INVESTIGATION OF RESISTANCE MECHANISMS AND THEIR TRANSMISSION IN EMERGING PATHOGENS APPLYING THE «ONE HEALTH» APPROACH

إنجليزي. This thesis aimed to explore antimicrobial resistance in Belgium in two critical clinical pathogens, Klebsiella pneumoniae and Aspergillus fumigatus, using a One Health approach. The One Health perspective emphasizes the interconnections between human, animal, and environmental health to address global health challenges like antimicrobial resistance (AMR). AMR occurs when microorganisms acquire resistance through genetic mutations or horizontal gene transfer, posing a global health crisis. K. pneumoniae is a leading cause of AMR-related deaths and has been identified as a priority pathogen by the world health organization (WHO) due to its resistance to carbapenems and 3rd-generation cephalosporins. Similarly, A. fumigatus is a critical fungal pathogen, with antifungal resistance exacerbated by cross-use of antifungals in healthcare and agriculture. Both pathogens highlight the necessity of a One Health approach to comprehensively manage AMR, ensuring health interventions in one domain (human, animal, or environmental) do not undermine others. This thesis underscores the urgent need for global strategies to combat the growing threat of AMR in these pathogens. The study is divided into five parts. The first section aimed at identifying the prevalence of K. pneumoniae in the food chain and in wastewater. Following this, the occurrence of antibiotic resistance in K. pneumoniae across the food chain, diseased animals, wastewater, and the clinical setting was investigated. Here, we mainly focused on the presence of K. pneumoniae resistant to third generation cephalosporins (3GC) and carbapenems. Whole genome sequencing was then conducted on a subset of K. pneumoniae isolates from all sectors to explore the resistance mechanisms and the potential transmission and genetic relatedness between sectors in the One Health framework. In the second study we describe a rare resistance mechanism that was linked to ertapenem resistance in a K. pneumoniae strain from a companion animal and elucidated the resistance mechanism using both short and long read sequencing techniques. For the third study, the prevalence of azole-resistant Aspergillus fumigatus in various environmental settings (agriculture, horticulture, and composting facilities) was analyzed to identify potential hotspots for azole resistance selection. Additionally, the occurrence of azole resistance in A. fumigatus from veterinary clinical samples, including food producing animals, pets and zoo animals, was examined in the fourth study. Lastly, a study was conducted to investigate the link between the environment and a high-risk Humboldt penguin population in a zoological setting. K. pneumoniae was detected in 18.6% of meat samples at the distribution level compared to 1.7% in healthy food producing animals. In 35.6% of wastewater samples K. pneumoniae was detected. Antimicrobial susceptibility testing (AST) revealed moderate to high resistance rates to azithromycin, high to extremely high resistance rates to ciprofloxacin and cefotaxime and low to moderate colistin resistance in human clinical and wastewater isolates. High rates of ertapenem resistance were observed Summary – résumé - samenvatting 26 in isolates resistant to third generation cephalosporins and carbapenems. Whole-genome sequencing identified significant genetic diversity among 286 isolates, with 141 distinct sequence types, including high-risk MDR clonal groups like CG258, CG15, CG147, and CG307. Transmission pathways were observed both within and across sectors, with notable clusters involving K. pneumoniae ST147 from the food chain at distribution level and wastewater, suggesting contamination during meat processing. In the second study, the K. pneumoniae strain (ST11) was isolated from a companion animal and showed resistance to critically important antibiotics, including, azithromycin, cefotaxime, ciprofloxacin, gentamicin and ertapenem. The strain carried three plasmids, including the IncFIB(K) plasmid harboring multiple antibiotic resistance genes (ARG’s), such as blaSCO-1, and the IncR plasmid with the blaDHA-1 gene. A mutation in outer membrane protein OmpK37 was observed. These genetic elements contributed to its ertapenem resistance. In the third study, environmental samples were collected in Belgium, including agriculture, horticulture, and composting facilities, to detect A. fumigatus and assess its azole resistance. Composting facilities showed a high prevalence of airborne A. fumigatus spores, though none were found in mature compost samples. In total, two colonies from composting facilities showed resistance to medical azoles. In horticulture, 4 azole resistant A. fumigatus (ARAf) colonies were isolated. Agriculture was confirmed as a coldspot. Across all samples, a prevalence of ARAf of 2.62% was detected, with TR34/L98H and TR46/Y121F/T289A mutations present in the resistant isolates. In study 4 and 5, azole resistance was assessed in veterinary clinical isolates. In study 4, isolates from food producing animals, pets, wild birds and the zoo were included. Azole resistance testing revealed five resistant isolates (3.3%), including two from pigeons, two from cows, and one from a cat. Three isolates exhibited pan-azole resistance, with the TR34/L98H mutation in the cyp51A gene found in 80% of the resistant cases. In study 5, environmental sampling inside a penguin enclosure revealed (non-significant) seasonal fluctuations in A. fumigatus contamination in the air. Between 2017 and 2022, 189 Humboldt penguins died, with 51 cases confirmed as pulmonary aspergillosis. Resistance was observed in 17.14% of clinical and 11.76% of environmental strains, mainly associated with the TR34/L98H mutation in the cyp51A gene. Microsatellite genotyping showed no identical genotypes between veterinary isolates, but closely related genotypes were found between some environmental and veterinary strains, suggesting possible transmission. The study emphasizes the importance of the One Health approach in tackling AMR, as it demonstrates the interconnectedness of human, animal, and environmental health. Clonal transmission of K. pneumoniae within and across sectors highlights the need for coordinated surveillance and intervention strategies to mitigate the spread of resistant strains. Furthermore, we confirmed that the environment is the main driver behind azole resistance selection in A. fumigatus and found the TR34/L98H to be the most prevalent mutation. Further research is necessary to identify hotspots in Belgium. The implementation of standardization in surveillance methods is crucial.

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