Aspergillus fumigatus is the main causative agent of avian aspergillosis and results in significant health problems in birds, especially those living in captivity. The fungal contamination by A. fumigatus in the environment of Humboldt penguins (Spheniscus humboldti), located in a Belgian zoo, was assessed through the analysis of air, water, sand and nest samples during four non-consecutive days in 2021–2022. From these samples, potential azole-resistant A. fumigatus (ARAF) isolates were detected using a selective culture medium. A total of 28 veterinary isolates obtained after necropsy of Humboldt penguins and other avian species from the zoo were also included. All veterinary and suspected ARAF isolates from the environment were characterized for their azole-resistance profile by broth microdilution. Isolates displaying phenotypic resistance against at least one medical azole were systematically screened for mutations in the cyp51A gene. A total of 14 (13.6%) ARAF isolates were identified from the environment (n = 8) and from Humboldt penguins (n = 6). The TR34/L98H mutation was observed in all resistant environmental strains, and in two resistant veterinary strains. To the best of our knowledge, this is the first description of this mutation in A. fumigatus isolates from Humboldt penguins. During the period 2017–2022, pulmonary aspergillosis was confirmed in 51 necropsied penguins, which reflects a death rate due to aspergillosis of 68.0%, mostly affecting adults. Microsatellite polymorphism analysis revealed a high level of diversity among environmental and veterinary A. fumigatus isolates. However, a cluster was observed between one veterinary isolate and six environmental strains, all resistant to medical azoles. In conclusion, the environment of the Humboldt penguins is a potential contamination source of ARAF, making their management even more complex.

Klebsiella pneumoniae of sequence type (ST) 11 is a hyper-epidemic nosocomial clone, which is spreading worldwide among humans and emerging in pets. This is the first report, to the best of our knowledge, of multidrug-resistant (MDR) K. pneumoniae ST11 carrying bla_SCO-1 and bla_DHA-1, isolated from a four-month-old dog in Belgium. Antimicrobial susceptibility testing (AST) of the isolate, performed via broth microdilution following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, revealed resistance to eight different classes of antimicrobials, including carbapenems, in particular ertapenem, third-generation cephalosporins and fluoroquinolones. A hybrid approach, combining long- and short-read sequencing, was employed for in silico plasmid characterization, multi-locus sequence typing (MLST) and the identification and localization of antimicrobial resistance (AMR) and virulence-associated genes. Three plasmids were reconstructed from the whole-genome sequence (WGS) data: the conjugative IncFIB(K), the non-mobilizable IncR and the mobilizable but unconjugative ColRNAI. The IncFIB(K) plasmid carried the bla_SCO-1 gene, whereas IncR carried bla_DHA-1, both alongside several other antimicrobial resistance genes (ARGs). No virulence genes could be detected. Here, we suggest that the resistance to ertapenem associated with susceptibility to imipenem and meropenem in K. pneumoniae could be related to the presence of bla_SCO-1 and bla_DHA-1, combined with permeability defects caused by point mutations in an outer membrane porin (OmpK37). The presence of the bla_SCO-1 gene on a conjugative IncFIB(K) plasmid is worrisome as it can increase the risk of transmission to humans, to animals and to the environment.

Azole resistance in Aspergillus fumigatus (ARAf) is becoming a worldwide health threat due to increasing occurrence in the environment. However, environmental surveillance programs are not commonly in place and are lacking in Belgium. Since no data on the occurrence of ARAf and the presence of hotspots for the selection of azole resistance is available in Belgium, a first study on the prevalence of ARAf in the environment was conducted. A total of 232 air and compost or soil samples were taken from two composting facilities, and from horticultural and agricultural crops. The azole susceptibility pattern was determined using the EUCAST method (E. Def. 9.4), and the cyp51A gene and its promotor region were sequenced in A. fumigatus isolates with phenotypic azole resistance. Six pan-azole-resistant A. fumigatus isolates were identified, originating from compost and horticultural crops. Four isolates carried the TR34/L98H mutation, and one isolate carried the TR46/Y121F/T289A mutation. However, we did not observe any ARAf isolates from agricultural crops. In conclusion, this study reported the first TR34/L98H and TR46/Y121F/T289A mutation isolated from a composting facility and horticulture in Belgium. The implementation of standardization in environmental surveillance of A. fumigatus on a European level would be beneficial in order to identify hotspots. The ubiquitous fungus Aspergillus fumigatus can cause serious invasive diseases in humans. Due to the extensive use of environmental azoles, an increase of clinical infections with azole-resistant A. fumigatus is seen. This pilot study aimed to estimate the prevalence of azole-resistant A. fumigatus in environmental reservoirs in Belgium.

