Bacterial strains and growth conditions
Samples were collected from a wide range of broiler farms with a history of diarrhea in Qalubiya governorate, Egypt. Fecal samples were collected and incubated overnight in buffered peptone water at 37°C, 100 µl of the overnight pre-enrichment broth was then inoculate a 10 ml Tetrathionate broth (Müller-Kaufmann) and again was incubated overnight at 37°C. Finally, 10 µl loop from the inoculated Tetrathionate broth was spread on Xylose Lysine Deoxycholate (XLD) agar plates and on Brilliant Green (BGA) agar plates, plates were then incubated overnight at 37°C. Salmonella like colonies were selected, subcultured and maintained for further identification. On XLD agar, a typical Salmonella colony has a slightly red transparent halo with black centers. On BGA agar, a typical Salmonella colony appear red in a red/pink color agar.
Isolated Salmonella serotypes were determined by identifying the outermost portion of the lipopolysaccharide “O” antigen and the flagellar protein “H” antigen. Five isolates of Salmonella enterica serovars were purified and stored at - 80°C in Brain-Heart-Infusion broth supplemented with 20% (v/v) glycerol.
Antibiotic susceptibility test
Antibiotics susceptibility test was performed using the disc diffusion method (Biemer, 1973) on Mueller-Hinton agar using a collection of ten antibiotics (Oxoid, UK); Ampicillin (AM 10 𝜇g), Amoxicillin + Clavulanic Acid (AMC 20 + 10 𝜇g), Ceftriaxone (CRO 30 𝜇g), Ciprofloxacin (CIP 5 𝜇g), Amikacin (AK 30 𝜇g), Chloramphenicol ( CL 30 𝜇g), Gentamycin (Gent 10 𝜇g), Tetracycline (TE 30 𝜇g), Kanamycin (KA 30 𝜇g) and Streptomycin (ST 10 𝜇g). The results were inferred according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (Wayne, 2019).
DNA Extraction and PCR amplification
Bacterial genomic DNA from pure isolates was extracted using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. DNA integrity was checked using 1% agarose gel electrophoresis and the image was captured using gel documentation system (Gel Doc. BioRad). Concentration and purity of purified DNA were measured by BioTek Epoch2 Microplate reader (Thermo Scientific, USA). For all samples, DNA purity was >1.8 ± 0.20 under absorbance ratio A260/A280.
PCR amplification was done based on bacterial genomic DNA using universal 16S rRNA primers 27F:5′-AGAGTTTGGATCMTGGCTCAG-3′ and 1492R: 5′-CGGTTACCTTGTTACGACTT-3′ (Badr et al., 2019). The expected PCR amplicon was almost 1.5 kb. PCR reaction was performed in a 25 μl mixture containing 0.2 μM of each primer with concentration of 10 pM, 200 μM of dNTPs mix, 2.5 μL of 10× PCR reaction buffer, 1.5 μM MgCl2, 1.25 units of TAKARA Taq DNA polymerase (Cat. #: R001AM), 2 μL of template DNA and the final volume was adjusted with sterilized double distilled water. PCR thermocycler (AriaMx) was used to amplify the reactions consisting of 95 °C for 3 min followed by 35 cycles at 95 °C for 50 s, 55 °C as annealing temperature for 1 min with an extension of 72 °C for 1 min followed by final extension temperature at 72 °C for 10 min. Amplified PCR products were stored at −20 °C for further purification and downstream application, then 5 μl of PCR amplicons was loaded on 2 % agarose gel electrophoresis stained with Ethidium bromide using GeneRuler™ 1 kb DNA ladder, then visualized using gel documentation system (Gel Doc. BioRad).
16S rDNA sequencing
PCR amplicons were purified according to the manufacturer's QIAquick Gel Extraction Kit (Cat. #: 28704). The purified PCR fragments were sequenced by Macrogen Company, South Korea applying automated Sanger Sequencing method. The obtained sequences for 16S rRNA gene were surveyed using Standard Nucleotide BLAST tool and registered at NCBI database under accession numbers [1], MW311328.1, MW310702.1, MN822653.1 and MN820824.1 for Salmonella enterica subsp. enterica serovar Enteritidis strains EG.SmE2, EG.SmE1 and Salmonella enterica subsp. enterica serovar Typhimurium strains EG.SmT3, EG.SmT2, EG.SmT1, respectively, (http://www.ncbi.nlm.nih.gov).
Pairwise alignment and Phylogenetic construction
NEbcuter software V2.0 was used to create a restriction map and to identify the GC content of the obtained sequences (Vincze et al. 2003, (http://nc2.neb.com/NEBcutter2/). Jalview software (Waterhouse et al. 2009) was used to show single nucleotide polymorphisms (SNPs) and consensus resulted from the alignment of the obtained sequence and the nearest strains in NCBI database (http:// www.jalview.org/). Construction of the phylogenetic tree was done using Clustal Omega tool (https://www.ebi.ac.uk/Tools/msa/clustalo/) and MEGA X software (Kumar et al. 2018).