Fermented table olives from Cyprus: Microbiota profile of three varieties from different regions through metabarcoding sequencing.

The knowledge about the microbial diversity of different olives varieties from diverse regions in the Mediterranean basin is limited. This work aimed to determine the microbial diversity of three different fermented olive varieties, collected from different regions in Cyprus, via Next Generation Sequencing (NGS) analysis. Olives were spontaneously fermented for 120 days, microbial DNA was extracted from the final products, and subjected to 16S rRNA gene and ITS1 loci metabarcoding analysis for the determination of bacterial and fungal communities, respectively. Results revealed that the bacterial profile of the studied varieties was similar, while no noteworthy differences were observed in olives from different regions. The bacterial profile was dominated by the co-existence of Lactobacillus and Streptococcus, while the genera Lactococcus and Salinivibrio and the family Leuconostocaceae were also present in increased relative abundances. Regarding fungal communities, the analysis indicated discrimination among the different varieties, especially in Kalamata ones. The most abundant fungi were mainly the genera Aspergillus, Botryosphaeria, Meyerozyma, and Zygosaccharomyces for Cypriot olives, the genera Botryosphaeria, Saccharomyces, Geosmithia, and Wickeromyces for Kalamata variety, while the dominant fungi in the Picual variety were mainly members of the genera Candida, Penicillium, Saccharomyces, Hanseniospora and Botryosphaeria. Potential microbial biomarkers that distinguish the three varieties are also proposed. Moreover, interaction networks analysis identified interactions among the key taxa of the communities. Overall, the present work provides useful information and sheds light on an understudied field, such as the comparison of microbiota profiles of different varieties from several regions in Cyprus. The study enriches our knowledge and highlights the similarities and the main differences between those aspects, booming in parallel the need for further works on this frontier, in the attempt to determine potentially olives' microbial terroir in Cyprus. Our work should be used as a benchmark for future works in this direction.

Metataxonomic Mapping of the Microbial Diversity of Irish and Eastern Mediterranean Cheeses.

The distinct sensorial characteristics of local cheeses influence consumer preferences, and make an essential contribution to the local economy. Microbial diversity in cheese is among the fundamental contributors to sensorial and qualitative characteristics. However, knowledge regarding the existence of microbial patterns associated with regional production practices in ripened cheeses remains limited. The present research was conducted to test the hypothesis that the background metagenome of cheeses could be used as a marker of their origin. We compared Irish versus Eastern Mediterranean cheeses-namely Greek and Cypriot-using High Throughput Sequencing (HTS). The study identified a significantly distinct separation among cheeses originating from the three different countries, in terms of the total microbial community composition. The use of machine learning and biomarkers discovery algorithms defined key microbes that differentiate each geographic region. Finally, the development of interaction networks revealed that the key species developed mostly negative interactions with the other members of the communities, highlighting their dominance in the community. The findings of the present research demonstrate that metagenome could indeed be used as a biological marker of the origin of mature cheeses, and could provide further insight into the dynamics of microbial community composition in ripened cheeses.

Current Status, Recent Advances, and Main Challenges on Table Olive Fermentation: The Present Meets the Future.

Table olives are among the most well-known fermented foods, being a vital part of the Mediterranean pyramid diet. They constitute a noteworthy economic factor for the producing countries since both their production and consumption are exponentially increasing year by year, worldwide. Despite its significance, olive's processing is still craft based, not changed since antiquity, leading to the production of an unstable final product with potential risk concerns, especially related to deterioration. However, based on industrial needs and market demands for reproducible, safe, and healthy products, the modernization of olive fermentation processing is the most important challenge of the current decade. In this sense, the reduction of sodium content and more importantly the use of suitable starter cultures, exhibiting both technological and potential probiotic features, to drive the process may extremely contribute to this need. Prior, to achieve in this effort, the full understanding of table olive microbial ecology during fermentation, including an in-depth determination of microbiota presence and/or dominance and its functionality (genes responsible for metabolite production) that shape the sensorial characteristics of the final product, is a pre-requisite. The advent of meta-omics technology could provide a thorough study of this complex ecosystem, opening in parallel new insights in the field, such as the concept of microbial terroir. Herein, we provide an updated overview in the field of olive fermentation, pointing out some important challenges/perspectives that could be the key to the olive sector's advancement and modernization.

