Antimicrobial activity, membrane interaction and structural features of short arginine-rich antimicrobial peptides.

Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its ß-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

Effects of Essential Oils and Hydrolates on the Infectivity of Murine Norovirus.

The use of natural substances with antiviral properties might reduce foodborne viral diseases. In this study, we evaluated the virucidal effect of Citrus limon and Thymus serpyllum essential oils (EOs) and of Citrus Limon, Thymus serpyllum and Thymus vulgaris hydrolates on murine norovirus (MNV), a human norovirus surrogate. To assess the virucidal effect of these natural substances, the reduction in viral infectivity was estimated by comparing the TCID50/mL of untreated viral suspension and the viral suspension treated with hydrolates and EOs at different concentrations. The results showed a natural loss of infectivity of the untreated virus after 24 h of approx. 1 log. The EO (1%) of T. serpyllum, and hydrolates (1% and 2%) of T. serpyllum and T. vulgaris immediately caused a reduction in MNV infectivity of about 2 log but did not provide a further significant decrease after 24 h. Instead, the EO (1%) and hydrolate (1% and 2%) of C. limon exerted an immediate reduction in the viral infectivity of about 1.3 log and 1 log, respectively, followed by a further reduction in infectivity of 1 log after 24 h for the hydrolate. These results will allow for the implementation of a depuration treatment based on the use of these natural compounds.

SARS-CoV-2 detection in nasopharyngeal swabs: Performance characteristics of a real-time RT-qPCR and a droplet digital RT-PCR assay based on the exonuclease region (ORF1b, nsp 14).

The emergence and spread of SARS-CoV-2 has led to a compelling request for accurate diagnostic tests. The aim of this study was assessing the performance of a real-time RT-qPCR (rt RT-qPCR) assay and of a droplet digital RT-PCR (dd RT-PCR) targeting the nsp14 genome region for the detection of SARS-CoV-2 in nasopharyngeal swabs. A total of 258 nasopharyngeal swabs were analyzed with the nsp14 assays and, for comparison, with a reference assay targeting the RdRp and E genes. Conflicting results were further investigated by two additional protocols, the Centers for Disease Control and Prevention (CDC) real-time targeting N1/N2, and a nested RT-PCR for the spike region. Agreement of results was achieved on 226 samples (156 positive and 70 negative), 8 samples were positive in the reference assay and in the nsp14 rt RT-qPCR but negative with the dd RT-PCR, and 24 samples provided different combinations of results with the three assays. Sensitivity, specificity and accuracy (95 %C.I.) of the nsp14 assays were: 100.0 % (97.4-100.0), 98.7 % (92.1-100.0), and 99.6 % (97.5-100.0) for the rt RT-qPCR; 92.4 % (87.4-95.6), 100.0 % (94.2-100.0), and 94.7 % (91.1-97.0) for the dd RT-PCR. The results of the study support the use of the nsp14 real-time RT-qPCR and ddPCR for the detection of SARS-CoV-2 in nasopharyngeal swabs.

Quantitative Real-Time PCR and Digital PCR to Evaluate Residual Quantity of HAV in Experimentally Depurated Mussels.

Kinetics of hepatitis A virus (HAV) accumulation and depuration from mussels (Mytilus galloprovincialis) was studied in an experimental depuration system. Different parameters likely to influence the rate of virus accumulation and elimination were evaluated. Analyses were carried out by both real-time RT-qPCR and digital PCR. Results demonstrated that the animals start to concentrate the virus already after one hour and reach the maximum level of contamination in 6 h of experiment. With respect to depuration, HAV showed a rapid reduction of the concentration (89%) during the first 24-48 h of experiment and a very slow virus decrement in the following days with a 1% residual RNA at the ninth day of depuration. When process parameters likely to increase the depuration rate (presence of ozone, microalgal feeding, presence of lactic bacteria, pre-treatment with digestive enzymes) were tested, no significant differences in the kinetics were observed. Only treatment with pancreatin seemed to positively affect depuration in the first two days of the experiment.

Occurrence and Trend of Hepatitis A Virus in Bivalve Molluscs Production Areas Following a Contamination Event.

