Susceptibility of the Different Oxygen-Sensing Probes to Interferences in Respirometric Bacterial Assays with Complex Media.

Respirometric microbial assays are gaining popularity, but their uptake is limited by the availability of optimal O2 sensing materials and the challenge of validating assays with complex real samples. We conducted a comparative evaluation of four different O2-sensing probes based on Pt-porphyrin phosphors in respirometric bacterial assays performed on standard time-resolved fluorescence reader. The macromolecular MitoXpress, nanoparticle NanO2 and small molecule PtGlc4 and PtPEG4 probes were assessed with E. coli cells in five growth media: nutrient broth (NB), McConkey (MC), Rapid Coliform ChromoSelect (RCC), M-Lauryl lauryl sulfate (MLS), and Minerals-Modified Glutamate (MMG) media. Respiration profiles of the cells were recorded and analyzed, along with densitometry profiles and quenching studies of individual media components. This revealed several limiting factors and interferences impacting assay performance, which include probe quenched lifetime, instrument temporal resolution, inner filter effects (mainly by indicator dyes), probe binding to lipophilic components, and dynamic and static quenching by media components. The study allowed for the ranking of the probes based on their ruggedness, resilience to interferences and overall performance in respirometric bacterial assays. The 'shielded' probe NanO2 outperformed the established MitoXpress probe and the small molecule probes PtGlc4 and PtPEG4.

A sensor-based system for rapid on-site testing of microbial contamination in meat samples and carcasses.

AIMS: To develop an oxygen sensor-based method for testing total aerobic viable counts (TVC) in raw meat samples and cattle carcass swabs, which is rapid, simple, affordable, provides good sensitivity and analytical performance and allows on-site use. METHODS AND RESULTS: The test uses the same sample preparation procedure as the established plate counting TVC method for meat samples and carcasses, ISO4833-1:2013. After this liquid samples are transferred into standard 25-ml vials with built-in phosphorescent O2  sensors and incubated on a block heater with hourly readings of sensor signals with a handheld reader, to determine signal threshold time (TT, hours) for each sample. The method is demonstrated with the quantification of TVC in industrial cuts of raw beef meat (CFU per g) and carcass swabs (CFU per cm2 ). Calibration curves were generated, which give the following analytical equations for calculating the TVC load in unknown samples from measured TT values: TVC [Log(CFU per cm2 )] = 7.83-0.73*TT(h) and TVC [Log(CFU per g)] = 8.74-0.70*TT(h) for the carcass swabs and meat samples respectively. The new tests show good correlation with the ISO methods, with correlation coefficients 0.85 and 0.83 respectively. The testing requires no dilutions, covers the ranges 2-7 Log(CFU per g) for the meat samples and 1-7 Log(CFU per cm2 ) for carcass swabs, and has time to result 1-10 h with faster detection of more contaminated samples. CONCLUSIONS: The sensor-based testing demonstrates simplicity, high speed, sample throughput and automation. It can provide a straightforward replacement for the conventional TVC tests, which are time consuming, laborious and have time to result of 48-72 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The method(s) can be adopted by the meat industry and research labs, and used to improve microbial quality and safety of meat products and processes.

A Simple Sensor System for Onsite Monitoring of O2 in Vacuum-Packed Meats during the Shelf Life.

Vacuum packaging (VP) is used to reduce exposure of retail meat samples to ambient oxygen (O2) and preserve their quality. A simple sensor system produced from commercial components is described, which allows for non-destructive monitoring of the O2 concentration in VP raw meat samples. Disposable O2 sensor inserts were produced by spotting small aliquots of the cocktail of the Pt-benzoporphyrin dye and polystyrene in ethyl acetate onto pieces of a PVDF membrane and allowing them to air-dry. These sensor dots were placed on top of the beef cuts and vacuum-packed. A handheld reader, FirestinGO2, was used to read nondestructively the sensor phase shift signals (dphi°) and relate them to the O2 levels in packs (kPa or %). The system was validated under industrial settings at a meat processing plant to monitor O2 in VP meat over nine weeks of shelf life storage. The dphi° readings from individual batch-calibrated sensors were converted into the O2 concentration by applying the following calibration equation: O2 (%) = 0.034 * dphi°2 - 3.413 * dphi° + 85.02. In the VP meat samples, the O2 levels were seen to range between 0.12% and 0.27%, with the sensor dphi signals ranging from 44.03° to 56.02°. The DIY sensor system demonstrated ease of use on-site, fast measurement time, high sample throughput, low cost and flexibility.

