Evelyne Meyer

Bovine mastitis remains a major driver of antimicrobial use in dairy production, highlighting the need for physiologically relevant in vitro models for preclinical safety assessment of novel intramammary therapeutics. Here, a previously established bovine epithelial cell line co-culture mammosphere model was adapted to a single-spheroid 96-well ultra-low attachment format, improving reproducibility and longitudinal monitoring. Mammospheres maintained epithelial organization and were further characterized for secretory function through assessment of casein expression. The suitability of the model for safety assessment was subsequently evaluated using complementary fluorescence microscopy, kinetic live-cell imaging, and flow cytometry following Triton X-100-induced cytotoxicity. Using this workflow, the safety of bacteriophage-derived recombinant lysin LysMK34 and antibiotic kanamycin, alone and in combination, was evaluated, with no cytotoxic effects observed, supporting the utility of the model for future preclinical intramammary safety assessment.

Designer cellulosomes (DCs) are precisely engineered multi-enzyme complexes aimed at lignocellulose saccharification. Achieving spontaneous designer cellulosome display on yeast cell surfaces has been a long-term objective to enhance consolidated bioprocesses with concurrent ethanol production. A “self-assembly” approach involves simultaneous scaffoldin display and docking enzyme secretion. However, challenges arise from the size and complexity of designer cellulosomes, coupled with the yeast cells’ limited capacity for heterologous protein expression. A comprehensive examination of Saccharomyces cerevisiae as a host for DC expression remains unaddressed. We meticulously examined the capability of S. cerevisiae to produce and display a fluorescent protein complex, which mimics the designer cellulosome architecture and allows for convenient detection of all individual components on the cell surface, using flow cytometry and confocal microscopy. Population-wide analysis revealed a fluorescent protein complex production efficiency of approximately 10%. Single-cell analysis highlighted a clear mutual influence between the expression of scaffoldin and docking proteins, impacting cellular fitness. Newly emerging buds were identified as hotspots for scaffoldin display. The finite capacity of yeast cells to produce heterologous proteins was identified as a major bottleneck. While distributing the cellular load among multiple hosts within a synthetic yeast consortium can alleviate this burden, the use of fluorescent protein complex surface display has visualized the heterogeneity and constraints of S. cerevisiae as a host for designer cellulosome expression at the population and single cell level. This study provides a realistic assessment of the challenges in achieving efficient S. cerevisiae -based DC display for consolidated bioprocessing. IMPORTANCE: Efficient and economically viable biomass conversion into fermentable sugars is a pivotal challenge in transitioning from a petroleum-based economy to a bio-economy. Drawing inspiration from nature, cellulosomes represent an exemplary solution for the effective digestion of lignocellulose. These multi-enzyme complexes can be precisely engineered to tailor their properties and transferred to the surface of yeast cells, which can subsequently ferment the sugars into bulk or fine chemicals. Achieving this transfer successfully necessitates a comprehensive understanding of how yeast cells can recombinantly produce and attach such multi-component complexes to their surface. This study employs a fluorescent surrogate to provide novel insights into the capabilities of yeast cells at both the single-cell and population levels.

The mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME), produced by Alternaria spp., are common contaminants of food and feed and are a potential threat to animal and human health. To date, the most prominent data gaps for their comprehensive risk assessment concern information on their in vivo absorption, distribution, metabolism and excretion (ADME) and toxicokinetic behavior. The aim of this study was to determine the absolute oral bioavailability, quantitative toxicokinetic characteristics and biotransformation of AOH and AME in vivo in pigs, using crossover trials with intravenous and oral administration of a single dose of both mycotoxins at 2 mg/kg b.w. Plasma profiles of the mycotoxins and phase I and II metabolites were studied in the vena jugularis using UPLC-MS/MS and LC-HRMS methods. Furthermore, plasma from the vena portae was analyzed to study presystemic biotransformation. Urine was collected to determine the urinary excretion and metabolite profiles. Results reveal a low absolute oral bioavailability of AOH (15%) and AME (9%), caused by a low absorption and/or extensive first-pass biotransformation in the liver to mainly phase II, and to a lesser extent phase I metabolites. Quantitative toxicokinetic modeling of the IV data showed a high total body clearance for both AOH and AME (12.9 and 16.8 L/(h*kg b.w.), respectively), a high volume of distribution (4.97 and 5.15 L/kg b.w., respectively) and a short elimination half-life of 0.16 and 0.21 h, respectively. These findings may contribute to the risk assessment of AOH and AME and to the development of candidate biomarkers of exposure in biomonitoring studies since pigs are considered a suitable animal model to extrapolate to humans.

