Gas chromatography-stable isotope ratio mass spectrometry prior solid phase microextraction and gas chromatography-tandem mass spectrometry: development and optimization of analytical methods to analyse garlic (Allium sativum L.) volatile fraction.

Garlic (Allium sativum L.) is not only appreciated for its flavour and taste, but it is also recognized for various health properties. The European Commission, through the attribution of the Protected Designation of Origin (PDO) certification mark, has officially recognized some specific varieties of garlic. To protect not only the commercial value but also the reputation of this appreciated product, effective tools are therefore required. For the first time, a new compound specific isotope analysis method based on carbon stable isotopic ratio measurement of the three major volatile garlic compounds allyl alcohol (AA), diallyl disulphide (DD) and diallyl trisulphide (DT) through head-space solid phase microextraction (HS-SPME) followed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was developed. A within-day standard deviation (Srwithin-day) of 0.3 ‰, 0.4 ‰ and 0.2 ‰ for δ(13C) and a between-day standard deviation (Srbetween-day) of 0.8 ‰, 1.0 ‰ and 0.6 ‰ of AA, DT and DD was estimated. For the first time, the ranges of isotopic variability for the three volatile compounds of red garlic from two neighbouring Italian regions (Abruzzo and Lazio) were defined analysing 30 samples. The same dataset was also considered in analysing the percentage composition of the previously mentioned three volatile compounds through HS-SPME followed by gas chromatography-tandem mass spectrometry (GC-MS/MS). The two analytical approaches were combined in this explorative study, aiming to provide potential parameters to discriminate garlic samples based on their geographical origin.

Cultivation and sequencing-free protocol for Serratia marcescens detection and typing.

Serratia marcescens is an opportunistic pathogen that survives in inhospitable environments causing large outbreaks, particularly in neonatal intensive care units (NICUs). Genomic studies revealed that most S. marcescens nosocomial infections are caused by a specific clone (here "Infectious clone"). Whole genome sequencing (WGS) is the only portable method able to identify this clone, but it requires days to obtain results. We present a cultivation-free hypervariable-locus melting typing (HLMT) protocol for the fast detection and typing of S. marcescens, with 100% detection capability on mixed samples and a limit of detection that can reach the 10 genome copies. The protocol was able to identify the S. marcescens infectious clone with 97% specificity and 96% sensitivity when compared to WGS, yielding typing results portable among laboratories. The protocol is a cost and time saving method for S. marcescens detection and typing for large environmental/clinical surveillance screenings, also in low-middle income countries.

Traceability and authentication in agri-food production: A multivariate approach to the characterization ofthe Italian food excellence elephant garlic (Allium ampeloprasum L.), a vasoactive nutraceutical.

A research platform for food authentication was set up by combining stable isotope ratio analysis, metabolomics by gas and liquid mass-spectrometry and NMR investigations, chemometric analyses for food excellences. This multi-analytical approach was tested on samples of elephant garlic (Allium ampeloprasum L.), a species belonging to the same genus of common garlic (Allium ampeloprasum L.), mainly produced in southern Tuscany-(Allium ampeloprasum). The isotopic composition allowed the product to be geographically characterized. Flavonoids, like (+)-catechin, cinnamic acids, quercetin glycosides were identified. The samples showed also a significant amount of dipeptides, sulphur-containing metabolites and glutathione, the latter of which could be considered a molecular marker of the analyzed elephant garlic. For nutraceutical profiling to reach quality labels, extracts were investigated in specific biological assays, displaying interesting vasorelaxant properties in rat aorta by mediating nitric oxide release from the endothelium and exhibited positive inotropic and negative chronotropic effects in rat perfused heart.

Discriminative power of DNA-based, volatilome, near infrared spectroscopy, elements and stable isotopes methods for the origin authentication of typical Italian mountain cheese using sPLS-DA modeling.