Antimicrobial Activity of Yeast Cell Wall Products Against Clostridium perfringens.

Yeast cell wall (YCW) products are used worldwide as alternatives to antibiotics growth promoters for health and performances improvement in livestock. The success of yeast and YCW products as feed additives in farm animals' nutrition relies on their capacity to bind enteropathogenic bacteria and on their immunomodulatory activity. In vivo studies report their anti-infectious activity on Gram-positive pathogens like clostridia. However, the in vitro antimicrobial activity of YCW products seems to be limited to some Gram-negative enteropathogens, and literature lacks in vitro evidences for antimicrobial effect of YCW products against Clostridium perfringens. This study aims to measure the antimicrobial activity of YCW products on C. perfringens. Five different YCW products were assayed for their capacity to inhibit the growth of C. perfringens, by analyzing the growth kinetics of the pathogen. All YCW products inhibited the growth of the pathogen, by reducing the growth rate and the maximum growth value and extending the lag phase duration. The effect on the growth parameters was product and dosage dependent. The most effective YCW (namely YCW2), at the minimum effective concentration of 1.25 mg/mL, increased the lag phase duration by 3.6 h, reduced the maximum growth rate by >50%, and reduced the final cell count by 102 colony-forming unit per milliliter in 24 h, with respect to the control. YCW products did not show a strain-dependent impact on C. perfringens growth when tested on different strains of the bacterium.

Equilibrium Isotherm Approach to Measure the Capability of Yeast Cell Wall to Adsorb Clostridium perfringens.

Yeast cell wall (YCW) products are currently used as substitutes to antibiotic growth promoters, to improve animal performances, and to reduce the incidence of infectious diseases in livestock. They are claimed to bind enteropathogens, thus interfering with their colonization in the intestinal mucosa. Although the anti-infectious activity of YCW products on Gram-positive pathogens like Clostridium perfringens has been reported in vivo, in vitro evidences on the adsorption of C. perfringens by YCW fractions are not yet available. Preliminary results showed that purified YCW products exert antimicrobial activity toward C. perfringens. Using the adsorption isotherm approach, we measured the ability of YCW products in adsorbing C. perfringens, thus reducing its viability. Dosages of YCW products >1 mg/mL adsorbed 4 Log colony-forming unit (CFU)/mL of C. perfringens in buffered solution. The maximum adsorption of the bacterium was reached in 3 h, whereas only one product of four YCW products retained the adsorption up to 6 h. The analysis of equilibrium isotherms and adsorption kinetics revealed that all products adsorb C. perfringens in a dose- and time-dependent manner, with high affinity and capacity, sequestering up to 4 Log CFU/mg of product. The determination of adsorption parameters allows to differentiate among adsorbents and select the most efficient product. This approach discriminated among YCW products more efficiently than the antimicrobial assay. In conclusion, this study suggests that the ability of YCW products in reducing C. perfringens viability can be the result of an adsorption mechanism.

Comparative efficacy of agricultural by-products in sequestering mycotoxins.

BACKGROUND: Biosorption using agricultural by-products has been proven as a low-cost and safe way to sequester mycotoxins. Few agricultural by-products have been studied for their efficacy in adsorbing simultaneously a large range of mycotoxins. The present work compared the ability of 51 agricultural by-products to adsorb mycotoxins from liquid mediums simulating physiological pH values, and it studied the mechanism for mycotoxin adsorption by isotherm adsorption experiments. RESULTS: Grape pomaces, artichoke wastes, and almond hulls were selected as promising biosorbents for mycotoxins, being quite effective towards aflatoxin B1 (AFB1 ), zearalenone (ZEA), and ochratoxin A (OTA). Their adsorption was not affected by medium pH, and the adsorbed fraction was not released when pH rose from acid to neutral values. Fumonisin B1 (FB1 ) was adsorbed to a lesser extent, and deoxynivalenol adsorption was not recorded. For the selected biosorbents, maximum adsorption capacity calculated by the best fitting model (Freundlich, Langmuir, or Sips equation) ranged from 1.2 to 2.9 µg mg-1 for AFB1 , 1.3 to 2.7 µg mg-1 for ZEA, 0.03 from 2.9 µg mg-1 for OTA, and 0.01-1.1 µg mg-1 for FB1 . CONCLUSION: This study confirms that some agricultural by-products can find technological applications as feed/food additives for mycotoxin reduction. © 2018 Society of Chemical Industry.

