Nonculture‐based identification of bacteria in milk by protein fingerprinting

Abstract

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI Sepsityper(TM) Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3) -10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (10(4) cfu mL(-1) ), bacterial identification could be performed after initial incubation at 37°C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products.

Evaluation of Intact Cell Matrix-Assisted Laser Desorption/Ionization Timeof- Flight Mass Spectrometry for Capillary Electrophoresis Detection of Controlled Bacterial Clumping

“…It has been shown that one loopful of bacterial cells per spot, or 1 mL of suspended solution (e.g., E. coli, E. faecalis), is sufficient for registering good quality spectra [1,8]. In the case of IC MALDI analysis of bacteria cell taken directly from a biological sample (blood, milk) the minimal value is 10 3 -10 5 cells per μL [8,9].…”

“…In addition, one of the advantages of IC MALDI method is the fact that only one (single) colony is needed for microbiological analysis [1,8]. Moreover, some released protocols enable direct IC MALDI analysis of bacteria cells from infected blood [8] or milk [9]. This procedure is based on analysis of the unique protein profile of a microorganism, known as molecular fingerprint (MF) [1,9].…”

“…The matrix serves as a medium for extraction and crystallization of proteins (and other components such as lipopolysaccharides), mainly bacterial ribosomal proteins, which are present in the largest amount in microorganism cells [9,10]. During evaporation of the solvent the extracted bacterial proteins undergo co-crystallization with the matrix and thus formed crystals are subsequently subjected to pulsed laser beam irradiation.…”

“…Moreover, some released protocols enable direct IC MALDI analysis of bacteria cells from infected blood [8] or milk [9]. This procedure is based on analysis of the unique protein profile of a microorganism, known as molecular fingerprint (MF) [1,9]. In this method, a pure culture of a microorganism is applied as a thin layer on a MALDI target.…”

Applicability of 2D gel electrophoresis and liquid chromatography in proteomic analysis of urine using mass spectrometry MALDI-TOF

“…For example, in the last few years comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of bacterial intramammary infection. The results of these researches provide the important information for therapy of these conditions (Addis et al 2011, Kim et al 2011, Barreiro et al 2012, Chiaradia et al 2013.…”