Origin authentication methods are pivotal in counteracting frauds and provide evidence for certification systems. For these reasons, geographical origin authentication methods are used to ensure product origin. This study focused on the origin authentication (i.e. at the producer level) of a typical mountain cheese origin using various approaches, including shotgun metagenomics, volatilome, near infrared spectroscopy, stable isotopes, and elemental analyses. DNA-based analysis revealed that viral communities achieved a higher classification accuracy rate (97.4 ± 2.6 %) than bacterial communities (96.1 ± 4.0 %). Non-starter lactic acid bacteria and phages specific to each origin were identified. Volatile organic compounds exhibited potential clusters according to cheese origin, with a classification accuracy rate of 90.0 ± 11.1 %. Near-infrared spectroscopy showed lower discriminative power for cheese authentication, yielding only a 76.0 ± 31.6 % classification accuracy rate. Model performances were influenced by specific regions of the infrared spectrum, possibly associated with fat content, lipid profile and protein characteristics. Furthermore, we analyzed the elemental composition of mountain Caciotta cheese and identified significant differences in elements related to dairy equipment, macronutrients, and rare earth elements among different origins. The combination of elements and isotopes showed a decrease in authentication performance (97.0 ± 3.1 %) compared to the original element models, which were found to achieve the best classification accuracy rate (99.0 ± 0.01 %). Overall, our findings emphasize the potential of multi-omics techniques in cheese origin authentication and highlight the complexity of factors influencing cheese composition and hence typicity.

High-Density Balsamic Vinegar: Application of Stable Isotope Ratio Analysis to Determine Watering Down.

Balsamic vinegar of Modena (ABM) is a product obtained from concentrated grape must with the addition of wine vinegar. It can be adulterated with the addition of exogenous water. The official method EN16466-3, based on the analysis of the stable isotope ratio δ18O of the water, is not applicable to ABM with high density (above 1.20 at 20 °C). In this work, for the first time, the official method was modified, providing for a prior dilution of the sample and applying a correction of the data in order to eliminate the isotopic contribution of the diluent, whereupon the within- and between-day standard deviations of repeatability (Sr) were estimated. Considering the limit values of δ18O for vinegar and concentrated must, the threshold limit of δ18O, below which the ABM product can be considered adulterated, has been identified.

Computational protocol to perform a spatiotemporal reconstruction of an epidemic.

Here, we present a computational protocol to perform a spatiotemporal reconstruction of an epidemic. We describe steps for using epidemiological data to depict how the epidemic changes over time and for employing clustering analysis to group geographical units that exhibit similar temporal epidemic progression. We then detail procedures for analyzing the temporal and spatial dynamics of the epidemic within each cluster. This protocol has been developed to be used on historical data but could also be applied to modern epidemiological data. For complete details on the use and execution of this protocol, please refer to Galli et al. (2023).1.

Ultrasound multifrequency strategy to estimate the lung surface roughness, in silico and in vitro results.

Lung ultrasound (LUS) is an important imaging modality to assess the state of the lung surface. Nevertheless, LUS is limited to the visual evaluation of imaging artifacts, especially the vertical ones. These artifacts are observed in pathologies characterized by a reduction of dimensions of air-spaces (alveoli). In contrast, there exist pathologies, such as chronic obstructive pulmonary disease (COPD), in which an enlargement of air-spaces can occur, which causes the lung surface to behave essentially as a perfect reflector, thus not allowing ultrasound penetration. This characteristic high reflectivity could be exploited to characterize the lung surface. Specifically, air-spaces of different sizes could cause the lung surface to have a different roughness, whose estimation could provide a way to assess the state of the lung surface. In this study, we present a quantitative multifrequency approach aiming at estimating the lung surface's roughness by measuring image intensity variations along the lung surface as a function of frequency. This approach was tested both in silico and in vitro, and it showed promising results. For the in vitro experiments, radiofrequency (RF) data were acquired from a novel experimental model. The results showed consistency between in silico and in vitro experiments.

Stable Isotope Ratio Analysis for Authentication of Natural Antioxidant Cannabidiol (CBD) from Cannabis sativa.

Cannabidiol (CBD) is a non-psychoactive cannabinoid of Cannabis sativa that exhibits several beneficial pharmacological effects, including anti-inflammatory and antioxidant properties. The molecule can be obtained via extraction from the plant or through a biosynthetic route. The two products have both advantages and disadvantages, thus necessitating the development of methods capable of distinguishing between the two products. In this study, for the first time, the analysis of the stable isotope ratios of oxygen and hydrogen demonstrated high efficiency in the discrimination of CBD of a totally natural origin from that obtained through chemical synthesis. Considering a probability level of 95%, it was possible to identify threshold values for δ2H and δ18O of the totally natural CBD of -215‱ and +23.4‱, respectively. Higher values may indicate a non-entirely natural origin of CBD (i.e., a biosynthetic molecule).

