RESUMO
The limits of intact cell-mass spectrometry (ICM-MS) were tested with regard to the minimum number of bacterial cells detectable and its power to discriminate mixed-bacterial cultures. The technique is a surface analysis tool, as is supported by evidence showing that mass fingerprints correspond to material desorbed directly from the cell wall. The brief exposure to solvents, which occurs during sample preparation, does not extract internal cellular material. Spectra were collected over the m/z range of 500 to 10,000. The UV absorbing matrices used were found to be highly specific to bacterial gram type: alpha-cyano-4-hydroxycinnamic acid for gram-negative bacteria and 5-chloro-2-mercaptobenzothiazole for gram-positive bacteria. This specificity allows mixed cultures of different gram types to be differentiated by ICM-MS. The minimum number of cells that could reliably give spectra of sufficient data was 10(4) cells (10(7) cells/mL).
Assuntos
Bactérias/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Biomarcadores , Parede Celular/química , Parede Celular/genética , Ácidos Cumáricos/química , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química , Indicadores e Reagentes , Espectrometria de Massas , Fenótipo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , Tiazóis/químicaRESUMO
Protocols for the identification of bacterial cells by intact cell matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (ICM-TOFMS) are presented. A mass range of 500 to 10,000 m/z is used. The use of formic acid and the crown ether 1, 4, 7, 10, 13, 16-hexaoxacyclooctadecane (18-crown-6) is described. Crown ether is useful for removing metal ion adducts, which degrade spectral purity, and formic acid promotes positive ions, improves spectral signal, and, hence, increases identification certainty.
Assuntos
Bactérias/química , Bactérias/classificação , Éteres de Coroa , Enterococcus/química , Éteres Cíclicos/química , Formiatos/química , Espectrometria de Massas , Metais/química , Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , UltrassomRESUMO
Rapid, accurate discrimination between methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving intact cell mass spectrometry (ICMS) is presented that shows promise for identification, discrimination of MSSA from MRSA and typing. In ICMS, cells from a bacterial colony are emulsified in a chemical matrix, added to a sample slide, dried and analysed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). This technique examines the chemistry of the intact bacterial cell surface, yielding spectra consisting of a series of peaks from 500 to 10000, which represent the mass:charge (m:z) ratios. Each peak corresponds to a molecular fragment released from the cell surface during laser desorption. Specimens can be prepared in a few seconds from plate cultures and a spectrum can be obtained within 2 min. ICMS spectra for 20 staphylococcal isolates showed characteristic peaks, some of which were conserved at species level, some at strain level and some were characteristic of the methicillin susceptibility status of the strain. ICMS may have potential for MRSA identification and typing, and may improve infection control measures.
