Search Results (23)

Virulence attributes and putative antibiotic resistance genes from enterococcal isolates from wastewater treatment facilities for sustainable reuse and the areas where they discharge treated water were assessed using phenotypic and molecular methods. This analysis was performed on 269 Enterococci, of which 202 were vancomycin-resistant Enterococcus (VRE). VRE strains show markedly higher resistance across multiple antibiotics, especially glycopeptides and beta-lactams, compared to the more susceptible profile observed in vancomycin-susceptible Enterococcus (VSE) strains. vanC was found in every instance of E. gallinarum among VRE and enterococci susceptible to vancomycin (VSE) isolates but not in VR E. faecium/faecalis. Among VRE, 127 (62.9%) possessed at least one of the tetK, tetL, tetM, or tetO, while 22 (17.3%) had two of these genes. The multidrug efflux pump gene emeA was detected in 27 out of 202 (13.4%) VRE isolates and 8 out of 67 (11.9%) VSE isolates. Exactly 69 (78.4%) possessed at least one of the virulence determinants tested, with 10 (11.4%) and seven (8%) positive for haemolysis and gelatinase activity respectively. The gelatinase gene, gelE, was detected in 16 (18.1%) isolates, while more isolates (n = 23; 26.1%) were positive for gelatinase activity. Cytolytic (cyl) genes (1.1%), Angiotensin-converting-enzyme genes (ace) (13.6%), endocarditis-specific antigen A genes (efaA) (25%), hyaluronidase (hyl) genes (9.1%), enterococcal surface protein (esp) genes (4.5%), among others, were detected. Gelatinase activity and the amplified virulence genes were further validated by sequencing the gel-positive amplicons, which were almost identical (98.97%), and the gelE gene of Enterococcus sp. strain SQ07C was deposited under the GenBank accession number PQ381122. Overall, our results showed that the enterococcal isolates were considered as potential pathogens of notable threat to human health via exposure through reuse, and there is a need for more stringent treatment protocols. Full article

Background: Equine influenza (EI) is a highly contagious viral disease of equids characterized by pyrexia and respiratory signs. Like other influenza A viruses, antigenic drift or shift could lead to a vaccine-induced immunity breakdown if vaccine strains are not updated. The aim of this study was to genetically characterize EIV strains circulating in Italy, detected in PCR-positive samples collected from suspected cases, especially in the absence of formal active surveillance. Methods: Between February and April 2019, blood samples and nasal swabs collected from each of the 20 symptomatic horses from North and Central Italy were submitted to the National Reference Centre for Equine Diseases in Italy to confirm preliminary analysis performed by other laboratories. Results: None of the sera analysed using haemagglutination inhibition and single radial haemolysis presented a predominant serological reactivity pattern for any antigen employed. All nasal swabs were positive with IAV RRT-PCR. Only one strain, isolated in an embryonated chicken egg from a sample collected from a horse of a stable located in Brescia, Lombardy, was identified as H3N8 Florida lineage clade 1 (FC1). In the constructed phylogenetic trees, this strain is located within the FC1, together with the virus isolated in France in 2018 (MK501761). Conclusions: This study reports the first detection of H3N8 FC1 in Italy, highlighting the importance of monitoring circulating EIV strains to verify the vaccine composition appropriateness for maximum efficacy. Full article

Certain Staphylococcus aureus strains harbour staphylococcal enterotoxin genes and hence can produce enterotoxin during their growth in food. Therefore, food can be a source of staphylococcal food poisoning, one of the most common food-borne diseases worldwide. Epidemiological data show that S. aureus is often present in raw milk cheeses, and consequently, cheeses are often the source of staphylococcal food poisoning outbreaks. The aim of this study was to determine the phenotypic characteristics of S. aureus isolates from fresh cheese, including antibiotic susceptibility; the presence of classical sea-see enterotoxin genes through molecular methods; and the isolate’s ability to produce SEA-SEE enterotoxins in vitro through reversed passive latex agglutination. A total of 180 coagulase-positive staphylococci were isolated from 18 out of 30 cheese samples, and 175 were confirmed as S. aureus through latex agglutination and API STAPH tests. All isolates possessed phenotypic characteristics typical for S. aureus, with certain variations in the egg yolk reaction (18.3% of the isolates showed a weak reaction and 28% no reaction at all) and haemolysis pattern (36.6% of the isolates produced double-haemolysis and 4.6% were non-haemolytic). Antibiotic resistance was observed in 1.1% of the isolates and to mupirocin only. Real-time PCR detected the sec gene in 34 (19.4%) isolates, but most isolates (80.6%) were not enterotoxigenic. For all 34 (19.4%) strains that carried the sec gene, the RPLA method detected the production of the SEC enterotoxin in vitro. For those enterotoxigenic strains, the possibility of enterotoxin production in fresh cheese could not be ruled out. Full article