Metataxonomic Analysis of Grape Microbiota During Wine Fermentation Reveals the Distinction of Cyprus Regional terroirs.

Wine production in Cyprus has strong cultural ties with the island's tradition, influencing local and foreign consumers' preferences and contributing significantly to Cyprus' economy. A key contributor to wine quality and sensorial characteristics development is the microbiota that colonizes grapes and performs alcoholic fermentation. Still, the microbial patterns of wines produced in different geographic regions (terroir) in Cyprus remain unknown. The present study investigated the microbial diversity of five terroirs in Cyprus, two from the PGI Lemesos region [Kyperounta (PDO Pitsilia) and Koilani (PDO Krasochoria)], and three from the PGI Pafos region [Kathikas (PDO Laona Akamas), Panayia, and Statos (PDO Panayia)], of two grape varieties, Xynisteri and Maratheftiko, using high-throughput amplicon sequencing. Through a longitudinal analysis, we examined the evolution of the bacterial and fungal diversity during spontaneous alcoholic fermentation. Both varieties were characterized by a progressive reduction in their fungal alpha diversity (Shannon index) throughout the process of fermentation. Additionally, the study revealed a distinct separation among different terroirs in total fungal community composition (beta-diversity) for the variety Xynisteri. Also, Kyperounta terroir had a distinct total fungal beta-diversity from the other terroirs for Maratheftiko. Similarly, a significant distinction was demonstrated in total bacterial diversity between the PGI Lemesos region and the PGI Pafos terroirs for grape juice of the variety Xynisteri. Pre-fermentation, the fungal diversity for Xynisteri and Maratheftiko was dominated by the genera Hanseniaspora, Aureobasidium, Erysiphe, Aspergillus, Stemphylium, Penicillium, Alternaria, Cladosporium, and Mycosphaerella. During and post-fermentation, the species Hanseniaspora nectarophila, Saccharomyces cerevisiae, Hanseniaspora guilliermondii, and Aureobasidium pullulans, became the predominant in most must samples. Regarding the bacterial diversity, Lactobacillus and Streptococcus were the predominant genera for both grape varieties in all stages of fermentation. During fermentation, an increase was observed in the relative abundance of some bacteria, such as Acetobacter, Gluconobacter, and Oenococcus oeni. Finally, the study revealed microbial biomarkers with statistically significant higher relative representation, associated with each geographic region and each grape variety, during the different stages of fermentation. The present study's findings provide an additional linkage between the grape microbial community and the wine terroir.

Cyprus Sausages' Bacterial Community Identification Through Metataxonomic Sequencing: Evaluation of the Impact of Different DNA Extraction Protocols on the Sausages' Microbial Diversity Representation.

Cyprus traditional sausages from the Troodos mountainous region of Pitsilia gained the protected geographical indication (PGI) designation from the European Committee (EU 2020/C 203/06). Still, we lack authentication protocols for the distinction of "Pitsilia" from industrially produced Cyprus sausages. Microbial activity is an essential contributor to traditional sausages' sensorial characteristics, but whether the microbial patterns might be associated with the area of production is unclear. In the present research, we applied high-throughput sequencing (HTS) to provide a linkage between the area of production and Cyprus sausages' bacterial diversity. To strengthen our findings, we used three different DNA extraction commercial kits: (i) the DNeasy PowerFood Microbial Kit (QIAGEN); (ii) the NucleoSpin Food Kit (MACHEREY-NAGEL); and (iii) the blackPREP Food DNA I Kit (Analytik Jena), in which we applied three different microbial cell wall lysis modifications. The modifications included heat treatment, bead beating, and enzymatic treatment. Results regarding metagenomic sequencing were evaluated in terms of number of reads, alpha diversity indexes, and taxonomic composition. The efficacy of each method of DNA isolation was assessed quantitatively based on the extracted DNA yield and the obtained copy number of (a) the 16S rRNA gene, (b) the internal transcribed spacer (ITS) region, and (c) three Gram-positive bacteria that belong to the genera Latilactobacillus (formerly Lactobacillus), Bacillus, and Enterococcus via absolute quantification using qPCR. Compared with some examined industrial sausages, Pitsilia sausages had significantly higher bacterial alpha diversity (Shannon and Simpson indexes). Principal coordinates analysis separated the total bacterial community composition (beta diversity) of the three Pitsilia sausages from the industrial sausages, with the exception of one industrial sausage produced in Pitsilia, according to the manufacturer. Although the eight sausages shared the abundant bacterial taxa based on 16S rDNA HTS, we observed differences associated with bacterial diversity representation and specific genera. The findings indicate that the microbial communities may be used as an additional tool for identifying of the authenticity of Cypriot sausages.

