Abstract
Escherichia coli (E. coli) are widely related to pyometra and cystitis in dogs, and these infections can occur simultaneously. The goal of this study was to determine genetic and pathogenic insights of 14 E. coli isolated simultaneously from pyometra content and bladder urine of seven bitches. To achieve this, in silico and in vitro comparative analyses were conducted. Whole-genome comparisons demonstrated that E. coli isolated from pyometra and urine of the same animal were predominantly genetic extraintestinal E. coli clones belonging to the same Sequence Type and phylogroup. The E. coli clones identified in this study included ST372, ST457, ST12, ST127, ST646, and ST961. Five isolates (35.7%) belonged to the ST12 complex. Except for two E. coli, all other isolates belonged to the B2 Clermont phylogroup. Interestingly, some genomes of E. coli from urine carried more virulence genes than those E. coli from pyometra. Both pyometra and urine E. coli isolates demonstrated a strong affinity for adhering to HeLa and T24 cells, with a low affinity for invading them. However, certain isolates from urine exhibited a greater tendency to adhere to T24 cells in qualitative and quantitative assays compared to isolates from pyometra. In conclusion, this study revealed the high genomic similarity between pyometra and urine E. coli isolates, as well as the virulent capacity of both to colonize endometrial and urothelial cells. The findings of this study underscore the importance of concurrently managing both infections clinically and could potentially contribute to future resources for the prevention of cystitis and pyometra.
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Acknowledgements
We thank Rede de Ciências Ômicas (RECOM), Dr. Claudio Wageck Canal, Dr. Ana Maria Oliveira Battastini, Dr. Annelise Kopp Alves, and Dr. Carlos Pérez Bergmann for the research collaboration. We also thank Lais Foresti for the support in sample collection and the Laboratory of Veterinary Bacteriology of the UFRGS team for technical support.
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This work was supported by the Coordination of Improvement of Higher Educational Personnel (CAPES) [grant number 001].
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Conceptualization: Cassiane Elisabete Lopes, Tania Aparecida Tardelli Gomes, and Franciele Maboni Siqueira; Methodology: Cassiane Elisabete Lopes, Tania Aparecida Tardelli Gomes, Mateus Matiuzzi da Costa, Rommel Thiago Jucá Ramos, Aristóteles Góes Neto, Vasco Ariston de Carvalho Azevedo, Bertram Brenig, and Franciele Maboni Siqueira; Formal analysis and investigation: Cassiane Elisabete Lopes, Tainara Soares Weyh, Camila Azevedo Moni, Maria Eduarda Dias, and Franciele Maboni Siqueira; Writing - original draft preparation: Cassiane Elisabete Lopes; Writing - review and editing: Franciele Maboni Siqueira; Resources: Mateus Matiuzzi da Costa, Rommel Thiago Jucá Ramos, Aristóteles Góes Neto, Vasco Ariston de Carvalho Azevedo, Bertram Brenig; Supervision: Franciele Maboni Siqueira. All authors reviewed the manuscript. All authors read and approved the final manuscript.
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ESM 1
Online Resource 2. Circular comparison among Escherichia coli genomes. A BLAST alignment was performed with the 14 genomes of this study and one characterized EnPEC genome (E. coli LBV005/17 - QLNJ00000000) against the reference UPEC genome (E. coli CFT 073 - AE014075.1) using the BRIG software. The image demonstrates a high similarity of the E. coli isolates with some punctual polymorphisms among them. Online Resource 3. Matrix of SNP differences among the Escherichia coli genomes of the study. The SNPs distance matrix was constructed by CSI Phylogeny v.1.4. The number of SNPs differences among the 14 assembled genomes of this study was demonstrated on each matrix box. A color scale was added to the matrix, in which the darker the green, the lower the number of SNPs among the genomes. E. coli CFT 073 (AE014075.1) and E. coli LBV005/17 (QLNJ00000000) were used as UPEC and EnPEC reference control genomes, respectively. Online Resource 4. Adhesion pattern of a set of pyometra and urine Escherichia coli isolates on HeLa cells by optical microscopy. The adherence pattern of the randomly chosen isolates E. coli LBV064/17_pyometra (A) and E. coli LBV064/17_urine (B) was obtained after a 3 h adhesion assay with HeLa cells. Light microscopy images (400X microscopic magnification) show bacterial clumps with affinity to HeLa cells (arrows).E. coli CFT 073 as positive control (C). E. coli EAEC 042 shows typical aggregative adhesion (D). Non-infected HeLa cells (E). Online Resource 5. Adhesion pattern of a set of pyometra and urine Escherichia coli isolates on T24 cells by optical microscopy. The adherence pattern of the randomly chosen isolatesE. coli LBV064/17_pyometra (A) and E. coli LBV064/17_urine (B) was obtained after a 3 h adhesion assay with the T24 cell line. E. coli UPEC CFT 073 as positive control (C). Light microscopy images (400X microscopic magnification) show affinity with the formation of bacterial clumps on T24 cells (arrows). E. coli EAEC 042 shows typical aggregative adhesion (D). Non-infected T24 cells (200X microscopic magnification) (E). Online Resource 6. Electron microscopy evaluation of a set of Escherichia coli isolates adhesion on HeLa cells compared to control strains. Electron microscopy images (3,300X microscopic magnification) were obtained after adhesion assays with 3 h of incubation. The adherence pattern of the randomly selected isolates E. coli LBV064/17_pyometra (A) and E. coli LBV064/17_urine (B) demonstrated diffuse adhesion with some grouped bacteria on HeLa surface. This adhesion pattern was very similar to the UPEC control strain CFT 073 (C). E. coli EAEC 042 exhibited typical aggregative adhesion (D). Online Resource 7. Electron microscopy evaluation of a set of Escherichia coli isolates adhesion on T24 cells compared to control strains. Electron microscopy images (3,300X microscopic magnification) were obtained after an adhesion assay with 3 h of incubation. The adherence pattern of the randomly selected isolates E. coli LBV064/17_pyometra (A) and E. coli LBV064/17_urine (B). Suggestive biofilm formation of E. coli LBV064/17_urine was frequently identified on the surface of T24 cells (arrows). The biofilm production was less frequent on the E. coli LBV064/17_pyometra sample. E. coli CFT 073 exhibited a high level of affinity to T24 cells (C). E. coli. EAEC 042 showcased typical aggregative adhesion (D) Online Resource 10. Virulence genes profile of the Escherichia coli isolated from bitch pyometra and urine. The graphic represents the frequency of each gene or family of genes found in the 14 E. coli genomes of this study. The genes were grouped by different pathogenic tools of E. coli (cytotoxins, microcins, biofilm production, host immune modulation, polysaccharide capsule production, and iron uptake systems). The search for genomic virulence genes was performed by PATRIC and Virulence Finder databases. (ZIP 358 MB)
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Lopes, C.E., Weyh, T.S., Moni, C.A. et al. Comparative genetic and pathogenic approaches of Escherichia coli isolated simultaneously from pyometra and urine of bitches. Vet Res Commun (2024). https://doi.org/10.1007/s11259-024-10355-7
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DOI: https://doi.org/10.1007/s11259-024-10355-7