Streptococcus uberis frequently causes bovine mastitis, an infectious udder disease with significant economic implications for dairy cows. Conventional antibiotics, such as cloxacillin, sometimes have limited success in eliminating S. uberis as a stand-alone therapy. To address this challenge, the study objective was to investigate the VersaTile engineered endolysin NC5 as a supplemental therapy to cloxacillin in a mouse model of bovine S. uberis mastitis. NC5 was previously selected based on its intracellular killing and biofilm eradicating activity. To deliver preclinical proof-of-concept of this supplemental strategy, lactating mice were intramammarily infected with a bovine S. uberis field isolate and subsequently treated with cloxacillin (30.0 μg) combined with either a low (23.5 μg) or high (235.0 μg) dose of NC5. An antibiotic monotherapy group, as well as placebo treatment, was included as controls. Two types of responders were identified: fast (n = 17), showing response after 4-h treatment, and slow (n = 10), exhibiting no clear response at 4 h post-treatment across all groups. The high-dose combination therapy in comparison with placebo treatment impacted the hallmarks of mastitis in the fast responders by reducing (i) the bacterial load 13,000-fold (4.11 ± 0.78 Δlog₁₀; p < 0.001), (ii) neutrophil infiltration 5.7-fold (p > 0.05), and (iii) the key pro-inflammatory chemokine IL-8 13-fold (p < 0.01). These mastitis hallmarks typically followed a dose response dependent on the amount of endolysin added. The current in vivo study complements our in vitro data and provides preclinical proof-of-concept of NC5 as an adjunct to intramammary cloxacillin treatment. Key points: • Engineered endolysin NC5 was preclinically evaluated as add-on to cloxacillin treatment. • Two types of mice (slow and fast responding) were observed. • The add-on treatment decreased bacterial load, neutrophil influx, and pro-inflammatory mediators.

Bacteriophage‐derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic‐resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis , Streptococcus agalactiae and Streptococcus dysgalactiae , also targeting intracellular survival and biofilm formation. For this purpose, high‐throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram‐positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time‐kill assays in phosphate‐buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis , also showing similar activity against S. agalactiae and S. dysgalactiae . This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta‐lactam penicillin used to intramammarily treat bovine Gram‐positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add‐on in the treatment of bovine streptococcal mastitis.

Streptococcus uberis is a major causative agent of bovine mastitis, an inflammation of the mammary gland with substantial economic consequences. To reduce antibiotic use in animal agriculture, alternative strategies to treat or prevent mastitis are being investigated. Bovine-associated non-aureus staphylococci are proposed in that respect due to their capacity to inhibit the in vitro growth of S. uberis. We demonstrate that priming the murine mammary gland with Staphylococcus chromogenes IM reduces S. uberis growth in comparison with non-primed glands. The innate immune system is activated by increasing IL-8 and LCN2, which may explain this decreased growth.

