Detection of fibrous microplastics and natural microfibers in fish species (Engraulis encrasicolus, Mullus barbatus and Merluccius merluccius) for human consumption from the Tyrrhenian sea.

The occurrence of natural/artificial and synthetic microfibers was assessed in three commercial fish species (Engraulis encrasicolus, Mullus barbatus, Merluccius merluccius) from the Tyrrhenian Sea sold for human consumption. The gastrointestinal tracts of n. 150 samples were analyzed, the isolated microfibers were classified applying a morphological approach, based on the analysis of their morphological features, coupled with the identification of the chemical composition of a subsample of microfibers. All the species contained microfibers at levels ranging from 0 to 49 items/individual and the number of ingested microfibers significantly differed between pelagic and demersal fishes. The evaluation of fiber morphologies highlighted that natural/artificial microfibers were the most numerous among the isolated microfibers, while the dominant colors were blue, black, and clear in all the species. Chemical characterization confirmed the morphological identification and indicated cellulose and polyester as the most common polymer types. Considering the analytical issues that may affect the evaluation of microfiber pollution, the results pointed out the importance of an accurate morphological approach that allows the distinction between different fiber types, before the spectroscopic analyses. Moreover, the implementation of fast and accessible methods to identify microfibers in fish species intended for human consumption will be beneficial also to make an adequate risk assessment to consumer health.

Selection and Multiplexing of Reverse Transcription-Quantitative PCR Tests Targeting Relevant Honeybee Viral Pathogens.

Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription-quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction.

In Vivo Functional Properties of Dairy Bacteria.

This literature review aimed to collect investigations on the in vivo evidence for bacteria associated with fermented dairy foods to behave as probiotics with beneficial effects in the prevention and treatment of various diseases. All main bacterial groups commonly present in high numbers in fermented milks or cheeses were taken into account, namely starter lactic acid bacteria (SLAB) Lactobacillus delbrueckii subsp. bulgaricus and lactis, L. helveticus, Lactococcus lactis, Streptococcus thermophilus, non-starter LAB (NSLAB) Lacticaseibacillus spp., Lactiplantibacillus plantarum, dairy propionibacteria, and other less frequently encountered species. Only studies regarding strains of proven dairy origin were considered. Studies in animal models and clinical studies showed that dairy bacteria ameliorate symptoms of inflammatory bowel disease (IBD), mucositis, metabolic syndrome, aging and oxidative stress, cancer, bone diseases, atopic dermatitis, allergies, infections and damage caused by pollutants, mild stress, and depression. Immunomodulation and changes in the intestinal microbiota were the mechanisms most often involved in the observed effects. The results of the studies considered indicated that milk and dairy products are a rich source of beneficial bacteria that should be further exploited to the advantage of human and animal health.

Special Issue "Functional Characterization of Lactic Acid Bacteria": Editorial.

Lactic acid bacteria (LAB) are a diverse group of microorganisms of the order Lactobacillales in the Bacillota phylum, subdivision Bacilli, comprising, at this stage of taxonomic descriptions six families (Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae and Streptococcaceae) [...].

Autochthonous Cultures to Improve Safety and Standardize Quality of Traditional Dry Fermented Meats.

Traditional dry fermented meat products are obtained artisanally in many countries, where they represent a gastronomic heritage well distinguished from industrial counterparts. This food category is most often obtained from red meat, a food commodity that is under attack because of evidence of increased risk of cancer and degenerative diseases with high consumption. However, traditional fermented meat products are intended for moderate consumption and gastronomic experience, and, as such, their production must be continued, which would also help safeguard the culture and economy of the geographical areas of origin. In this review, the main risks attributed to these products are considered, and how these risks are reduced by the application of autochthonous microbial cultures is highlighted by reviewing studies reporting the effects of autochthonous lactic acid bacteria (LAB), coagulase negative staphylococci (CNS), Debaryomyces hansenii and Penicillium nalgiovense on microbiological and chemical safety and on sensory attributes. The role of dry fermented sausages as a source of microorganisms that can be beneficial to the host is also considered. From the results of the studies reviewed here it appears that the development of autochthonous cultures for these foods can ensure safety and stabilize sensory characteristics and has the capacity to be extended to a larger variety of traditional products.

