Parvalbumin Gene: A Valuable Marker for Pike Authentication and Allergen Risk Assessment.

Fish from the pike (Esox) genus are valued in gastronomy for their superior meat quality. However, they can cause allergic reactions in sensitive consumers. This work aimed to fill the gap in the detection of pike allergens using molecular-biological techniques. New, fast, and accurate loop-mediated isothermal amplification (LAMP) and real-time PCR (qPCR) assays were designed to detect pike DNA using the parvalbumin gene as a marker. LAMP was assessed by electrophoresis, SYBR green optical detection, and real-time fluorescence detection. The latter was the most sensitive, detecting as little as 0.78 ng of pike DNA; the qPCR detection limit was 0.1 ng. The LAMP analysis took 20-70 min, which is significantly faster than qPCR. The study provides reliable detection and quantification of the parvalbumin gene in both fresh and processed samples and further highlights the versatility of the use of the parvalbumin gene for the authentication of food products and consumer protection via refined allergen risk assessment that is independent of the type of tissue or food processing method used.

cor1 Gene: A Suitable Marker for Identification of Opium Poppy (Papaver somniferum L.).

This paper discusses the development of rapid, reliable, and accurate polymerase chain reaction (PCR) assays for detecting opium poppy (Papaver somniferum L.) in food. Endpoint, quantitative, and digital PCRs were compared based on the amplification of a newly developed DNA marker targeting the NADPH-dependent codeinone reductase (COR) gene. Designed assays were shown to be highly specific and sensitive in discriminating opium poppy from other plant species, even in heat-treated and food samples. Digital PCR was the most sensitive, with a detection limit of up to 5 copies, i.e., approximately 14 pg of target DNA per reaction. Quantitative and digital PCR further allowed the quantification of opium poppy in up to 1.5 ng and 42 pg (15 copies) of target DNA in a sample, respectively. In addition, two duplex PCRs have been developed for the simultaneous detection of opium poppy DNA and representatives of (i) the Papaveraceae family or (ii) the Plantae kingdom. Finally, all designed assays were successfully applied for analysis of 15 commercial foodstuffs; two were suspected of being adulterated. The study results have an important impact on addressing food fraud and ensuring the safety and authenticity of food products. Beyond food adulteration, the study may also have significant implications for forensics and law enforcement.

Influence of Fiber Diameter of Polycaprolactone Nanofibrous Materials on Biofilm Formation and Retention of Bacterial Cells.

To develop microbiologically safe nanofibrous materials, it is crucial to understand their interactions with microbial cells. Current research indicates that the morphology of nanofibers, particularly the diameter of the fibers, may play a significant role in biofilm formation and retention. However, it has not yet been determined how the fiber diameter of poly-ε-caprolactone (PCL), one of the most widely used biopolymers, affects these microbial interactions. In this study, two nanofibrous materials electrospun from PCL (PCL45 and PCL80) with different fiber diameter and characteristic distance δ between fibers were compared in terms of their ability to support or inhibit bacterial biofilm formation and retain bacterial cells. Strains of Escherichia coli (ATCC 25922 and ATCC 8739) and Staphylococcus aureus (ATCC 25923 and ATCC 6538) were used as model bacteria. Biofilm formation rate and retention varied significantly between the E. coli and S. aureus strains (p < 0.05) for the tested nanomaterials. In general, PCL showed a lower tendency to be colonized by the tested bacteria compared to the control material (polystyrene). Fiber diameter did not influence the biofilm formation rate of S. aureus strains and E. coli 25922 (p > 0.05), but it did significantly impact the biofilm formation rate of E. coli 8739 and biofilm morphology formed by all of the tested bacterial strains. In PCL45, thick uniform biofilm layers were formed preferably on the surface, while in PCL80 smaller clusters formed preferably inside the structure. Further, fiber diameter significantly influenced the retention of bacterial cells of all the tested strains (p < 0.001). PCL45, with thin fibers (average fiber diameter of 376 nm), retained up to 7 log (CFU mL-1) of staphylococcal cells (100% retention). The overall results indicate PCL45's potential for further research and highlight the nanofibers' morphology influence on bacterial interactions and differences in bacterial strains' behavior in the presence of nanomaterials.

Uncovering the microbial diversity of Czech Republic archives: A study of metabolically active airborne microbes.