Multiple Technology Approach Based on Stable Isotope Ratio Analysis, Fourier Transform Infrared Spectrometry and Thermogravimetric Analysis to Ensure the Fungal Origin of the Chitosan.

Chitosan is a natural polysaccharide which has been authorized for oenological practices for the treatment of musts and wines. This authorization is limited to chitosan of fungal origin while that of crustacean origin is prohibited. To guarantee its origin, a method based on the measurement of the stable isotope ratios (SIR) of carbon δ13C, nitrogen δ15N, oxygen δ18O and hydrogen δ2H of chitosan has been recently proposed without indicating the threshold authenticity limits of these parameters which, for the first time, were estimated in this paper. In addition, on part of the samples analysed through SIR, Fourier transform infrared spectrometry (FTIR) and thermogravimetric analysis (TGA) were performed as simple and rapid discrimination methods due to limited technological resources. Samples having δ13C values above -14.2‱ and below -125.1‱ can be considered as authentic fungal chitosan without needing to analyse other parameters. If the δ13C value falls between -25.1‱ and -24.9‱, it is necessary to proceed further with the evaluation of the parameter δ15N, which must be above +2.7‱. Samples having δ18O values lower than +25.3‱ can be considered as authentic fungal chitosan. The combination of maximum degradation temperatures (obtained using TGA) and peak areas of Amide I and NH2/Amide II (obtained using FTIR) also allows the discrimination between the two origins of the polysaccharide. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) based on TGA, FTIR and SIR data successfully distributed the tested samples into informative clusters. Therefore, we present the technologies described as part of a robust analytical strategy for the correct identification of chitosan samples from crustaceans or fungi.

A spatiotemporal reconstruction of the 1630 plague epidemic in Milan.

In 1630, a devastating plague epidemic struck Milan, one of the most important Italian cities of that time, deeply affecting its demography and economy for decades. The lack of digitized historical data strongly limits our comprehension of that important event. In this work, we digitized and analyzed the Milan death registers of 1630. The study revealed that the epidemic evolved differently among the areas of the city. Indeed, we were able to group the parishes of the city (comparable with modern neighborhoods) in two groups based on their epidemiological curves. These different epidemiological progressions could reflect socio-economical and/or demographic features specific of the neighborhoods, opening questions about the relationship between these features and the evolution of epidemics in the pre-modern period. The study of historical records, like the one presented here, can help us to better understand European history and pre-modern epidemics.

Myo-Inositol, Scyllo-Inositol, and Other Minor Carbohydrates as Authenticity Markers for the Control of Italian Bulk, Concentrate, and Rectified Grape Must.

Myo-inositol polyalcohol is a characteristic component of natural and concentrated grape musts (CMs), and Regulation (EU) no. 1308/2013 prescribes its presence as a marker of the authenticity of rectified concentrated must (RCM). Other polyalcohols besides myo-inositol, such as scyllo-inositol or minor sugars, could be considered authenticity markers, but an extensive search in the literature yielded no exhaustively investigated study of their concentration variability in genuine products. The aim of this study was to create an extensive national data bank of minor carbohydrates profiles and investigate the impact of the geographical origin and the different vintages on the concentration of these compounds; to this end, 450 authentic Italian grape musts of different varieties were sampled and analyzed during the harvest season in 2019, 2020, and 2021. The grape musts from the Italian wine-growing areas CII and CIIIb had myo- and scyllo-inositol contents always higher than 756 and 39 mg/kg of sugar, respectively. Conversely, also considering other mono- and disaccharides, sucrose, sorbitol, lactose, maltose, and isomaltose showed contents always lower than 534, 1207, 390, 2222, and 1639 mg/kg of sugar, respectively. The general applicability to the CM and RCM of the proposed authenticity thresholds, established in the must, was demonstrated by studying the influence of must concentration on the myo- and scyllo-inositol content. Inter-laboratory comparison experiments were also conducted to harmonize and characterize laboratory methods and validate the analytical dataset. Based on the obtained results, the text of the EU legislation (Reg. (EU) 1308/2013), which defines the characteristics of the must and the CRM product, should be revised.

