A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads

Viruses are present at low concentrations in wastewater, and therefore an effective concentration of virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the concentrated over that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency became significantly higher when calculated based on virus titers than genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk due to wastewater contaminated with infectious enteric viruses. PCR inhibitors were not concentrated by PGM-MBs, suggesting this tool will be successful for use with environmental samples. The PGM-MBs method is cost-effective (0.43 USD/sample) and fast turnaround (3 hours from virus concentration to genome quantification), and thus this method can be implemented for high throughput facilities. Based on good performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, we are confident that the PGM-MBs method can be applied for successful WBE and ultimately provides valuable public health information.Graphical abstract

Interactions of β-Lactoglobulin with Bovine Submaxillary Mucin vs. Porcine Gastric Mucin: The Role of Hydrophobic and Hydrophilic Residues as Studied by Fluorescence Spectroscopy

The aim of this study was to investigate binding interactions between β-lactoglobulin (BLG) and two different mucins, bovine submaxillary mucins (BSM) and porcine gastric mucin (PGM), using intrinsic and extrinsic fluorescence spectroscopies. Intrinsic fluorescence spectra showed an enhanced decrease of fluorescence intensity of BLG at all pH conditions when BLG was mixed with PGM rather than with BSM. We propose that, unlike BSM, the tertiary structure of PGM changes and the hydrophobic regions are exposed at pH 3 due to protonation of negatively charged residues. Results suggest that PGM also facilitated the structural unfolding of BLG and its binding with PGM by a hydrophobic interaction, especially at acidic pH, which was further supported by extrinsic fluorescence spectroscopy. Hydrophobic interaction is suggested as the dominant interaction mechanism between BLG and PGM at pH 3, whereas electrostatic interaction is the dominant one between BLG and BSM.

Impact of artificial sputum media formulation on Pseudomonas aeruginosa secondary metabolite production

In vitro culture media are being developed to understand how host site-specific nutrient profiles influence microbial pathogenicity and ecology. To mimic the cystic fibrosis (CF) lung environment, a variety of artificial sputum media (ASM) have been created. However, the composition of these ASM vary in the concentration of key nutrients, including amino acids, lipids, DNA, and mucin. In this work, we used feature-based molecular networking (FBMN) to perform comparative metabolomics of Pseudomonas aeruginosa , the predominant opportunistic pathogen infecting the lungs of people with CF, cultured in nine different ASM. We found that the concentration of aromatic amino acids and iron from mucin added to the media contribute to differences in the production of P. aeruginosa virulence-associated secondary metabolites. IMPORTANCE Different media formulations aiming to replicate in vivo infection environments contain different nutrients, which affects interpretation of experimental results. Inclusion of undefined components, such as commercial porcine gastric mucin (PGM), in an otherwise chemically defined medium can alter the nutrient content of the medium in unexpected ways and influence experimental outcomes.

Impact of mucin on the anti-erosive/anti-abrasive efficacy of chitosan and/or F/Sn in enamel in vitro

The application of stannous ions in combination with fluoride (F/Sn) is one of the central strategies in reducing erosive tooth wear. F/Sn efficacy can be enhanced by adding chitosan, a positively charged biopolymer. For patients with low saliva flow, this efficacy, however, is not sufficient, making further improvement desirable. This could be achieved by combining chitosan with other molecules like mucin, which together might form multilayers. This in-vitro study aimed to investigate the effect of chitosan, mucin, F/Sn and combinations thereof on enamel erosion and erosion-abrasion. Human enamel samples (n = 448, 28 groups) were cyclically eroded or eroded-abraded (10 days; 6 × 2 min erosion and 2 × 15 s/200 g abrasion per day). Samples were treated 2 × 2 min/day with solutions containing either, chitosan (50 or 500 mPas), porcine gastric mucin, F/Sn or combinations thereof after abrasive challenge. Tissue loss was measured profilometrically, interaction between hard tissue and active agents was assessed with energy dispersive spectroscopy and scanning electron microscopy. Chitosan and F/Sn showed the expected effect in reducing tissue loss under erosive and under erosive-abrasive conditions. Neither mucin alone nor the combinations with mucin showed any additional beneficial effect.