Despite the diligent efforts of libraries, archives, and similar institutions to preserve cultural monuments, biodeterioration continues to pose a significant threat to these objects. One of the main sources of microorganisms responsible for the biodeterioration process is the presence of airborne microorganisms. Therefore, this research aims to monitor and compare outcomes of both culture-dependent (utilising various cultivation strategies) and culture-independent approaches (RNA-based sequencing) to identifying metabolically active airborne microorganisms in archives in the Czech Republic. Through this study, several species that have the potential to pose risks to both cultural heritage objects and the health of institution employees were found. Additionally, the efficacy of different cultivation media was demonstrated to be varied across archive rooms, highlighting the necessity of employing multiple cultivation media for comprehensive analyses. Of noteworthy importance, the resuscitating-promoting factor (Rpf) proved to be a pivotal tool, increasing bacterial culturability by up to 30% when synergistically employed Reasoner's 2A agar (R2A) and R2A + Rpf media. Next, the study emphasises the importance of integrating both culture-dependent and culture-independent approaches. The overlap between genera identified by the culture-dependent approach and those identified also by the culture-independent approach varied from 33% to surpassing 94%, with the maximum alignment exceeding 94% in only one case. Our results highlight the importance of actively monitoring and assessing levels of microbial air contamination in archives to prevent further deterioration of cultural heritage objects and to promote improved conditions for employees in archives and similar institutions.

Prevalence of SARS-CoV-2 variants in Prague wastewater determined by nanopore-based sequencing.

The early detection of upcoming disease outbreaks is essential to avoid both health and economic damage. The last four years of COVID-19 pandemic have proven wastewater-based epidemiology is a reliable system for monitoring the spread of SARS-CoV-2, a causative agent of COVID-19, in an urban population. As this monitoring enables the identification of the prevalence of spreading variants of SARS-CoV-2, it could provide a critical tool in the fight against this viral disease. In this study, we evaluated the presence of variants and subvariants of SARS-CoV-2 in Prague wastewater using nanopore-based sequencing. During August 2021, the data clearly showed that the number of identified SARS-CoV-2 RNA copies increased in the wastewater earlier than in clinical samples indicating the upcoming wave of the Delta variant. New SARS-CoV-2 variants consistently prevailed in wastewater samples around a month after they already prevailed in clinical samples. We also analyzed wastewater samples from smaller sub-sewersheds of Prague and detected significant differences in SARS-CoV-2 lineage progression dynamics among individual localities studied, e.g., suggesting faster prevalence of new variants among the sites with highest population density and mobility.

Genotyping of Campylobacter jejuni and prediction tools of its antimicrobial resistance.

Although Campylobacter jejuni is the pathogen responsible for the most common foodborne illness, tracing of the infection source remains challenging due to its highly variable genome. Therefore, one of the aim of the study was to compare three genotyping methods (MLST, PFGE, and mP-BIT) to determine the most effective genotyping tool. C. jejuni strains were divided into 4 clusters based on strain similarity in the cgMLST dendrogram. Subsequently, the dendrograms of the 3 tested methods were compared to determine the accuracy of each method compared to the reference cgMLST method. Moreover, a cost-benefit analysis has showed that MLST had the highest inverse discrimination index (97%) and required less workflow, time, fewer consumables, and low bacterial sample quantity. PFGE was shown to be obsolete both because of its low discriminatory power and the complexity of the procedure. Similarly, mP­BIT showed low separation results, which was compensated by its high availability. Therefore, our data showed that MLST is the optimal tool for genotyping C. jejuni. Another aim was to compare the antimicrobial resistance to ciprofloxacin, erythromycin, and tetracycline in C. jejuni strains isolated from human, water, air, food, and animal samples by two gene sequence-based prediction methods and to compare them with the actual susceptibility of C. jejuni strains using the disc diffusion method. Both tools, ResFinder and RGI, synchronously predict the antimicrobial susceptibility of C. jejuni and either can be used.

Plant Growth-Promoting Endophytic Bacteria Isolated from Miscanthus giganteus and Their Antifungal Activity.

Modern technologies can satisfy human needs only with the use of large quantities of fertilizers and pesticides that are harmful to the environment. For this reason, it is possible to develop new technologies for sustainable agriculture. The process could be carried out by using endophytic microorganisms with a (possible) positive effect on plant vitality. Bacterial endophytes have been reported as plant growth promoters in several kinds of plants under normal and stressful conditions. In this study, isolates of bacterial endophytes from the roots and leaves of Miscanthus giganteus plants were tested for the presence of plant growth-promoting properties and their ability to inhibit pathogens of fungal origin. Selected bacterial isolates were able to solubilize inorganic phosphorus, fix nitrogen, and produce phytohormones, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and siderophore. Leaf bacterial isolate Pantoea ananat is 50 OL 2 had high production of siderophores (zone ≥ 5 mm), and limited phytohormone production, and was the only one to show ACC deaminase activity. The root bacterial isolate of Pseudomonas libanensis 5 OK 7A showed the best results in phytohormone production (N6-(Δ2-isopentenyl)adenine and indole-3-acetic acid, 11.7 and 12.6 ng·mL-1, respectively). Four fungal cultures-Fusarium sporotrichioides DBM 4330, Sclerotinia sclerotiorum SS-1, Botrytis cinerea DS 90 and Sphaerodes fimicola DS 93-were used to test the antifungal activity of selected bacterial isolates. These fungal cultures represent pathogenic families, especially for crops. All selected root endophyte isolates inhibited the pathogenic growth of all tested fungi with inhibition percentages ranging from 30 to 60%. Antifungal activity was also tested in two forms of immobilization of selected bacterial isolates: one in agar and the other on dextrin-coated cellulose carriers. These results demonstrated that the endophytic Pseudomonas sp. could be used as biofertilizers for crops.

