Multidrug-Resistant High-Risk Escherichia coli and Klebsiella pneumoniae Clonal Lineages Occur in Black-Headed Gulls from Two Conservation Islands in Germany

Abstract

Multidrug-resistant (MDR) Enterobacterales, including extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, not only emerge in healthcare settings but also in other habitats, such as livestock and wildlife. The spread of these pathogens, which often combine resistance with high-level virulence, is a growing problem, as infections have become increasingly difficult to treat. Here, we investigated the occurrence of ESBL-producing E. coli and K. pneumoniae in fecal samples from two black-headed gull colonies breeding on two nature conservation islands in Western Pomerania, Germany. In addition to cloacal samples from adult birds (n = 211) and their nestlings (n = 99) during the 2021 breeding season, collective fecal samples (n = 29) were obtained. All samples were screened for ESBL producers, which were then subjected to whole-genome sequencing. We found a total of 12 ESBL-producing E. coli and K. pneumoniae consisting of 11 E. coli and 1 K. pneumoniae, and including the international high-risk E. coli sequence types (ST)131, ST38, and ST58. Eight of the investigated strains had a MDR genotype and carried a large repertoire of virulence-associated genes, including the pap operon, which is important for urinary tract infections. In addition, we identified many genes associated with adherence, biofilm formation, iron uptake, and toxin production. Finally, our analysis revealed the close phylogenetic relationship of ST38 strains with genomes originating from human sources, underlining their zoonotic and pathogenic character. This study highlights the importance of the One Health approach, and thus the interdependence between human and animal health and their surrounding environment.

Keywords: One Health; antimicrobial resistance; black-headed gulls; next-generation sequencing; virulence; wildlife.

Figure 1

Both black-headed gull colonies are located on the German Baltic Sea coast. The large map shows the coastline of the Baltic Sea and the North Sea. Blue areas indicate the German federal state of Mecklenburg–Western Pomerania, where both black-headed gull colonies are located (yellow). The map was created using https://www.mapchart.net (accessed on 19 August 2022).

Figure 2

Geno- and phenotypic resistance of the 12 ESBL-producing strains. The corresponding gene content and resistant phenotypes are summarized in a presence–absence matrix. The predictions of resistance (genes and point mutations) (gray) were based on alignments of the sequences from the AMRFinderPlus database (default settings of identity ≥90.0% and coverage ≥50.0%). Resistant phenotypes (blue) were defined according to the published breakpoints for Enterobacterales of the Clinical and Laboratory Standards Institute (CLSI). Uncolored boxes indicate the absence of the genotype or phenotype. CIP, ciprofloxacin; COL, colistin; CTX, cefotaxime; GEN, gentamicin; TET, tetracycline.

Figure 3

Overview of the virulence-associated genes (VAGs) carried by the 12 ESBL-producing strains. Predictions of VAGs (gray) were based on the alignment of sequences from the virulence factor database (threshold for coverage and identity, ≥65.0%).

Figure 4

ST38 phylogeny. Phylogenetics revealed the close relationship of PBIO3541 and PBIO3584 with publicly available genomes (n = 30). The genomes included here were selected on the basis of the most shared k-mers using a Mash-based approach. The phylogenetic tree was constructed using a maximum likelihood-based approach and based on a core single nucleotide polymorphism alignment, with PBIO3541 as the reference. The tree was midpoint-rooted and the circumference of the circles on the branches indicates bootstrapping support of ≥50%. Labels indicate the accession numbers (except for PBIO3541 and PBIO3584, which are highlighted in bold). In addition, the three-letter country code indicates the sample’s origin (https://www.iso.org/obp/ui/#search, accessed on 19 September 2022). Different colors indicate the collection date and source. Further information includes the encoded ESBL genes and either their chromosomal or plasmid location. In the case of an unclear assignation, both the chromosome and plasmid locations are indicated (n = 6). NA, not available (i.e., no metadata provided); ST, sequence type.