Highly Virulent and Multidrug-Resistant Escherichia coli Sequence Type 58 from a Sausage in Germany

Abstract

Studies have previously described the occurrence of multidrug-resistant (MDR) Escherichia coli in human and veterinary medical settings, livestock, and, to a lesser extent, in the environment and food. While they mostly analyzed foodborne E. coli regarding phenotypic and sometimes genotypic antibiotic resistance and basic phylogenetic classification, we have limited understanding of the in vitro and in vivo virulence characteristics and global phylogenetic contexts of these bacteria. Here, we investigated in-depth an E. coli strain (PBIO3502) isolated from a pork sausage in Germany in 2021. Whole-genome sequence analysis revealed sequence type (ST)58, which has an internationally emerging high-risk clonal lineage. In addition to its MDR phenotype that mostly matched the genotype, PBIO3502 demonstrated pronounced virulence features, including in vitro biofilm formation, siderophore secretion, serum resilience, and in vivo mortality in Galleria mellonella larvae. Along with the genomic analysis indicating close phylogenetic relatedness of our strain with publicly available, clinically relevant representatives of the same ST, these results suggest the zoonotic and pathogenic character of PBIO3502 with the potential to cause infection in humans and animals. Additionally, our study highlights the necessity of the One Health approach while integrating human, animal, and environmental health, as well as the role of meat products and food chains in the putative transmission of MDR pathogens.

Keywords: CTX-M-1; Enterobacterales; Escherichia coli; IncI1; One Health; antimicrobial resistance; food safety.

Figure 1

PBIO3205 exhibits a highly virulent phenotype. (a) Biofilm formation on polystyrene surfaces as determined by crystal violet staining (n = 4). Results are expressed as growth-adjusted specific biofilm formation. The line within the box marks the median value, while the boxplot represents the 25th to 75th percentile of the data set. The whiskers mark the minimum and maximum values, respectively. (b) Morphology of long-term colonies examining the expression of the biofilm-associated extracellular matrix components curli fibers and cellulose. Congo red was used to specifically stain curli fibers, and cellulose was visually interpreted based on the textured surface. PBIO1435 and W3110 were used as references. (c) Cellulose formation was examined by staining with Calcofluor and measuring the fluorescence intensity of bound Calcofluor (n = 3). Results are given as mean values of fluorescence intensity and standard errors. (d) The extent of secreted siderophores is expressed as the mean of the percent unit of siderophore production and standard error (n = 3–5). (e) Survival in 50% human serum (n = 3–5). Results are given as means and standard errors of log₂ fold change in CFU mL⁻¹ after 4 h of incubation in the presence of human serum. (f,g) Kaplan–Meier plot of mortality in the Galleria mellonella larvae infection model (n = 3). Results are expressed as mean percent mortality after injection of 10⁴ CFU per larva (g) and 10⁵ CFU per larva, respectively. NC, negative control. PC, positive control.

Figure 2

Phylogenetics reveals close relationship of PBIO3502 with publicly available genomes (n = 159). Included genomes were selected based on most shared k-mers using Mash. The phylogenetic tree was constructed using a maximum likelihood-based approach and is based on a core singe-nucleotide polymorphism alignment (4984 sites). The circular tree was midpoint-rooted and the circles on the branches indicate bootstrap support of ≥50% from 1000 replicates. The labels indicate the accession number (except PBIO3502) and are colored according to their source/host, as indicated in the legend. Annotations indicate (from inner to outer circle): encoded CTX-M (cefotaximase) allele, presence of genes encoding aerobactin and tellurite resistance. NA, not applicable (i.e., no metadata provided).