Interpolymer Complexes Based on Carbopol® and Poly(2-ethyl-2-oxazoline) as Carriers for Buccal Delivery of Metformin

Introduction. Buccal drug delivery has a number of advantages over oral administration: ease of application, good blood supply to the buccal mucosa, drug can enter the systemic circulation directly, avoiding the "first pass effect through the liver", and are not exposed to the acidic environment of the gastric juice and the destructive action of digestive enzymes. The use of interpolymer complexes (IPCs) makes it possible not only to ensure adhesion to the mucosal membranes of the oral cavity, but also to achieve a prolonged release of drugs.Aim. Development of carriers based on interpolymer complexes using Carbopol® 971 NF (C971) and poly(2-ethyl-2-oxazoline) (POZ) of different molecular weights for buccal delivery of metformin (MF).Materials and methods. The study of IPC adhesion was carried out using a TA.XTplus texture analyzer (Stable Micro Systems, UK); mucin compacts with a diameter of 13 mm were used as a substrate; these were prepared by compressing porcine gastric mucin powder using a manual hydraulic press for IR spectroscopy (PerkinElmer, USA) at a pressure of 2.45 MPa. The study of the swelling capacity was carried out by placing polymer matrices in an artificial saliva medium, with constant thermostating at a temperature of 37.0 ± 0.5 °C for 5 hours. The study of the release of MF from the matrices based on IPC was carried out using a DFZ II apparatus (Erweka, Germany) according to the Flow Through Cell method (USP IV) with cells for tablets (22.6 mm) and adaptors for ointments, creams and gels in a medium simulating saliva. The concentration of MF in the samples from the dissolution tests was determined with UV-spectrophotometry (Lambda, PerkinElmer, USA) at 232.8 nm.Results and discussion. In a comparative study of the mucoadhesive properties of polymer samples, IPC compacts showed a mucoadhesion capacity comparable to that of poly(2-ethyl-2-oxazoline); at the same time, compacts from physical mixtures (PM) and C971 are inferior in terms of the separation force to IPC samples, however, POZes dissolve in an artificial saliva medium, that is, they are not suitable as dosage forms for buccal delivery. For 5 hours of the experiment to assess the swelling capacity, the IPC matrices did not change significantly, which can ensure their comfortable use as carriers for buccal delivery. When evaluating the release of metformin from polymer matrices (with weight ratio MF/IPC 1: 0.5), the most complete release (more than 90 %) is observed from both IPC matrices compared to matrices of PM and individual polymers.Conclusion. Polycomplex matrix systems based on C971-POZ (50 kDa) and C971-POZ (500 kDa) are suitable for buccal delivery of metformin.

Artificial Sputum Medium Formulation Impacts Pseudomonas aeruginosa Phenotype and Chemotype

ABSTRACTArtificial sputum medium (ASM) is a class of in vitro bacterial culture medium intended to mimic the nutritional environment of cystic fibrosis (CF) pulmonary mucus. One of the most commonly studied microbes in ASM is Pseudomonas aeruginosa, a prevalent and dangerous pathogen of the CF pulmonary microbiome. Many ASM formulations have been reported in literature, with differing nutrient concentrations and availability. Here, we show that common formulations of ASM yield different phenotypes and chemotypes of P. aeruginosa. Further, we demonstrate that iron in commercial porcine gastric mucin (PGM) is sufficient to alter production of P. aeruginosa siderophores in the chemically defined ASM, synthetic CF medium 1 (SCFM1). These results highlight that the choice of ASM formulation for in vitro investigations of microbial pathogenicity, physiology, and interactions should be carefully considered.IMPORTANCEIn vitro culture media are being developed to resemble the in vivo nutritional environment more closely. These culture media are used to investigate microbial pathogenicity and ecology in environments that are more reflective of disease states. In cystic fibrosis (CF), a number of different artificial sputum media (ASM) formulations have been created to recapitulate the CF lung environment. However, these ASM have different sources and concentrations of nutrients. Here, we cultured Pseudomonas aeruginosa in nine different formulations of ASM. P. aeruginosa is the primary pathogen causing lung infection in CF.We show that different ASM formulations lead to different phenotypes and chemotypes by P. aeruginosa and one component of ASM, mucin, contains high levels of iron, which may affect P. aeruginosa physiology.

