Development and in vitro evaluation of an alternative thermo-tolerant antimicrobial polymer for the prevention and treatment of bovine mastitis

Abstract

Despite advancements in veterinary medicine, bovine mastitis remains one of the most significant and prevalent diseases in Irish dairy herds. Raw milk represents one of Ireland’s main food exports and as DAFM Food-Wise 2025 aims to increase food exports by 85%, coupled with the removal of the EU Milk Quota in March 2015, there is need for improved milk yields from Irish farms. Bovine mastitis is an inflammatory infection of the mammary gland, which is a major constraint on milk production, causing animals discomfort, health complications, production of poor-quality milk and when treated using conventional means, requires their removal from the herd. There are several prevention plans being implemented but the use of antibiotics remains the only recognised means of treatment, which requires a cool-off period to ensure no traces of antibiotics enter the food supply. Due to emerging antibiotic resistance, there is an ever-increasing demand for antibiotic-free treatments in all fields of medicine; thus, highlighting the need for developing new or alternative treatments targeting infectious diseases such as mastitis. In this study, four thermo-tolerant bioactives (nisin, silver nitrate, zinc oxide and chitosan) were evaluated in order to determine their suitability as alternatives for antibiotics in the treatment of bovine mastitis. They will be assessed for their antimicrobial capabilities as well as their suitability for polymer incorporation through hot-melt extrusion (HME), which would greatly open their potential applications and uses. Well established methods in the evaluation of antibiotics and other antimicrobial compounds were utilised to allow comparisons with published literature and already well-established antimicrobials. Additional methods have also been implemented and developed to assess the antibiofilm capabilities of the bioactives, and other methods commonly used in pharmaceutical studies for determining combinational potential of the compounds in terms of a synergy assessment. Methods: The four bioactives were first incorporated into a polymer matrix of PVP-VA64 through hot-melt extrusion (HME) as a means to access this novel route of therapeutic delivery. Broth microdilution assays were utilized to evaluate the microbial growth inhibition capabilities of each bioactive, before and after HME, and to determine their individual minimum inhibitory concentration (MIC) against four bacterial strains, E. coli, S. aureus, S. epidermidis and P. aeruginosa. Growth inhibition was determined by measuring absorbance 3 while secondary analysis was carried out by use of the extra-cellular dye, resazurin. MIC studies were conducted on a number of veterinary isolate strains, some of which have demonstrated antimicrobial resistance (AMR). The biofilm disruptive capabilities of each bioactive was assessed against S. aureus and P. aeruginosa biofilms. A novel protocol was developed to assess bioactive effects upon the three stages of biofilm development, attachment, growth, and maturity. Thereafter, the bioactives were assessed in arrangements of two, three and four drug combinations to determine their synergistic capabilities for inhibiting E. coli, S. aureus and S. epidermidis growth. The synergistic relationship between bioactive activity was evaluated by a new analytical system developed using the Python coding language, allowing for high-throughput evaluation and quantification. In vitro experiments were then conducted in order to determine the cytotoxic and inflammatory potential of the bioactives against bovine mammary epithelial (BME) cells. To assess the bioactive cytotoxicity, BME cells were treated with each bioactive at a range of concentrations, and cell viability was determined by use of the MTT assay. Cells were also treated and assessed to determine their inflammatory response, by determining changes in the expression of the cytokines TNFα, IL-1β, IL-6 and IL-8. Relative expression was determined by use of RT-qPCR using β-actin as the reference housekeeping gene. Results: The four bioactives were found to exhibit satisfactory antimicrobial inhibitory effects, before and after their incorporation into a polymer matrix against E. coli, S. aureus, S. epidermidis and P. aeruginosa. Bioactive studies against veterinary and reference strain bacterial isolates also exhibited promising findings in terms of addressing AMR. Assessment of the biofilm disruptive capabilities highlighted varying degrees of bioactive efficacy. Furthermore, this study generated novel data that will advance the field of biofilm innovation. The inclusion of a novel anti-attachment step in the anti-biofilm assessment process holds great potential as a pivotal source of data, informing future preventative measures against biofilms and biofilm-mediated infections. The novel anti-attachment step included in the present study generated important revealing the bioactives effect upon the initial stages of biofilm development. While some results varied greatly with some treatments (such as AgNO3) causing notable decreases in biofilm growth against P. aeruginosa, but causing increased S. aureus biofilm growth, the study shows the import of considering such scenarios. 4 Synergistic combinational assessments yielded enormous amounts of data for the named bioactives tested, with the majority showing clear synergistic capabilities when combined. This data also exhibited interesting trends such as how therapeutic behaviour can change unpredictably depending upon the combination of bioactives used. Results show how two or more drugs can interact differently with each other when they are at the active site of treatment, and how their activity can change. While the primary intention is for positive therapeutic reinforcement of activity (i.e. synergy), certain bioactives were shown to inhibit each other (i.e. antagonism). While antagonistic interactions are not favourable, they are equally valuable data points to consider when preparing combinational therapies as these combinations can be reduced to limit such antagonism. The most interesting finding from this section involved the use of nisin, a lantibiotic unable to affect gram-negative bacteria, which was shown to have a definite effect when used in combination with other bioactives (primarily AgNO3 and Chitosan) which was a hugely significant finding that carried the hypothesis of the combination studies. Previously used combination/synergy predictive models would claim there would be no effect between these compounds, whereas in the present study it was proven that there was an effect. This constitutes the first study that exploits a three-drug combinational study for these bioactives and the only four-drug conducted study of any therapeutic compounds. Cytotoxic assessment of the bioactives reported negative effects when using AgNO3 and ZnO with BME cells. Both AgNO3 and ZnO were quite toxic to the cells in vitro, however as per results from other studies carried out during this project, both bioactives still hold great potential for inclusion in a final treatment solution. For example, combinational report much lower required concentrations of each which enables the potential of using sub-toxic concentrations of each compound that may still hold notable anti-microbial effect. Furthermore, inflammatory assessment revealed interesting effects upon cytokine expressions. AgNO3 was noted to produce a strong anti-inflammatory response, as noted by the reduced expression of the pro-inflammatory cytokines, TNFα, IL-1β, IL-6 and IL-8. Reduced expression of such cytokines is a key indicator of reduced inflammation, which holds great promise for the treatment of inflammatory diseases, such as mastitis. Nisin, chitosan and ZnO also reduced the expression of some target cytokines. Notably, there was no major increase in expression of targeted cytokines using the aforementioned bioactives which indicates that 5 they did not elicit an inflammatory response in the cells. This too is an important characteristic required for treatment of inflammatory diseases. The findings of this research were published in leading journals that will inform solutions for AMR crisis where this cross-cutting area aligns with the new TUS Strategic Plan 2023-2026.