Novobiocin

For experiments with linear cells, we grew cells in liquid M9 minimum medium (Fluka Analytical) supplemented with 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glycerol (Sigma-Aldrich), and 0.1% protein hydrolysate amicase (PHA) (Fluka Analytical) overnight at 30 °C to reach late exponential phase. On the day of the experiment, the overnight culture was refreshed (1:100 vol) for 2 h on fresh M9 medium at 30 °C. We then pipetted 1 μl culture onto a cover glass and immediately covered the cells with a flat agarose pad, containing the above composition of M9 medium as well as 3% agarose. The cover glass was then placed onto a baseplate and sealed with parafilm to prevent evaporation. The baseplate was placed onto the microscope inside a 40 °C incubator for 2 h to stop the cells from replicating and to let them grow longer. To reinitiate DNA replication, the baseplate was moved to room-temperature for 10 min before placing it back onto the microscope inside the 40 °C chamber for imaging.
To obtain circular chromosomes, we used the same protocol as described above, with minor changes: We grew cells in liquid M9 minimum medium (Fluka Analytical) supplemented with 2 mM MgSO₄, 0.1 mM CaCl₂, 0.4% glycerol (Sigma-Aldrich), and 0.01% PHA (Fluka Analytical) overnight at 30 °C to reach late exponential phase. On the day of the experiment, the overnight culture was refreshed (1:100 vol) for 2 h on fresh M9 minimal medium at 30 °C. We then pipetted 1 μl culture onto a cover glass and immediately covered the cells with a flat agarose pad, containing the above composition of M9 medium, A22 (final 3 μg/ml), as well as 3% agarose. The cover glass was then placed onto a baseplate and sealed with parafilm to prevent evaporation. The baseplate was placed onto the microscope inside a 40 °C incubator for 2.5 h to stop the cells from replicating and to let them grow into round shapes. To reinitiate DNA replication, the baseplate was moved to room-temperature for 10 min before placing it back onto the microscope (inside 40 °C chamber) for imaging.
For treatment of replicating cells with Novobiocin, we first grew the cells in the presence of A22 as described above for 2.5 h to ensure they reach desired size and shape. Then we moved the baseplate to room-temperature for 10 min and afterwards added 10 µl of Novobiocin (~50 µg/ml final) to the agarose pad during replication initiation phase. Finally the cells were moved back to 40 °C chamber and imaged.

Theileria annulata-infected cells TaNM1 were cultured in 12-well culture plates at 10⁵ cells per well and treated with 200 ng/ml (stock 2 mg/ml in ethanol) buparvaquone (BW720c, Sigma). Meanwhile, cells were treated with the same amount of ethanol that used for dissolve BW720c as the control group. HeLa cells were also treated with the same concentration of BW720c to test its effect on the protein level of telomerase reverse transcriptase.
5 × 10⁵ TaNM1 cells were treated with 30 nmol/ml BIBR, 300 nmol/ml novobiocin, and 1 nmol/ml Geldanamycin (MedChemExpress), respectively. The equal volume of DMSO was used to treat the cells as control group.

Assays involving the DNA gyrase inhibitor novobiocin (Sigma-Aldrich) were performed as follows: bacteria containing the fimA-lacZ transcriptional fusion were screened for their Lac phenotypes on MacConkey lactose indicator medium as described previously [27 (link)]. Distinctly phase ON (red/Lac⁺) or phase OFF (white/Lac⁻) colonies were used to inoculate 2 ml LB (lysogeny broth) in test tubes and grown overnight. These were used to inoculate 250 ml flasks containing 25 ml of LB. These cultures were grown aerobically at 200 r.p.m. until they reached an optical density of approximately 0.1 at 600 nm. At this point novobiocin (aqueous stock solution 100 mg ml⁻¹) was added to a final concentration of 0, 12.5, 25, 50, 75, or 100 μg ml⁻¹. Cultures were incubated for a further 20 h before sampling to determine the orientation of the fimS switch in the chromosome.

On Day 0, pigs were randomly assigned to one of three treatments according to BW with ten replicates of one animal per treatment. Treatments consisted of no ST inoculation (Basal), 1 × 10⁸ CFU of ST inoculation, and 1.5 × 10⁸ CFU of ST inoculation.
The Basal group was inoculated by oral gavage with 5-mL of brain heart infusion (BHI, CM 1135; Oxoid, Thermo Fisher Scientific, Hampshire, England) broth solution without ST and kept in a separate facility to avoid cross-contamination. The ST-challenged groups received an oral inoculum with 5-mL of BHI broth solution containing 1 × 10⁸ CFU of ST or 1.5 × 10⁸ CFU of ST. The inoculum was administrated into the mouth of the animals using a 5-mL syringe coupled with an orogastric tube. The liquid was slowly pushed into the esophagus according to the pigs’ deglutition. The gilts were fasted for six hours and had no water consumption for 1-h prior to inoculation.
For inoculation, the oral dose of Salmonella enterica subsp. enterica serovar Typhimurium was prepared from a sample of Salmonella Typhimurium (RL0971/09) originally isolated from swine feces and naturally resistant to nalidixic acid (Nal+). This ST sample was obtained from the Laboratory of Ornitopathology of the Department of Veterinary Pathology (FCAV/UNESP, Jaboticabal-SP). The inoculum strain was tested for its antimicrobial sensitivity on Mueller–Hinton agar (CM0337; Oxoid, Basingstoke, Hampshire, UK), with the Kirby–Bauer method [21 (link)], using commercial discs impregnated with the following antibiotics: gentamicin, florfenicol, cefotaxime, cephalothin, ciprofloxacin, ceftriaxone, polymyxin B, tetracycline, streptomycin, nalidixic acid, ampicillin, chloramphenicol, sulfamethoxazole + trimethoprim, and novobiocin. The antibiogram test was also performed to evaluate if the Salmonella excreted by pigs, after inoculation, had the same pattern of sensitivity or resistance to the antibiotics tested with the strain used in the preparation of the inoculum.
The inoculum was prepared according to the recommendations of Wood et al. [22 ] and Oliveira et al. [23 (link)]. Briefly, the inoculum was prepared from a frozen culture inoculated in BHI overnight at 37 °C. On the following day, the bacterial culture was diluted in phosphate-buffered saline (to contain 10⁸ CFU) and incubated for 24h at 37 °C. Then, the inoculum concentration was confirmed by the drop-counting technique.
After inoculation, to mimic a commercial housing condition, and to assist in exacerbating the inflammatory response and maintaining the challenge as long as possible, no cleaning routine was adopted in the ST-challenged facility. On the other hand, the Basal group facility was cleaned twice a day, and it was washed with a bleach solution (1:10) once a week as part of the biosecurity protocol. During the experimental period, the team members were required to wear clean and disinfected clothing and footwear was cleaned with a bleach solution (1:10) when entering the facility.