Gaps in Knowledge About SARS-CoV-2 & COVID-19 Among University Students Are Associated With Negative Attitudes Toward People With COVID-19: A Cross-Sectional Study in Cyprus.

University students represent a highly active group in terms of their social activity in the community and in the propagation of information on social media. We aimed to map the knowledge, attitudes, and perceptions of University students in Cyprus about severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Coronavirus disease 2019 (COVID-19) to guide targeted future measures and information campaigns. We used a cross-sectional online survey targeting all students in conventional, not distance-learning, programs in five major universities in the Republic of Cyprus. Students were invited to participate through the respective Studies and Student Welfare Office of each institution. The survey was made available in English and Greek on REDCap. Participation was voluntary and anonymous. The questionnaire was developed based on a consensus to cover the main factual information directed by official channels toward the general public in Cyprus at the time of the survey. In addition to sociodemographic information (N = 8), the self-administered questionnaire consisted of 19 questions, assessing the knowledge regarding the characteristics of SARS-CoV-2 and COVID-19, infection prevention and control measures (N = 10), perceptions related to COVID-19, for instance, whether strict travel measures are necessary (N = 4), and attitudes toward a hypothetical person infected (N = 2). Furthermore, participants were asked to provide their own assessment of their knowledge about COVID-19 and specifically with regard to the main symptoms and ways of transmission (N = 3). The number of students who completed the survey was 3,641 (41% studying Health/Life Sciences). Amongst them, 68.8% responded correctly to at least 60% of knowledge-related questions. Misconceptions were identified in 30%. Only 29.1% expressed a positive attitude toward a hypothetical person with COVID-19 without projecting judgment (9.2%) or blame (38%). Odds of expressing a positive attitude increased by 18% (95% CI 13-24%; p < 0.001) per unit increase in knowledge. Postgraduate level education was predictive of better knowledge (odds ratio (OR) 1.81; 95% CI 1.34-2.46; p < 0.001 among doctoral students] and positive attitude [OR 1.35; 95% CI 1.01-1.80; p = 0.04). In this study, we show that specific knowledge gaps and misconceptions exist among University students about SARS-CoV-2 and COVID-19 and their prevalence is associated with negative attitudes toward people with COVID-19. Our findings highlight the integrated nature of knowledge and attitude and suggest that improvements to the former could contribute to improvements in the latter.

Evolution of Bacterial Communities, Physicochemical Changes and Sensorial Attributes of Natural Whole and Cracked Picual Table Olives During Spontaneous and Inoculated Fermentation.