Primaquine for radical cure of Plasmodium vivax malaria poses a potentially life-threatening risk of haemolysis in G6PD-deficient patients. Herein, we review five events of acute haemolytic anaemia following the administration of primaquine in four malaria trials from Indonesia, the Solomon Islands, and Vietnam. Five males aged 9 to 48 years were improperly classified as G6PD-normal by various screening procedures and included as subjects in trials of anti-relapse therapy with daily primaquine. Routine safety monitoring by physical examination, urine inspection, and blood haemoglobin (Hb) assessment were performed in all those trials. Early signs of acute haemolysis, i.e., dark urine and haemoglobin drop >20%, occurred only after day 3 and as late as day 8 of primaquine dosing. All patients were hospitalized and fully recovered, all but one following blood transfusion rescue. Hb nadir was 4.7 to 7.9 g/dL. Hospitalization was for 1 to 7 days. Hb levels returned to baseline values 3 to 10 days after transfusion. Failed G6PD screening procedures in these trials led G6PD-deficient patients to suffer harmful exposures to primaquine. The safe application of primaquine anti-relapse therapy requires G6PD screening and anticipation of its failure with a means of prompt detection and rescue from the typically abrupt haemolytic crisis. Full article

The search for novel microRNA (miRNA) biomarkers in plasma is hampered by haemolysis, the lysis and subsequent release of red blood cell contents, including miRNAs, into surrounding fluid. The biomarker potential of miRNAs comes in part from their multicompartment origin and the long-lived nature of miRNA transcripts in plasma, giving researchers a functional window for tissues that are otherwise difficult or disadvantageous to sample. The inclusion of red-blood-cell-derived miRNA transcripts in downstream analysis introduces a source of error that is difficult to identify posthoc and may lead to spurious results. Where access to a physical specimen is not possible, our tool will provide an in silico approach to haemolysis prediction. We present DraculR, an interactive Shiny/R application that enables a user to upload miRNA expression data from a short-read sequencing of human plasma as a raw read counts table and interactively calculate a metric that indicates the degree of haemolysis contamination. The code, DraculR web tool and its tutorial are freely available as detailed herein. Full article

Equine influenza (EI) is a fast-spreading respiratory disease of equids caused by equine influenza A virus (EIV), often resulting in high morbidity and a huge economic impact on the equine industry globally. In this cross-sectional study to determine the seroprevalence of EI and its associated risk factors, sera from 830 horses bled on a single occasion in Northwest Nigeria between October 2019 and January 2020 were screened for antibodies to A/equine/Richmond/1/2007 (H3N8) using the single radial haemolysis (SRH) assay. Antibodies were detected in 71.3% (592/830, 95% CI: 68–74%) of horses (SRH area ≥ 0.5 mm²). Although there were statistically significant univariable associations between seropositivity and age, sex, breed, purpose and coat colour, only age remained significant when included with each of the other variables in bivariable analyses. There was a clear trend for increasing odds of seropositivity with increasing age: OR 1.6, 95% CI: 1.05–2.40 (p = 0.03) for 5–14-year-olds and OR 8.13, 95% CI: 2.75–24.1 (p < 0.001) for ≥15-year-olds compared to horses <5 years old. The mean SRH value was 78.2 mm² (median = 88 mm², interquartile range = 0–121 mm²) with only 9% of the horses having an SRH value > 150 mm², considered sufficient to protect against clinical disease and virus shedding. Comparative screening of a subset of the horses (n = 118) with a 2019 H3N8 virus (A/equine/Worcestershire/2019) revealed a significantly greater seropositivity (p = 0.0001) than A/equine/Richmond/1/2007 consistent with exposure of the population during a widespread outbreak of EI in the region in 2019. In conclusion, there was an insufficient level of protection against EI in the region and introduction of a vaccination programme with vaccines containing recently circulating virus is recommended to mitigate against further outbreaks of EI in Nigeria. Full article

