Table 1.
Characteristics of strains selected for whole genome sequencing in this study.
Fig 1.
Histological examination of mice mammary glands.
A, non-pyrogenic phosphate-buffered saline (negative control), 1 DPC, no histological changes (x10); B, strain K71, 1 DPC, focal light inter-alveolar infiltrate with no signs of inflammation of the gland (x4); C, strain P4, 1 DPC, focal intra-alveolar PMN infiltration (x4); D, strain P4, 2 DPC, recovery of the gland and no inflammatory infiltrate or significant damage (x10); E, strain VL2874, 1 DPC, extensive intra-alveolar and ductal PMN infiltration (x4); F, strain VL2874, 2 DPC, partially destructed non-lactating alveoli, protein deposit and necrosis (x10); G, strain VL2732, 1 DPC, diffuse inter and intra-alveolar PMN infiltration (x10); H, strain VL2732, 2 DPC, inter-alveolar inflammatory infiltrate, necrosis, hyperemia and loss of alveoli architecture (x10); I, strain VL2732, 5 DPC, degenerative PMN, diffuse necrosis, hemorrhage, protein deposit and extensive loss of alveoli architecture (x10); J-K, strain K-12 MG1655, 1 DPC, diffuse intra-alveolar PMN infiltration without alterations to the gland structure and numerous lactating alveoli (x4-x20); L, representative picture of intra-alveolar PMN infiltration (x20). Eosine-hematoxylin stain.
Table 2.
Gross and histopathological examinations of mammary glands injected by three mammary pathogenic and one non-pathogenic, environmental strain.
Fig 2.
Heatmap of the area under the curve of phenotype microarray.
Heatmap of the area under the curve (AUC) parameter extracted from kinetic data over 24 h in phenotype microarray with validation by 100 bootstrap repetitions. Numerals after the strain name indicate technical replicates of the same strain. The heatmap color indicates the AUC; yellow for higher values and blue for lower.
Table 3.
Positive reactions for utilization of nutrient sources by category.
In brackets, the percentage of positive reactions in a particular nutrient category is shown.
Fig 3.
Distribution of functional annotation into RAST subsystems.
Table 4.
Main functional features present in the three MPEC strains and lacking in the non-mammary pathogenic strain K71 and their expected effects on virulence.
Fig 4.
Alignment of predicted sequence of conjugative plasmid pP4 of strain P4 aligned to plasmid F of strain K-12 and to plasmid p1303_109 of mastitis strain 1303.
Fig 5.
VL2732 Pathogenicity Island containing the yersiniabactin and invasin Inv clusters, resembling the Yersinia high pathogenicity island.
The figure shows the alignment of the PAI region in the genome of strain VL2732 and the genomes of MPEC strains VL2874 and P4, showing the insertion site of the yersiniabactin elements.
Fig 6.
Whole genome SNP based phylogenetic analysis.
Phylogenetic analysis of SNP extracted from whole genome alignment of the three MPEC strains (VL2874, VL2732 and P4), the environmental, non-mammary pathogenic strain K71 and representative strains of diverse E. coli pathotypes and non-pathogenic strains. Overall, the strains clustered according to their phylogenetic groups, indicated here by different colors. Confidence values are shown over each node.
Fig 7.
Genome-to-genome distance matrix.
GGD was calculated between the four studied strains and various E. coli genomes from diverse pathotypes and non-pathogenic strains. The three mastitis pathogenic genomes were mostly similar, whereas notable genomic distances were found to other pathotypes and to the environmental, non-mammary pathogenic strain K71.
Fig 8.
Relative Manhattan distance of predicted proteomes.
Predicted proteomes of three mammary pathogenic E. coli and representatives of diverse E. coli pathotypes and non-pathogenic strains. Dendrogram generated on the basis of gene presence/absence considering all genes, including singletons, and validated after 100 bootstraps repetitions (depicted in red). Genes were clustered by 80% sequence identity over 80% sequence length. The three mammary pathogenic strains are closely related, and significantly distant from the environmental, non-mammary pathogenic strain K71.
Fig 9.
Growth rate in milk of MPEC (VL2874, VL2732 and P4), K-12 MG1655 and the environmental, non-mammary pathogenic strain K71.
Error bars show SD of triplicate experiments. Statistically significant differences at the same time point are indicated by letters.