• Our research suggest that anthocyanins are promising anti-bacterial agents • The antimicrobial effects are highly dependent on the source of the anthocyanin-extract • Rose-anthocyanins appear to posses the strongest anti-bacterial effects • Gram-positive strains appear to be more sensitive compared to gram-negative strains • Future research efforts should focus on different anthocyanin entities
Manure application can spread antimicrobial resistance (AMR) from manure to soil and surface water. This study evaluated the role of the soil texture on the dynamics of antimicrobial resistance genes (ARGs) in soils and surrounding surface waters. Six dairy farms with distinct soil textures (clay, sand, and peat) were sampled at different time points after the application of manure, and three representative ARGs sul1, erm(B), and tet(W) were quantified with qPCR. Manuring initially increased levels of erm(B) by 1.5 ± 0.5 log copies/kg of soil and tet(W) by 0.8 ± 0.4 log copies/kg across soil textures, after which levels gradually declined. In surface waters from clay environments, regardless of the ARG, the gene levels initially increased by 2.6 ± 1.6 log copies/L, after which levels gradually declined. The gene decay in soils was strongly dependent on the type of ARG (erm(B) < tet(W) < sul1; half-lives of 7, 11, and 75 days, respectively), while in water, the decay was primarily dependent on the soil texture adjacent to the sampled surface water (clay < peat < sand; half-lives of 2, 6, and 10 days, respectively). Finally, recovery of ARG levels was predicted after 29–42 days. The results thus showed that there was not a complete restoration of ARGs in soils between rounds of manure application. In conclusion, this study demonstrates that rather than showing similar dynamics of decay, factors such as the type of ARG and soil texture drive the ARG persistence in the environment.
MULTIFILE
Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.
MULTIFILE
To treat microbial infections, antibiotics are life-saving but the increasing antimicrobial resistance is a World-wide problem. Therefore, there is a great need for novel antimicrobial substances. Fruit and flower anthocyanins have been recognized as promising alternatives to traditional antibiotics. How-ever, for future application as innovative alternative antibiotics, the full potential of anthocyanins should be further investigated. The antimicrobial potential of anthocyanin mixtures against different bacterial species has been demonstrated in literature. Preliminary experiments performed by our laboratories, using grape, rose and red cabbage anthocyanins against S. aureus and E. coli confirmed the antimicrobial potential of these substances. Hundreds of different anthocyanin entities have been described. However, which of these entities hold antimicrobial effects is currently unknown. Our preliminary data show that an-thocyanins extracted from grape, rose and red cabbage contain different collections of anthocyanin entities with differential antimicrobial efficacies. Our focus is on the extraction and characterization of anthocyanins from various crop residues. Grape peels are residues in the production of wine, while red rose and tulip leaves are residues in the production of tulip bulbs and regular horticulture. The presence of high-grade substances for pharmacological purposes in these crops may provide an innovative strategy to add value to other-wise invaluable crop residues. This project will be performed by the collaborative effort of our institute together with the Medi-cal Microbiology department of the University Medical Center Groningen (UMCG), 'Wijnstaete', a small-scale wine-producer (Lemelerveld) and Imenz Bioengineering (Groningen), a company that develops processes to improve the production of biobased chemicals from waste products. Within this project, we will focus on the antimicrobial efficacy of anthocyanin-mixtures from sources that are abundantly and locally available as a residual waste product. The project is part of a larger re-search effect to further characterize, modify and study the antimicrobial effects of specific anthocy-anin entities.
Worldwide over- and misuse of antibiotics has contributed to the development of antibiotic-resistance. The occurrence and increase of antibiotic-resistance is one of the most pressing global health care issues of the 21st century. Recently it has been recognized that fruit and flower anthocyanins have antimicrobial activity and thereby the potential to function as novel antibiotics. At the Hanze University of Applied Science, we were able to confirm the antimicrobial efficacy of purified Rosa and Tulipa anthocyanin extracts against an array of microbial species. Using our optimized extraction methods, anthocyanins can easily be extracted and purified from floral residual streams. Once marketed as novel antimicrobials, this valorization of residual streams to high-value compounds contributes to the transition towards a circular economy. However, for future application in different antimicrobial products, it is necessary to identify and characterize single antimicrobial anthocyanin molecules. Moreover, analysis of pilot-scale extraction- and fractionation-yields and antimicrobial bench-mark doses will provide information on their market and application potential. In the current project we propose to develop a strategy composed of fractionation and state-of-the-art characterization methods to identify anthocyanin-molecules with potent antimicrobial effects. To our knowledge this is the first strategy that combines in-depth chemical characterization of anthocyanins in relation to their antimicrobial efficacy. Once developed, this strategy will allow us to single out anthocyanin molecules with antimicrobial properties. The development of the proposed fractionation and characterization strategy is the first step towards the development of single anthocyanin molecules as novel plant-based antibiotics.