Aerial drone technology is currently being investigated worldwide for the delivery of blood components. Although it has been demonstrated to be safe, the delivered medical substances still need to be analyzed at the end of the flight mission to assess the level of haemolysis and pH prior to the use in a patient. This process can last up to 30 min and prevent the time saved using drone delivery. Our study aims to integrating an innovative sensor for the haemolysis and pH detection into the Smart Capsule, an already demonstrated technology capable of managing transfusion transport through drones. In the proposed scenario, the haemolysis is evaluated optically by a minilysis device using LED–photodetector combination. The preliminary validation has been demonstrated for both the thermal stability of the Smart Capsule and the haemolysis detection of the minilysis device prototype. Firstly, the onboard temperature test has shown that the delivery system is capable of maintaining proper temperature, even though the samples have been manipulated to reach a higher temperature before inserting into the Smart Capsule. Then, in the laboratory haemolysis test, the trend of linear regression between the outputs from the spectrophotometer and the minilysis prototype confirmed the concept design of the minilysis device. Full article

The abundance of cell-free microRNA (miRNA) has been measured in blood plasma and proposed as a source of novel, minimally invasive biomarkers for several diseases. Despite improvements in quantification methods, there is no consensus regarding how haemolysis affects plasma miRNA content. We propose a method for haemolysis detection in miRNA high-throughput sequencing (HTS) data from libraries prepared using human plasma. To establish a miRNA haemolysis signature we tested differential miRNA abundance between plasma samples with known haemolysis status. Using these miRNAs with statistically significant higher abundance in our haemolysed group, we further refined the set to reveal high-confidence haemolysis association. Given our specific context, i.e., women of reproductive age, we also tested for significant differences between pregnant and non-pregnant groups. We report a novel 20-miRNA signature used to identify the presence of haemolysis in silico in HTS miRNA-sequencing data. Further, we validated the signature set using firstly an all-male cohort (prostate cancer) and secondly a mixed male and female cohort (radiographic knee osteoarthritis). Conclusion: Given the potential for haemolysis contamination, we recommend that assays for haemolysis detection become standard pre-analytical practice and provide here a simple method for haemolysis detection. Full article

Plasma extraction from blood is essential for diagnosis of many diseases. The critical process of plasma extraction requires removal of blood cells from whole blood. Fluid viscoelasticity promotes cell migration towards the central axis of flow due to differences in normal stress and physical properties of cells. We investigated the effects of altering fluid viscoelasticity on blood plasma extraction in a serpentine microchannel. Poly (ethylene oxide) (PEO) was dissolved into blood to increase its viscoelasticity. The influences of PEO concentration, blood dilution, and flow rate on the performance of cell focusing were examined. We found that focusing performance can be significantly enhanced by adding PEO into blood. The optimal PEO concentration ranged from 100 to 200 ppm with respect to effective blood cell focusing. An optimal flow rate from 1 to 15 µL/min was determined, at least for our experimental setup. Given less than 1% haemolysis was detected at the outlets in all experimental combinations, the proposed microfluidic methodology appears suitable for applications sensitive to haemocompatibility. Full article

This study aimed to determine the prevalence of several pathovirotypes and evaluate the association of haemolysis with the virotypes of pathogenic E. coli isolated from post-weaning piglets in South Korea from 2015 to 2019. We isolated 890 E. coli and tested for O-serogroups, virulence genes, haemolysis, and multilocus sequence typing. The predominant virotypes were STb:EAST1:AIDA-I, F18b:Stx2e:AIDA-I, F18:STa:STb:Stx2e, and eae:Paa in enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), ETEC/STEC, and enteropathogenic E. coli (EPEC), respectively. Regarding serogroups, O139, O149, O141, and O121 were mostly detected in F18:Stx2e:AIDA-I, F4:LT:STb:EAST1, F18:STa:STb, and F18:Stx2e:EAST1, respectively. There was a significant change in the frequency of the O141:F18ac:STa:STb (an increase from 1.6% to 10.1%) and O139:F18ab:Stx2e:AIDA-I (a decrease from 13.0% to 5.3%) virotypes in ETEC and STEC, respectively, from 2015 to 2019. The O141:F18ac:STa:STb virotype was mostly detected in the central area and was spreading to the southern area. The odds ratios between haemolysis and virotypes were 11.0, 6.25, and 8.57 in F18:STa:STb, F18:Stx2e:AIDA-I, and F4:LT:STb:EAST1, respectively. Our findings provide insights regarding the recent prevalence of pathogenic E. coli in South Korea and could be used for the development of vaccines for E. coli responsible for PWD and ED in post-weaning piglets. Full article