Recent Trends of Antibiotic Resistance in Staphylococcus aureus Causing Clinical Mastitis in Dairy Herds in Abruzzo and Molise Regions, Italy.

This study aimed to investigate the recent trends of antibiotic resistance (AR) prevalence in Staphylococcus aureus isolated from the milk of animals with clinical mastitis in areas of the Abruzzo and Molise regions in Central Italy. Fifty-four S. aureus isolates were obtained from routine testing for clinical mastitis agents carried out in the author institution in the years 2021 and 2022 and were analyzed for phenotypic resistance to eight antibiotics recommended for testing by European norms and belonging to the antibiotic classes used for mastitis treatment in milk-producing animals. Moreover, the presence of 14 transferable genetic determinants encoding resistance to the same antibiotics was analyzed using qPCR tests developed in this study. Phenotypic resistance to non-ß-lactams was infrequent, with only one 2022 isolate resistant to clindamycin. However, resistance to the ß-lactam cefoxitin at concentrations just above the threshold of 4 µg/mL was observed in 59.2% of isolates in both years, making these isolates classifiable as methicillin-resistant. The AR genotypes detected were the blaZ gene (50% of 2021 isolates and 44.4% of 2022 isolates), aphA3-blaZ- ermC/T (one 2021 isolate), aphA3-ant6-blaZ-ermC/T (one 2021 isolate), blaZ-ermB (one 2022 isolate) and mecA-mph (one 2022 isolate). An inquiry into the veterinarians who provided the samples, regarding the antimicrobials prescribed for mastitis treatment and criteria of usage, indicated a possible causal relation with the AR test results. The occurrence of AR genotypes did not increase in time, most probably reflecting how mastitis was treated and prevented in farms. However, the frequently observed cefoxitin resistance needs to be explained genotypically, further monitored and limited by modifying antibiotic usage practices. The identification of a mecA-positive isolate in 2022 suggests further investigation if this genotype is emerging locally.

Recent Records on Bacterial Opportunistic Infections via the Dietary Route.

This narrative review was aimed at identifying the opportunistic bacterial pathogens that can be transmitted by contaminated food and represent a current threat for patients particularly susceptible to infections because of underlying conditions or predisposing factors. The analysis was focused on recent case or outbreak reports and systematic reviews published in the years 2019 to 2023 and resulted in sorting 24 bacterial groups comprising the genera or species able to cause a variety of systemic or invasive infections if ingested with food or drinking water. These included both bacteria known to cause mild infections in immunocompetent persons and bacteria considered to be innocuous, which are used in food fermentation or as probiotics. No recent cases of infections transmitted through dietary routes were reported for the critical nosocomial pathogens widely found in food products, primarily Acinetobacter baumannii and Klebsiella pneumoniae. However, the very first sources of their introduction into the clinical environment still need to be established. In many instances, risky dietary habits, such as eating raw fish, seafood, raw meat, unpasteurized milk, and their derived products or the lack of control in fermentation processes, has led to the reported illnesses, pointing out the necessity to improve the hygiene of production and consumer awareness of the risks.

