Bacterial strains, purification, and molecular validation:
A total of 131 P. multocida isolates were examined, isolated from Sick or seemingly healthy animals from diverse geographical regions of Iran. Among them are cattle (11), sheep (44), goats (24), cats (21), dogs (16), and poultry (15). From February 2014 to September 2014, these isolates were collected.Isolated from cattle, sheep and goats from Fars province and Shiraz city with pneumonic involvement, as well as from the oral cavity and tonsils of cats and dogs in Tehran and Shiraz, also obtained isolates from blood samples and liver samples of suspicious birds, including ducks, chickens, and turkeys from Tehran and Shiraz, as well as cattle blood samples from Tehran and Varamin that had septicemic symptoms. In aseptic and ideal conditions, samples were taken to the laboratory in sampling containers. They are stored as standard in the freezer at a temperature of minus 80°C. Blood agar plates (Oxoid, United Kingdom) supplemented with 5% defibrinated fresh sheep blood were used to culture and purify these isolates and were incubated aerobically for 24 hours at 37°C. For confirming morphological and biochemical properties, standard bacteriology methods like Munir et al.'s work were used (Munir et al., 2006). After DNA extraction using Evers et al.'s boiling method (Ewers et al., 2006). Perform special PCR tests for P. multocida according to the method of Tausand et al.'s (Townsend et al., 1998), KMT1T7, and KMT1SP6 (Sinacolon, Iran) were used as reverse and forward primers. (that the PCR program shown in Table <link rid="tb1">1</link>–1). Electrophoresis was performed on 1% agarose gel with safe staining to detect the amplification products. Products were then photographed under UV light. As a positive control, P. multocida strain 85020 (wild type, type B:2) was used.
Detecting the presence of plasmid DNA
We used alkaline lysis to isolate plasmid DNA from bacteria and to assess plasmid presence or absence in isolates. Using this method, user can analyze their plasmid products by gel electrophoresis,andthismethod enabled us to select five isolates with definite plasmids. This method relies on alkaline denaturation; basically, the alkaline conditions of this method lead to the denaturation and linearization of chromosomal DNA and plasmid DNAs. As soon as the neutral condition is formed, the plasmid DNA renatures faster than chromosomal DNA because of its small size, creating the initial loop state. The chromosomal DNA, as a result of its high molecular weight, will not have the opportunity to be renatured, and so it will be selectively deposited and removed from the reaction.
We were inspired by Sambrook et al., which was developed in 1989 (Sambrook et al.,1989). but we modified it slightly. A single bacterial colony was cultured in 5 ml of LB or BHI broth culture medium to extract plasmid DNA. Overnight, the tube was placed in a shaker incubator at 37°C. An Eppendorf tube was filled with approximately 1.5 ml of the culture medium and Microfuge for 1 minute in order to obtain a bacterial pellet. Repeat this until the entire volume of the culture medium has been spun down in one tube. The pellet was dissolved in 100 µl of Alkaline lysis solution I, and then 20 µl of 10 mg/ml lysozyme enzyme was added to the suspension and mixed well. After this, the tube was left at room temperature for 2 min. Then, 200 µl of Alkaline lysis solution II was added and gently mixed (by turning the tube upside down), then placed on ice for 5 minutes. The next step was to add 150 ml of alkaline lysis solution III to the suspension, gently mixed to form small white clumps. After that, place it on ice for 5 minutes. Ideally, we should incubate on ice for no more than 5 minutes so that we don't introduce chromosomal DNA into our final sample. Genomic DNA and plasmid DNA are renatured under these neutral conditions and thus appear in the final sample. It should be noted that turning the tube upside down very slowly will reduce the risk of plasmid DNA fragmentation.
