The current work investigated the discriminatory potential of MALDI-TOF MS fingerprinting towards most-relevant major (Streptococcus agalactiae, S. dysgalactiae, S. uberis) and minor (S. canis, S. ...parauberis, S. salivarius, S. equinus and S. gallolyticus) streptococci involved in bovine mastitis (BM), in comparison to 16S rRNA gene sequencing (GS)-based identification. The MALDI-TOF MS-generated spectral fingerprints were recruited for eliciting a detailed proteomic map that demonstrated clear variability for inter- and intra-species-specific biomarkers. Besides, a phyloproteomic dendrogram was evolved and comparatively analyzed against the phylogenetic one obtained from 16S rRNA GS in order to assess the differentiation of streptococci of bovine origin based on variability of protein fingerprints versus the variation of 16S rRNA gene homology. Results showed that the discrimination of BM-implicated streptococci can be obtained by both approaches; however MALDI-TOF MS was superior, achieving more variability at both intra- and sub-species levels. MALDI-TOF MS spectral analytics revealed that Streptococcus spp. exhibited three genus-specific biomarkers (peaks with m/z values at 2112, 4452 and 5955) and all streptococci exhibited spectral variability at both species and subspecies levels. Remarkably, MALDI-TOF MS fingerprinting was found to be at least as robust as 16S rRNA GS-based identification, allowing much cheaper and faster analysis, and additionally exhibiting high reliability for characterization of BM-implicated streptococci, thus proving to be a powerful tool that can be used independently within dairy diagnostics.
•Discrimination of BM-implicated streptococci was facilitated by both 16S rRNA GS and by MALDI-TOF MS fingerprinting•MALDI-TOF MS contributed to the discovery of protein biomarkers of streptococci, which is crucial for mastitis control.•MALDI-TOF MS also provided more information at the intra-species level for certain streptococci compared to 16S rRNA GS.•MALDI-TOF MS can be used efficiently and independently for the diagnosis of BM-implicated Streptococcus spp.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The characterization of four novel bacteriocin-producing enterococcal strains, isolated from nonfermented animal foods, was carried out with a view to evaluate their potential application as ...probiotics in raw and processed foodstuffs. 16S rRNA sequencing and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis allowed the identification and intra-specific grouping of Enterococcus faecium strains, which inhibited the growth of four relevant food-borne pathogenic and spoilage species. Enterococcus faecium strains exhibited remarkable probiotic profiles, being able to survive to pH 3·0 and to the presence of bile salts, pancreatin and pepsin. Enterococcus faecium strains evaluated did not exhibit bile salt hydrolase or haemolytic activity, but showed good adhesion properties, also exhibiting sensitivity to clinically relevant antimicrobial agents. In our study, DNA sequencing of the 16S rRNA gene and RAPD-PCR analysis were equally discriminatory for typing E. faecium strains. This study also confirmed the potential tolerance and survival of E. faecium strains isolated from nonfermented animal foods to the gastrointestinal tract. This study represents the first report on potential probiotic E. faecium strains isolated from nonfermented meat and fish. Their moderate heat resistance opens the way to their potential use as probiotics in minimally processed foods subjected to moderate heat processing.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
► A new primer-probe set for the detection and quantification of
Bacillus cereus,
Bacillus licheniformis and
Bacillus subtilis by real-time PCR (Rti-PCR) in a single assay was developed. ► 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. ► 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food ...processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
The resistance rates of intestinal Escherichia coli populations from poultry were determined during treatment and withdrawal period with 3 antimicrobial agents commonly used as therapeutics in ...poultry medicine. A total of 108 chickens were considered: 18 were treated orally with enrofloxacin, 18 with doxycycline, and 18 with sulfonamides, whereas another 18 chickens were maintained as controls for each antimicrobial group. Fecal samples were taken during the treatment and after the withdrawal period, and E. coli were isolated through Fluorocult media plating. A total of 648 E. coli strains (216 per antimicrobial tested) were isolated and identified though biochemical methods. Minimal inhibitory concentrations to the antimicrobials used were also determined using a broth microdilution method. The resistance rates of intestinal E. coli to all of the antimicrobials tested significantly increased during the course of the therapeutic treatment. In addition, significant differences (P = 0.0136) in resistance rates persisted between the intestinal E. coli of the enrofloxacin-treated and control batches until the end of the withdrawal period, but this difference was not observed for the cases of doxycycline or sulfonamides treatments. Antimicrobial use in poultry medicine seems to select for antimicrobial-resistant strains of pathogenic bacterial species such as E. coli. In some cases, the higher frequencies of resistant strains may persist in the avian intestinal tract until the end of the withdrawal period, when it is legal to use these animals for human consumption.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mean counts of Enterobacteriaceae were determined for 30 samples each of organic chicken meat, conventional chicken meat and conventional turkey meat to assess differences in contamination. Two ...strains from each sample were isolated to obtain a total of 180 strains, which were examined for resistance to ampicillin, chloramphenicol, cephalothin, doxycycline, ciprofloxacin, gentamicin, nitrofurantoin, and sulfisoxazole. The mean counts of Enterobacteriaceae from organic chicken meat were significantly higher than those obtained from conventional chicken (
P
<
0.0001) or conventional turkey (
P
<
0.0001) meat. However, the resistance data obtained showed that isolates from organic chicken meat were less resistant than isolates from conventional chicken meat to ampicillin (
