Third-generation cephalosporin (3GC) resistance and its association with Extra-intestinal pathogenic Escherichia coli (ExPEC). Focus on broiler carcasses.

The worldwide spread of Extra-intestinal Pathogenic Escherichia coli (ExPEC), together with the antimicrobial resistance linked with extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpCs) are pressing threats for public health. This study aimed to investigate the presence of ExPEC genes in third-generation cephalosporin (3 GC)-resistant E. coli and to study their distribution in broiler carcasses at the slaughterhouse after the chilling process. To this purpose, isolates from a collection of 3 GC-resistant E. coli from carcasses of broilers originating from twelve broiler farms and three production chains were investigated. Several multivariate statistical approaches were adopted to elucidate the relationships among features. Phylogroup F was predominant in all broiler batches and was mainly associated with blaTEM and ESBL genes but less correlated to ExPEC genes. Another remarkable finding was the predominance of ExPEC strains assigned to uncommon phylogroups, such as B2, D, E and Clade I, commonly found into the environment. This study represents a first step for a comprehensive characterization of ExPEC genes harboured by 3 GC-resistant E. coli. These findings may be valuable for the identification of potential risks associated to broiler carcasses as source of uncommon E. coli phylogroups.

Employment of Phenolic Compounds from Olive Vegetation Water in Broiler Chickens: Effects on Gut Microbiota and on the Shelf Life of Breast Fillets.

Olive vegetation water (OVW) is a by-product with a noticeable environmental impact; however, its polyphenols may be reused food and feed manufacture as high-value ingredients with antioxidant/antimicrobial activities. The effect of dietary supplementation with OVW polyphenols on the gut microbiota, carcass and breast quality, shelf life, and lipid oxidation in broiler chickens has been studied. Chicks were fed diets supplemented with crude phenolic concentrate (CPC) obtained from OVW (220 and 440 mg/kg phenols equivalent) until reaching commercial size. Cloacal microbial community (rRNA16S sequencing) was monitored during the growth period. Breasts were submitted to culture-dependent and -independent microbiological analyses during their shelf-life. Composition, fatty acid concentration, and lipid oxidation of raw and cooked thawed breasts were measured. Growth performance and gut microbiota were only slightly affected by the dietary treatments, while animal age influenced the cloacal microbiota. The supplementation was found to reduce the shelf life of breasts due to the growth of spoilers. Chemical composition and lipid oxidation were not affected. The hydroxytyrosol (HT) concentration varied from 178.6 to 292.4 ug/kg in breast muscle at the beginning of the shelf-life period. The identification of HT in meat demonstrates that the absorption and metabolism of these compounds was occurring efficiently in the chickens.

Depuration processes affect the Vibrio community in the microbiota of the Manila clam, Ruditapes philippinarum.

As filter-feeders, bivalve molluscs accumulate Vibrio into edible tissues. Consequently, an accurate assessment of depuration procedures and the characterization of the persistent Vibrio community in depurated shellfish represent a key issue to guarantee food safety in shellfish products. The present study investigated changes in the natural Vibrio community composition of the Ruditapes philippinarum microbiota with specific focus on human pathogenic species. For this purpose, the study proposed a MLSA-NGS approach (rRNA 16S, recA and pyrH) for the detection and identification of Vibrio species. Clam microbiota were analysed before and after depuration procedures performed in four depuration plants, using culture-dependent and independent approaches. Microbiological counts and NGS data revealed differences in terms of both contamination load and Vibrio community between depuration plants. The novel MLSA-NGS approach allowed for a clear definition of the Vibrio species specific to each depuration plant. Specifically, depurated clam microbiota showed presence of human pathogenic species. Ozone treatments and the density of clams in the depuration tank probably influenced the level of contamination and the Vibrio community composition. The composition of Vibrio community specific to each plant should be carefully evaluated during the risk assessment to guarantee a food-safe shellfish-product for the consumer.