Virulence Characteristics and Distribution of the Pathogen Listeria ivanovii in the Environment and in Food

Listeria ivanovii and L. monocytogenes, are the only pathogenic species of the genus Listeria and share many virulence factors and mechanisms of pathogenicity. L. ivanovii shows host tropism towards small ruminants and rodents and much lower virulence for humans compared to L. monocytogenes. However, severe infections caused by L. ivanovii, resulting in bacteremia, abortion and stillbirth, occasionally occurred in immunocompromised persons and in pregnant women, while in immunocompetent hosts L. ivanovii can cause gastroenteritis. In this review, the updated knowledge on virulence aspects and distribution of L. ivanovii in the environment and in food is summarized. Recent research on its virulence characters at genome level gave indications on how pathogenicity evolved in this bacterial species. As for L. monocytogenes, L. ivanovii infections occurred after the ingestion of contaminated food, so an overview of reports regarding its distribution in food products was carried out to obtain indications on the categories of foods exposed to contamination by L. ivanovii. It was found that a wide variety of food products can be a source of this microorganism and that, like L. monocytogenes, L. ivanovii is able to persist in the food production environment. Studies on its ability to grow in enrichment and isolation media suggested that its occurrence in nature might be underestimated. Moreover, virulence varies among strains for differences in virulence character regulation, presence/absence of genetic regions and the possible instability of a Listeria pathogenicity genomic island, LIPI-2, which is unique to L. ivanovii. We can conclude that L. ivanovii, as a possible pathogen for animals and humans, requires more focused investigations regarding its occurrence in the environment and in food and on intra-species variability of pathogenic potential.

Biochemical and structural analysis of a cytosolic sulfotransferase of the malaria vector Anopheles gambiae overexpressed in the reproductive tissues.

The temporary or permanent chemical modification of biomolecules is a crucial aspect in the physiology of all living species. However, while some modules are well characterised also in insects, others did not receive the same attention. This holds true for sulfo-conjugation that is catalysed by cytosolic sulfotransferases (SULT), a central component of the metabolism of endogenous low molecular weight molecules and xenobiotics. In particular, limited information is available about the functional roles of the mosquito predicted enzymes annotated as SULTs in genomic databases. The herein described research is the first example of a biochemical and structural study of a SULT of a mosquito species, in general, and of the malaria vector Anopheles gambiae in particular. We confirmed that the AGAP001425 transcript displays a peculiar expression pattern that is suggestive of a possible involvement in modulating the mosquito reproductive tissues physiology, a fact that could raise attention on the enzyme as a potential target for insect-containment strategies. The crystal structures of the enzyme in alternative ligand-bound states revealed elements distinguishing AgSULT-001425 from other characterized SULTs, including a peculiar conformational plasticity of a discrete region that shields the catalytic cleft and that could play a main role in the dynamics of the reaction and in the substrate selectivity of the enzyme. Along with further in vitro biochemical studies, our structural investigations could provide a framework for the discovery of small-molecule inhibitors to assess the effect of interfering with AgSULT-001425-mediated catalysis at the organismal level.

Variability of Genetic Characters Associated with Probiotic Functions in Lacticaseibacillus Species.

This study aims to explore the intra-species distribution of genetic characteristics that favor the persistence in the gastrointestinal tract (GIT) and host interaction of bacteria belonging to species of the Lacticaseibacillus genus. These bacterial species comprise commercial probiotics with the widest use among consumers and strains naturally occurring in GIT and in fermented food. Since little is known about the distribution of genetic traits for adhesion capacity, polysaccharide production, biofilm formation, and utilization of substrates critically important for survival in GIT, which influence probiotic characteristics, a list of genetic determinants possibly involved in such functions was created by a search for specific genes involved in the above aspects in the genome of the extensively characterized probiotic L. rhamnosus GG. Eighty-two gene loci were retrieved and their presence and variability in other Lacticaseibacillus spp. genomes were assessed by alignment with the publicly available fully annotated genome sequences of L. casei, L. paracasei, L. rhamnosus, and L. zeae. Forty-nine of these genes were found to be absent in some strains or species. The remaining genes were conserved and covered almost all the functions considered, indicating that all strains of the genus may exert some probiotic effects. Among the variable loci, a taurine utilization operon and a α-L-fucosidase were examined for the presence/absence in 26 strains isolated from infant feces by PCR-based tests. Results were variable among the isolates, though their common origin indicated the capacity to survive in the intestinal niche. This study indicated that the capacity to exert probiotic actions of Lacticaseibacillus spp. depends on a conserved set of genes but variable genetic factors, whose role is only in part elucidated, are more numerous and can explain the enhanced probiotic characteristics for some strains. The selection of the most promising probiotic candidates to be used in food is feasible by analyzing the presence/absence of a set of variable traits.