European consumers have a growing interest in new fruits and flavours, which has promoted the production and commercialisation of exotic fruits such as goldenberry (Physalis peruviana L., Fam. Solanaceae). Colombia and South Africa are currently the main producers of this fruit, but it can be cultivated in almost all the highlands of the tropics and in several parts of the subtropics, given its ability to adapt to a wide range of agroecological conditions [¹]. Consumer demand for this small orange berry has also been driven by its nutritional value and health-promoting effects [²]. Although there have been different studies about this fruit, the available information is still scarce. Furthermore, the fruit quality attributes can vary depending on the agricultural practices used and the edaphoclimatic conditions of the growing sites. Therefore, this study was carried out to evaluate the nutritional composition and in vitro antioxidant and antimicrobial activity of goldenberry cultivated in the northeast region of Portugal. Goldenberry samples were lyophilised and analysed to determine their proximate composition (moisture, protein, fat, ash, dietary fibre, and carbohydrates), following official methods of food analysis [³]. Their profiles of sugars, organic acids, fatty acids, and tocopherols were characterised by standardised chromatographic methods [³]. The powdered goldenberry sample was also prepared in a hydroethanolic extract used for the evaluation of antioxidant activity (by oxidative haemolysis and lipid peroxidation inhibition assays) and antimicrobial effects against foodborne bacteria and fungi (by serial microdilution methods) [³]. The nutritional analysis revealed high levels of carbohydrates (manly fructose and glucose) and fibre and a lipid fraction consisting mainly of polyunsaturated fatty acids. Citric and ascorbic acids were detected in high amounts, as well as the four tocopherol isoforms. The hydroethanolic berry extract showed capacity for inhibiting haemolytic oxidation and lipid peroxidation, antibacterial effects against Staphylococcus aureus and Bacillus cereus, and antifungal activity against Aspergillus and Penicillium strains. Overall, this work highlighted the nutritional value of goldenberry, which has been noted as a functional fruit with a growing presence in the Portuguese market. Full article

Biostimulants are a novel and eco-friendly agronomic tool with practical applications in alleviating negative effects of environmental stressors. The present work studied the effects of three biostimulant products (Nomoren (N), Twin-Antistress (TW), and X-Stress (XS)) under normal irrigation (W+) and water deficit irrigation conditions (W−) on the nutritional, chemical composition and bioactive properties of common bean fresh pods. A variable effect of biostimulants and water deficit irrigation was observed on nutritional value parameters, while fructose and sucrose were the main detected sugars, especially in NW+ and CW− treatments. Oxalic, malic, and citric acid were the main detected organic acids, while γ- and total tocopherol content was the highest in TWW+. (+)-Catechin and (−)-epicatechin were the most abundant phenolic compounds, especially in the NW− treatment. A variable antioxidant capacity was observed for the Thiobarbituric Acid Reactive Substances (TBARS) and Oxidative Haemolysis assays (OxHLIA), while TWW+ extracts showed the best overall results against the tested fungi. In conclusion, the tested biostimulants had a positive effect on chemical composition and bioactivities of purple bean depending on the irrigation regime. Full article

