Inmaculada C. Fernández-No

Species Differentiation of Seafood Spoilage and Pathogenic Gram-Negative Bacteria by MALDI-TOF Mass Fingerprinting

Species differentiation is important for the early detection and identification of pathogenic and food-spoilage microorganisms that may be present in fish and seafood products. The main 26 species of seafood spoilage and pathogenic Gram-negative bacteria, including Aeromonas hydrophila, Acinetobacter baumanii, Pseudomonas spp., and Enterobacter spp. among others, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of low molecular weight proteins extracted from intact bacterial cells by a fast procedure. From the acquired spectra, a library of specific mass spectral fingerprints was constructed. To analyze spectral fingerprints, peaks in the mass range of 2000-10 000 Da were considered and representative mass lists of 10-35 peak masses were compiled. At least one unique biomarker peak was observed for each species, and various genus-specific peaks were detected for genera Proteus, Providencia, Pseudomonas, Serratia, Shewanella, and Vibrio. Phyloproteomic relationships based on these data were compared to phylogenetic analysis based on the 16S rRNA gene, and a similar clustering was found. The method was also successfully applied for the identification of three bacterial strains isolated from seafood by comparing the spectral fingerprints with the created library of reference fingerprints. Thus, the proteomic approach demonstrated to be a competent tool for species identification.

Rapid species identification of seafood spoilage and pathogenic Gram-positive bacteria by MALDI-TOF mass fingerprinting

The rapid identification of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Seafood contaminated with pathogenic bacteria is one of the major causes of food intoxications, and the rapid spoilage of seafood products results in high economic losses. In this study, a collection of the main seafood pathogenic and spoilage Gram‐positive bacteria was compiled, including Bacillus spp., Listeria spp., Clostridium spp., Staphylococcus spp. and Carnobacterium spp. The strains, belonging to 20 different species, were obtained from the culture collections and studied by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). A reference library was created, including the spectral fingerprints of 32 reference strains and the extracted peak lists with 10–30 peak masses. Genus‐specific as well as species‐specific peak masses were assigned and could serve as biomarkers for the rapid bacterial identification. Furthermore, the peak mass lists were clustered with the web‐application SPECLUST to show the phyloproteomic relationships among the studied strains. Afterwards, the method was successfully applied to identify six strains isolated from seafood by comparison with the reference library. Additionally, phylogenetic analysis based on the 16S rRNA gene was carried out and contrasted with the proteomic approach. This is the first time MALDI‐TOF MS fingerprinting is applied to Gram‐positive bacterial identification in seafood, being a fast and accurate technique to ensure seafood quality and safety.

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R(2) values of 0.9969 and 0.9958 respectively. Linear correlations between the log(10) input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10(1) CFU/mL to 1.65 × 10(6) CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.

Differential characterization of biogenic amine-producing bacteria involved in food poisoning using MALDI-TOF mass fingerprinting

Histamine poisoning is caused by the consumption of fish and other foods that harbor bacteria possessing histidine decarboxylase activity. With the aim of preventing histamine formation, highly specific mass spectral fingerprints were obtained from the 16 major biogenic amine‐producing enteric and marine bacteria by means of MALDI‐TOF MS analysis. All bacterial strains analyzed exhibited specific spectral fingerprints that enabled its unambiguous differentiation. This technique also identified peaks common to certain bacterial groups. Thus, two protein peaks at m/z 4182±1 and 8363±6 were found to be present in all Enterobacteriaceae species analyzed except for Morganella morganii. Peaks at m/z 3635±1 and 7267±2 were specific to both M. morganii and Proteus spp. Biogenic amine‐forming Proteus spp. exhibited three genus‐specific peaks at m/z 3980, 7960±1 and 9584±2. The genus Photobacterium also showed three genus‐specific peaks at m/z 2980±1, 4275±1 and 6578±1. The two histamine‐producing Gram‐positive bacteria Lactobacillus sp. 30A and Staphylococcus xylosus exhibited a few protein peaks in the 2000–7000 m/z range and could be easily distinguished from biogenic amine‐forming Gram‐negative bacteria. Clustering based on MALDI‐TOF MS also exhibited a good correlation with phylogenetic analysis based on the 16S rRNA gene sequence, validating the ability of the MALDI‐TOF technique to establish relationships between microbial strains and species. The approach described in this study leads the way toward the rapid and specific identification of major biogenic amine‐forming bacteria based on molecular protein markers with a goal to the timely prevention of histamine food poisoning.

Identification and classification of seafood-borne pathogenic and spoilage bacteria: 16S rRNA sequencing versus MALDI-TOF MS fingerprinting

The present study aims to compare two molecular technologies, 16S rRNA sequencing and MALDI‐TOF MS, for bacterial species identification in seafood. With this aim, 70 reference strains from culture collections, including important seafood‐borne pathogenic and spoilage bacterial species, and 50 strains isolated from commercial seafood products, were analysed by both techniques. Genomic analysis only identified the species of 50% of the isolated strains, proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI‐TOF MS fingerprinting identified 76% of the strains at the species level. The mass spectral data were submitted to the SpectraBank database (http://www.spectrabank.org), making this information available to other researchers. Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences. However, the MALDI‐TOF MS analysis demonstrated more discriminating potential that allowed for better classification, especially for the Pseudomonas and Bacillus genera. This is of importance with respect to the varying pathogenic and spoilage character at the intragenus and intraspecies level. In this sense, MALDI‐TOF MS demonstrated to be a competent bacterial typing tool that extends phenotypic and genotypic approaches, allowing a more ample classification of bacterial strains.

SpectraBank: an open access tool for rapid microbial identification by MALDI-TOF MS fingerprinting.

