Bacteria were grown overnight at 37°C on blood agar (Tryptose Blood Agar Base, Becton-Dickinson, Sparks, MD, USA, enriched with 5% washed sheep red blood cells). Bacterial DNA was extracted using MagNa Pure Compact Nucleic Acid Isolation Kit I and libraries were prepared with GS FLX Titanium Rapid Library Preparation Kit (Roche Applied science, Mannheim, Germany). Genomes were sequenced using Roche 454 GS FLEX Titanium technology (Dynlabs, Zerifin, Israel). Sequences were assembled de novo using Newbler 2.6. The presence of plasmids was assessed by plasmid DNA extraction with QIAprep Miniprep (QIAGEN) for small plasmids, and a heat lysis method previously described [33 (link)] to include large plasmids. Briefly, overnight cultures of E. coli were pelleted and lysed in 3% SDS and 50 mM Tris (pH 12.6) at 55°C for one hour, followed by phenol-chloroform DNA extraction. Plasmids were visualized with gel electrophoresis.
Tryptose blood agar base
Manufactured by BD
Sourced in United States
Tryptose Blood Agar Base is a microbiological growth medium used for the cultivation and isolation of a variety of fastidious bacteria. It provides the necessary nutrients and growth factors required for the growth of these organisms.
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5 protocols using tryptose blood agar base
1
Bacterial Genome Sequencing and Plasmid Characterization
2
Precise Intra-Mammary E. coli Challenge
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Three different E. coli strains were used for the intra-mammary challenge: VL2874 (isolated from per-acute mastitis), VL2732 (isolated from persistent mastitis) and P4 (widely used as model strain for mammary pathogenic E coli, isolated from clinical mastitis in the UK). Strains typing, phenotypic and genomic characteristics were extensively studied and described before2 (link),7 (link),8 (link). Bacteria were recovered from frozen stocks (kept at −80 °C in a mixture of brain-heart infusion and 25% glycerol) on blood agar (Tryptose Blood Agar Base; Becton-Dickinson, Sparks, MD, USA, with 5% washed sheep erythrocytes) and incubated aerobically at 37 °C overnight. For inoculum preparation, bacteria were harvested and washed in pyrogenic-free saline (PFS), suspended in PFS and stored at 4 °C for 10 h. Aliquots were taken for colony forming units (CFU) count on blood agar. CFU number was adjusted with PFS right before challenge, aiming to an inoculum of ~50 CFU in 3 mL. Final CFU number in the inoculum ranged from 10–30 CFU/gland, as determined from aliquots separated just prior to challenge. The small CFU counts in the inoculum aimed to better represent a natural infection in which it is expected that only a small number of bacteria enter the mammary gland and initiate the infection.
3
Preparation of Bacterial Strains for Mastitis Study
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The following bacteria were prepared for inoculation: E.coli strain P4, isolated from acute mastitis [22 (link)] and widely used as a model strain in mastitis research, was obtained from NCIMB (catalogue no. 702070) and propagated once before storage; field-isolated S. aureus ZO3984 (β hemolytic) [23 (link),24 (link)] and E. coli VL2874 were previously genotyped and analyzed by whole-genome sequencing and phenotyping [25 (link),26 ,27 ]. Bacteria were recovered from stock (-80°C in brain heart infusion, 25% w/v glycerol) on blood agar (tryptose blood agar base; Becton-Dickinson, Sparks, MD, with 5% washed sheep erythrocytes), and incubated aerobically at 37°C overnight. Bacteria were harvested and washed in pyrogen-free saline (PFS). Bacterial concentration was determined by colony counting. For in vivo inoculation, bacteria were suspended in PFS and stored at 4°C for 10 h. Bacterial concentration was adjusted with PFS before challenge, aiming for an inoculum of about 30–100 CFU in 3 mL PFS. Final bacterial concentrations assessed from inoculum aliquots separated just prior to challenge ranged between 10 and 30 CFU per 3 mL PFS. For in vitro inoculation, bacteria were suspended at 105 CFU/mL Roswell Park Memorial Institute (RPMI) media.
4
Murine Mammary Gland Pathogenicity Evaluation
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Pathogenicity in the mammary gland (or lack thereof, for strain K71) was evaluated in vivo in a model of intra-mammary challenge in female mice as previously described [28 (link)], with modifications as follows. Briefly, bacteria were grown to log phase in nutrient broth (Merck, Darmstadt, Germany) at 37°C and washed in non-pyrogenic phosphate-buffered saline (PBS). An aliquot was serially diluted and plated on blood agar plates (Tryptose Blood Agar Base; Becton-Dickinson, Sparks, MD, USA, enriched with 5% washes sheep red cells) for colony forming units (CFU) counting. An average of 5x102 CFU (range 4-7x103 CFU) in 5 μL were injected subcutaneously into the left abdominal mammary gland (L4) of Swiss female mice 7–10 days post-partum using a 30-G needle, with care not to injure blood vessels. This route of injection was chosen to avoid injuring the teat, which could lead to unspecific reaction in the gland. Mice were euthanized one, two and five days post-challenge (DPC). The experiment was performed in triplicates. Injected glands were observed for gross pathology externally and internally in comparison to the contralateral mammary gland, then removed, weighted and examined by bacterial culture and histopathology. Animal experiments were approved by the Kimron Veterinary Institute Committee of Animal Experimentation.
5
Antibiotic Susceptibility Profiling of Equine Isolates
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All available frozen archived S equi isolates from 16 clinical samples submitted to the Laboratory for Bacteriology in the Kimron Veterinary Institute in the years 2008–2012 were analysed in this study. Isolates were cultured on blood agar (Tryptose Blood Agar Base; Becton-Dickinson, Sparks, Maryland, USA, enriched with 5 per cent sheep blood). DNA was extracted using QIAGEN DNeasy as per the manufacturer's protocol (QIAGEN, Germany). An antibiogram was performed by the disc diffusion test and interpreted following the Clinical & Laboratory Standards Institute veterinary standards (Clinical & Laboratory Standards Institute, 2013 ).
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