Next, the suspension was centrifuged for five minutes at 4°C. Then the supernatant is transferred into a clean tube. After adding 400 µl of phenol-chloroform mixture and mixing, it was centrifuged for 2 minutes. Once the microfuge is complete, transfer the aqueous phase into a new microtube. Afterward, 1 ml of pure ethanol (EtOH) was added, mixed, and allowed to stand at room temperature for two minutes. Next, centrifuged for 5 minutes using a refrigerated microfuge. A white pellet will form at the end of the eppendorf tube after centrifugation. Removed all ethanol and was allowed the ethanol residue to evaporate at room temperature. In the end, the end pellet containing the plasmid is dissolved in 50 µl of TE solution and stored at minus 20°C for later use. 100 ml of alkaline lysis solution I contains 25.0 ml of 1 M Tris-Cl at 0.8 pH, 2.0 ml of EDTA 0.5 M, 5.0 ml of 1 M glucose, 68.0 ml of sterile distilled water (SDW). 1 ml of 10 mg/ml lysozyme solution contains 0.01 g lysozyme in 1 ml of 0.250 M Tris pH 8.0. 1 ml of alkaline lysis solution II contains 50.0 µl of 20% SDS, 20 µl NaOH 10N, 930.0 µl of SDW. 100 ml of alkaline lysis solution III contains 60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic acid, 28.0 ml of SDW (5 M potassium acetate: 49.075 g potassium acetate fill to 100.0 ml with SDW).
Gel electrophoresis can be conducted to determine if plasmid is present in your bacterial sample. Nano-Drops were performed on the plasmid sample to determine the concentration of dsDNA.
During plasmid curing, the bacteria that contain the plasmid will lose the plasmid, and the colony will subsequently be plasmid-free. Caro et al., 1984, used the same plasmid curing method as we do with a few modifications (Caro et al., 1984). As part of this method, a known plasmid-removing agent, ethidium bromide (at final concentration is 25 µg/ml), was added to 10 ml of BHI broth. In this medium, a single colony of the bacterium was cultured and incubated for 18 hours in a shaker incubator at 42°C. Following incubation, ten-fold diluted in fresh BHI broth, 500 µl each of the dilutions 6 to 10 were spread on BHI agar plates with the help of a sterile spreader. Colonies were screened for loss of plasmid DNA after overnight incubations at 37°C. In the end, colonies without plasmids were stored (-20°C) for use in later stages of the study. In 2005, Jamal et al. successfully removed the plasmid of the P. multocida using a similar process to ours (Jamal et al., 2005).
We tested the ability of endonuclease restriction enzymes to digest plasmids derived from our five samples. The restriction endonucleases BglII, SspI, and AluI were used under conditions recommended by the supplier (Thermos Scientific, USA). Electrophoresis of restricted DNA was performed in 1.2% agarose gel.
Disk-Diffusion test for antibiotic susceptibility:
The work process and measurements were performed in accordance with CLSI guideline M45-2015 and EUCAST-Version 10.0. Five plasmid-containing and five plasmid-free organisms were sub-cultured on blood agar plates supplemented with 5% defibrinated fresh sheep blood and incubated at 37°C overnight. After making a bacterial suspension equal to 0.5 McFarland standard, it was used to inoculate plates containing Mueller-Hinton blood agar with 5% defibrinated sheep blood. After the antibiotic discs were placed on the inoculated plates, they were incubated at 37°C for 18 hours; the antibiotic discs (Padtanteb, Iran) are: amoxicillin (25µg), chloramphenicol (30µg), trimethoprim-sulfamethoxazole (1.25/23.75µg), erythromycin (15µg), oxytetracycline(30µg), gentamycin (10µg), streptomycin (10µg), tylosin(30µg), enrofloxacin(5µg), imipenem(10µg), cephalothin(30µg), knamycin(30µg), cefalexin(30µg), florfenicol(30µg), cefotaxime(15µg) and tetracycline (30µg). We measured and recorded the diameter of the no-growth zone. As in some cases, when the areas overlapped and were very large, it is necessary to measure each area's radius and double it to determine the diameter of each area.
As quality control organisms, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used (Following the articles of association).