Aspergillosis causes significant health risks to both birds and mammals. The outcome of these infections is often poor due to delayed diagnosis and treatment failure. We investigated 152 cases of aspergillosis from birds and mammals in Belgium. Most samples originated from the taxonomic orders Artiodactyla (40.1 %) and Columbiformes (19.7 %). Five isolates (3.3 %) showed phenotypical resistance against at least one medical azole. Three of these isolates were pan-azole resistant bearing the TR34/L98H mutation. The predominance of this resistance mutation supports an environmental route for exposure and resistance selection, highlighting the importance of the One Health concept.

Klebsiella pneumoniae is an important reservoir and disseminator of antimicrobial resistance (AMR) genes. Multidrug-resistant lineages, particularly extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant strains, represent an important public health threat. This study characterized antimicrobial resistance and genomic diversity of K. pneumoniae across One Health compartments in Belgium and investigated genomic relatedness within and across sectors. Between 2019 and 2022, 888 presumptive K. pneumoniae isolates were collected from diseased animals ( n = 311), wastewater ( n = 299), the food chain ( n = 178), and human clinical samples ( n = 100). Following selection based on isolation source and AMR characteristics, 286 WGS-confirmed K. pneumoniae isolates were characterized for AMR and virulence determinants, plasmid replicons, population structure, and phylogenomic relatedness. Phenotypic and genotypic resistance showed high concordance for third-generation cephalosporins (93.4%) and carbapenems (89.2%). Carbapenemase genes encoding KPC, NDM, OXA-48-like, and VIM enzymes were detected in 67 isolates from humans ( n = 56) and wastewater ( n = 11), but not in food-chain or diseased-animal isolates. Population structure was highly diverse, with ST307, ST15, ST147, and ST11 representing the most frequently detected high-risk lineages. Thirteen novel STs were identified in 18 K. pneumoniae isolates. Seventeen genomic clusters were identified, including 13 within-sector and four cross-sector clusters. Notably, an ST147 cluster comprising two poultry-meat isolates and a human clinical isolate showed no SNP differences, indicating very close genomic relatedness across sectors. Overall, our findings demonstrate a genetically diverse K. pneumoniae population alongside the circulation of clinically relevant high-risk lineages across One Health compartments and support integrated genomic surveillance to better understand their dissemination.

Although research on foodborne diseases is well documented for certain microorganisms, less research has been undertaken on cross-contamination with Enterobacteriaceae from food within the kitchen environment. In this study, we analysed the presence of Klebsiella spp. and Raoultella spp. and their antibiotic resistance in 100 used kitchen sponges. In a domestic environment, kitchen sponges are commonly used by consumers for doing the dishes and/or cleaning kitchen surfaces. Given their multipurpose use, their often high humidity and presence of organic residuals they are considered a favourable habitat for various groups of microorganisms and could serve as a vehicle in transmission of foodborne pathogens.^1,2 A total of 100 kitchen sponges alongside a questionnaire regarding hygienic parameters were randomly collected from domestic environments and analysed within 24 h after arrival in the lab. Kitchen sponges were immersed in 100 mL buffered peptone water, homogenized and incubated at 37°C for 24 h ± 2 h. Ten microlitres of this enrichment was plated on MacConkey agar (Bio-Rad) and incubated at 37°C for 24 h ± 2 h. Presumptive Klebsiella spp. and Raoultella spp. were isolated and confirmed by MALDI-TOF MS. Antibiotic resistance testing (ART) was performed on all Klebsiella spp. and Raoultella spp. isolates following the EUCAST guidelines. WGS was performed using the Illumina platform. A total of 65% of the kitchen sponges were positive for Klebsiella spp. or Raoultella spp. The species Klebsiella oxytoca was detected in 78.5% of positive samples, followed by Klebsiella pneumoniae (12.31%), Raoultella ornithinolytica (4.62%), Klebsiella variicola (1.54%), Klebsiella aerogenes (1.54%) and Raoultella planticola (1.54%). To the best of our knowledge this is the first study performed on detection of Klebsiella spp. in kitchen sponges from Belgium and has comparable results with previous studies.^1–3 We hypothesize the high positivity rate is caused by cross-contamination. ART revealed the presence of one carbapenem-resistant isolate, with resistance to ertapenem and meropenem. Reduced susceptibility to carbapenems was caused by the presence of bla_SHV-36 combined with porin deficiency. Klebsiella spp. can be part of the commensal flora of human intestines but are considered to be opportunistic pathogens. Klebsiella spp. might present antimicrobial resistance and carbapenem-resistant Enterobacteriaceae are taken up in the WHO global priority list of antibiotic-resistant bacteria. High prevalence of Klebsiella spp. and a carbapenem-resistant isolate in kitchen sponges highlight the occurrence of cross-contamination from food and the possible risks for foodborne disease associated with this kitchen tool. These findings advocate for good hygienic measures within a household setting.