Genomic and Phenomic Study of Mammary Pathogenic Escherichia coli

Abstract

Escherichia coli is a major etiological agent of intra-mammary infections (IMI) in cows, leading to acute mastitis and causing great economic losses in dairy production worldwide. Particular strains cause persistent IMI, leading to recurrent mastitis. Virulence factors of mammary pathogenic E. coli (MPEC) involved pathogenesis of mastitis as well as those differentiating strains causing acute or persistent mastitis are largely unknown. This study aimed to identify virulence markers in MPEC through whole genome and phenome comparative analysis. MPEC strains causing acute (VL2874 and P4) or persistent (VL2732) mastitis were compared to an environmental strain (K71) and to the genomes of strains representing different E. coli pathotypes. Intra-mammary challenge in mice confirmed experimentally that the strains studied here have different pathogenic potential, and that the environmental strain K71 is non-pathogenic in the mammary gland. Analysis of whole genome sequences and predicted proteomes revealed high similarity among MPEC, whereas MPEC significantly differed from the non-mammary pathogenic strain K71, and from E. coli genomes from other pathotypes. Functional features identified in MPEC genomes and lacking in the non-mammary pathogenic strain were associated with synthesis of lipopolysaccharide and other membrane antigens, ferric-dicitrate iron acquisition and sugars metabolism. Features associated with cytotoxicity or intra-cellular survival were found specifically in the genomes of strains from severe and acute (VL2874) or persistent (VL2732) mastitis, respectively. MPEC genomes were relatively similar to strain K-12, which was subsequently shown here to be possibly pathogenic in the mammary gland. Phenome analysis showed that the persistent MPEC was the most versatile in terms of nutrients metabolized and acute MPEC the least. Among phenotypes unique to MPEC compared to the non-mammary pathogenic strain were uric acid and D-serine metabolism. This study reveals virulence factors and phenotypic characteristics of MPEC that may play a role in pathogenesis of E. coli mastitis.

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.

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.

Fig 3. Distribution of functional annotation into RAST subsystems.

Fig 4. Plasmid pP4 of strain P4.

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.