Mucin-derived O-glycans supplemented to diet mitigate diverse microbiota perturbations

Microbiota-accessible carbohydrates (MACs) are powerful modulators of microbiota composition and function. These substrates are often derived from diet, such as complex polysaccharides from plants or human milk oligosaccharides (HMOs) during breastfeeding. Host-derived mucus glycans on gut-secreted mucin proteins serve as a continuous endogenous source of MACs for resident microbes; here we investigate the potential role of purified, orally administered mucus glycans in maintaining a healthy microbial community. In this study, we liberated and purified O-linked glycans from porcine gastric mucin and assessed their efficacy in shaping the recovery of a perturbed microbiota in a mouse model. We found that porcine mucin glycans (PMGs) and HMOs enrich for taxonomically similar resident microbes. We demonstrate that PMGs aid recovery of the microbiota after antibiotic treatment, suppress Clostridium difficile abundance, delay the onset of diet-induced obesity, and increase the relative abundance of resident Akkermansia muciniphila. In silico analysis revealed that genes associated with mucus utilization are abundant and diverse in prevalent gut commensals and rare in enteric pathogens, consistent with these glycan-degrading capabilities being selected for during host development and throughout the evolution of the host–microbe relationship. Importantly, we identify mucus glycans as a novel class of prebiotic compounds that can be used to mitigate perturbations to the microbiota and provide benefits to host physiology.

Evaluation of Copper Alloy Surfaces for Inactivation of Tulane Virus and Human Noroviruses

ABSTRACT This study evaluated the efficacy of copper alloy surfaces for inactivation of Tulane virus (TV), assessed by plaque assay and porcine gastric mucin–conjugated magnetic bead (PGM-MB) binding assay, followed by quantitative reverse transcription PCR (PGM-MB–RT-qPCR assay). In addition, the efficacy of a copper surface for inactivation of human norovirus (HuNoV) GII.4 Sydney and GI.3B Potsdam strains was evaluated by PGM-MB–RT-qPCR assay. Results of time-dependent inactivation of viruses on copper, bronze, and brass coupons revealed that 15 min of surface treatments of each of the copper and copper alloys achieved >4-log reduction of purified TV, as assessed by plaque assay, while up to 20 min of copper alloy surface treatments only achieved ∼2-log reduction, as assessed by PGM-MB–RT-qPCR assay. As assessed by PGM-MB–RT-qPCR assay, 10 min of copper surface treatments achieved reductions of 3 and 4 log units for HuNoVs GII.4 Sydney and GI.3B Potsdam, respectively. Results from this study suggest that even though PGM-MB–RT-qPCR assay underestimated the efficacy of copper alloy surface inactivation of TV, copper alloy surfaces were able to effectively inactivate TV and HuNoVs. Therefore, copper alloys can be used as a preventive measure to prevent HuNoV infection and are an effective surface treatment for HuNoVs. HIGHLIGHTS

The Basis of Peracetic Acid Inactivation Mechanisms for Rotavirus and Tulane Virus under Conditions Relevant for Vegetable Sanitation

ABSTRACT We determined the disinfection efficacy and inactivation mechanisms of peracetic acid (PAA)-based sanitizer using pH values relevant for vegetable sanitation against rotavirus (RV) and Tulane virus (TV; a human norovirus surrogate). TV was significantly more resistant to PAA disinfection than RV: for a 2-log10 reduction of virus titer, RV required 1 mg/liter PAA for 3.5 min of exposure, while TV required 10 mg/liter PAA for 30 min. The higher resistance of TV can be explained, in part, by significantly more aggregation of TV in PAA solutions. The PAA mechanisms of virus inactivation were explored by quantifying (i) viral genome integrity and replication using reverse transcription-quantitative PCR (RT-qPCR) and (ii) virus-host receptor interactions using a cell-free binding assay with porcine gastric mucin conjugated with magnetic beads (PGM-MBs). We observed that PAA induced damage to both RV and TV genomes and also decreased virus-receptor interactions, with the latter suggesting that PAA damages viral proteins important for binding its host cell receptors. Importantly, the levels of genome-versus-protein damage induced by PAA were different for each virus. PAA inactivation correlated with higher levels of RV genome damage than of RV-receptor interactions. For PAA-treated TV, the opposite trends were observed. Thus, PAA inactivates each of these viruses via different molecular mechanisms. The findings presented here potentially contribute to the design of a robust sanitation strategy for RV and TV using PAA to prevent foodborne disease. IMPORTANCE In this study, we examined the inactivation mechanisms of peracetic acid (PAA), a sanitizer commonly used for postharvest vegetable washing, for two enteric viruses: Tulane virus (TV) as a human norovirus surrogate and rotavirus (RV). PAA disinfection mechanisms for RV were mainly due to genome damage. In contrast, PAA disinfection in TV was due to damage of the proteins important for binding to its host receptor. We also observed that PAA triggered aggregation of TV to a much greater extent than RV. These studies demonstrate that different viruses are inactivated via different PAA mechanisms. This information is important for designing an optimal sanitation practice for postharvest vegetable washing to minimize foodborne viral diseases.