Moniek Ringenier

Background: Raw-meat-based diets (RMBDs) for companion animals have gained popularity but may serve as vehicles for antimicrobial-resistant (AMR) bacteria, posing risks to animal and public health. This study investigated the occurrence and risk factors of AMR in indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium) and Campylobacter spp. from commercial RMBD products. Methods: In 2023, 50 RMBD samples were collected in Belgium, representing 21 brands from five countries. After both selective and non-selective isolation and MALDI-TOF identification, antimicrobial susceptibility testing of the isolates was performed using broth microdilution. Results: From non-selective media, E. coli was found in 45 samples (90.0%), E. faecalis in 31 samples (62.0%), E. faecium in 23 samples (46.0%), and Campylobacter spp. in 3 samples (6.0%). Among these, one E. faecalis strain with acquired resistance to vancomycin and daptomycin was isolated. Multidrug resistance (MDR) was identified in 17 isolates from 15 samples (30.0%), including 14 MDR E. coli, 1 MDR E. faecalis, and 2 MDR E. faecium. From selective media, presumptive ESBL/AmpC-producing E. coli were detected in 17 samples (34.0%), and 5 E. faecium from linezolid-supplemented media were confirmed by the broth microdilution method. Samples from Belgian origin showed significantly higher E. faecium prevalence (76.5%) compared to Dutch samples (21.4%) (OR = 11.9, p < 0.001). Minor livestock sources were associated with increased MDR risk (OR = 5.52, p = 0.016). Conclusions: Commercial RMBDs in Belgium exhibit widespread bacterial contamination with concerning AMR patterns. These findings highlight the need for improved production standards in the RMBD industry and the need to raise awareness in pet owners.

Background: Antimicrobial resistance poses a global health threat, driven by antimicrobial use (AMU). In veterinary medicine, antibiotics are being prescribed in both livestock and companion animals. Therefore, promoting responsible AMU across both sectors is essential. Methods: This qualitative study explored the determinants of antibiotic prescribing among Belgian veterinarians using focus groups and thematic analysis. Results: Forty‐two veterinarians specialised in companion animals, poultry, pigs or cattle participated in eight focus groups. Six themes were identified: (i) diagnostic assessments as the basis for antibiotic decision making, (ii) evaluating therapeutic options, (iii) the veterinarian's role in decision making, (iv) client characteristics, (v) initiatives promoting responsible AMU, and (vi) veterinarian–client communication. Antibiotic prescribing involves complex decision making, mainly guided by clinical expertise. Veterinarians value guidelines, but prioritise therapeutic autonomy. A shift from routine AMU to preventive care was found, especially among livestock veterinarians, while companion animal veterinarians are still adopting this mindset. Despite progress, challenges remain, including client expectations, prescribing habits and limited awareness. Limitations: Although data saturation was not confirmed for pig and poultry, broader themes reached saturation, reinforcing the relevance of the findings. Conclusion: This study provides a framework for enhancing antimicrobial stewardship in both livestock and companion animals.

Introduction: Sustained resistance against fluoroquinolones (FQ), without the use of FQ in broilers, raises important questions about other possible factors contributing to the persistence in farms. Therefore, the current study investigates the prevalence of FQ non-susceptibility in Escherichia coli on broiler farms and examines the roles of day-old chicks, the farm environment, and the antimicrobial use (AMU) in the dynamics of the within-flock spread.Methods: On 29 Belgian broiler farms, AMU was monitored, while environmental and day-old chick intestinal samples (day 0) were collected before their arrival. On days 3 and 36 of the production round, 30 cloacal swabs were taken per farm. In all samples, total E. coli and FQ non susceptible E. coli isolates were quantified by non-selective and FQ selective isolation. A selection of the isolates was analyzed using whole-genome sequencing to characterize their resistance and virulence-associated determinants and to investigate their phylogenetic relatedness using core-genome multi-locus sequence typing (cgMLST) and whole-genome single-nucleotide polymorphism (SNP) analysis.Results: Before entering the stable, day-old chicks carried FQ non-susceptible E. coli on 79.3% of the farms, while FQ non-susceptible E. coli were found in 48.3% of the sampled environments. According to cgMLST, identical FQ non-susceptible isolates were found on day 0 and 36, suggesting that FQ non-susceptible isolates present in the environment at the start of a production round or in day-old chicks, can remain present until slaughter, even when no FQs were used. Total AMU was positively correlated with the proportion of FQ non-susceptible E. coli [r = 0.42, 95% CI (0.06, 0.68), p = 0.03], often also multidrug-resistant, at the end of the production cycle.Conclusion: The continued presence of FQ non-susceptible E. coli in broiler farms is likely the result of both a historical contamination at the farm level and a continuous influx along the production chain. AMU contributes to the continued presence of FQ non-susceptible E. coli in broiler farms, but accounted for only a small proportion of the variability in FQ non-susceptibility in currently investigated farms. The role of certain virulence-associated genes in the persistence of FQ non-susceptible E. coli in broiler farms deserves more in-depth research.

To explore potential factors contributing to high fluoroquinolone resistance levels, it is essential to develop analytical methods capable of detecting residues and trace amounts of antibiotic use in broilers. The aim of the present study was to develop and in-house validate a sensitive UHPLC-MS/MS method capable of determining enrofloxacin (ENR) and flumequine (FLU) residues at slaughter age (day 45) when the animals were treated with these antimicrobials one day after hatching. Residue depletion of ENR and FLU in feathers was also assessed. Two experimental trials were performed, both consisting of 5 different treatment groups. In the first trial animals were treated with ENR and in the second one with FLU. The developed method was successfully validated and was found to be sensitive enough to detect residues of fluoroquinolones in the feathers up until slaughter age in all treatment groups. Average ENR concentration on day 45 was 10 ng g^-1 feather after drinking water treatment, with all concentrations above the limit of quantification (LOQ) of 5 ng g^-1 feather. For FLU average concentration on day 45 after drinking water administration was 4 ng g^-1 feather, with an LOQ of 1 ng g^-1 feather. Therefore, the method is suited for application to monitor fluoroquinolone use in broilers.