MALDI‐TOF MS has proved to be an accurate, rapid, and cost‐effective technique for microbial identification in which the spectral fingerprint of an unknown strain can be compared to a database of spectra from reference strains. Most of the existing databases are private and often costly to access, and little spectral information is shared among researchers. The objective of the present communication is to introduce the SpectraBank database (http://www.spectrabank.org), which provides open access MALDI‐TOF mass spectra from a variety of microorganisms. This work aims to familiarize readers with the SpectraBank database, from the sample preparation, data collection, and data analysis to how the spectral reference data can be used for microbial species identification. The database currently includes more than 200 MALDI‐TOF MS spectra from more than 70 bacterial species and links to the freely available web‐based application SPECLUST (http://bioinfo.thep.lu.se/speclust.html) to allow comparisons of the obtained peak mass lists and evaluate phyloproteomic relationships. The SpectraBank database is intended to be expanded by the addition of new spectra from microbial strains, obtained in our laboratory and by other researchers.

Characterization of different food‐isolated Enterococcus strains by MALDI‐TOF mass fingerprinting

Enterococcus is a controversial genus due to its great variability; this genus includes pathogenic strains, spoilage strains, and apparently safe strains including some probiotic strains. Previous studies focused on the characterization of strains of Enterococcus spp. involved in nosocomial infections. However, little research has been conducted on Enterococcus strains in foodstuffs. In the present work, 36 strains of different species of Enterococcus have been characterized by means of MALDI‐TOF MS, resulting in highly specific mass spectral fingerprints. Characteristic peak masses common to certain bacterial species of Enterococcus have been identified. Thus, a peak at m/z 4426 ± 1 was assigned as a genus‐specific biomarker. In addition, phyloproteomic relationships based on the mass spectral data were compared to the results of a phylogenetic analysis based on the 16S rRNA gene sequence. A better grouping at the species level was observed in the phyloproteomic tree, especially for the Enterococcus faecium group. Presumably, the assortment of some strains or ecotypes could be related to their ecological niche specialization. The approach described in this study leads the way toward the rapid and specific identification of different strains and species of Enterococcus in food based on molecular protein markers, aiming at the early detection of pathogenic strains and strains implicated in food poisoning or food spoilage.

The Immunology of Mammary Gland of Dairy Ruminants between Healthy and Inflammatory Conditions

The health of dairy animals, particularly the milk-producing mammary glands, is essential to the dairy industry because of the crucial hygienic and economic aspects of ensuring production of high quality milk. Due to its high prevalence, mastitis is considered the most important threat to dairy industry, due to its impacts on animal health and milk production and thus on economic benefits. The MG is protected by several defence mechanisms that prevent microbial penetration and surveillance. However, several factors can attenuate the host immune response (IR), and the possession of various virulence and resistance factors by different mastitis-causing microorganisms greatly limits immune defences and promotes establishment of intramammary infections (IMIs). A comprehensive understanding of MG immunity in both healthy and inflammatory conditions will be an important key to understand the nature of IMIs caused by specific pathogens and greatly contributes to the development of effective control methods and appropriate detection techniques. Consequently, this review aims to provide a detailed overview of antimicrobial defences in the MG under healthy and inflammatory conditions. In this sense, we will focus on pathogen-dependent variations in IRs mounted by the host during IMI and discuss the potential ramifications of these variations.

Recent Patents on Bacteriocins: Food and Biomedical Applications

Most types of bacteria produce bacteriocins, which are proteinaceous extracellular compounds that can inhibit the growth of other undesirable microorganisms. Bacteriocins are receiving increasing attention, due to their many applications, ranging from their initial application in strategies for food preservation to more recent proposed uses in biomedical strategies aimed at fighting certain bacterial infections. Thus, while nisin has a long history of use as a safe additive in certain food products for the purpose of food preservation, certain bacteriocin-producing lactic acid bacteria, which are generally recognised as safe microorganisms, or their extracellular extracts are receiving increased attention as protective cultures or antimicrobial extracts in minimally processed food products. More recently, a number of these bacteriocinproducing cultures have been proposed for use in other applications, such as in probiotics, for the inhibition of biofilms in the food industry, or even as coadjuvants of combined therapeutical strategies along with other antimicrobial agents in biomedical applications. This review aims to provide a brief overview of the most relevant recent patents in this field.

Comparative analysis of protein extraction methods for the identification of seafood-borne pathogenic and spoilage bacteria by MALDI-TOF mass spectrometry

Species differentiation of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been applied to species identification of microorganisms, proving to be a rapid and cost-effective technique and allowing species differentiation due to the highly specific spectral profiles obtained. In this work, bacterial strains from our laboratory intern collection of seafood-borne pathogenic and spoilage species were studied by MALDI-TOF MS. Different sample preparation protocols were applied and compared to each other. Two methods were based on the analysis of whole bacterial cells that were suspended in an organic solvent or applied directly to the sample target. In a different sample preparation technique, cell extracts were obtained from intact bacterial cells by a dissolution/centrifugation step. The protocol applied for the study of cell extracts was shown to be very fast and simple, allowing the standardization of sample preparation. Furthermore, the analysis of cell extracts had several advantages with respect to the analysis of suspensions of whole bacterial cells. Thus, spectral profiles obtained from cell extracts showed less noise and more reproducible peaks as compared to spectra obtained from intact cells. The analysis of cell extracts by MALDI-TOF MS was also applied to create a mass spectral library of the main pathogenic and spoilage bacteria potentially present in seafood and was demonstrated to be a rapid and accurate method for microbial species differentiation, as well as for the classification of unknown strains isolated from seafood.