Improved SARS-CoV-2 RNA recovery in wastewater matrices using a CTAB-based extraction method.

Wastewater-based epidemiology has allowed tracking the magnitude and distribution of SARS-CoV-2 in communities, allowing public health officials to prepare for impending outbreaks. While many factors influence recovery of SARS-CoV-2 from wastewater, proper extraction, concentration, and purification of RNA are key steps to ensure accurate detection of viral particles. The aim of this study was to compare the efficiency of four commonly used RNA extraction methods for detection of the SARS-CoV-2 RNA genome in sewage samples artificially inoculated with the virus, in order to identify a protocol that improves viral recovery. These methods included CTAB-based, TRIzol-based, and guanidinium thiocyanate (GTC)-based extraction procedures coupled with silica spin column-based purification, and an automated extraction/purification protocol using paramagnetic particles. Following RNA extraction, virus recovery rates were compared using RT-qPCR-based detection. The CTAB-based approach yielded the highest recovery rates and was the only method to consistently demonstrate stable virus recovery percentages regardless of the specific physicochemical characteristics of the samples tested. The TRIzol method proved to be the second most effective, yielding significantly higher recovery rates compared to both the GTC-based and the automated extraction methods. These results suggest that the CTAB-based approach could be a useful tool for the recovery of viral RNA from complex wastewater matrices.

Meta-analysis of the prevalence of norovirus and hepatitis a virus in berries.

Norovirus (NoV) and hepatitis A virus (HAV) stand as the predominant agents associated with viral foodborne infections. Outbreaks have been documented to be caused by various types of food items, including fresh and/or frozen berries. Comprehensive data concerning crucial viral pathogens in berries remain limited and are not currently available in aggregate form. Consequently, the present study aimed to compile the existing information regarding the prevalence of NoV and HAV in this matrix. Records of foodborne viruses were systematically extracted from database repositories up to December 2022, adhering to PRISMA standards and were subjected to a multilevel random effect meta-analysis model to determine the mean occurrence rate of NoV and HAV. A high heterogeneity across studies was observed (I2 = 82 %), reflecting variations in the prevalence of sampling locations, years, berry types, and sample conditions, among other potential contributing factors. The prevalence of NoV and HAV in berries was calculated at 2.12 % (95 % CI 1.74-2.59 %), and no statistically differences were observed among the viral types or genogroup categories. However, it is important to clarify that this estimate should be taken with caution given the high heterogeneity. There was no discernible correlation between viral prevalence and any particular berry type. However, there was a temporal correlation observed with the year of sampling, revealing a significantly decreasing trend throughout the study period. A significant influence of the sample condition (fresh or frozen) was observed in relation to the prevalence of NoV GII and HAV. Overall higher viral prevalences were identified in berries originating from African countries as compared to those sourced from other continents. It was also noted that the prevalence of NoV GI was significantly higher in samples collected directly from farms compared to those obtained from retailers. The outcomes of this comprehensive meta-analysis propose that while viral contamination of berries is diminishing in more recent times, the prevalence remains substantial in certain African countries, having a significant risk for foodborne infections. It is imperative to implement intervention strategies in these regions to enhance product safety.

Meta-analysis of the prevalence of the main human pathogens in vegetables, with emphasis on lettuce.

This meta-analysis aims to summarize the available information on the prevalence of the main human pathogenic microorganisms in vegetables, with emphasis on lettuce (Lactuca sativa). The database searches included scientific papers from 1980 to 2019, without language restrictions. Inclusion criteria were prevalence or incidence studies published in peer-reviewed journals reporting the total number of vegetable samples studied and the number of samples positive for the presence of the studied pathogens. The target pathogens were grouped into the following categories: bacteria, parasites and viruses. Results of different vegetable types, years of sampling, analyzed regions or species of microorganisms reported in the same article were considered as different studies. Therefore, each scientific article may contain several studies. Multilevel random-effect meta-analysis models were fitted to estimate the mean occurrence rate of pathogenic microorganisms and to compare them with different factors potentially associated with the outcome. Overall, the prevalence of bacterial, parasitic and viral pathogens in vegetables was relatively low. The mean prevalence of bacterial hazards was < 0.023, with the exception of S. aureus, whose prevalence was estimated at 0.096. The mean occurrence rates of parasites and viruses were 0.067 (95 % CI: 0.056-0.080) and 0.079 (95 % CI: 0.054-0.113), respectively. The prevalence of pathogenic E. coli and parasites increased as the year of publication of the scientific articles progressed, whereas the prevalence of the other bacterial pathogens and enteric viruses was steady. The types of vegetables evaluated did not affect pathogen prevalence. The prevalence of pathogenic microorganisms differed according to the continent of origin, except for E. coli O157:H7 and parasites. The prevalence of pathogens in vegetables is of public health importance, especially in vegetable types that are eaten raw, without thermal treatment to inactivate pathogens. This meta-analysis results show the need to apply proper sanitation methods to treat raw vegetables in order to avoid foodborne infections.