Lactobacilli Infection Case Reports in the Last Three Years and Safety Implications.

Lactobacilli constitute the dominant microbiota in many fermented foods and comprise widely used probiotics. However, these bacteria cause rare infections mostly in diabetic and immunocompromised subjects in presence of risk factors such as prosthetic hearth valves and dental procedures or caries. The scope of this survey was re-assessing the pathogenic potential of lactobacilli based on the infection case reports published in the last three years. In 2019, 2020, and 2021, total of 17, 15, and 16 cases, respectively, including endocarditis, bacteremia, and other infections, were reported. These annual numbers are higher than those observed previously. Lacticaseibacillus rhamnosus (13 cases), comprising strain GG (ATCC 53103) with established applications in healthcare, L. paracasei (7 cases), Lactobacillus acidophilus (5 cases), L. jensenii (5 cases), Lactiplantibacillus plantarum (3 cases), L. paraplantarum, L. delbrueckii subsp. delbrueckii, L. gasseri, L. paragasseri, Limosilactobacillus fermentum, and L. reuteri (1 case each) were involved. Virulence characterization of two strains that caused infections, a derivative of L. rhamnosus GG and L. paracasei LP10266, indicated that increased biofilm-forming capacity favors pathogenicity and it is determined by variable genetic traits. This survey highlights that the strains of lactobacilli that cause infections are little characterized genetically. Instead, to avoid that these bacteria become a hazard, genetic stability should be periodically re-evaluated by whole genome sequencing (WGS) to ensure that only non-pathogenic variants are administered to vulnerable individuals.

Draft Genome Sequence of Serratia rubidaea, a Potential Opportunistic Pathogen Isolated from Food in Italy.

Serratia rubidaea has emerged in recent years as an opportunistic nosocomial pathogen. Here, we present the draft genome sequence of an isolate derived from an industrial meat food product purchased in a large-scale retail store that revealed fluoroquinolone, ß-lactam, and aminoglycoside resistance genes and two different host-unspecific prophages.

The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate.

SNAP-tag ® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag ® and its relative thermostable version (SsOGT-H5 ) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag.

Effect of Water Stress on Reproduction and Colonization of Podosphaera aphanis of Strawberry.

In a number of pathosystems involving the powdery mildews (Erysiphales), plant stress is associated with decreased disease susceptibility and is detrimental to pathogen growth and reproduction. However, in strawberry, anecdotal observations associate severe powdery mildew (Podosphaera aphanis) with water stress. In a 2017 survey of 42 strawberry growers in Norway and California, 40 growers agreed with a statement that water-stressed strawberry plants were more susceptible to powdery mildew compared with nonstressed plants. In repeated in vitro and in vivo experiments, we found that water stress was consistently and significantly unfavorable to conidial germination, infection, and increases in disease severity. Deleterious effects on the pathogen were observed from both preinoculation and postinoculation water stress in the host. Soil moisture content in the range from 0 to 50% was correlated (R2 = 0.897) with germinability of conidia harvested from extant colonies that developed on plants growing at different levels of water stress. These studies confirm that P. aphanis fits the norm for biotrophic powdery mildews and hosts under stress. Mild water stress, compared with a state of optimal hydration, is likely to decrease rather than increase susceptibility of strawberry to P. aphanis. We believe it is possible that foliar symptoms of leaf curling due to diffuse and inconspicuous infection of the lower leaf surfaces by P. aphanis could easily be mistakenly attributed to water stress, which we observed as having a nearly identical leaf curling symptom in strawberry.

Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair.

Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis (TB), an ancient disease which still today causes 1.4 million deaths worldwide per year. Long-term, multi-agent anti-tubercular regimens can lead to the anticipated non-compliance of the patient and increased drug toxicity, which in turn can contribute to the emergence of drug-resistant MTB strains that are not susceptible to first- and second-line available drugs. Hence, there is an urgent need for innovative antitubercular drugs and vaccines. A number of biochemical processes are required to maintain the correct homeostasis of DNA metabolism in all organisms. Here we focused on reviewing our current knowledge and understanding of biochemical and structural aspects of relevance for drug discovery, for some such processes in MTB, and particularly DNA synthesis, synthesis of its nucleotide precursors, and processes that guarantee DNA integrity and genome stability. Overall, the area of drug discovery in DNA metabolism appears very much alive, rich of investigations and promising with respect to new antitubercular drug candidates. However, the complexity of molecular events that occur in DNA metabolic processes requires an accurate characterization of mechanistic details in order to avoid major flaws, and therefore the failure, of drug discovery approaches targeting genome integrity.

Crystal structure of Haemophilus influenzae 3-isopropylmalate dehydrogenase (LeuB) in complex with the inhibitor O-isobutenyl oxalylhydroxamate.

3-isopropylmalate dehydrogenases (LeuB) belong to the leucine biosynthetic pathway and catalyze the irreversible oxidative decarboxylation of 3IPM to 2-ketoisocaproate that is finally converted into leucine by a branched-chain aminotransferase. Since leucine is an essential amino acid for humans, and it is also vital for the growth of many pathogenic bacteria, the enzymes belonging to this pathway can be considered as potential target sites for designing of a new class of antibacterial agents. We have determined the crystal structure of the Haemophilus influenzae LeuB in complex with the cofactor NAD+ and the inhibitor O-IbOHA, at 2.1 Å resolution; moreover, we have investigated the inhibitor mechanism of action by analyzing the enzyme kinetics. The structure of H. influenzae LeuB in complex with the intermediate analog inhibitor displays a fully closed conformation, resembling the previously observed, closed form of the equivalent enzyme of Thiobacillus ferrooxidans in complex with the 3IPM substrate. O-IbOHA was found to bind the active site by adopting the same conformation of 3IPM, and to induce an unreported repositioning of the side chain of the amino acids that participate in the coordination of the ligand. Indeed, the experimentally observed binding mode of O-IbOHA to the H. influenzae LeuB enzyme, reveals aspects of novelty compared to the computational binding prediction performed on M. tuberculosis LeuB. Overall, our data provide new insights for the structure-based rational design of a new class of antibiotics targeting the biosynthesis of leucine in pathogenic bacteria.

Occurrence, Diversity of Listeria spp. Isolates from Food and Food-Contact Surfaces and the Presence of Virulence Genes.

This study evaluates the hazards posed by foodborne bacteria of the Listeria genus by analyzing the occurrence, diversity and virulence of Listeria spp.in food and food-manufacturing plants. Seventy-five isolates obtained from the routine analysis of 653 samples taken by three diagnostic laboratories in Northern Italy were genotypically differentiated by Repetitive Extragenic Palindrome (rep) PCR, with the GTG5 primer identified by sequencing the 16S rRNA gene and examined by specific PCR tests for the presence of L. monocytogenes virulence determinants occasionally found to occur in other species of the genus. Within this sample, 76% (n = 57) isolates were identified as L. innocua, 16% (n = 12) as L. monocytogenes, 6.6 % (n = 5) as L. welshimeri and 1.3% (n = 1) as L. seeligeri. All L. monocytogenes isolates belonged to the serotype 1/2a and were predicted to be virulent for the presence of the inlJ internalin gene. Potentially virulent strains of L. innocua, L. seeligeri and L. welshimeri, carrying the L. monocytogenesinlA gene and/or hly gene, were identified, and most isolates were found to possess the toxin-antitoxin system mazEF for efficient adaptation to heat shock. Results indicated the need to reinforce food-contamination-prevention measures against all Listeria species by defining efficiently their environmental distribution.