Application of near-infrared spectroscopy for frozen-thawed characterization of cuttlefish (Sepia officinalis).

Few works to date have reported the application of near-infrared spectroscopy (NIRS) for the characterization and authentication of cephalopods. This study investigated the feasibility of a portable NIRS instrument for the non-destructive freshness evaluation of fresh (F) and frozen-thawed (FT) cuttlefish (Sepia officinalis). Samples were examined by chemical, microbiological and sensorial analyses, during 13 days of conservation at 3 °C. The spectral data were collected on lateral mantle of cuttlefish, and different partial least squares discriminant analyses (PLS-DA) were applied for classification purposes. The interpretation of spectra was also investigated by applying the specific water coordinates, using aquaphotomics. Few significant differences in the wet chemistry and microbiological data were detected between F and FT during storage. The quality index method and microbiological analyses suggested similar behavior between F and FT samples until to near 9 days of shelf life. PLS-DA models with the spectral range 900-1650 nm achieved a classification precision of 0.91 between F and FT, while the performances for the prediction of storage days were less effective. The results of aquaphotomics plotted in aquagrams were suitable for the interpretation of the main physicochemical changes of cuttlefish throughout the shelf life. The water coordinates suggested a different molecular conformation of water species in the FT than F samples, with more free water molecules and a lower amount of bound species and the water solvation shell, respectively.

Versatile nano-platform for tailored immuno-magnetic carriers.

Custom immuno-magnetic devices are desirable tools for biomedical and biotechnological applications. Herein, surface active maghemite nanoparticles (SAMNs) are proposed as a versatile platform for developing tailored immuno-magnetic nano-carriers by simple wet reactions. Two examples for conjugating native and biotinylated antibodies were presented along with their successful applications in the recognition of specific foodborne pathogens. Nanoparticles were functionalized with rhodamine B isothiocyanate (RITC), leading to a fluorescent nano-conjugate, and used for binding anti-Campylobacter fetus antibodies (SAMN@RITC@Anti-Cf). The microorganism was selectively captured in the presence of two other Campylobacter species (C. jejuni and C. coli), as verified by PCR. Alternatively, SAMNs were modified with avidin, forming a biotin-specific magnetic nano-carrier and used for the immobilization of biotinylated anti-Listeria monocytogenes antibodies (SAMN@avidin@Anti-Lm). This immuno-magnetic carrier was integrated in piezoelectric quartz crystal microbalance (QCM) sensor for the detection of L. monocytogenes in milk, showing a detection limit of 3 bacterial cells. The present work presents a new category of customized immuno-magnetic nano-carriers as a competitive option for suiting specific applications. Graphical abstract ᅟ.

Edible processed insects from e-commerce: Food safety with a focus on the Bacillus cereus group.

Edible insects are increasing in popularity as a trendy new food item in many countries worldwide. However, little information is available regarding the associated foodborne pathogens, in particular, the spore-forming bacteria that may be present in these processed foods. In this study, mealworms, crickets, mole crickets and silkworms that are widely available online were investigated for microbial quality traits. Whereas water activity and pH results revealed a stable product, microbiota characterisation highlighted wide variability among the analysed insect species. Among the microbial targets considered in this study, total viable count, total aerobic spores and Bacillus cereus count were the most suitable for describing the food safety and microbial quality of these products. Endospore-forming bacteria ascribed to the B. cereus group were identified with genetic methods and putative virulence patterns. Three major clusters were delineated according to the phylogenetic analysis of the B. cereus group: B. cereus, B. thuringiensis and B. cytotoxicus, with virulence patterns particular to the enterotoxin genes found in each cluster. This work represents a first step in the hazard identification of B. cereus group bacteria isolated from edible insects. The presented results should also be considered with respect to domestic handling and rehydration of such products prior to consumption.

Enlightening mineral iron sensing in Pseudomonas fluorescens by surface active maghemite nanoparticles: Involvement of the OprF porin.

