Antibiotic free raising

The contribution of antibiotic-free production of broiler chickens is constantly growing. Addressing the issue of the success of large-scale production without the usage of antimicrobials comparative studies are performed. Samples collected from antibiotic-free and conventional farms are examined for the estimation of exploring the prevalence of antibiotic-resistant E. coli. Data concerning levels of drug-resistant strains are correlated with particular approaches implemented on individual farms, which will allow accurate identification of key factors contributing to antibiotic-free chicken production. Currently, results obtained from comparative studies indicate significantly lower levels of resistant E. coli characterized pheno- and genotypically. Outcomes of field studies will be prerequisites in education on important drivers for the policy regarding animal health and the use of antibiotics by poultry producers and veterinarians.

Therapeutic Alternative to the Use of Antibiotics in Chicken Farming

An experimental study will be performed in three batches of chickens: One batch will not be treated, the second one will be treated with classical antibiotics and a third one treated with a therapeutic alternative (medicinal plant: thym and/or lavender). Each batch will be repeated 10 times. The same approach will also take place in three small farms located in rural areas to offer them solutions to counteract the problems of bacterial infections (colibacillosis) with the lower risk of developing resistance to antibiotics.

Zootechnical parameters such as daily weight gain, feed intake as well as results of histological sections will be monitored from the experimental study for each chicken. AMR bacteria and genes will be investigated in all live animals and in the chicken meat after slaughter. 

Vaccination

Vaccines are used to prevent animals and humans from infectious diseases and vaccination of broiler chickens against pathogenic E. coli is successfully applied. A so far neglected concept is the potential of live vaccines on the reduction or prevention in colonization of broiler chickens with antimicrobial resistant bacteria. The approach of colonization reduction/prevention is called Competitive Exclusion (CE) and was already demonstrated for various bacterial species like Salmonella spp., Campylobacter spp. and Escherichia spp. in broiler chickens. Using complex and non-defined CE cultures high reductions in colonization with unwanted bacteria was shown and defined single CE strains were able to reduce colonization to a certain extent. However, as an authorization of non-defined CE cultures is not possible in Germany our approach is to determine the CE potential of an approved E. coli live vaccine against antimicrobial resistant bacteria in broiler chickens.

Currently, the research is focused on investigation of antimicrobial resistance of ESBL/AmpC E. coli strains isolated from various samples of vaccinated and non-vaccinated broiler flocks. AMR testing is caried out against 15 antibiotics (PX, CRO, AMS, AUG, CIP, MRP, CN, AK, CXT, TE, ATM, IMI, F, FOS, CS). Based on preliminary results we have identified 31 and 45 different antimicrobial resistance profiles of ESBL/AmpC E. coli within two different broiler growth cycles. B1 phylogenetic group of ESBL/AmpC E. coli is predominated in two one after the other broiler growth cycles. Sixty ESBL/AmpC E. coli strains from the first growing cycle were chosen for whole-genome sequencing based on their source of isolation, vaccination status, antibiotic resistance profile, and phylogenetic group. The results indicate, that the sequence types of ST-162 and ST-1011 of ESBL/AmpC E.coli  are predominant. Approx. 50 additional ESBL/AmpC E. coli genomes will be analysed representig  the second and third broler growth cycles.

Phages

Bacteriophages, or simply phages, are viruses that specifically target and infect bacteria. Unlike antibiotics, which often face resistance due to bacterial mutations, phages can evolve rapidly to counteract bacterial resistance. This unique property makes them a promising alternative or complement to traditional treatment with antibiotics. Evaluation of the ability of phages cocktail to reduce antibiotic-resistant E. coli strains of poultry origin was examined on experimental flock. In this study, UPWr_E124 phage cocktail significantly decreased the number of drug-resistant E. coli in chicken excreta.

Treatment of Manure

Antimicrobial resistant (AMR) bacteria that are excreted with broiler feces and the manure are used as organic fertilizer on agricultural land. After manure application on the field, AMR bacteria can contaminate the environment by dispersal in soil, surface water, plants and air. The effectiveness of different chicken manure treatments to reduce antimicrobial resistant bacteria will be tested experimentally (in the laboratory) and standardized conditions and in the field (on the broiler farms) under practical conditions. From the obtained results, feasible interventions for reducing AMR transmission from chicken manure into the environment will be deduced.