Eubiotics for Food Security at Farm Level: Yeast Cell Wall Products and Their Antimicrobial Potential Against Pathogenic Bacteria.

The population increase in the last century was the first cause of the industrialization of animal productions, together with the necessity to satisfy the high food demand and the lack of space and land for the husbandry practices. As a consequence, the farmers moved from extensive to intensive agricultural systems and introduced new practices, such as the administration of antimicrobial drugs. Antibiotics were then used as growth promoters and for disease prevention. The uncontrolled and continuous use of antibiotics contributed to the spread of antibiotic resistance in animals, and this had adverse impacts on human health. This emergence led the European Union, in 2003, to ban the marketing and use of antibiotics as growth promoters, and for prophylaxis purposes from January 2006. This ban caused problems in farms, due to the decrease in animal performances (weight gain, feed conversion ratio, reproduction, etc.), and the rise in the incidence of certain diseases, such as those induced by Clostridium perfringens, Salmonella, Escherichia coli, and Listeria monocytogenes. The economic losses due to the ban increased the interest in researching alternative strategies for the prophylaxis of infectious diseases and for health and growth promotion, such as feed additives. Yeast-based materials, such as cell wall extract, represent promising alternatives to antibiotics, on the base of their prebiotic activity and their claimed capacity to bind enteropathogenic bacteria. Several authors reported examples of the effectiveness of yeast cell wall products in adsorbing bacteria, but there is a lack of knowledge on the mechanisms involved in this interaction. The purpose of this review is to provide an overview of the current approaches used for the control of pathogenic bacteria in feed, with a particular focus on the use of yeast-derived materials proposed to control zoonoses at farm level, and on their effect on animal health.

Combining the oxygen sensor based respirometry and 16S rRNA amplicon sequencing for the analysis of microbiota in commercial mince products.

In this study, rapid respirometric microbial testing was combined with 16S rRNA amplicon sequencing, to assess the composition of microbiota in a total of 64 samples of commercial beef, turkey, lamb and pork mince. The O2 sensor-based respirometry system, while producing the anticipated total aerobic viable counts (TVC) data and patterns for most samples, also revealed unusual (linear) respiration profiles for some samples, mostly lamb and pork mince. The TVC values for beef mince, produced by respirometry and calculated using the available calibration equation, correlated well with the conventional plate counting method, ISO 4833-1:2013, 2013, while for the other species the correlation was less good. These effects, not observed in previous studies employing various food matrices, require further investigation. Using the same samples (crude homogenates) as in respirometry, the whole microbiome was also analysed by 16S rRNA amplicon sequencing for each mince-type. The sequencing showed an overall decrease in alpha diversity over shelf-life, with lamb and pork mince maintaining a proportion of rare taxa. Some taxa exhibited significant changes in abundance over shelf-life and after the respirometric analysis, with beef mince exhibiting a decrease in aerobic bacteria and an increase in facultative anaerobes. Beta diversity was also seen to depend on mince-type. Thus, the combined use of respirometry and sequencing techniques shows promise as a useful and unique analytical approach for food quality and safety evaluation, However, more data points and in-depth analysis are required to back up the findings of this initial study.

Simultaneous Removal of Mycotoxins by a New Feed Additive Containing a Tri-Octahedral Smectite Mixed with Lignocellulose.

Simultaneous removal of mycotoxins has been poorly addressed, and a limited number of studies have reported the efficacy of feed additives in sequestering a large spectrum of mycotoxins. In this study, a new mycotoxin-adsorbing agent was obtained by properly mixing a tri-octahedral smectite with a lignocellulose-based material. At a dosage of 1 mg mL-1, these materials simultaneously adsorbed frequently occurring mycotoxins and did not exert a cytotoxic effect on intestinal cells. Chyme samples obtained by a simulated GI digestion did not affect the viability of Caco-2TC7 cells as measured by the MTT test. In addition, the chyme of the lignocellulose showed a high content of polyphenols (210 mg mL-1 catechin equivalent) and good antioxidant activity. The properties of the individual constituents were maintained in the final composite, and were unaffected by their combination. When tested with a pool of seven mycotoxins at 1 µg mL-1 each and pH 5, the composite (5 mg mL-1) simultaneously sequestered AFB1 (95%), FB1 (99%), ZEA (93%), OTA (80%), T-2 (63%), and DON (22%). HT-2 adsorption did not occur. Mycotoxin adsorption increased exponentially as dosage increased, and occurred at physiological pH values. AFB1, ZEA and T-2 adsorption was not affected by pH in the range 3-9, whereas OTA and FB1 were adsorbed at pH values of 3-5. The adsorbed amount of AFB1, ZEA and T-2 was not released when pH rose from 3 to 7. FB1 and OTA desorption was less than 38%. Langmuir adsorption isotherms revealed high capacity and affinity for adsorption of the target mycotoxins. Results of this study are promising and show the potential of the new composite to remove mycotoxins in practical scenarios where several mycotoxins can co-occur.