BACKGROUND: Mineral iron(III) recognition by bacteria is considered a matter of debate. The peculiar surface chemistry of novel naked magnetic nanoparticles, called SAMNs (surface active maghemite nanoparticles) characterized by solvent exposed Fe(3+) sites on their surface, was exploited for studying mineral iron sensing in Pseudomonas fluorescens. METHODS: SAMNs were applied for mimicking Fe(3+) ions in solution, acting as magnetically drivable probes to evaluate putative Fe(3+) recognition sites on the microorganism surface. Culture broths and nano-bio-conjugates were characterized by UV-Vis spectroscopy and mass spectrometry. RESULTS: The whole heritage of a membrane porin (OprF) of P. fluorescens Ps_22 cells was recognized and firmly bound by SAMNs. The binding of nanoparticles to OprF porin was correlated to a drastic inhibition of a siderophore (pyoverdine) biosynthesis and to the stimulation of the production and rate of formation of a secondary siderophore. The analysis of metabolic pathways, based on P. fluorescens Ps_22 genomic information, evidenced that this putative secondary siderophore does not belong to a selection of the most common siderophores. CONCLUSIONS: In the scenario of an adhesion mechanism, it is plausible to consider OprF as the biological component deputed to the mineral iron sensing in P. fluorescens Ps_22, as well as one key of siderophore regulation. GENERAL SIGNIFICANCE: The present work sheds light on mineral iron sensing in microorganisms. Peculiar colloidal naked iron oxide nanoparticles offer a useful approach for probing the adhesion of bacterial surface on mineral iron for the identification of the specific recognition site for this iron uptake regulation in microorganisms.

Molecular Typing of Vibrio parahaemolyticus Strains Isolated from Mollusks in the North Adriatic Sea.

BACKGROUND: Vibrio parahaemolyticus is an emerging foodborne pathogen in the Mediterranean, usually associated with shellfish consumption. The increase in the number of outbreaks in Europe is primarily associated with the global warming of the ocean that has a great impact on the spread and genetic selection of waterborne pathogens. The primary role of Italy in Europe's mollusk production, together with the fact that cases of infections with V. parahaemolyticus are not always notified to the European community, highlighted the necessity of acquiring new information about the epidemiological involvement of shellfish products. OBJECTIVE: The aim of the study was to provide useful insights into the first steps of the Risk Assessment associated with V. parahaemolyticus through the molecular characterization of isolates from commercialized mollusks. MATERIALS AND METHODS: A total of 102 strains identified as V. parahaemolyticus were investigated as part of a larger sampling (1-year survey) from several shellfish species collected from the Venice lagoon and the North Adriatic sea. All strains were characterized by multilocus sequence typing and tested for the presence of virulence genes (trh and tdh). The study of sampling/environmental factors and epidemiological analyses was performed to describe the behaviors of the different genetic populations. RESULTS: The population structure analysis highlighted three genetic clusters that could be subject to temperature selection during cold (≤15°C) and warm (>16°C) seasons. Moreover, other factors, such as molluscan species (clams/mussels), probably played a role in the distribution of genetic clusters. Although few strains carried the virulence factors (n = 6 trh+), epidemiological links with clinical isolates and a local dissemination of some sequence types were underlined. CONCLUSION: This work provides a useful background on the genotype spread as a first step in the Hazard Identification in light of future climate changes.

Aeromonas spp.: ubiquitous or specialized bugs?

The genus Aeromonas comprises ubiquitous bacteria that are known to play several roles in the environment. These bacteria were first described as fish pathogens, but their presence was documented in other reservoirs, such as animals and humans. Today, these bacteria are described as emerging pathogens, but their effective role in human pathogenicity is still controversial. In addition, their taxonomy is heavily debated, as species distinction is often difficult to achieve. To study the interspecies relationships and to investigate their connection with the environment, a multilocus sequence typing scheme previously developed for Aeromonas spp. was applied to 258 strains, and the genetic data were analysed by population software. Sampling was a fundamental step, including several of the main sources of Aeromonas: fish, food products and human cases of disease. The objective was to characterize the isolates and to find potential associations among them according to the following: species, sharing of virulence factors, source and adaptation to a specific habitat. The strains were characterized and demonstrated exceptionally high nucleotide variability in the Aeromonas genus. Among the sampled sources, different species distributions were found, highlighting the occurrence of adaptation processes towards specific habitats.