Characterization of Beef Coming from Different European Countries through Stable Isotope (H, C, N, and S) Ratio Analysis.

The need to guarantee the geographical origin of food samples has become imperative in recent years due to the increasing amount of food fraud. Stable isotope ratio analysis permits the characterization and origin control of foodstuffs, thanks to its capability to discriminate between products having different geographical origins and derived from different production systems. The Framework 6 EU-project "TRACE" generated hydrogen (2H/1H), carbon (13C/12C), nitrogen (15N/14N), and sulphur (34S/32S) isotope ratio data from 227 authentic beef samples. These samples were collected from a total of 13 sites in eight countries. The stable isotope analysis was completed by combining IRMS with a thermal conversion elemental analyzer (TC/EA) for the analysis of δ(2H) and an elemental analyzer (EA) for the determination of δ(13C), δ(15N), and δ(34S). The results show the potential of this technique to detect clustering of samples due to specific environmental conditions in the areas where the beef cattle were reared. Stable isotope measurements highlighted statistical differences between coastal and inland regions, production sites at different latitudes, regions with different geology, and different farming systems related to the diet the animals were consuming (primarily C3- or C4-based or a mixed one).

Authentication and Geographical Characterisation of Italian Grape Musts through Glucose and Fructose Carbon Isotopic Ratios Determined by LC-IRMS.

The authenticity of grape musts is normally checked through a time-consuming stable isotopic analysis of carbon (δ13C) after fermentation and distillation by following the official OIV MA AS-312-06 method. In this study, the alternative use of a technique based on δ13C isotopic analysis of the major sugars of the grape must by liquid chromatography coupled with isotope ratio mass spectrometry (LC-IRMS) is provided. It allows not only the detection of the fraudulent addition to grape must of exogenous glucose and fructose deriving from C4 plants but also the characterisation of it based on its geographical origin. In order to discriminate between musts from different areas of Italy, a preliminary dataset was considered; the δ13C isotopic ratios of glucose and fructose of around 100 authentic samples were analysed. The two analysed parameters, ranging from -29.8‱ to -21.9‱, are well correlated (R2 = 0.7802) and the northern regions showed significantly more negative δ13C values for both sugars than the rest of the dataset.

Stable Isotope Ratio Analysis for the Authentication of Natural Antioxidant Curcuminoids from Curcuma longa (Turmeric).

Curcuminoid complex, a mixture of curcumin, demethoxycurcumin and didemethoxycurcumin, is one of the most popular antioxidants of natural origin, and it has a multitude of other health benefits. It is threatened by the proliferation of counterfeit products on the market containing synthetic curcuminoids whose addition is difficult to identify as they present the three curcuminoid forms in the correct ratios. Consequently, the necessity to detect this fraudulent practice is escalating. Carbon-14 analysis is the most effective available method, but it is also expensive and difficult to implement. This paper describes the first attempt to characterize natural curcuminoids and their synthetic form, used as an adulterant, through the analysis of stable isotope ratios of carbon and hydrogen (expressed as δ13C and δ2H). Carbon values greater than -28.6‱ and hydrogen values greater than -71‱ may indicate the addition of synthetic curcuminoids to the natural ones.

Insights into the stable isotope ratio variability of hybrid grape varieties: a preliminary study.

BACKGROUND: Official stable isotope databases, based on the analysis of (D/H)I ethanol , (D/H)II ethanol , δ13 Cethanol and δ18 Owater of wine, are an indispensable tool for establishing the limits beyond which the mislabeling or the addition of sugar and/or water in wine production can be detected. The present study investigates, for the first time, whether the use of hybrid varieties instead of European Vitis vinifera for wine production can have an impact on the stable isotope ratios. RESULTS: The analyses were performed by isotope ratio mass spectrometry and site-specific natural isotope fractionation by nuclear magnetic resonance, in accordance with the official methods of the International Organization of Grapes and Wine. The comparison shows the tendency of some stable isotope ratios of hybrid varieties, in particular (D/H)I , to deviate from the regional averages of the V. vinifera samples. Notably, Baron, Monarch and Regent showed significantly different values at one of the two sampling sites. Particularly high δ13 C values characterize Helios compared to other hybrid varieties. CONCLUSION: For the first time, and from an isotopic point of view, the present study investigates the wine obtained from hybrid varieties, showing that further attention should be paid to their interpretation, on the basis of the database established according to the European Regulation 2018/273. © 2022 Society of Chemical Industry.