Table olives are one of the most well-known traditionally fermented products, and their global consumption is exponentially increasing. In direct brining, table olives are produced spontaneously, without any debittering pre-treatment. Up to date, fermentation process remains empirical and inconstant, as it is affected by the physicochemical attributes of the fruit, tree and fruit management of pro and post-harvest. In the present study, whole and cracked Picual table olives were fermented at industrial scale for 120 days, using three distinct methods (natural fermentation, inoculation with lactic acid bacteria (LAB) at a 7 or a 10% NaCl concentration). Microbial, physicochemical and sensorial alterations monitored during the whole process, and several differences were observed between treatments. Results indicated that in all treatments, the dominant microflora were LAB. Yeasts also detected in noteworthy populations, especially in non-inoculated samples. However, LAB population was significantly higher in inoculated compared to non-inoculated samples. Microbial profiles identified by metagenomic approach showed meaningful differences between spontaneous and inoculated treatments. As a result, the profound dominance of starter culture had a severe effect on olives fermentation, resulting in lower pH and higher acidification, which was mainly caused by the higher levels of lactic acid produced. Furthermore, the elimination of Enterobacteriaceae was shortened, even at lower salt concentration. Although no effect observed concerning the quantitated organoleptic parameters such as color and texture, significantly higher levels in terms of antioxidant capacity were recorded in inoculated samples. At the same time, the degradation time of oleuropein was shortened, leading to the production of higher levels of hydroxytyrosol. Based on this evidence, the establishment of starter culture driven Picual olives fermentation is strongly recommended. It is crucial to mention that the inoculated treatment with reducing sodium content was highly appreciated by the sensory panel, enhancing the hypothesis that the production of Picual table olives at reduced NaCl levels is achievable.

Newly Developed System for the Robust Detection of Listeria monocytogenes Based on a Bioelectric Cell Biosensor.

Human food-borne diseases caused by pathogenic bacteria have been significantly increased in the last few decades causing numerous deaths worldwide. The standard analyses used for their detection have significant limitations regarding cost, special facilities and equipment, highly trained staff, and a long procedural time that can be crucial for foodborne pathogens with high hospitalization and mortality rates, such as Listeria monocytogenes. This study aimed to develop a biosensor that could detect L. monocytogenes rapidly and robustly. For this purpose, a cell-based biosensor technology based on the Bioelectric Recognition Assay (BERA) and a portable device developed by EMBIO Diagnostics, called B.EL.D (Bio Electric Diagnostics), were used. Membrane engineering was performed by electroinsertion of Listeria monocytogenes homologous antibodies into the membrane of African green monkey kidney (Vero) cells. The newly developed biosensor was able to detect the pathogen's presence rapidly (3 min) at concentrations as low as 102 CFU mL-1, demonstrating a higher sensitivity than most existing biosensor-based methods. In addition, lack of cross-reactivity with other Listeria species, as well as with Escherichia coli, was shown, thus, indicating biosensor's significant specificity against L. monocytogenes.

High Throughput Sequencing Technologies as a New Toolbox for Deep Analysis, Characterization and Potentially Authentication of Protection Designation of Origin Cheeses?

Protected Designation of Origin (PDO) labeling of cheeses has been established by the European Union (EU) as a quality policy that assures the authenticity of a cheese produced in a specific region by applying traditional production methods. However, currently used scientific methods for differentiating and establishing PDO are limited in terms of time, cost, accuracy and their ability to identify through quantifiable methods PDO fraud. Cheese microbiome is a dynamic community that progressively changes throughout ripening, contributing via its metabolism to unique qualitative and sensorial characteristics that differentiate each cheese. High Throughput Sequencing (HTS) methodologies have enabled the more precise identification of the microbial communities developed in fermented cheeses, characterization of their population dynamics during the cheese ripening process, as well as their contribution to the development of specific organoleptic and physio-chemical characteristics. Therefore, their application may provide an additional tool to identify the key microbial species that contribute to PDO cheeses unique sensorial characteristics and to assist to define their typicityin order to distinguish them from various fraudulent products. Additionally, they may assist the cheese-makers to better evaluate the quality, as well as the safety of their products. In this structured literature review indications are provided on the potential for defining PDO enabling differentiating factors based on distinguishable microbial communities shaped throughout the ripening procedures associated to cheese sensorial characteristics, as revealed through metagenomic and metatranscriptomic studies. Conclusively, HTS applications, even though still underexploited, have the potential to demonstrate how the cheese microbiome can affect the ripening process and sensorial characteristics formation via the catabolism of the available nutrients and interplay with other compounds of the matrix and/or production of microbial origin metabolites and thus their further quality enhancement.

Benefits of the Use of Lactic Acid Bacteria Starter in Green Cracked Cypriot Table Olives Fermentation.