Members of the Lactobacillus Genus Complex (LGC) as Opportunistic Pathogens: A Review.

Microorganisms belonging to the Lactobacillus genus complex (LGC) are naturally associated or deliberately added to fermented food products and are widely used as probiotic food supplements. Moreover, these bacteria normally colonize the mouth, gastrointestinal (GI) tract, and female genitourinary tract of humans. They exert multiple beneficial effects and are regarded as safe microorganisms. However, infections caused by lactobacilli, mainly endocarditis, bacteremia, and pleuropneumonia, occasionally occur. The relevance of Lactobacillus spp. and other members of the LGC as opportunistic pathogens in humans and related risk factors and predisposing conditions are illustrated in this review article with more emphasis on the species L. rhamnosus that has been more often involved in infection cases. The methods used to identify this species in clinical samples, to distinguish strains and to evaluate traits that can be associated to pathogenicity, as well as future perspectives for improving the identification of potentially pathogenic strains, are outlined.

Study of Anopheles gambiae 3-hydroxykynurenine transaminase activity and inhibition by LC-MS/MS method.

In Anopheles gambiae, the most efficient vector of the malaria parasite Plasmodium falciparum, 3-hydroxykynurenine is endowed with a toxic potential. In adult mosquitoes, the excess of 3-hydroxykynurenine is removed by a specific transaminase (3-hydroxykynurenine transaminase, 3-HKT) which converts the compound into the more stable xanthurenic acid. Interfering with 3-hydroxykynurenine metabolism in A. gambiae is a potential approach for the development of transmission-blocking drugs and insecticides. Hence, the aims of this work were to develop and validate a new LC-MS/MS method for the evaluation of A. gambiae 3-hydroxykynurenine transaminase (Ag-HKT) activity and the determination of the potency of inhibitors of the enzyme. We set up a LC-MS/MS based enzymatic assay for the determination of kinetic constants values of the recombinant Ag-HKT enzyme and for the evaluation of Ag-HKT inhibition by a known protein inhibitor used as reference and a newly synthesized compound. The chromatographic separation was performed in a gradient mode on a Phenomenex Synergi Polar-RP (150 mm × 2.0, 4 µm) with methanol and water containing both 0.2% formic acid. Mass spectrometric detection was achieved with an ion trap equipped with an ESI source, in positive ionization scan, operating in SRM mode. The LC-MS/MS method was validated in terms of selectivity, linearity, precision and accuracy.

The Synthesis of Kynurenic Acid in Mammals: An Updated Kynurenine Aminotransferase Structural KATalogue.

Kynurenic acid (KYNA) is a bioactive compound that is produced along the kynurenine pathway (KP) during tryptophan degradation. In a few decades, KYNA shifted from being regarded a poorly characterized by-product of the KP to being considered a main player in many aspects of mammalian physiology, including the control of glutamatergic and cholinergic synaptic transmission, and the coordination of immunomodulation. The renewed attention being paid to the study of KYNA homeostasis is justified by the discovery of selective and potent inhibitors of kynurenine aminotransferase II, which is considered the main enzyme responsible for KYNA synthesis in the mammalian brain. Since abnormally high KYNA levels in the central nervous system have been associated with schizophrenia and cognitive impairment, these inhibitors promise the development of novel anti-psychotic and pro-cognitive drugs. Here, we summarize the currently available structural information on human and rodent kynurenine aminotransferases (KATs) as the result of global efforts aimed at describing the full complement of mammalian isozymes. These studies highlight peculiar features of KATs that can be exploited for the development of isozyme-specific inhibitors. Together with the optimization of biochemical assays to measure individual KAT activities in complex samples, this wealth of knowledge will continue to foster the identification and rational design of brain penetrant small molecules to attenuate KYNA synthesis, i.e., molecules capable of lowering KYNA levels without exposing the brain to the harmful withdrawal of KYNA-dependent neuroprotective actions.