A Novel High Discriminatory Protocol for the Detection of Borrelia afzelii, Borrelia burgdorferi Sensu Stricto and Borrelia garinii in Ticks.

Bacteria of the Borrelia burgdorferi sensu lato complex are the causative agents of Lyme borreliosis (LB). Even if the conventional diagnosis of LB does not rely on the species itself, an accurate species identification within the complex will provide a deepened epidemiological scenario, a better diagnosis leading to a more targeted therapeutic approach, as well as promote the general public's awareness. A comparative genomics approach based on the 210 Borrelia spp. genomes available in 2019 were used to set up three species-specific PCR protocols, able to detect and provide species typing of Borrelia afzelii, Borrelia burgdorferi sensu stricto (s.s.) and Borrelia garinii, the three most common and important human pathogenic Lyme Borrelia species in Europe. The species-specificity of these protocols was confirmed on previously identified B. afzelii, B. burgdorferi s.s. and B. garinii specimens detected in Ixodes ricinus samples. In addition, the protocols were validated on 120 DNA samples from ticks collected in Sweden, showing 88% accuracy, 100% precision, 72% sensitivity and 100% specificity. The proposed approach represents an innovative tool in epidemiological studies focused on B. burgdorferi s.l. occurrence in ticks, and future studies could suggest its helpfulness in routine diagnostic tests for health care.

Hypervariable-Locus Melting Typing: a Novel Approach for More Effective High-Resolution Melting-Based Typing, Suitable for Large Microbiological Surveillance Programs.

Pathogen typing is pivotal to detecting the emergence of high-risk clones in hospital settings and to limit their spread. Unfortunately, the most commonly used typing methods (i.e., pulsed-field gel electrophoresis [PFGE], multilocus sequence typing [MLST], and whole-genome sequencing [WGS]) are expensive or time-consuming, limiting their application to real-time surveillance. High-resolution melting (HRM) can be applied to perform cost-effective and fast pathogen typing, but developing highly discriminatory protocols is challenging. Here, we present hypervariable-locus melting typing (HLMT), a novel approach to HRM-based typing that enables the development of more effective and portable typing protocols. HLMT types the strains by assigning them to melting types (MTs) on the basis of a reference data set (HLMT-assignment) and/or by clustering them using melting temperatures (HLMT-clustering). We applied the HLMT protocol developed on the capsular gene wzi for Klebsiella pneumoniae on 134 strains collected during surveillance programs in four hospitals. Then, we compared the HLMT results to those obtained using wzi, MLST, WGS, and PFGE typing. HLMT distinguished most of the K. pneumoniae high-risk clones with a sensitivity comparable to that of PFGE and MLST+wzi. It also drew surveillance epidemiological curves comparable to those obtained using MLST+wzi, PFGE, and WGS typing. Furthermore, the results obtained using HLMT-assignment were consistent with those of wzi typing for 95% of the typed strains, with a Jaccard index value of 0.9. HLMT is a fast and scalable approach for pathogen typing, suitable for real-time hospital microbiological surveillance. HLMT is also inexpensive, and thus, it is applicable for infection control programs in low- and middle-income countries. IMPORTANCE In this work, we describe hypervariable-locus melting typing (HLMT), a novel fast approach to pathogen typing using the high-resolution melting (HRM) assay. The method includes a novel approach for gene target selection, primer design, and HRM data analysis. We successfully applied this method to distinguish the high-risk clones of Klebsiella pneumoniae, one of the most important nosocomial pathogens worldwide. We also compared HLMT to typing using WGS, the capsular gene wzi, MLST, and PFGE. Our results show that HLMT is a typing method suitable for real-time epidemiological investigation. The application of HLMT to hospital microbiology surveillance can help to rapidly detect outbreak emergence, improving the effectiveness of infection control strategies.