Vibrio trends in the ecology of the Venice lagoon.

Vibrio is a very diverse genus that is responsible for different human and animal diseases. The accurate identification of Vibrio at the species level is important to assess the risks related to public health and diseases caused by aquatic organisms. The ecology of Vibrio spp., together with their genetic background, represents an important key for species discrimination and evolution. Thus, analyses of population structure and ecology association are necessary for reliable characterization of bacteria and to investigate whether bacterial species are going through adaptation processes. In this study, a population of Vibrionaceae was isolated from shellfish of the Venice lagoon and analyzed in depth to study its structure and distribution in the environment. A multilocus sequence analysis (MLSA) was developed on the basis of four housekeeping genes. Both molecular and biochemical approaches were used for species characterization, and the results were compared to assess the consistency of the two methods. In addition, strain ecology and the association between genetic information and environment were investigated through statistical models. The phylogenetic and population analyses achieved good species clustering, while biochemical identification was demonstrated to be imprecise. In addition, this study provided a fine-scale overview of the distribution of Vibrio spp. in the Venice lagoon, and the results highlighted a preferential association of the species toward specific ecological variables. These findings support the use of MLSA for taxonomic studies and demonstrate the need to consider environmental information to obtain broader and more accurate bacterial characterization.

Interactions between prokaryotic polysaccharides and colloidal magnetic nanoparticles for bacteria removal: A strategy for circumventing antibiotic resistance.

Highly stable, colloidal iron oxide nanoparticles with an oxyhydroxide-like surface were used as bacteria-capturing nano-baits. Peptidoglycan isolated from Listeria spp was used as bacteria polysaccharide model, and the nanoparticle binding was characterized showing a Langmuir isotherm constant, KL, equal to 50 ± 3 mL mg-1. The chemical affinity was further supported by dynamic light scattering, transmission electron microscopy, and infrared and UV-Vis data, pointing at the occurrence of extended, coordinative multiple point bindings. The interaction with Gram (+) (Listeria spp) and Gram (-) (Aeromonas veronii) bacteria was shown to be effective and devoid of any toxic effect. Moreover, a real sample, containing a population of several oligotrophic bacteria strains, was incubated with 1 g L-1 of nanoparticle suspension, in the absence of agitation, showing a 100 % capture efficiency, according to plate count. A nanoparticle regeneration method was developed, despite the known irreversibility of such bacterial-nanosurface binding, restoring the bacteria capture capability. This nanomaterial represents a competitive option to eliminate microbiological contamination in water as an alternative strategy to antibiotics, aimed at reducing microbial resistance dissemination. Finally, beyond their excellent features in terms of colloidal stability, binding performances, and biocompatibility this nanoparticle synthesis is cost effective, scalable, and environmentally sustainable.

Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon.

Lactic acid bacteria (LAB) are widely used in the food industry. Pediococcus spp. belong to the LAB group and include several species that are essential for the quality of fermented food. Pediococcus pentosaceus is the species that is most frequently isolated from fermented food and beverages but its uncontrolled growth during food fermentation processes can contribute to undesired flavours. Hence, the characterisation of these bacteria at the strain level is of great importance for the quality of fermented products. Despite their importance, misidentification at the species level is common for members of the genus Pediococcus. To clarify the taxonomic relationships among strains, a multilocus sequencing approach was developed for the characterisation of a collection of 29 field strains, 1 type strain and 1 reference strain of P. pentosaceus isolated from food. These strains were also tested for several phenotypic properties of technological interest and for the production of bacteriocins. The chromosomal operon involved in the synthesis of the bacteriocin penocin was also investigated. The present study enabled a good genomic characterisation, identifying 17 sequence types, with an overview of phenotypic characteristics related to different technological abilities, and also provides a thorough characterisation of the operon involved in penocin production.

