Genome-Wide Identification of Fitness Factors in Mastitis-Associated Escherichia coli

• Published in: Applied and environmental microbiology Vol. 84; no. 2
• Main Authors: Olson, Michael A, Siebach, Timothy W, Griffitts, Joel S, Wilson, Eric, Erickson, David L
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 15.01.2018
• Subjects: Animals; Bacteria; Breasts; Cattle; Clonal deletion; Cow's milk; E coli; Escherichia coli; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - pathogenicity; Escherichia coli Infections - microbiology; Escherichia coli Proteins - genetics; Female; Fitness; Gene deletion; Gene sequencing; Genes; Genetic Fitness; Genetics and Molecular Biology; Genome, Bacterial; Genomes; Genomic Islands; Glands; Infections; Insertion; Iron; Iron - metabolism; Lactation; Larva - microbiology; Larvae; Mammary glands; Mammary Glands, Animal - microbiology; Mastitis; Mastitis, Bovine - microbiology; Mice; Milk; Milk - microbiology; Moths - microbiology; Mutagenesis; Polysaccharides, Bacterial - genetics; Receptors, Cell Surface - deficiency; Receptors, Cell Surface - genetics; Scavenging; Virulence; Virulence factors; Whole Genome Sequencing; capsule; fecA; mastitis; MPEC; Escherichia coli; milk; Galleria mellonella; mammary gland; ferric dicitrate; ExPEC
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.02190-17

Virulence factors of mammary pathogenic (MPEC) have not been identified, and it is not known how bacterial gene content influences the severity of mastitis. Here, we report a genome-wide identification of genes that contribute to fitness of MPEC under conditions relevant to the natural history of the disease. A highly virulent clinical isolate (M12) was identified that killed at low infectious doses and that replicated to high numbers in mouse mammary glands and spread to spleens. Genome sequencing was combined with transposon insertion site sequencing to identify MPEC genes that contribute to growth in unpasteurized whole milk, as well as during and mouse mastitis infections. These analyses show that strain M12 possesses a unique genomic island encoding a group III polysaccharide capsule that greatly enhances virulence in Several genes appear critical for MPEC survival in both and in mice, including those for nutrient-scavenging systems and resistance to cellular stress. Insertions in the ferric dicitrate receptor gene caused significant fitness defects under all conditions (in milk, , and mice). This gene was highly expressed during growth in milk. Targeted deletion of from strain M12 caused attenuation in larvae and reduced growth in unpasteurized cow's milk and lactating mouse mammary glands. Our results confirm that iron scavenging by the ferric dicitrate receptor, which is strongly associated with MPEC strains, is required for MPEC growth and may influence disease severity in mastitis infections. Mastitis caused by inflicts substantial burdens on the health and productivity of dairy animals. Strains causing mastitis may express genes that distinguish them from other strains and promote infection of mammary glands, but these have not been identified. Using a highly virulent strain, we employed genome-wide mutagenesis and sequencing to discover genes that contribute to mastitis. This extensive data set represents a screen for mastitis-associated fitness factors and provides the following contributions to the field: (i) global comparison of genes required for different aspects of mastitis infection, (ii) discovery of a unique capsule that contributes to virulence, and (iii) conclusive evidence for the crucial role of iron-scavenging systems in mastitis, particularly the ferric dicitrate transport system. Similar approaches applied to other mastitis-associated strains will uncover conserved targets for prevention or treatment and provide a better understanding of their relationship to other pathogens.