P
=
0.0001), chloramphenicol (
P
=
0.0004), doxycycline (
P
=
0.0013), ciprofloxacin (
P
=
0.0034), gentamicin (
P
=
0.0295) and sulfisoxazole (
P
=
0.0442), and were less resistant than isolates from turkey meat to doxycycline (
P
=
0.0014) and sulfisoxazole (
P
=
0.0442). Multidrug resistant isolates were found in every group tested, but rates of multidrug resistant strains were higher in conventional chicken (63.3%) and turkey (56.7%) than organic chicken (41.7%) meat. The rates obtained for antimicrobial resistance support the theory that although organic chicken meat contains more Enterobacteriaceae contamination, organic farming practices contribute to decreased dissemination of antibiotic resistance.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Bacillus genus includes foodborne pathogenic and spoilage-associated species, such as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus. Bacillus is also a heterogeneous ...genus that includes closely related species that are difficult to discriminate among, especially when well-conserved genes such as 16S rRNA and 23S rRNA are considered. The main goal of the present work was to study the usefulness of three housekeeping genes, the TU elongation factor (tuf), the DNA gyrase β subunit (gyrB) and the RNA polymerase β subunit (rpoB) genes, for use in differentiating among the most important foodborne Bacillus spp. sequences from 20 foodborne isolated Bacillus strains, and sequences belonging to different Bacillus spp. retrieved from the GenBank were analysed. In general terms, gyrB, rpoB and tuf gene regions for the strains considered in this study exhibited interspecific similarities of 57.8%, 67.23% and 77.66% respectively. Novel tufGPF and tufGPR universal primers targeted to the tuf gene were designed and proved to be useful for the amplification of all Bacillus spp considered. In conclusion, the tuf gene can be considered to be a good target for the differential characterisation of foodborne Bacillus species, especially for differentiating B. subtilis and B. cereus from other closely related species.
•Bacillus genus contains closely related species difficult to discriminate.•We study the usefulness of tuf, gyrB and rpoB genes.•The tuf gene can be a good target for characterisation of Bacillus spp.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The state-of-the-art and future trends of the application of proteomics to seafood and other marine products are reviewed. Consumers' demands for seafood products have increased in the recent years ...and this situation has underlined the need to guarantee the safety, traceability, authenticity, and health benefits of such products. The increasing presence of commercially available aquaculture products has also prompted the seafood industry to face newer challenges. In this sense, a review of the present status and perspectives of the application of proteomics in the development of newer biotechnology products of marine origin is given. Keywords: proteomics • marine • aquaculture • seafood • authenticity • biotechnology • fish
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IJS, KILJ, NUK, PNG, UL, UM
The Bacillus genus includes species such as Bacillus cereus, Bacillus licheniformis and Bacillus subtilis, some of which may be pathogenic or causative agents in the spoilage of food products. The ...main goal of this work was to apply matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass fingerprinting to the classification of these Bacillus species. Genetic analyses were also compared to phyloproteomic analyses. A collection of 57 Bacillus strains isolated from fresh and processed food and from culture collections were studied and their mass spectra compiled. The resulting mass fingerprints were compared and characteristic peaks at the strain and species levels were assigned. The results showed that MALDI-TOF was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate classification of Bacillus species, especially for differentiating B. subtilis and B. cereus from Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. MALDI-TOF was also found to provide valuable information at both intra- and interspecies levels in the Bacillus species studied.
► MALDI-TOF was compared to genetic analysis for the identification of Bacillus spp. ► MALDI-TOF allowed a better differentiation of Bacillus subtilis from Bacillus amyloliquefaciens. ► MALDI-TOF allowed a better differentiation of Bacillus cereus from Bacillus thuringiensis. ► Genetic analysis of Bacillus spp is complemented with MALDI-TOF fingerprinting.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Streptococcus parauberis is known as an etiological agent of mastitis in cows and for producing streptococcosis in farmed fish, although its presence in foods has seldom been reported. In this work, ...two bacterial isolates were recovered from a spoiled vacuum-packaged refrigerated seafood product. Both isolates were identified by 16S rRNA gene sequencing, exhibiting 99% homology with respect to S. parauberis. Both isolates were also characterized by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Genetic analysis revealed the clonal homogeneity of the isolates and their grouping together with other S. parauberis strains in a different cluster with respect to Streptococcus uberis strains. Proteomic analysis by MALDI-TOF MS allowed for the identification of five mass peaks in the range of 2200–6000 m/z that resulted to be specific to the species S. parauberis and allowed its rapid and direct identification with respect to other pathogenic and spoilage bacteria potentially present in seafood and other food products. This study represents, to our knowledge, the first report of S. parauberis in seafood in general and in vacuum-packed food products in particular. Moreover, it provides a rapid method based on MALDI-TOF MS for the identification of S. parauberis.
► Two strains of Streptococcus parauberis were isolated from vacuum-packed seafood. ► The full characterization of S. parauberis was performed by genomics and proteomics. ► MALDI-TOF MS allowed its rapid detection and identification in seafood products. ► This study represents the first report of S. parauberis in seafood so far.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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