Occurrence of Norovirus, Rotavirus, Hepatitis a Virus, and Enterovirus in Berries in Argentina.

Berries have been implicated as the probable vehicle of infection in multiple outbreaks of norovirus and hepatitis A virus (HAV). These foods often receive minimal or no processing and may be exposed to virus contamination at each stage of production. In an increasingly globalized world, berries have a wide distribution and can give rise to the spread of diseases in distant parts of the world. With the aim of describing the virological quality of the berries cultivated in Argentina, a total of 184 soft fruits of different varieties (strawberries, blueberries, raspberries, blackberries, currants, pomegranate arils, cassis, and elder) were collected during the periods 2016-2018 and 2020. Viral particles were eluted and concentrated by polyethylene glycol precipitation according to ISO 15216-2:2019 guidelines. Genome detection of norovirus (NoV) genogroups I (GI) and II (GII), HAV, rotavirus, and enterovirus was performed by real-time RT-PCR with TaqMan probes. Positive samples were amplified by conventional RT-PCR and the amplicons were purified and sequenced in both directions. Phylogenetic analysis was performed using the Neighbor-Joining method based on the evolutionary model Kimura-2-parameters. NoV GII.6 was detected in 1/184 (0.5%) of the soft fruits, corresponding to a raspberry sample obtained during the fall of 2017. No presence of other human enteric viruses was found in the other berries analyzed. The collected data are the first in Argentina in relation to the prevalence of enteric viruses in berries and is useful as reference data for a risk assessment of soft fruits as vehicles of foodborne pathogenic viruses.

Evaluation of viral concentration methods for SARS-CoV-2 recovery from wastewaters.

Wastewater-based epidemiology (WBE) is a useful tool that has the potential to act as a complementary approach to monitor the presence of SARS-CoV-2 in the community and as an early alarm system for COVID-19 outbreak. Many studies reported low concentrations of SARS-CoV-2 in sewage and also revealed the need for methodological validation for enveloped viruses concentration in wastewater. The aim of this study was to evaluate different methodologies for the concentration of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. A total of 11 concentration techniques based on different principles were evaluated: adsorption-elution protocols with negatively charged membranes followed by polyethylene glycol (PEG) precipitation (Methods 1-2), PEG precipitation (Methods 3-7), aluminum polychloride (PAC) flocculation (Method 8), ultrafiltration (Method 9), skim milk flocculation (Method 10) and adsorption-elution with negatively charged membrane followed by ultrafiltration (Method 11). To evaluate the performance of these concentration techniques, feline calicivirus (FCV) was used as a process control in order to avoid the risk associated with handling SARS-CoV-2. Two protocols, one based on PEG precipitation and the other on PAC flocculation, showed high efficiency for FCV recovery from wastewater (62.2% and 45.0%, respectively). These two methods were then tested for the specific recovery of SARS-CoV-2. Both techniques could recover SARS-CoV-2 from wastewater, PAC flocculation showed a lower limit of detection (4.3 × 102 GC/mL) than PEG precipitation (4.3 × 103 GC/mL). This work provides a critical overview of current methods used for virus concentration in wastewaters and the analysis of sensitivity for the specific recovery of SARS-CoV-2 in sewage. The data obtained here highlights the viability of WBE for the surveillance of COVID-19 infections in the community.

Potential of Lactic Acid Bacteria Isolated From Different Forages as Silage Inoculants for Improving Fermentation Quality and Aerobic Stability.

We aimed at isolating lactic acid bacteria (LAB) from different plant materials to study their crossed-fermentation capacity in silos and to find strains able to confer enhanced aerobic stability to silage. A total of 129 LAB isolates were obtained from lucerne (alfalfa), maize, sorghum, ryegrass, rice, barley, canola, Gatton panic, Melilotus albus, soy, white clover, wheat, sunflower, oat, and moha. Four Lactiplantibacillus plantarum subsp. plantarum strains (isolated from oat, lucerne, sorghum, or maize) were selected for their growth capacity. Identity (16S sequencing) and diversity (RAPD-PCR) were confirmed. Fermentative capacity (inoculated at 104, 105, 106, 107 CFU/g) was studied in maize silage and their cross-fermentation capacity was assessed in oat, lucerne, sorghum, and maize. Heterofermentative strains with the highest acetic acid production capacity conferred higher aerobic stability to maize silages. Regardless the source of isolation, L. plantarum strains, inoculated at a rate of 106 CFU/g, were effective to produce silage from different plant materials. From more than 100 isolates obtained, the application of a succession of experiments allowed us to narrow down the number of potential candidates of silage inoculants to two strains. Based on the studies made, L. plantarum LpM15 and Limosilactobacillus fermentum LfM1 showed potential to be used as inoculants, however further studies are needed to determine their performance when inoculated together. The former because it positively influenced different quality parameters in oat, lucerne, sorghum, and maize silage, and the latter because of its capacity to confer enhanced aerobic stability to maize silage. The rest of the strains constitute a valuable collection of autochthonous strains that will be further studied in the future for new applications in animal or human foods.