The experimental testing of chicken manure treatments will cover composting, storing, fermentation and the addition of an additional carbon source (molasses). Thereby, parameters as temperature, pH, moisture, and duration will be varied and their influence on AMR bacterial survival will be tested. Different bacterial species and their resistance against different antibiotics will be studied. AMR bacteria and resistance genes will be investigated by classical microbiological methods on selection media, genotyping and whole genome sequencing of single bacteria. A minimum of 100 isolates will be analyzed before and after each treatment.

Currently, ATB is focusing on the reduction of antibiotic resistant bacteria in chicken manure by anaerobic digestion. During anaerobic digestion treatment, biowaste is degraded by several group of bacteria without oxygen access. By this process biogas is produced, which serves as an alternative energy source, and pathogens and bacteria are reduced. Our goal is to investigate the effects of temperature and C/N ratio on the abundance of total Escherichia coli, ESBL-producing and Fluoroquinolone-resistant E. coli during anaerobic digestion. Resistant and total E. coli show the same reduction kinetics and temperature was identified as the major factor influencing the bacterial decrease. In parallel, we monitor parameters as biogas production and chemical composition changes during the treatments. The characteristics of the input material are important because they have a main impact on the process and biogas quality. Chicken manure has a naturally low C/N ratio compared to other types of manure. Based on preliminary results, E. coli reduction is fast at 37°C compared to 30°C and biogas production (CH₄:CO₂ ratio) is more efficient in samples with a higher C:N ratio compared to the natural ratio.

Decontamination of Farm Effluents

Antibiotic residues are found in the environment as runoff of domestic, agricultural, and industrial effluents. Conventional water and wastewater treatment technologies based on biological treatment, filtration, etc. can only achieve partial elimination. However, main constraints of these methods are related to application cost, catalyst management and residual toxicity in treated effluents and resulting by-products. Such issues have motivated active research in recent years to develop new alternative technologies that are simple and more efficient in eliminating antibiotics from the bodies of water. The adsorption approach provides various advantages compared to other treatment technologies. Various adsorbents have been successfully developed for the removal of antibiotics from aqueous environments.

The main objective of this project is the elaboration of new adsorbent phases from a natural bio-product to remove a wide variety of antibiotics from water. The elaborated phases can also be used for the concentration of antibiotics for analytical purpose. First, the adsorption efficiency of powder polymers to adsorb a wide range of antibiotics in distilled water will be tested on a laboratory scale. The second phase will also be carried out in the laboratory, but using effluents from two industrial farms. The idea will be to compare the concentrations of the antibiotics studied before and after treatment with the adsorbent matrix.

Existing Knowledge Synthesis

A comprehensive synthesis of existing knowledge (peer reviewed and grey literature) in the field of environmental antimicrobial resistance in poultry production and environment, especially focusing on hazards relevant to this project (i.e. ESBL-, colistin-, and quinolone-resistant Enterobacteriaceae or resistance-genes), will be carried out. This will directly inform the risk assessment model, by providing data on those model parameters that are not covered by the intervention studies in the ENVIRE consortium.

Risk Assessment Modeling

A stochastic quantitative risk assessment model will be developed accounting for the selection of antimicrobial resistance in animals (chicken), its release by the animals, its spread through food and the environment and the subsequent exposure of humans. Two quantitative risk assessment modules will be developed to evaluate the effect of selected interventions on the reduction of human exposure to antimicrobial resistance from chicken origin, via two routes of major interest: i) foodborne (i.e. via consumption of chicken products) and ii) occupational (e.g. via direct contact with positive flocks) routes. The third module –the environmental module – of the QRA model will map the pathways of environmental exposure to risks from chicken farms, including recreational exposure to contaminated surface water (via recreational swimming), consumption of contaminated drinking water and fresh produce contaminated via chicken manure spread. The three modules will be joined to represent the full process of antimicrobial resistance selection, release, spread through food and the environment and human exposure to antimicrobial resistance from various routes of chicken origin (Figure 1).