Mineral Composition and Bioaccessibility in Rocket and Purslane after Zn Biofortification Process.

Zinc (Zn) is an essential key nutrient in different biochemical and physiological processes. The nutritional deficit of this mineral element is estimated to affect the health of over 3 billion people worldwide. Several strategies are available to reduce the negative impact of mineral malnutrition; among them, biofortification is the practice of deliberately increasing the nutrients and healthy compounds in the edible parts of vegetables. This study aims to evaluate Zn bioaccessibility in biofortified and non-biofortified rocket and purslane using an in vitro gastrointestinal digestion process and measure the concentration of other mineral elements (Al, B, Ca, Fe, K, Mg, Mn, and Sr) released during the digestion process from rocket and purslane biofortified with Zn. The bioaccessible Zn in biofortified rocket and purslane ranged from 7.43 to 16.91 mg/kg, respectively. In addition, the daily intake, the RDA coverage (%), and the hazard quotient (HQ) for the intake of Zn (resulting from the consumption of 100 g of rocket and purslane) were calculated. The calculated HQ highlights the safety of these baby leaf vegetables. The study confirms that it is possible to obtain Zn-biofortified rocket and purslane with high Zn bioaccessibility by adopting an appropriate mineral plant nutrition solution enriched in Zn.

Facile biosensor-based system for on-site quantification of total viable counts in food and environmental swabs.

We describe a new biosensor platform for rapid and simple quantification of total aerobic viable counts of bacteria (TVC) in food and environmental swabs by oxygen respirometry. The system uses disposable swab vials with phosphorescent oxygen sensors integrated in the bottom part, a small block heater/incubator and a handheld sensor reader. In the testing, groups of 1-20 swabs samples were prepared using the standard method (ISO, 18593:2018) in sensor vials, which were then incubated at 30 °C and measured hourly in a contactless, non-invasive manner. The measurements reveal time profiles of dissolved O2 in each sample vial, from which Threshold Time of sensor signal was determined and then TVC values (CFU/cm2) were calculated using the calibration equation. The method covers the range of 0.65-7.87 Log (CFU/cm2) and produces results in 1-8 hrs. The test was validated with swab samples from surfaces contaminated with E. coli, with whole meat microbiota, and with real environmental swabs. The results showed no statistically significant difference with the reference method which takes 48-72 h. The swab testing platform is fast and accurate, simple (sample-and-measure), portable, low cost (<$5k), requires no serial dilutions and is suitable for on-site deployment and use.

Development of a DNA-based biosensor for the fast and sensitive detection of ochratoxin A in urine.

In this paper, a novel DNA-based biosensor is proposed, which is based on paramagnetic microbeads carrying an ochratoxin A (OTA) capture aptamer. A sandwich-like detection complex is linked to the capture aptamer and is able to trigger, in presence of OTA, an isothermal rolling circle amplification (RCA) reaction. This latter generated autocatalytic units with a peroxidase activity (DNAzyme) that, in presence of a proper substrate, gave a blue-coloured product visible by the naked eye. The capture aptamer, blocked onto magnetic beads, allowed the specific capture of OTA in liquid samples. The modified detection aptamer, annealed to a circularized probe, was then used to detect the toxin capture event. Indeed, in the presence of OTA and an isothermal enzyme, the circular DNA was amplified, producing a single-stranded and tandem repeated long homologous copy of its sequence. In the DNA strand, a self-catalytic structure was formed with hemin as the catalytic core, inducing the development of blue colour in the presence of ABTS and hydrogen peroxide. The results showed that the biosensor has high sensitivity and selectivity for the detection of OTA, as low as 1.09 × 10-12 ng/mL. Moreover, the proposed biosensor was successfully used for the detection of OTA in naturally contaminated rat urine. Accuracy and repeatability data obtained in recovery experiments were satisfying, being recoveries >95% with relative standard deviations in the range 3.6-15%. For the first time, an aptasensor was successfully applied to detect OTA in biological fluids. It can be used for mycotoxin biomonitoring and assessment of individual exposure.