Gas Chromatography Combustion Isotope Ratio Mass Spectrometry to Detect Differences in Four Compartments of Simmental Cows Fed on C3 and C4 Diets.

Fatty acids (FAs) metabolism in animals represents an important field of study since they influence the quality and the properties of the meat. The aim of this study is to assess the possibility to discriminate the diets of cows in different animal compartments and to study the fate of dietary FAs in the bovine organism, using carbon isotopic ratios. Five FAs, both essential (linoleic and linolenic) and non-essential (palmitic, stearic, and oleic) in four compartments (feed, rumen, liver, meat) of animals fed two different diets (based on either C3 or C4 plants) were considered. For all compartments, the carbon isotopic ratio (δ13C) of all FAs (with few exceptions) resulted significantly lower in cows fed on C3 than C4 plants, figuring as a powerful tool to discriminate between different diets. Moreover, chemical reactions taking place in each animal compartment result in fraction processes affecting the δ13C values. The δ13CFAs tendentially increase from feed to meat in group C3. On the other hand, the δ13CFAs generally increase from rumen to liver in group C4, while δ13CFAs of rumen and meat are mostly not statistically different. Different trends in the δ13CFAs of the two groups suggested different FAs fates depending on the diet.

Extra Virgin Olive Oil Extracts of Indigenous Southern Tuscany Cultivar Act as Anti-Inflammatory and Vasorelaxant Nutraceuticals.

Extra virgin olive oil (EVOO) is the typical source of fats in the Mediterranean diet. While fatty acids are essential for the EVOO nutraceutical properties, multiple biological activities are also due to the presence of polyphenols. In this work, autochthonous Tuscany EVOOs were chemically characterized and selected EVOO samples were extracted to obtain hydroalcoholic phytocomplexes, which were assayed to establish their anti-inflammatory and vasorelaxant properties. The polar extracts were characterized via 1H-NMR and UHPLC-HRMS to investigate the chemical composition and assayed in CaCo-2 cells exposed to glucose oxidase or rat aorta rings contracted by phenylephrine. Apigenin and luteolin were found as representative flavones; other components were pinoresinol, ligstroside, and oleuropein. The extracts showed anti-inflammatory and antioxidant properties via modulation of NF-κB and Nrf2 pathways, respectively, and good vasorelaxant activity, both in the presence and absence of an intact endothelium. In conclusion, this study evaluated the nutraceutical properties of autochthonous Tuscany EVOO cv., which showed promising anti-inflammatory and vasorelaxant effects.

Antimicrobial Susceptibility, Virulence, and Genomic Features of a Hypervirulent Serotype K2, ST65 Klebsiella pneumoniae Causing Meningitis in Italy.

The rise of a new hypervirulent variant of Klebsiella pneumoniae (hvKp) was recently reported, mainly linked to the ST23 lineage. The hvKp variants can cause severe infections, including hepatic abscesses, bacteremia, and meningitis, with a particularly disconcerting propensity to cause community-acquired, life-threatening infection among young and otherwise healthy individuals. The present study aimed to report the clinical characteristics of a hypermucoviscous K. pneumoniae strain isolated in Italy and sustaining recurrent meningitis in a patient of Peruvian origin. A further objective was to retrospectively investigate, by means of whole-genome sequencing (WGS) analysis, the genomic features of such an isolate. The hypermucoviscosity phenotype of the strain (sk205y205t) was determined using the string test. Genomic information was obtained by WGS (Illumina) and bioinformatic analysis. Strain sk205y205t was susceptible to most antibiotics, despite the presence of some resistance genes, including blaSHV-11, blaSHV-67, fosA, and acrR. The isolate belonged to ST65 and serotype K2, and exhibited several virulence factors related to the hvKp variant. Among these, were the siderophore genes entB, irp2, iroN, iroB, and iucA; the capsule-regulating genes rmpA and rmpA2; and the type 1 and 3 fimbriae fimH27 and mrkD, respectively. A further operon, encoding the genotoxin colibactin (clbA-Q), was also identified. The virulence plasmids pK2044, pRJA166b, and pNDM. MAR were also detected. Phylogenetic investigation showed that this Italian strain is highly similar to a Chinese isolate, suggesting a hidden circulation of this hvKp ST65 K2 lineage.