Table olives are one of the most established Mediterranean vegetables, having an exponential increase consumption year by year. In the natural-style processing, olives are produced by spontaneous fermentation, without any chemical debittering. This natural fermentation process remains empirical and variable since it is strongly influenced by physicochemical parameters and microorganism presence in olive drupes. In the present work, Cypriot green cracked table olives were processed directly in brine (natural olives), using three distinct methods: spontaneous fermentation, inoculation with lactic acid bacteria at a 7% or a 10% NaCl concentration. Sensory, physicochemical, and microbiological alterations were monitored at intervals, and major differences were detected across treatments. Results indicated that the predominant microorganisms in the inoculated treatments were lactic acid bacteria, while yeasts predominated in control. As a consequence, starter culture contributed to a crucial effect on olives fermentation, leading to faster acidification and lower pH. This was attributed to a successful lactic acid fermentation, contrasting the acetic and alcoholic fermentation observed in control. Furthermore, it was established that inhibition of enterobacteria growth was achieved in a shorter period and at a significantly lower salt concentration, compared to the spontaneous fermentation. Even though no significant variances were detected in terms of the total phenolic content and antioxidant capacity, the degradation of oleuropein was achieved faster in inoculated treatments, thus, producing higher levels of hydroxytyrosol. Notably, the reduction of salt concentration, in combination with the use of starter, accented novel organoleptic characteristics in the final product, as confirmed from a sensory panel; hence, it becomes obvious that the production of Cypriot table olives at reduced NaCl levels is feasible.

Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and innate immunity.

Plant innate immune response to pathogen infection includes an elegant signaling pathway leading to reactive oxygen species generation and resulting hypersensitive response (HR); localized programmed cell death in tissue surrounding the initial infection site limits pathogen spread. A veritable symphony of cytosolic signaling molecules (including Ca(2+), nitric oxide [NO], cyclic nucleotides, and calmodulin) have been suggested as early components of HR signaling. However, specific interactions among these cytosolic secondary messengers and their roles in the signal cascade are still unclear. Here, we report some aspects of how plants translate perception of a pathogen into a signal cascade leading to an innate immune response. We show that Arabidopsis thaliana CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/DND1) conducts Ca(2+) into cells and provide a model linking this Ca(2+) current to downstream NO production. NO is a critical signaling molecule invoking plant innate immune response to pathogens. Plants without functional CNGC2 lack this cell membrane Ca(2+) current and do not display HR; providing the mutant with NO complements this phenotype. The bacterial pathogen-associated molecular pattern elicitor lipopolysaccharide activates a CNGC Ca(2+) current, which may be linked to NO generation due to buildup of cytosolic Ca(2+)/calmodulin.

Quorum-sensing regulation governs bacterial adhesion, biofilm development, and host colonization in Pantoea stewartii subspecies stewartii.

The phytopathogenic bacterium Pantoea stewartii subsp. stewartii synthesizes stewartan exo/capsular polysaccharide (EPS) in a cell density-dependent manner governed by the EsaI/EsaR quorum-sensing (QS) system. This study analyzes biofilm development and host colonization of the WT and QS regulatory mutant strains of P. stewartii. First, we show that the cell density-dependent synthesis of stewartan EPS, governed by the EsaI/EsaR QS system, is required for proper bacterial adhesion and development of spatially defined, 3D biofilms. Second, a nonvirulent mutant lacking the esaI gene adheres strongly to surfaces and develops densely packed, less structurally defined biofilms in vitro. This strain appears to be arrested in a low cell density developmental mode. Exposure of this strain to exogenous N-acyl-homoserine lactone counteracts this adhesion phenotype. Third, QS mutants lacking the EsaR repressor attach poorly to surfaces and form amorphous biofilms heavily enmeshed in excess EPS. Fourth, the WT strain disseminates efficiently within the xylem, primarily in a basipetal direction. In contrast, the two QS mutant strains remain largely localized at the site of infection. Fifth, and most significantly, epifluorescence microscopic imaging of infected leaf tissue and excised xylem vessels reveals that the bacteria colonize the xylem with unexpected specificity, particularly toward the annular rings and spiral secondary wall thickenings of protoxylem, as opposed to indiscriminate growth to fill the xylem lumen. These observations are significant to bacterial plant pathogenesis in general and may reveal targets for disease control.