Cuttlefish Species Authentication: Advancing Label Control through Near-Infrared Spectroscopy as Rapid, Eco-Friendly, and Robust Approach.

Accurate species identification, especially in the fishery sector, is critical for ensuring food safety, consumer protection and to prevent economic losses. In this study, a total of 93 individual frozen-thawed cuttlefish samples from four different species (S. officinalis, S. bertheloti, S. aculeata, and Sepiella inermis) were collected from two wholesale fish plants in Chioggia, Italy. Species identification was carried out by inspection through morphological features using dichotomic keys and then through near-infrared spectroscopy (NIRS) measurements. The NIRS data were collected using a handled-portable spectrophotometer, and the spectral range scanned was from 900-1680 nm. The collected spectra were processed using principal component analysis for unsupervised analysis and a support vector machine for supervised analysis to evaluate the species identification capability. The results showed that NIRS classification had a high overall accuracy of 93% in identifying the cuttlefish species. This finding highlights the robustness and effectiveness of spectral analysis as a tool for species identification, even in complex spatial contexts. The findings emphasize the potential of NIRS as a valuable tool in the field of fishery product authentication, offering a rapid and eco-friendly approach to species identification in the post-processing stages.

Characterization of the archaeal community in foods: The neglected part of the food microbiota.

Despite the large number of studies conducted on archaea associated with extreme environments, the archaeal community composition in food products is still poorly known. Here, we investigated a new insight into exploring the archaeal community in several food matrices, with a particular focus on determining whether living archaea were present. A total of 71 samples of milk, cheese and its derived brine, honey, hamburger, clam, and trout were analyzed by high-throughput 16S rRNA sequencing. Archaea were detected in all the samples, ranging from 0.62 % of microbial communities in trout to 37.71 % in brine. Methanogens dominated 47.28 % of the archaeal communities, except for brine, which was dominated by halophilic taxa affiliated with the genus Haloquadratum (52.45 %). Clams were found to be a food with high richness and diversity of archaea and were targeted for culturing living archaea under different incubation time and temperature conditions. A subset of 16 communities derived from culture-dependent and culture-independent communities were assessed. Among the homogenates and living archaeal communities, the predominant taxa were distributed in the genera Nitrosopumilus (47.61 %) and Halorussus (78.78 %), respectively. A comparison of the 28 total taxa obtained by culture-dependent and culture-independent methods enabled their categorization into different groups, including detectable (8 out of 28), cultivable (8 out of 28), and detectable-cultivable (12 out of 28) taxa. Furthermore, using the culture method, the majority (14 out of 20) of living taxa grew at lower temperatures of 22 and 4 °C during long-term incubation, and few taxa (2 out of 20) were found at 37 °C during the initial days of incubation. Our results demonstrated the distribution of archaea in all analyzed food matrices, which opens new perspectives to expand our knowledge on archaea in foods and their beneficial and detrimental effects.

Evaluating Escherichia coli contamination in bivalve mollusks using the impedance method: a comparison with most probable number analyses and correlation with environmental parameters.

