and high-throughput analyses.
Rapid Communications in Mass Spectrometry 03/2006; 20(5):785-9. DOI:10.1002/rcm.2378 · 2.64 Impact Factor