title: A method for the detection of antibiotic resistance markers in clinical strains of Escherichia coli using MALDI mass spectrometry.
abstract: Matrix-assisted laser-desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass spectrometry based approaches for bacterial identification and classification. The relatively simple sample preparation requirements and the speed of analysis which can usually be completed within a few minutes have resulted in the adoption and assimilation of MALDI-TOF MS into the routine diagnostic workflow of Clinical microbiology laboratories worldwide. This study describes the facilitation of bacterial discrimination based on antibiotic resistance markers through the implementation of MALDI-TOF MS. The periplasmic compartment of whole bacterial cells contains several proteins which confer antibiotic resistance in the Enterobacteriaceae. In order to reduce the complexity of the sample to be analysed via MALDI-TOF MS, the periplasm was extracted and subjected to in solution tryptic digestion followed by nano-LC separation. This method, established that peptide sequence biomarkers from several classes of antibiotic resistance proteins could be predicted using protein/peptide database tools such as Mascot. Biomarkers for a CTX-M-1 group extended spectrum β-lactamase, CMY-2 an Amp-C β-lactamase, VIM a metallo-β-lactamase, TEM a β-lactamase and KanR an aminoglycoside modifying enzyme were detected. This allowed for discrimination at a species level and at an almost identical strain level where the only difference between strains was the carriage of a modified antibiotic resistance carrying plasmid. This method also was able to detect some of these biomarkers in clinical strains where multiple resistance mechanisms were present.
authors: Shimadzu, Wharfside, Trafford Wharf Road, Manchester M17 1GP, UK.
authors: Philippa J
authors: PJ
authors: Hart
authors: Royal Free Hospital NHS Foundation Trust, UK.
authors: Emmanuel
authors: E
authors: Wey
authors: UCL Centre for Clinical Microbiology, Division of Infection and Immunity, UK.
authors: Timothy D
authors: TD
authors: McHugh
authors: Royal Free Hospital NHS Foundation Trust, UK.
authors: Indran
authors: I
authors: Balakrishnan
authors: Shimadzu, Wharfside, Trafford Wharf Road, Manchester M17 1GP, UK. Electronic address: omar.belgacem@kratos.co.uk.
authors: Omar
authors: O
authors: Belgacem
mesh_terms: D000595:Amino Acid Sequence; D015415:Biomarkers / Q000032:analysis; D030562:Databases, Protein; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000737:chemistry* / Q000145:classification / Q000187:drug effects* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000302:isolation & purification; D010455:Peptides; D019897:Periplasm / Q000737:chemistry; D033903:Periplasmic Proteins / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D001618:beta-Lactamases / Q000302:isolation & purification
chemical_list: D015415:Biomarkers; D029968:Escherichia coli Proteins; D010455:Peptides; D033903:Periplasmic Proteins; C103022:beta-lactamase CMY-2; D001618:beta-Lactamases
keywords: Antibiotic resistance; Bacterial identification; MALDI-TOF MS