Antimicrobial properties of rosin acids-loaded nanoparticles against antibiotic-sensitive and antibiotic-resistant foodborne pathogens.

Rosin acids (RA) from coniferous trees are used in folk medicine for healing various skin infections. Despite the antimicrobial potential of RA, their poor solubility in aqueous media may limit their use. In this work RA-loaded polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticles (RA-NPs) with enhanced antimicrobial properties against foodborne bacterial pathogens were produced. RA-NPs were prepared by solvent displacement technique and characterized for relevant colloidal features by dynamic light scattering, laser Doppler anemometry and transmission electron microscopy. Association of RA to NPs occurred with high yields (86% w/w). RA and RA-NPs (∼130 nm) were strongly active against antibiotic-sensitive Gram + pathogens, i.e. Clostridium perfringens, Listeria monocytogenes and antibiotic-resistant Staphylococcus aureus. However, both failed in inhibiting the growth of Gram - pathogens (Campylobacter jejuni, Campylobacter coli, Escherichia coli and Salmonella enterica). Association to NPs enhanced the antimicrobial activity of RA. MIC, IC50, IC90, and MBC values of RA-NPs were ten-times lower than RA. RA-NPs did not change the intrinsic toxicity potential of RA. This is the first study on the enhancement of the antimicrobial activity of RA when associated to nanocarriers. This approach may be an effective strategy to produce aqueous-based RA solutions with enhanced antimicrobial activity against antibiotic-sensitive and antibiotic-resistant Gram + pathogens.

Infection cycle of Artichoke Italian latent virus in tobacco plants: meristem invasion and recovery from disease symptoms.

Nepoviral infections induce recovery in fully expanded leaves but persist in shoot apical meristem (SAM) by a largely unknown mechanism. The dynamics of infection of a grapevine isolate of Artichoke Italian latent virus (AILV-V, genus Nepovirus) in tobacco plants, including colonization of SAM, symptom induction and subsequent recovery of mature leaves from symptoms, were characterized. AILV-V moved from the inoculated leaves systemically and invaded SAM in 7 days post-inoculation (dpi), remaining detectable in SAM at least up to 40 dpi. The new top leaves recovered from viral symptoms earliest at 21 dpi. Accumulation of viral RNA to a threshold level was required to trigger the overexpression of RDR6 and DCL4. Consequently, accumulation of viral RNA decreased in the systemically infected leaves, reaching the lowest concentration in the 3rd and 4th leaves at 23 dpi, which was concomitant with recovery of the younger, upper leaves from disease symptoms. No evidence of virus replication was found in the recovered leaves, but they contained infectious virus particles and were protected against re-inoculation with AILV-V. In this study we also showed that AILV-V did not suppress initiation or maintenance of RNA silencing in transgenic plants, but was able to interfere with the cell-to-cell movement of the RNA silencing signal. Our results suggest that AILV-V entrance in SAM and activation of RNA silencing may be distinct processes since the latter is triggered in fully expanded leaves by the accumulation of viral RNA above a threshold level rather than by virus entrance in SAM.

Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants.

The quantification of messenger RNA expression levels by real-time reverse-transcription polymerase chain reaction requires the availability of reference genes that are stably expressed regardless of the experimental conditions under study. We examined the expression variations of a set of eight candidate reference genes in tomato leaf and root tissues subjected to the infection of five taxonomically and molecularly different plant viruses and a viroid, inducing diverse pathogenic effects on inoculated plants. Parallel analyses by three commonly used dedicated algorithms, geNorm, NormFinder and BestKeeper, showed that different viral infections and tissues of origin influenced, to some extent, the expression levels of these genes. However, all algorithms showed high levels of stability for glyceraldehyde 3-phosphate dehydrogenase and ubiquitin, indicated as the most suitable endogenous transcripts for normalization in both tissue types. Actin and uridylate kinase were also stably expressed throughout the infected tissues, whereas cyclophilin showed tissue-specific expression stability only in root samples. By contrast, two widely employed reference genes, 18S ribosomal RNA and elongation factor 1α, demonstrated highly variable expression levels that should discourage their use for normalization. In addition, expression level analysis of ascorbate peroxidase and superoxide dismutase showed the modulation of the two genes in virus-infected tomato leaves and roots. The relative quantification of the two genes varied according to the reference genes selected, thus highlighting the importance of the choice of the correct normalization method in such experiments.