The vibrant colours of many plants are due to secondary metabolites, such as nitrogen-containing compounds, where betacyanins are included. These compounds can be found in plants such as Amaranthus caudatus L. that, due to their high nutritional benefits, have been overproduced, which leads to the accumulation of large amounts of bio-residues. Among these bio-residues, the flowers which have a very intense pink colour and present no economic value or subsequent destination can be exploited as sources of natural colouring agents (betacyanins). This work aimed at characterising the flower’s extract in terms of bioactive molecules such as tocopherols, organic acids, but essentially in terms of betacyanins, in order to obtain a natural colouring agent. For the extraction, ultrasound-assisted extraction (UAE) ideal conditions were obtained using the Response Surface Methodology (RSM), allowing the attainment of an enriched extract of betacyanins in high yields and purity. The obtained extracts were analysed for their bioactive potential, namely antioxidant, antimicrobial and cytotoxic properties. From the obtained results, three isoforms of tocopherols were detected, β-tocopherol (0.884 ± 0.003 mg/100 g dry weight (dw)) being the most abundant one. Regarding the organic acids, oxalic (2.48 ± 0.05 mg/100 g dw), shikimic (0.170 ± 0.003 mg/100 g dw) and traces of fumaric acid were found. Four betacyanins were identified and quantified, namely: amaranthine (171 ± 1 mg/g extract), isoamaranthine (38 ± 1 mg/g extract), betanin (1.6 ± 0.1 mg/g), and isobetanin (1.3 ± 0.1 mg/g extract). The obtained extract also presented antioxidant activity with inhibition concentration (IC₅₀ values) of 29.0 ± 0.4 μg/mL and 114 ± 4 μg/mL for Δt of 60 min and 120 min, respectively in the oxidative haemolysis inhibition assay (OxHLIA) assay. The obtained extract also presented an interesting antibacterial activity with minimum inhibitory concentrations ranging from 5 to 20 mg/mL against pathogenic bacteria and revealed no toxicity for normal cells. Full article

In this research, our aim was to assess the occurrence of Staphylococcus aureus in a Hungarian large-scale dairy farm during the S. aureus control program conducted in the course of our studies. Furthermore, the phenotypic and genotypic properties of the isolates (type of haemolysis, antibiotic susceptibility, staphylococcal enterotoxin (SE) gene carrying ability and spa type) were determined. S. aureus was detected in all bulk tank milk samples collected during this study. Two different spa types were identified among the 17 strains isolated in the farm. A total of 14 of the 17 studied strains (82%) showed β-haemolysis on blood agar, 2/17 strains (12%) expressed double zone and 1/17 strains (6%) showed weak β-haemolysis. All strains were susceptible to most antibiotics tested (cefoxitin, chloramphenicol, clindamycin, erythromycin, gentamicin, tetracycline and trimethoprim/sulphamethoxazole), but all strains were resistant to penicillin G. A total of 11 of the 17 strains (65%) were found to harbour seg, sei, selm, seln, selo genes; 4/17 strains (24%) harboured sei, selm, seln, selo genes and 2/17 strains (11%) harboured sei gene. Since the new SEs/SEls can also cause foodborne outbreaks potentially and all strains were found to be resistant to penicillin G, it is essential to decrease and keep the prevalence of S. aureus low in the dairy farm and the implementation of the S. aureus control program is also highly justified. The results showed that the S. aureus count decreased by the end of our studies, so the control program was proved to be effective. Full article

Hereditary factor XIII (FXIII) deficiency is a rare autosomal bleeding disorder which can cause life-threatening bleeding. Acquired deficiency can be immune-mediated or due to increased consumption or reduced synthesis. The most commonly used screening test is insensitive, and widely used quantitative assays have analytical limitations. The present study sought to validate Technofluor FXIII Activity, the first isopeptidase-based assay available on a routine coagulation analyser, the Ceveron s100. Linearity was evidenced throughout the measuring range, with correlation coefficients of >0.99, and coefficients of variation for repeatability and reproducibility were <5% and <10%, respectively. A normally distributed reference range of 47.0–135.5 IU/dL was derived from 154 normal donors. Clinical samples with Technofluor FXIII Activity results between 0 and 167.0 IU/dL were assayed with Berichrom^® FXIII Activity, a functional ammonia release assay, and the HemosIL^™ FXIII antigen assay, generating correlations of 0.950 and 0.980, respectively. Experiments with a transglutaminase inhibitor showed that Technofluor FXIII Activity can detect inhibition of enzymatic activity. No interference was exhibited by high levels of haemolysis and lipaemia, and interference by bilirubin was evident at 18 mg/dL, a level commensurate with severe liver disease. Technofluor FXIII Activity is a rapid, accurate and precise assay suitable for routine diagnostic use with fewer interferents than ammonia release FXIII activity assays. Full article