The application of an electrochemical (impedance) tool for monitoring Escherichia coli contamination in shellfish was evaluated after 13 months of observation. The primary aim of the present study was to compare the standard most probable number (MPN) and µ-trac 4200 (log imped/100 g) for the assessment of E. coli contamination (log MPN/100 g) in non-depurated bivalve mollusks (BM) from five sampling areas of the Veneto-Emilian coast (Italy) (118 samples). The secondary aim was to evaluate the correlation between E. coli concentrations in BM and environmental factors on a large data set (690). The methods showed a moderate, positive correlation (0.60 and 0.69 Pearson and Spearman coefficients, respectively; P<0.01) in Ruditapes philippinarum. The McNemar test indicated analogous sample classification between methods, and the impedance method overestimated the most contaminated class (P=0.03; >4,600 MPN/100 g). The results highlighted the suitability of the impedance method for a faster evaluation and routine use especially in clams, while in Mytilus it seemed less effective. Different models built by multivariate permutational variance analysis and multinomial logistic regression selected the suitable environmental features able to predict the E. coli load. Overall, salinity and season affected the E. coli contamination, whereas locally it was mainly influenced by hydrometry and salinity. The application of the impedance method coupled with environmental data analysis could help purification phase management to adhere to legal limits and could represent an advantage for local control authorities to define actions, considering extreme meteorological events' effects as a proactive reaction to climate change.

Retrospective analysis of Vibrio spp. isolated from marketed crustaceans using multilocus sequence analysis.

The genus Vibrio includes bacteria with different morphological and metabolic characteristics responsible for different human and animal diseases. An accurate identification is essential to assess the risks in regard to aquatic organisms and consequently to public health. The Multilocus Sequence Analysis (MLSA) scheme developed on the basis of 4 housekeeping genes (gyrB, pyrH, recA and atpA) was applied to identify 92 Vibrio strains isolated from crustaceans in 2011. Concatenated sequences were used for the phylogenetic and population analyses and the results were compared with those from biochemical identification tests. From the phylogenetic analysis, 10 clusters and 4 singletons emerged, whereas the population analysis highlighted 12 subpopulations that were well supported by phylogeny with few exceptions. The retrospective analysis allowed correct re-attribution of isolated species, indicating how, for some pathogens, there may be an overestimation of phenotypic identification (e.g. V. parahaemolyticus). Use of the PubMLST Vibrio database highlighted a possible genetic link between Sequence Type (ST) 529 and ST195 (V. alginolyticus) isolated from a human case in Norway during 2018. In addition to the identification of major risk groups of V. cholerae, V. vulnificus and V. parahaemolyticus, MLSA could be a valid support for species considered a minor risk, such as V. alginolyticus, V. mimicus and V. fluvialis. Due to the increased incidence of vibriosis in Europe, the application of different tools will also have to be considered to investigate the possible epidemiological links of the various species in the perspective of Open Science to protect the consumer.

Quality Evaluation of Fair-Trade Cocoa Beans from Different Origins Using Portable Near-Infrared Spectroscopy (NIRS).

Determining cocoa bean quality is crucial for many players in the international supply chain. However, actual methods rely on a cut test protocol, which is limited by its subjective nature, or on time-consuming, expensive and destructive wet-chemistry laboratory procedures. In this context, the application of near infrared (NIR) spectroscopy, particularly with the recent developments of portable NIR spectrometers, may represent a valuable solution for providing a cocoa beans' quality profile, in a rapid, non-destructive, and reliable way. Monitored parameters in this work were dry matter (DM), ash, shell, fat, protein, total polyphenols, fermentation index (FI), titratable acidity (TA) and pH. Different chemometric analyses were performed on the spectral data and calibration models were developed using modified partial least squares regression. Prediction equations were validated using a fivefold cross-validation and a comparison between the different prediction performances for the portable and benchtop NIR spectrometers was provided. The NIRS benchtop instrument provided better performance of quantification considering the whole than the portable device, showing excellent prediction capability in protein and DM quantification. On the other hand, the NIRS portable device, although showing lower but valuable performance of prediction, can represent an appealing alternative to benchtop instruments for food business operators, being applicable in the field.

Determination of microbial diversity of Aeromonas strains on the basis of multilocus sequence typing, phenotype, and presence of putative virulence genes.

