Nonculture-based identification of bacteria in milk by protein fingerprinting

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.     

Classification and identification of bacteria by mass spectrometry and computational analysis

Background

In general, the definite determination of bacterial species is a tedious process and requires extensive manual labour. Novel technologies for bacterial detection and analysis can therefore help microbiologists in minimising their efforts in developing a number of microbiological applications.

Methodology

We present a robust, standardized procedure for automated bacterial analysis that is based on the detection of patterns of protein masses by MALDI mass spectrometry. We particularly applied the approach for classifying and identifying strains in species of the genus Erwinia. Many species of this genus are associated with disastrous plant diseases such as fire blight. Using our experimental procedure, we created a general bacterial mass spectra database that currently contains 2800 entries of bacteria of different genera. This database will be steadily expanded. To support users with a feasible analytical method, we developed and tested comprehensive software tools that are demonstrated herein. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used genotyping of single nucleotide polymorphisms by mass spectrometry to unambiguously determine closely related strains that are difficult to distinguish by only relying on protein mass pattern detection.

Conclusions

With the method for bacterial analysis, we could identify fire blight pathogens from a variety of biological sources. The method can be used for a number of additional bacterial genera. Moreover, the mass spectrometry approach presented allows the integration of data from different biological levels such as the genome and the proteome.     

Selective Isolation of Glycoproteins and Glycopeptides for MALDI-TOF MS Detection Supported by Magnetic Particles

Glycosylation is the most common form of posttranslational modification of proteins (50–80%). The isolation, discovery, and subsequent identification of glycosylated peptides and proteins is becoming more and more important in glycoproteomics and diagnosis. MALDI-TOF mass spectrometry is an ideal technique for identifying peptides and proteins and their corresponding modifications. The enrichment of glycosylated peptides and proteins from different sources can be attained by affinity chromatography supported by functionalized magnetic particles. Covalent coating of magnetic beads with Concanavalin A (ConA) and diboronic acid was performed by carbodiimide and poly-glutaraldehyde methods, respectively. The functionalized beads were employed to establish and optimize protocols for the binding and detection of glycosylated peptides and proteins with respect to an automated workflow and the subsequent detection and identification by MALDI-TOF mass spectrometry. For several model proteins, the capture and identification could be demonstrated by SDS-PAGE and MALDI-TOF mass spectrometry. According to the type of glycosylation (high man-nose, hybrid, or complex type) the different proteins were enriched by ConA or boronic acid–functionalized beads.     

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.     

Antioxidative activity of flavonoids in stimulated human neutrophils

The release and production of oxidative products generated by the respiratory burst under the influence of natural flavonoids: quercetin, kaempferol and isorhamnetin derivatives have been studied in the polymorphonuclear neutrophils (PMNs) from healthy human donors. Flavonoids were tested in vitro at concentration range 1-100 microM. The antioxidative potential of flavonoids was compared to the activity of a food preservative, butylated hydroxyanisole. Two methods were applied to the measurement of the PMNs respiratory burst: flow cytometry using dichlorofluorescein diacetate and luminol-dependent chemiluminescence. It was found that the studied products decreased the neutrophil hydrogen peroxide production in concentration-dependent manner. The highest degree of inhibition was registered for concentration of 100 microM, although in the chemiluminescence method the metabolic activity inhibition was more prominent. Antioxidative activity of flavonoids depended on the number of hydroxyl groups. These results provide useful data for establishing methods used to assess the respiratory burst in phagocytic cells.     

Dopaminergic denervation enhances susceptibility to hydroxyl radicals in rat neostriatum

To determine if greater amounts of hydroxyl radical (*OH) are formed by dopamine (DA) denervation and treatment with L-dihydroxyphenylalanine (L-DOPA), the neostriatum was DA denervated (99% reduction in DA content) by 6-hydroxydopamine treatment (134microg icv, desipramine pretreatment) of neonatal rats. At 10 weeks the peripherally restricted dopa decarboxylase inhibitor carbidopa (12.5mg/kg i.p.) was administered 30min before vehicle, L-DOPA (60mg/kg i.p.), or the known generator of reactive oxygen species, 6-hydroxydopa (6-OHDOPA) (60mg/kg i.p.); and this was followed 30min later (and 15 min before termination) by the spin trap, salicylic acid (8micromoles icv). By means of a high performance liquid chromatographic method with electrochemical detection, we found a 4-fold increase in the non-enzymatically formed spin trap product, 2,3-dihydroxybenzoic acid (2,3-DHBA), with neither L-DOPA nor 6-OHDOPA having an effect on 2,3-DHBA content of the neostriatum. Basal content of 2,5-DHBA, the enzymatically formed spin trap product, was 4-fold higher vs. 2,3-DHBA in the neostriatum of untreated rats, while L-DOPA and 6-OHDOPA each reduced formation of 2,5-DHBA. We conclude that DA innervation normally suppresses *OH formation, and that the antiparkinsonian drug L-DOPA has no effect (2,3-DHBA) or slightly reduces (2,5-DHBA) *OH formation in the neostriatum, probably by virtue of its bathing the system of newly formed *OH.     

Fish bioenergetics model used for estimation of food consumption in a navigation canal with heavy traffic

Fish were sampled in two successive years in a canal in north-west England with heavy boat traffic (10000 mh^–1yr^–1). An energy budget was used to calculate the food consumption of the two most dominant species, roach Rutilus rutilus and gudgeon Gobio gobio. For two subdominant (perch, Perca fluviatilisand ruffe, Gymnocephalus cernuus) and four rare species living there, parameters of the energy budget and prey consumption were estimated using ecological indices calculated for both dominants. The food conversion efficiency was low compared with values for the same species in other habitats, probably because of detritus and scarcity of macrobenthos in the diet. This may have been a result of the heavy boat traffic.