Saccharomyces uvarum isolated from patagonian ciders shows excellent fermentative performance for low temperature cidermaking.

Saccharomyces uvarum has been recovered from natural habitats and traditionally fermented beverages (apple chicha) in Patagonia. However, this species has never been obtained from industrially relevant beverages like wine or cider in the same region. In this work, different strains belonging to the cryotolerant species S. uvarum were recovered from spontaneous cider fermentations carried out at low temperature in Red Delicious apple must. The strain S. uvarum NPCC1420 obtained from this cider and selected for its physiological and technological features, evidenced a better adaptation to the cidermaking process than a previously selected strain obtained from a less industrialized product called apple chicha. Some differences, like a higher ethanol and sulphite tolerance, seemed to be associated with differential domestication pressures suffered by each different strain. Moreover, the most important fermentative features of the strain NPCC1420 were a higher competition capacity than the strain NPCC1314 in non-sterile apple must, as well as significantly higher amounts of glycerol, 2-phenylethanol and 2-phenylethyl acetate than the strain isolated from apple chicha.

Saccharomyces cerevisiae × Saccharomyces uvarum hybrids generated under different conditions share similar winemaking features.

Interspecific hybrids among species in the Saccharomyces genus are frequently detected in anthropic habitats and can also be obtained easily in the laboratory. This occurs because the most important genetic barriers among Saccharomyces species are post-zygotic. Depending on several factors, including the involved strains, the hybridization mechanism and stabilization conditions, hybrids that bear differential genomic constitutions, and hence phenotypic variability, can be obtained. In the present study, Saccharomyces cerevisiae × Saccharomyces uvarum hybrids were constructed using genetically and physiologically different S. uvarum parents at distinct temperatures (13 and 20°C). The effect of those variables on the main oenological features of the wines obtained with these hybrids was evaluated. Hybrids were successfully obtained in all cases. However, genetic stabilization based on successive fermentations in white wine at 13°C was significantly longer than that at 20°C. Our results demonstrated that, irrespective of the S. uvarum parent and temperature used for hybrid generation and stabilization, similar physicochemical and aromatic features were found in wines. The hybrids generated herein were characterized by low ethanol production, high glycerol synthesis and the capacity to grow at low temperature and to produce malic acid with particular aroma profiles. These features make these hybrids useful for the new winemaking industry within the climate change era frame. Copyright © 2017 John Wiley & Sons, Ltd.

Physiological characterization of Saccharomyces uvarum and Saccharomyces eubayanus from Patagonia and their potential for cidermaking.

A diversity of yeast strains belonging to the cryotolerant fermentative species S. uvarum and S. eubayanus have been recovered from natural habitats and traditional fermentations in North Patagonia. The aim of this work was to evaluate the most relevant physiological features in a set of Patagonian strains belonging to S. uvarum and S. eubayanus, in order to analyze their potentiality to be used as starter cultures for cidermaking elaborated at low temperature. We evidenced that S. uvarum strains isolated from natural habitats (Araucaria araucana bark) showed similar physiological features to S. eubayanus strains obtained from the same habitat, and different from S. uvarum strains from fermentative environments (apple chichas). We also confirm the capacity of S. uvarum to produce high glycerol levels, low acetic acid and elevated production of the higher alcohol 2-phenylethanol and 2-phenylethyl acetate and demonstrated similar properties in S. eubayanus. Finally, we evidenced for the first time the antagonistic activity of S. eubayanus and selected three strains (two S. uvarum and one S. eubayanus) bearing the best combination of features to be used as a starter culture in cidermaking.

Fate of Alicyclobacillus spp. in enrichment broth and in juice concentrates.