Acute kidney injury (AKI) is common after pediatric cardiac surgery (CS). Several urine biomarkers have been validated to detect AKI earlier. The objective of this study was to evaluate urine CHI3L1, NGAL, TIMP-2, IGFBP7, and NephroCheck^® as predictors for AKI ≥ 1 in pediatric CS after 48 h and AKI ≥ 2 after 12 h. Pediatric patients (age < 18 year; body weight ≥ 2 kg) requiring CS were prospectively included. Urine CHI3L1, NGAL, TIMP-2, IGFBP7, and NephroCheck^® were measured during surgery and intensive care unit (ICU) stay and corrected for urine dilution. One hundred and one pediatric patients were included. AKI ≥ 1 within 48 h after ICU admission occurred in 62.4% and AKI ≥ 2 within 12 h in 30.7%. All damage biomarkers predicted AKI ≥ 1 within 48 h after ICU admission, when corrected for urine dilution: CHI3L1 (AUC-ROC: 0.642 (95% CI, 0.535–0.741)), NGAL (0.765 (0.664–0.848)), TIMP-2 (0.778 (0.662–0.868)), IGFBP7 (0.796 (0.682–0.883)), NephroCheck^® (0.734 (0.614–0.832)). Similarly, AKI ≥ 2 within 12 h was predicted by all damage biomarkers when corrected for urine dilution: uCHI3L1 (AUC-ROC: 0.686 (95% CI, 0.580–0.780)), NGAL (0.714 (0.609–0.804)), TIMP-2 (0.830 (0.722–0.909)), IGFBP7 (0.834 (0.725–0.912)), NephroCheck^® (0.774 (0.658–0.865)). After pediatric cardiac surgery, the damage biomarkers urine CHI3L1, NGAL, TIMP-2, IGFBP7, and NephroCheck^® reliably predict AKI after correction for urine dilution.

In this paper, we describe a new quantitative method to evaluate and quantify in vitro growth inhibition of mastitis-related bacteria. Colony-forming units of Staphylococcus (S.) aureus (n = 10), Escherichia (E.) coli (n = 10), and Streptococcus (S.) uberis (n = 10) were quantified after their growth on top of layers of trypticase soy agar (TSA) containing six different concentrations (varying from 10² to 10⁷ CFU/mL) of bovine non-aureus staphylococci (NAS), i.e., S. chromogenes (n = 3) and S. simulans (n = 3) isolates. Growth inhibition of the mastitis-related major bacterial pathogens, including E. coli, was confirmed by all NAS, an effect that varied highly among NAS isolates and was not evident from the semiquantitative method with which the new method was compared. By subsequent application of the new method on a larger set of 14 bovine NAS isolates, we observed that S. simulans and NAS originating from teat apices (especially S. epidermidis) required lower concentrations to inhibit both methicillin-sensitive (MSSA) (n = 5) and methicillin-resistant S. aureus (MRSA) isolates (n = 5) originating from milk. Therefore, the new assay is a promising tool to precisely quantify the intra- and inter-species differences in growth inhibition between NAS.

In a temperate climate, the mycotoxins deoxynivalenol (DON), nivalenol (NIV), enniatin B (ENN B), mycophenolic acid (MPA), roquefortine C (ROC) and zearalenone (ZEN) are often found in maize silage. Although rumen microbiota are able to degrade some mycotoxins (e.g. DON), others are known to stay mainly intact (e.g. ROC). In addition, mycotoxin degradation can be hampered by a low ruminal pH or decrease in rumen microbial activity. Hence, these mycotoxins can reach the small intestine and exert a cytotoxic effect on intestinal epithelial cells. In this study, a real-time kinetic analysis of the cytotoxicity of these six mycotoxins and some of their metabolites (α- and β-zearalenol, α- and β-ZOL) was performed in a calf small intestinal epithelial cell line (CIEB). Confluency as well as the cell death parameters apoptosis and necrosis were determined to evaluate the mycotoxin-induced cytotoxicity. A combination of Annexin-V green and Cytotox red staining was used to determine early and late apoptosis as well as necrosis. Six different concentrations were tested ranging from 0.78 to 12.5 μM. Compared to cells not exposed to mycotoxins, DON and NIV exert a fast toxic effect with DON being more toxic than NIV within the first hours of incubation, whereas the inverse was observed at 16 h of incubation. On the other hand, MPA and ZEN induced increased Annexin V green positive cells within several hours of incubation with higher toxicity over time. Increased Annexin V green and Cytotox red positive cells were seen for ROC only at the highest concentration tested. For ENN B, increased Annexin V green positive cells were observed only after 12 h and α- and β-ZOL did not show cytotoxic effects. Hence, mycotoxin exposure causes either severe (DON and NIV) or more limited (ZEN, ROC, MPA, and ENN B) risk of bovine intestinal epithelial damage.