The aim of this study was to assess the trend of hepatitis A virus (HAV) in a coastal zone impacted by a contamination event, providing data for the development of management strategies. A total of 352 samples, including four bivalve mollusc species (Mytilus galloprovincialis, Solen vagina, Venus gallina and Donax trunculus), were taken over a period of 6 months from 27 production areas of the coast and analysis were performed according to ISO/TS 15216-1:2013. HAV presence was detected in 77 samples from 11 production areas and all positive results were related to samples collected in the first 3 months of the surveillance, during which HAV prevalence was 39.9% and values as high as 5096 genome copies/g were detected. A progressive reduction of viral contamination was evident during the first trimester of the monitoring, with prevalence decreasing from 78.8% in the first month, to 37.8% in the second and 3.9% in the third and quantitative levels reduced from an average value of 672 genome copies/g to 255 genome copies/g over a period of 4 weeks (virus half-life: 21.5 days). A regression analysis showed that, during the decreasing phase of the contamination, the data fitted a reciprocal quadratic model (Ra2 = 0.921) and, based on the model, a residual presence of HAV could be estimated after negativization of the production areas. The statistical analysis of the results per shellfish species and per production area showed that there were limited differences in contamination prevalence and levels among diverse bivalve species, while a statistically significant difference was present in quantitative levels of one production area. These data could be useful for the development of both risk assessment models and code of practice for the management of viral contamination in primary production.

Development of a colony hybridization method for the enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in shellfish.

Vibrio parahaemolyticus is a marine microorganism, recognized as cause of gastroenteritis outbreaks associated with seafood consumption. In this study the development and the in-house validation of a colony hybridization method for the enumeration of total and potentially pathogenic V. parahaemolyticus is reported. The method included a set of three controls (process, hybridization and detection control) for the full monitoring of the analytical procedure. Four digoxigenin-labeled probes were designed for pathogenic strains enumeration (tdh1, tdh2, trh1 and trh2 probes) and one for total V. parahaemolyticus count (toxR probe). Probes were tested on a panel of 70 reference strains and 356 environmental, food and clinical isolates, determining the inclusivity (tdh: 96.7%, trh: 97.8%, toxR: 99.4%) and the exclusivity (100% for all probes). Accuracy and linearity of the enumeration were evaluated on pure and mixed cultures: slopes of the regression lines ranged from 0.957 to 1.058 depending on the target gene and R(2) was greater than or equal to 0.989 for all reactions. Evaluation was also carried on using four experimentally contaminated seafood matrices (shellfish, finfish, crustaceans and cephalopods) and the slopes of the curves varied from 0.895 (finfish) to 0.987 (cephalopods) for the counts of potentially pathogenic V. parahaemolyticus (R(2)≥0.965) and from 0.965 to 1.073 for total V. parahaemolyticus enumeration (R(2)≥0.981). Validation was performed on 104 naturally contaminated shellfish samples, analyzed in parallel by colony hybridization, ISO/TS 21872-1 and MPN enumeration. Colony hybridization and ISO method showed a relative accuracy of 86.7%, and a statistically significant correlation was present between colony hybridization enumeration and MPN results (r=0.744, p<0.001). The proposed colony hybridization can be a suitable alternative method for the enumeration of total and potentially pathogenic V. parahaemolyticus in seafood.

Detection and quantification of Vibrio parahaemolyticus in shellfish from Italian production areas.

Vibrio parahaemolyticus is a marine microorganism, recognized as an important cause of foodborne illness particularly in Asia, South America and United States. Outbreaks are rarely reported in Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e. carrying the tdh and/or the trh genes) but currently there is a lack of occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian regions (Sardinia and Veneto) were analyzed for Escherichia coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic V. parahaemolyticus strains using a new DNA colony hybridization method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus were 84 and 73 CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7 × 10(3)CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120 CFU/g and most of the positive samples (n=17) showing values equal or below 20 CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency. The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help in defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs.

Evaluation of different polymerase chain reaction methods for the identification of Vibrio parahaemolyticus strains isolated by cultural methods.

Control of contamination by Vibrio parahaemolyticus in fishery products is often hampered by the lack of standardized methods and by the uncertainty associated with biochemical identification of the isolates. In this study, 5 polymerase chain reaction (PCR) methods for the identification of V. parahaemolyticus to the species level were evaluated by using 25 Vibrio reference strains and 163 isolates from fishery products, environmental sources, and clinical samples. Sequence targets of the methods were toxR, gyrB, and tlh genes (tested with 2 protocols), and the fragment pR72H. Isolate identification was confirmed by sequencing of the 16S rRNA gene and by PCR protocols for the identification of other Vibrio species. The PCR assay targeting the toxR gene achieved the highest performance (100% inclusivity and exclusivity). The 2 PCR protocols based on tlh gene detection, although showing the same inclusivity (100%), differed in the exclusivity (50 and 91%, respectively). Finally, the results provided by the PCR assays targeting the gyrB gene and pR72H fragment were less reliable and, in some cases, difficult to assess. According to the results of this study, the characteristics of accuracy expressed by the toxR identification method make it a suitable candidate as a reference method for the molecular identification of V. parahaemolyticus strains.