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Genomic and Phenomic Study of Mammary Pathogenic Escherichia coli.

Blum SE, Heller ED, Sela S, Elad D, Edery N, Leitner G - PLoS ONE (2015)

Bottom Line: 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.Among phenotypes unique to MPEC compared to the non-mammary pathogenic strain were uric acid and D-serine metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, Rehovot, Israel; National Mastitis Reference Center, Department of Bacteriology, Kimron Veterinary Institute, Bet Dagan, Israel; Department of Bacteriology, Kimron Veterinary Institute, Bet Dagan, Israel.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0136387.g009: 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.

Mentions: From the GGD and proteome analysis described above it is noteworthy that MPEC strains are closely related to strain K-12. The GGD results could be explained by the fact that the MPEC strains studied here share with K-12 the same genotypic background in terms of phylogenetic group (group A) and sequence type in MLST (ST10). The similarity between MPEC and K-12 was recently described also for other E. coli strains isolated from mastitis, based on phylogenetic analysis of conserved (core) genomic regions [65]. However, the proteome comparison presented here was based on presence/absence of genes considering all the genes found in the genomes, including non-conserved (accessory) regions, and is not necessarily in accord to phylogeny. The proteome comparison actually shows that the gene contents of the MPEC studied here are relatively close to that of K-12, suggesting two possible explanations. First, that only few genes in MPEC over the "basic" genes repertoire present in strain K-12 are necessary for pathogenicity in the mammary gland. Second, that the genome of K-12 may actually include genes promoting pathogenicity in the mammary gland. Due to the similarity between K-12 and MPEC, and since K-12 is widely considered a non-pathogenic strain, it was interesting to examine if K-12 could be pathogenic in the mammary gland. For this purpose, the growth in milk phenotype of K-12 was tested at first. As shown in Fig 9, K-12 is able to grow in milk in a rate similar to MPEC, and significantly different from K71, which consistently shows a slow growth rate in milk [21]. Growth in milk is a phenotype highly conserved in MPEC. As previously shown by Blum et al. [21], and later confirmed with a larger collection of MPEC and environmental strains from different farms (unpublished data), all MPEC strains are able to grow in milk at particularly high rates, whereas several E. coli strains present in the environment have slow growth rates in milk (like strain K71). Differences in growth rate were observed specifically in milk as all strains showed similar growth rates when bacteria were inoculated in regular nutrient broth (data not shown). Even though growth in milk is a phenotype of pivotal importance in E. coli pathogenicity in the mammary gland [6], this assay by itself does not confirm actual virulence potential in the gland. Thus, K-12 was also tested with the murine IMI model described above. In mice mammary glands, K-12 elicited a clear inflammatory response after 1 DPC, characterized by diffuse intra-alveolar neutrophil infiltration, but no observable inter-alveolar reaction or extensive damage to the gland tissue (Fig 1), and in fact a considerable number of milking alveoli was observed after challenge. No inflammation or tissue alterations were observed at 2 DPC, and milk producing alveoli remained conserved. K-12 bacteria were isolated from challenged mammary glands at 1 DPC. The inflammation elicited by K-12 was therefore somewhat milder than that of the MPEC strains studied here and that were actually isolated from mastitis. However, the potential of K-12 to cause mastitis cannot be discarded at this time. Hence it is possible that K-12 harbors genes that allow for pathogenicity in the mammary gland, partially explaining the close relatedness of MPEC and K-12 in the predicted proteome analysis.


Genomic and Phenomic Study of Mammary Pathogenic Escherichia coli.

Blum SE, Heller ED, Sela S, Elad D, Edery N, Leitner G - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556653&req=5

pone.0136387.g009: 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.
Mentions: From the GGD and proteome analysis described above it is noteworthy that MPEC strains are closely related to strain K-12. The GGD results could be explained by the fact that the MPEC strains studied here share with K-12 the same genotypic background in terms of phylogenetic group (group A) and sequence type in MLST (ST10). The similarity between MPEC and K-12 was recently described also for other E. coli strains isolated from mastitis, based on phylogenetic analysis of conserved (core) genomic regions [65]. However, the proteome comparison presented here was based on presence/absence of genes considering all the genes found in the genomes, including non-conserved (accessory) regions, and is not necessarily in accord to phylogeny. The proteome comparison actually shows that the gene contents of the MPEC studied here are relatively close to that of K-12, suggesting two possible explanations. First, that only few genes in MPEC over the "basic" genes repertoire present in strain K-12 are necessary for pathogenicity in the mammary gland. Second, that the genome of K-12 may actually include genes promoting pathogenicity in the mammary gland. Due to the similarity between K-12 and MPEC, and since K-12 is widely considered a non-pathogenic strain, it was interesting to examine if K-12 could be pathogenic in the mammary gland. For this purpose, the growth in milk phenotype of K-12 was tested at first. As shown in Fig 9, K-12 is able to grow in milk in a rate similar to MPEC, and significantly different from K71, which consistently shows a slow growth rate in milk [21]. Growth in milk is a phenotype highly conserved in MPEC. As previously shown by Blum et al. [21], and later confirmed with a larger collection of MPEC and environmental strains from different farms (unpublished data), all MPEC strains are able to grow in milk at particularly high rates, whereas several E. coli strains present in the environment have slow growth rates in milk (like strain K71). Differences in growth rate were observed specifically in milk as all strains showed similar growth rates when bacteria were inoculated in regular nutrient broth (data not shown). Even though growth in milk is a phenotype of pivotal importance in E. coli pathogenicity in the mammary gland [6], this assay by itself does not confirm actual virulence potential in the gland. Thus, K-12 was also tested with the murine IMI model described above. In mice mammary glands, K-12 elicited a clear inflammatory response after 1 DPC, characterized by diffuse intra-alveolar neutrophil infiltration, but no observable inter-alveolar reaction or extensive damage to the gland tissue (Fig 1), and in fact a considerable number of milking alveoli was observed after challenge. No inflammation or tissue alterations were observed at 2 DPC, and milk producing alveoli remained conserved. K-12 bacteria were isolated from challenged mammary glands at 1 DPC. The inflammation elicited by K-12 was therefore somewhat milder than that of the MPEC strains studied here and that were actually isolated from mastitis. However, the potential of K-12 to cause mastitis cannot be discarded at this time. Hence it is possible that K-12 harbors genes that allow for pathogenicity in the mammary gland, partially explaining the close relatedness of MPEC and K-12 in the predicted proteome analysis.

Bottom Line: 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.Among phenotypes unique to MPEC compared to the non-mammary pathogenic strain were uric acid and D-serine metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Sciences, Robert H. Smith Faculty of Agriculture, Food and Environment, Rehovot, Israel; National Mastitis Reference Center, Department of Bacteriology, Kimron Veterinary Institute, Bet Dagan, Israel; Department of Bacteriology, Kimron Veterinary Institute, Bet Dagan, Israel.

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.

No MeSH data available.


Related in: MedlinePlus