In this study, the fate of Alicyclobacillus acidoterrestris spores in different types of juice concentrates stored under different conditions was investigated. In addition, the impact of dilution procedures during the enrichment step for the detection of Alicyclobacillus in lemon juice concentrates was studied. Pear, red grape, mango, tangerine, carrot and lemon juice concentrates (50-69.4°Brix, pH1.7-4.3) were inoculated with A. acidoterrestris spores (10(3) spore/mL) and stored at 4 °C and 20 °C, after which the spores were counted at 0, 2, 5, 9, 17, 21, 28, 36, 43, and 50 days. No significant differences in the number of Alicyclobacillus spores were observed at storage temperatures of 4 °C and 20 °C (p>0.05). The results also indicated that the number of spores of A. acidoterrestris remained stable in all types of juice concentrates during the storage period, except in lemon juice concentrate. In lemon juice concentrate, a decline in A. acidoterrestris spore populations of 0.3-0.8 log CFU/mL was observed within 5-10 days of storage. The decline in A. acidoterrestris spore populations was more pronounced in cloudy lemon juice concentrate, which contained higher concentrations of flavonoids (mainly eriocitrin and hesperidin) than clarified lemon juice concentrate. It was also found that dilution of lemon juice concentrate samples in the proportion of 1:19 allowed the germination of A. acidoterrestris spores and the growth of populations of up to 10(7) CFU/mL. In contrast, the proportion (1:9) recommended in internationally recognized methods led to a reduction in the population of this microorganism that would yield false negative results. Data presented in this study demonstrated that Alicyclobacillus spores remain stable in most juice concentrates during storage, but that natural antimicrobial compounds present in some of them may decrease spore counts and inhibit their recovery by detection procedures.

Flavorings as new sources of contamination by deteriogenic Alicyclobacillus of fruit juices and beverages.

This study aimed to report the incidence of Alicyclobacillus and Alicyclobacillus acidoterrestris in apple and pear flavorings (n=42) and to assess the effect of guaiacol-producing A. acidoterrestris strains on apple flavorings stored at 4, 20 and 45 °C. A real-time polymerase chain reaction (RT-PCR) method was used for simultaneous confirmation of alicyclobacilli. A total of six isolates were identified as A. acidoterrestris, and only one strain was not able to produce guaiacol. The storage of apple flavoring at the optimum growth temperature of A. acidoterrestris (45 °C) resulted in the reduction in the spores' counts within 30 days of storage. On the other hand, during chilling (4 °C) and ambient storage conditions (20 °C) the counts of spores that remained stable for up to 60 days. An A. acidoterrestris strain inoculated in flavoring and further added to apple juice was able to grow and produce guaiacol in high amounts between 1-7 days of storage at 30 °C. In the current study it was shown that flavorings may be contaminated by deteriogenic A. acidoterrestris strains that are able to survive during storage in a wide range of temperature for long periods and further contaminate and spoil formulated fruit juices and beverages. A novel potential source of fruit juices and beverages contamination by deteriogenic Alicyclobacillus was shown. To the best of the author's knowledge, this is the first report on the incidence and fate of Alicyclobacillus and A. acidoterrestris in flavorings.

Use of a multivariate approach to assess the incidence of Alicyclobacillus spp. in concentrate fruit juices marketed in Argentina: results of a 14-year survey.

The purpose of this study was to determine the incidence of Alicyclobacillus spp. in fruit/vegetable juices (concentrated pulps and clarified and non-clarified juices) marketed in Argentina between 1996 and 2009. The presence of Alicyclobacillus was determined in a total of 8556 samples of fruit and vegetable juices (apple, pear, grape, peach, blend of juices, tangerine, pineapple, orange, mango, plum, guava, apricot, lemon, banana, kiwi, carrot, strawberry, grapefruit, and beetroot) collected in seven Argentinean provinces. Multiple factor analysis (MFA) was carried out on a data matrix that contained the percentage of positive samples, type of juice, raw material and production year. Except for kiwi and orange, Alicyclobacillus was found in juices from all the evaluated raw materials. The highest percentage of positive samples was found for beetroot, strawberry, banana, peach, mango, carrot and plum juices. The percentage of positive samples for these juices ranged from 100% to 24%. Furthermore, the application of multivariate techniques provided an insight on the relationship between the incidence of Alicyclobacillus and production variables. This approach enabled the identification of the most relevant variables that increased the percentage of positive samples among the juices, which could help in developing strategies to avoid the incidence of this bacterium. By means of hierarchical cluster analysis seven groups (clusters) of juices which showed different percentages of positive samples for Alicyclobacillus spp. were identified. This analysis showed that pineapple, peach, strawberry, mango and beetroot juices had higher rates of positivity for Alicyclobacillus than the rest of the evaluated juices. MFA analysis also showed that some clear relationships could be highlighted between the percentage of samples positive for Alicyclobacillus and five types of fruit juices (strawberry, beetroot, grapefruit, pineapple and mango). It was observed that a large proportion of juices produced in 2000, 2005 and 2008 were located in clusters with higher incidence of Alicyclobacillus spp., whereas a larger proportion of clarified concentrate juice and concentrate pulp samples showed higher probability of incidence of Alicyclobacillus in these products. Data presented in this study brings a contribution to the ecology of Alicyclobacillus in fruit/vegetable juices marketed in Argentina. This information would be useful to enhance the microbiological stability of fruit juices regarding the presence of Alicyclobacillus spp.