RESUMEN
E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli.
Asunto(s)
Técnicas Bacteriológicas/métodos , Escherichia coli/química , Escherichia coli/clasificación , Shigella/química , Shigella/clasificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Tipificación Molecular , Shigella/genética , Factores de TiempoRESUMEN
Shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to detect ß-lactamases in clinical Acinetobacter baumannii isolates. The correlation of the detection of ß-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.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/enzimología , Carbapenémicos/farmacología , Ceftazidima/farmacología , Farmacorresistencia Bacteriana Múltiple , beta-Lactamasas/análisis , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/química , Antibacterianos/farmacología , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Técnicas de Tipificación Bacteriana , Cromatografía Liquida , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mapeo Peptídico/métodos , Proteómica/métodos , Espectrometría de Masas en Tándem , beta-Lactamasas/químicaRESUMEN
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.
Asunto(s)
Espectrometría de Masas/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Manejo de Especímenes/métodos , Virosis/diagnóstico , Virus/clasificación , Virus/aislamiento & purificación , Humanos , Virus de la Influenza A , Gripe Humana , Metapneumovirus , Virus Sincitiales Respiratorios , Infecciones del Sistema Respiratorio/virología , Sensibilidad y Especificidad , Factores de Tiempo , Virosis/virología , Virus/químicaRESUMEN
BACKGROUND: Cholera 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. RESULTS: The 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. CONCLUSIONS: The 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.