The genus Aeromonas has been described as comprising several species associated with the aquatic environment, which represents their principal reservoir. Aeromonas spp. are commonly isolated from diseased and healthy fish, but the involvement of such bacteria in human infection and gastroenteritis has frequently been reported. The primary challenge in establishing an unequivocal link between the Aeromonas genus and pathogenesis in humans is the extremely complicated taxonomy. With the aim of clarifying taxonomic relationships among the strains and phenotypes, a multilocus sequencing approach was developed and applied to characterize 23 type and reference strains of Aeromonas spp. and a collection of 77 field strains isolated from fish, crustaceans, and mollusks. All strains were also screened for putative determinants of virulence by PCR (ast, ahh1, act, asa1, eno, ascV, and aexT) and the production of acylated homoserine lactones (AHLs). In addition, the phenotypic fingerprinting obtained from 29 biochemical tests was submitted to the nonparametric combination (NPC) test methodology to define the statistical differences among the identified genetic clusters. Multilocus sequence typing (MLST) achieved precise strain genotyping, and the phylogenetic analysis of concatenated sequences delineated the relationship among the taxa belonging to the genus Aeromonas, providing a powerful tool for outbreak traceability, host range diffusion, and ecological studies. The NPC test showed the feasibility of phenotypic differentiation among the majority of the MLST clusters by using a selection of tests or the entire biochemical fingerprinting. A Web-based MLST sequence database (http://pubmlst.org/aeromonas) specific for the Aeromonas genus was developed and implemented with all the results.

Fast and Green Method to Control Frauds of Geographical Origin in Traded Cuttlefish Using a Portable Infrared Reflective Instrument.

An appropriate seafood origin identification is essential for labelling regulation but also economic and ecological issues. Near infrared (NIRS) reflectance spectroscopy was employed to assess the origins of cuttlefish caught from five fishing FAO areas (Adriatic Sea, northeastern and eastern central Atlantic Oceans, and eastern Indian and western central Pacific Oceans). A total of 727 cuttlefishes of the family Sepiidae (Sepia officinalis and Sepiella inermis) were collected with a portable spectrophotometer (902-1680 nm) in a wholesale fish plant. NIR spectra were treated with standard normal variate, detrending, smoothing, and second derivative before performing chemometric approaches. The random forest feature selection procedure was executed to select the most significative wavelengths. The geographical origin classification models were constructed on the most informative bands, applying support vector machine (SVM) and K nearest neighbors algorithms (KNN). The SVM showed the best performance of geographical classification through the hold-out validation according to the overall accuracy (0.92), balanced accuracy (from 0.83 to 1.00), sensitivity (from 0.67 to 1.00), and specificity (from 0.88 to 1.00). Thus, being one of the first studies on cuttlefish traceability using NIRS, the results suggest that this represents a rapid, green, and non-destructive method to support on-site, practical inspection to authenticate geographical origin and to contrast fraudulent activities of cuttlefish mislabeled as local.

Combining Culture-Dependent and Culture-Independent Methods: New Methodology Insight on the Vibrio Community of Ruditapes philippinarum.

Vibrios represent a natural contaminant of seafood products. V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus are the most hazardous species to human health. Given the worldwide consumption of mollusc products, reliable detection of Vibrio species is recommended to prevent human vibriosis. In this study, culture-dependent and -independent methods were compared and integrated to implement knowledge of the Manila clam Vibrio community composition. Here, 16S and recA-pyrH metabarcoding were applied to compare the microbial communities of homogenate clam samples (culture-independent method) and their culture-derived samples plated on three different media (culture-dependent method). In addition, a subset of plated clam samples was investigated using shotgun metagenomics. Homogenate metabarcoding characterized the most abundant taxa (16S) and Vibrio species (recA-pyrH). Culture-dependent metabarcoding detected the cultivable taxa, including rare species. Moreover, marine agar medium was found to be a useful substrate for the recovery of several Vibrio species, including the main human pathogenic ones. The culture-dependent shotgun metagenomics detected all the main human pathogenic Vibrio species and a higher number of vibrios with respect to the recA-pyrH metabarcoding. The study revealed that integration of culture-dependent and culture-independent methods might be a valid approach for the characterization of Vibrio biodiversity.