Antimicrobial resistance is a global concern, posing risks to human and animal health. This research quantified antimicrobial resistance (AMR) in E. coli isolates from poultry fecal and environmental samples in Bangladesh and explored their association with antimicrobial use (AMU). We screened 725 fecal and 250 environmental samples from 94 conventional broilers and 51 Sonali farms for E. coli presence using MALDI-TOF mass spectrometry. AMU data were collected at flock levels, expressed as treatment incidence (TI), while minimum inhibitory concentrations (MIC) for 14 antibiotics were determined on five fecal E. coli isolates per farm and on all environmental isolates. MIC results were interpreted using human clinical breakpoints and EUCAST epidemiological cut-off values (ECOFFs). Acquired resistance against commonly used antimicrobial agents such as ciprofloxacin, tetracycline and ampicillin, was extremely high and predominantly clinically relevant. There was a moderate correlation between fecal and environmental antibiotic resistance index (ARI), but there was no significant correlation between AMU and AMR, suggesting that the observed AMR prevalence is unrelated to current AMU in poultry, but may be due to high historical AMU. A high level of multidrug resistance, including against critically important antimicrobials, was found in both farm types. Therefore, an AMR/AMU surveillance program is urgently needed in the poultry production sector of Bangladesh.

Optimal intestinal health is a prerequisite for sustainable poultry production. Intestinal health can be evaluated by scoring macroscopic intestinal abnormalities and by histological measurements. The aim of this study was to evaluate correlations between macroscopic gut appearance scoring (GAS), coccidiosis lesion scoring, histological measurements and performance parameters in broilers under field conditions. Therefore, a cross-sectional study was performed on 50 broiler farms where birds were sampled at 28 days of age. The GAS was determined by scoring the absence or presence of 10 macroscopically visible parameters of the gastrointestinal tract, yielding a GAS between 0 and 10, with 0 meaning no gut abnormalities. On individual bird level, when birds had a GAS score of 6 or higher, significantly shorter villi were found in the duodenum. Also, CD3+ T-lymphocyte area percentage in the duodenal mucosa was significantly negatively correlated to villus length. On farm level, the average feed conversion ratio (FCR) was 1.59 ± 0.04 [1.52–1.73]. However, no correlations were found between the GAS at the age of 28 days and the prevalence of coccidiosis, nor did coccidiosis lesion scoring correlate with the FCR. Also, a higher GAS could not be associated with a higher FCR. In conclusion, on all farms a certain degree of macroscopic visible gut and coccidiosis lesions were present in birds of 28 days of age, while this did not correlate with the FCR. This suggests that broilers are able to cope with a certain level of gut damage before it influences the overall performance.

Background. The use of antibiotics in food production selects for resistant bacteria and may cause a threat to human and animal health. Belgium and the Netherlands have one of the highest densities of broilers and pigs in Europe, making active monitoring of antibiotic use and resistance in this region vital. Objectives. This study aimed to quantify ESBL-producing (ESBL-E. coli), carbapenem- and ciprofloxacin-resistant (CiproR) Escherichia coli in animal feces on broiler and pig farms with a history of high antibiotic use in Belgium and the Netherlands. Methods. A total of 779 broiler and 817 pig fecal samples, collected from 29 conventional broiler and 31 multiplier pig farms in the cross-border region of Belgium and the Netherlands, were screened for the presence of antibiotic-resistant E. coli using selective culturing. Results. Carbapenem-resistant E. coli were not detected. ESBL-E. coli were remarkably more prevalent in samples from Belgian than Dutch farms. However, CiproR-E. coli were highly prevalent in broilers of both countries. The percentage of samples with ESBL- and CiproR-E. coli was lower in pig compared to poultry farms and varied between farms. No clear association with the on-farm antibiotic use in the year preceding sampling was observed. Multidrug resistance was frequently observed in samples from both countries, but ESBL-production in combination with ciprofloxacin resistance was higher in samples from Belgium. Conclusions. This study demonstrated marked differences in antibiotic resistance between countries, farms and within farms. The observed variation cannot be explained straightforward by prior quantity of antibiotic use suggesting that it results from more complex interactions that warrant further investigation.

A reduction in antimicrobial use (AMU) is needed to curb the increase in antimicrobial resistance in broiler production. Improvements in biosecurity can contribute to a lower incidence of disease and thereby lower the need for AMU. However, veterinary advice related to AMU reduction or biosecurity is often not complied with, and this has been linked to the attitudes of farmers. Behavior change promoted by coaching may facilitate uptake and compliance regarding veterinary advice. Thirty broiler farms in Belgium and the Netherlands with high AMU were included in this study for 13 months. For each farmer, the attitude towards AMU reduction was quantified using an adjusted Awareness, Desire, Knowledge, Ability, and Reinforcement (ADKAR^®) change management model, and farm biosecurity was assessed with the Biocheck.UGent^™ tool. Subsequently, farmers were coached to improve disease prevention and antimicrobial stewardship. After the individual coaching of farmers, there was a change in their attitudes regarding AMU, reflected by an increase in ADKAR^® scores. Biosecurity levels improved by around 6% on average, and AMU was reduced by 7% on average without negative effects on performance parameters. Despite these improvements, no significant association could be found between higher ADKAR^® scores and lower AMU. Further investigation into sociological models is needed as a tool to reduce AMU in livestock production.