Monitoring of monkeypox viral DNA in Prague wastewater.

Monkeypox virus (Mpxv) is a dsDNA virus that has become a global concern for human health in 2022. As both infected people and non-human hosts can shed the virus from their skin, faeces, urine and other body fluids, and the resulting sewage contains viral load representative of the whole population, it is highly promising to detect the spread of monkeypox virus in municipal wastewater. We established a methodology for sewage-based monitoring of Mpxv in Prague and analysed samples (n = 24) already early August-October of 2022 in a municipality with 1.4 million inhabitants that only reported 29 cumulative cases in this period. We isolated Mpxv DNA with the Wizard Enviro Total Nucleic Acid Kit, and thereafter detected Mpxv DNA using the EliGene® Monkeypox RT-PCR Kit. Prague wastewater was positive for Mpxv (in total 9 positive samples in periods with 1-9 new cases per week, coinciding with a weekly incidence of 0.07-0.64 per 100,000 inhabitants. The method for confirmation of wastewater positivity via semi-nested PCR and Sanger sequencing was successfully confirmed on positive controls including Mpxv particles and Mpxv-positive wastewater from the Netherlands. However, for Prague wastewater samples, amplification of Mpxv DNA via semi-semi-nested PCR was unsuccessful. This was probably due to extremely low case count, leading to the amplification of non-target bacterial DNA. Compared to other studies with much higher Mpxv prevalence, we show the outstanding sensitivity of our approach for monitoring the spread of monkeypox using wastewater.

Bacterial Diversity on Historical Audio-Visual Materials and in the Atmosphere of Czech Depositories.

Microbial contamination in cultural heritage storage facilities is undoubtedly still a huge problem and leads to the biodeterioration of historical objects and thus the loss of information for future generations. Most studies focus on fungi that colonize materials, which are the primary agents of biodeterioration. However, bacteria also play crucial roles in this process. Therefore, this study focuses on identifying bacteria that colonize audio-visual materials and those present in the air in the archives of the Czech Republic. For our purposes, the Illumina MiSeq amplicon sequencing method was used. Using this method, 18 bacterial genera with an abundance of higher than 1% were identified on audio-visual materials and in the air. We also evaluated some factors that were assumed to possibly influence the composition of bacterial communities on audio-visual materials, of which locality was shown to be significant. Locality also explained most of the variability in bacterial community structure. Furthermore, an association between genera colonizing materials and genera present in the air was demonstrated, and indicator genera were evaluated for each locality. IMPORTANCE The existing literature on microbial contamination of audio-visual materials has predominantly used culture-based methods to evaluate contamination and has overlooked the potential impact of environmental factors and material composition on microbial communities. Furthermore, previous studies have mainly focused on contamination by microscopic fungi, neglecting other potentially harmful microorganisms. To address these gaps in knowledge, our study is the first to provide a comprehensive analysis of bacterial communities present on historical audio-visual materials. Our statistical analyses demonstrate the critical importance of including air analysis in such studies, as airborne microorganisms can significantly contribute to the contamination of these materials. The insights gained from this study are not only valuable in developing effective preventive measures to mitigate contamination but also valuable in identifying targeted disinfection methods for specific types of microorganisms. Overall, our findings highlight the need for a more holistic approach to understanding microbial contamination in cultural heritage materials.

Identification of Fish Species and Targeted Genetic Modifications Based on DNA Analysis: State of the Art.

Food adulteration is one of the most serious problems regarding food safety and quality worldwide. Besides misleading consumers, it poses a considerable health risk associated with the potential non-labeled allergen content. Fish and fish products are one of the most expensive and widely traded commodities, which predisposes them to being adulterated. Among all fraud types, replacing high-quality or rare fish with a less valuable species predominates. Because fish differ in their allergen content, specifically the main one, parvalbumin, their replacement can endanger consumers. This underlines the need for reliable, robust control systems for fish species identification. Various methods may be used for the aforementioned purpose. DNA-based methods are favored due to the characteristics of the target molecule, DNA, which is heat resistant, and the fact that through its sequencing, several other traits, including the recognition of genetic modifications, can be determined. Thus, they are considered to be powerful tools for identifying cases of food fraud. In this review, the major DNA-based methods applicable for fish meat and product authentication and their commercial applications are discussed, the possibilities of detecting genetic modifications in fish are evaluated, and future trends are highlighted, emphasizing the need for comprehensive and regularly updated online database resources.