The rapid identification of existing and emerging respiratory viruses is crucial in combating outbreaks and epidemics. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and reliable identification method in bacterial diagnostics, but has not been used in virological diagnostics. Mass spectrometry systems have been investigated for the identification of respiratory viruses. However, sample preparation methods were laborious and time-consuming. In this study, a reliable and rapid sample preparation method was developed allowing identification of cultured respiratory viruses. Tenfold serial dilutions of ten cultures influenza A strains, mixed samples of influenza A virus with human metapneumovirus or respiratory syncytial virus, and reconstituted clinical samples were treated with the developed sample preparation method. Subsequently, peptides were subjected to MALDI-TOF MS and liquid chromatography tandem mass spectrometry (LC-MS/MS). The influenza A strains were identified to the subtype level within 3h with MALDI-TOF MS and 6h with LC-MS/MS, excluding the culturing time. The sensitivity of LC-MS/MS was higher compared to MALDI-TOF MS. In addition, LC-MS/MS was able to discriminate between two viruses in mixed samples and was able to identify virus from reconstituted clinical samples. The development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry.
c Shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to detect -lactamases in clinical Acinetobacter baumannii isolates. The correlation of the detection of -lactamase proteins (rather than PCR detection of the corresponding genes) with the resistance phenotypes demonstrated an added value for LC-MS/MS in antimicrobial susceptibility testing. Rapid detection and identification of -lactamase-related resistance are complicated by the increasing variety of -lactamases (with differences in substrate specificity), as well as the lack of information on the expression of particular -lactamases for DNA-based detection. Recently, methods based on mass spectrometry, either to identify resistance-conferring proteins (1, 2) or to assay their activity (3, 4), have been developed. In this study, liquid chromatographytandem mass spectrometry (LC-MS/MS) was employed to detect and to identify oxacillinases and other -lactamases in clinical isolates of the nosocomial pathogen Acinetobacter baumannii, in order to evaluate its potential as a rapid and generic method for the detection of -lactamase-related resistance. Emerging multidrug resistance in A. baumannii, resulting from innate resistance to multiple classes of antimicrobials along with a large capacity for acquiring resistance, is an increasing concern in hospitals; the recent rapid development and spread of resistance against carbapenems are of particular concern (5, 6). Resistance is usually caused by the activity of intrinsic or acquired carbapenem-hydrolyzing class D -lactamases, also known as oxacillinases. Although oxacillinases are considered weak carbapenem hydrolyzers, strains become resistant when the genes are strongly expressed (7). Similarly, A. baumannii resistance against ceftazidime resulting from overexpression of Acinetobacter-derived cephalosporinase (ADC), a chromosomally encoded, AmpC-type -lactamase in A. baumannii, has been reported (8). Elevated expression of -lactamase genes in A. baumannii is often associated with the presence of an insertion element (IS) (in particular, ISAba1) upstream of the -lactamase gene, providing strong promoter elements (9). The LC-MS/MS results in this study were compared with the results of susceptibility tests, PCR tests for the presence of different -lactamase genes and insertion elements, and sequencing of detected -lactamase genes.A total of 29 A. baumannii isolates from blood and wound infections, collected at the San Antonio Military Medical Center (San Antonio, TX) between 2006 and 2008, were studied. Pulsedfield gel electrophoresis (PFGE) analysis revealed that these clustered into 15 different PFGE types, with a maximum of 5 isolates belonging to a PFGE type (see Fig. S1 in the supplemental material) (10, 11). Resistance to the carbapenems imipenem and meropenem and the third-generation cephalosporin ceftazidime was determined by broth microdilution testing (12) ( Table 1). Fifteen isolates were resistant to both carbapenems, 11 were susceptible, and three...
BackgroundCholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks.ResultsThe use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay.ConclusionsThe extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved.
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