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      Postgenomics Characterization of an Essential Genetic Determinant of Mammary Pathogenic Escherichia coli


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          Escherichia coli are major bacterial pathogens causing bovine mastitis, a disease of great economic impact on dairy production worldwide. This work aimed to study the virulence determinants of mammary pathogenic E. coli (MPEC). By whole-genome sequencing analysis of 40 MPEC and 22 environmental (“dairy-farm” E. coli [DFEC]) strains, we found that only the fec locus ( fecIRABCDE) for ferric dicitrate uptake was present in the core genome of MPEC and that it was absent in DFEC genomes ( P < 0.05). Expression of the FecA receptor in the outer membrane was shown to be citrate dependent by mass spectrometry. FecA was overexpressed when bacteria were grown in milk. Transcription of the fecA gene and of the inner membrane transport component fecB gene was upregulated in bacteria recovered from experimental intramammary infection. The presence of the fec system was shown to affect the ability of E. coli to grow in milk. While the rate of growth in milk of fec-positive ( fec +) DFEC was similar to that of MPEC, it was significantly lower in DFEC lacking fec. Furthermore, deletion of fec reduced the rate of growth in milk of MPEC strain P4, whereas fec-transformed non-mammary gland-pathogenic DFEC strain K71 gained the phenotype of the level of growth in milk observed in MPEC. The role of fec in E. coli intramammary pathogenicity was investigated in vivo in cows, with results showing that an MPEC P4 mutant lacking fec lost its ability to induce mastitis, whereas the fec + DFEC K71 mutant was able to trigger intramammary inflammation. For the first time, a single molecular locus was shown to be crucial in MPEC pathogenicity.


          Bovine mastitis is the major infectious disease in dairy cows and the leading cause of economic loss to the global dairy industry, directly contributing to the price of dairy products on supermarket shelves and the financial hardships suffered by dairy farmers. Mastitis is also the leading reason for the use of antibiotics in dairy farms. Good farm management practices in many countries have dramatically reduced the incidence of contagious mastitis; however, the problems associated with the incidence of environmental mastitis caused by bacteria such as Escherichia coli have proven intractable. E. coli bacteria cause acute mastitis, which affects the health and welfare of cows and in extreme cases may be fatal. Here we show for the first time that the pathogenicity of E. coli causing mastitis in cows is highly dependent on the fecIRABCDE ferric citrate uptake system that allows the bacterium to capture iron from citrate. The Fec system is highly expressed during infection in the bovine udder and is ubiquitous in and necessary for the E. coli bacteria that cause mammary infections in cattle. These results have far-reaching implications, raising the possibility that mastitis may be controllable by targeting this system.

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          Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

          Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.
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            Rapid procedure for detection and isolation of large and small plasmids.

            Procedures are described for the detection and isolation of plasmids of various sizes (2.6 to 350 megadaltons) that are harbored in species of Agrobacterium, Rhizobium, Escherichia, Salmonella, Erwinia, Pseudomonas, and Xanthomonas. The method utilized the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) that is released from cells under conditions that denature chromosomal DNA by using alkaline sodium dodecyl sulfate (pH 12.6) at elevated temperatures. Proteins and cell debris were removed by extraction with phenol-chloroform. Under these conditions chromosomal DNA concentrations were reduced or eliminated. The clarified extract was used directly for electrophoretic analysis. These procedures also permitted the selective isolation of plasmid DNA that can be used directly in nick translation, restriction endonuclease analysis, transformation, and DNA cloning experiments.
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              Redundancy and specificity of Escherichia coli iron acquisition systems during urinary tract infection.

              Uropathogenic Escherichia coli (UPEC), the predominant cause of uncomplicated urinary tract infection (UTI), utilizes an array of outer membrane iron receptors to facilitate siderophore and heme import from within the iron-limited urinary tract. While these systems are required for UPEC in vivo fitness and are assumed to be functionally redundant, the relative contributions of specific receptors to pathogenesis are unknown. To delineate the relative roles of distinct UPEC iron acquisition systems in UTI, isogenic mutants in UPEC strain CFT073 or 536 lacking individual receptors were competed against one another in vivo in a series of mixed infections. When combinations of up to four mutants were coinoculated using a CBA/J mouse model of ascending UTI, catecholate receptor mutants (ΔfepA, Δiha, and ΔiroN mutants) were equally fit, suggesting redundant function. However, noncatecholate siderophore receptor mutants, including the ΔiutA aerobactin receptor mutant and the ΔfyuA yersiniabactin receptor mutant, were frequently outcompeted by coinoculated mutants, indicating that these systems contribute more significantly to UPEC iron acquisition in vivo. A tissue-specific preference for heme acquisition was also observed, as a heme uptake-deficient Δhma ΔchuA double mutant was outcompeted by siderophore receptor mutants specifically during kidney colonization. The relative contribution of each receptor to UTI only partially correlated with in vivo levels of receptor gene expression, indicating that other factors likely contributed to the observed fitness differences. Overall, our results suggest that UPEC iron receptors provide both functional redundancy and niche specificity for this pathogen as it colonizes distinct sites within the urinary tract.

                Author and article information

                Role: Editor
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                3 April 2018
                Mar-Apr 2018
                : 9
                : 2
                : e00423-18
                [a ]National Mastitis Center, Division of Bacteriology, Kimron Veterinary Institute, Bet Dagan, Israel
                [b ]IB3, Heriot Watt University, Edinburgh, United Kingdom
                [c ]Institute of Infection, Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
                [d ]ISP, INRA, Université Tours, Nouzilly, France
                [e ]Moredun Research Institute, Edinburgh, Midlothian, United Kingdom
                GSK Vaccines
                Author notes
                Address correspondence to Shlomo E. Blum, shlomobl@ 123456moag.gov.il , or shlomo.blum@ 123456mail.huji.ac.il .

                Present address: Robert J. Goldstone, The Francis Crick Institute, London, United Kingdom.

                S.E.B., R.J.G., G.L., and D.G.E.S. contributed equally to this article.

                Author information
                Copyright © 2018 Blum et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

                : 20 February 2018
                : 27 February 2018
                Page count
                supplementary-material: 7, Figures: 3, Tables: 0, Equations: 0, References: 32, Pages: 11, Words: 7991
                Funded by: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC), https://doi.org/10.13039/501100000268;
                Award Recipient : Award Recipient : Award Recipient : Award Recipient :
                Funded by: Israeli Dairy Board (IDB), https://doi.org/10.13039/501100009439;
                Award Recipient : Award Recipient :
                Research Article
                Custom metadata
                March/April 2018

                Life sciences
                escherichia coli,bovine,ferric citrate,mammary gland,mastitis,milk,pathogenesis,whole-genome sequencing


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