2.1. Study area: Giza is one of the of Egyptian governorates which located in the center of Egypt at 29.26°N 29.67°E. It situated at the west branch of the Nile River opposite to Cairo. It includes a stretch of the left bank of the Nile Valley around Giza, and constitute a large stretch of Egypt's Western Desert. According to population estimates in 2018 the majority of populations are live in urban areas, with 60.9% urbanization rate and the total number of population was estimated 8,759,000 people and about 5,332,000 people live in urban areas as opposed to only 3,428,000 in rural areas(wikipedia, 2012)
2.2. Animals And Samples:
Two hundred buffaloes reared in a private farm in Giza governorate were examined and sixty mastitis milk samples were collected from clinical cases of mastitis buffaloes from the period from October 2021 until March 2022. The clinical examination and palpation of udder was done according to (Islam et al., 2012). The acute mastitis sings were assessed according to (fever, inflamed udder &edema, abnormal milk secretion as clotted and flacks milk). All the samples were aseptically collected then transported in a cool condition in icebox at 40C to the laboratory for further bacteriological examination.
2.3. Phenotypic characterization of P. aeruginosa
The collected samples were subculture onto specific medium for P. aeruginosa (Pseudomonas selective agar and blood agar) and incubated aerobically at 37 C for 24-48h. The characteristic pure colonies appear as bluish green color and confirmation was carried out based on the pigment production as well as biochemical tests include; oxidase, catalase, coagulase, citrate, OF (Oxidative-Fermentative), urease, Arginine dehydrolase, cetrimide test and nitrate reduction and lipase activity as described by (Arai et al., 1970; Cookson & Stirk, 2019).Data obtained were stored on excel sheet and descriptive analysis was carried out.
2.4.Antimicrobial suscptability test of P. aeruginosa isolated from buffoles
Disk diffusion method was used to test five P. aeruginosa isolates against 10 antibiotics of different antimicrobials classes; (Oxoid Ltd., Basingstoke, UK): 100 IU penicillin, 20/10 µg, amoxicillin/clavulanic acid, 30µg cefotaxime, 10 µg norfloxacin, 30 µg chloramphenicol, 30 µg tetracycline, 10 µg gentamicin, 100 µg spectinomycin, 30 µg streptomycin, 30µg amikacin and 1.25/23.75 µg sulfamethoxazole/trimethoprim. A suspension of the bacterial suspension was prepared to equal the turbidity of a 0.5 McFarland standard (1.5 × 108 colony forming units (CFU) ml − 1). The results was determined as resistance, sensitive, and intermediate according to (CLSI, 2018).
2.5. Determination of the combined effect between amikacin and norfloxacin against resistant P.aeruginosa by checker broad dilution assay : This test is a standard technique used to determine the synergism effect between two antibiotics and was done using of the checker broad dilution assay and estimation the FIC index as described by (Bellio et al., 2021).
2.6. Molecular detection of P. aeruginosa virulence associated genes.
DNA extraction. DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56OC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.
Oligonucleotide Primer. Primers used were supplied from Metabion (Germany) as described previously by the following authors (Bratu et al., 2006; Frana et al., 2001; Matar et al., 2002; Robicsek et al., 2006; Winstanley et al., 2005; Xu et al., 2004) for lasI gene, aadB gene, toxA gene, qrnA and qnrS genes, exoT and exoS, oprL gene respectively. For toxA the initial denaturation step at 94 0C for 5 minutes followed by 35 cycles with denaturation at 940C for 30sec, annealing at 550C for 40 sec and extension at 720C for 40 sec and the final extension was at 720C for 10 minute; exoT gene, initial denaturation step at 94 0C for 5 minutes followed by 35 cycles with secondary denaturation at 940C for 30sec, annealing at 580C for 30 sec and extension at 720C for 30 sec and the final extension was at 720C for 7minute; for exoS the initial denaturation step at 94 0C for 5 minutes followed by 35 cycles, each cycle composed of secondary denaturation at 940C for 30sec, annealing at 550C for 30 sec and extension at 720C for 30 sec and the final extension was at 720C for 7minute; for oprL gene the initial denaturation step at 94 0C for 5 minutes followed by 35 cycles, each cycle composed of denaturation at 940C for 30sec, annealing at 550C for 40 sec and extension at 720C for 45 sec and the final extension was at 720C for 10 minute; For lasI gene the initial denaturation step at 94 0C for 5 minutes followed by 35 cycles, each cycle composed of denaturation at 940C for 30sec, annealing at 560C for 40 sec and extension at 720C for 45 sec and the final extension was at 720C for 10 minute; while, qnrA and qnrS genes, The initial denaturation step at 94 0C for 5 minutes followed by 35 cycles, each cycle composed of denaturation at 940C for 30sec, annealing at 560C for 40 sec and extension at 720C for 40–45 sec and the final extension was at 720C for 10 minute. For aadB gene the initial denaturation step at 94 0C for 5 minutes followed by 35 cycles, each cycle composed of denaturation at 940C for 30sec, annealing at 580C for 30 sec and extension at 720C for 30 sec and the final extension was at 720C for 7 minute For mPCR, Primers were utilized in a 25- µl reaction containing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an Applied biosystem 2720 thermal cycler. For mPCR, primers were utilized in a 50- µl reaction containing 25 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 13 µl of water, and 8 µl of DNA template. The reaction was performed in an Applied biosystem 2720 thermal cycler.
Analysis of the PCR Products.
The PCR products were electrophoresis on 1% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. Followed by gel analysis, 40 µl of the mPCR products were loaded in each gel slot. Generuler 100 bp ladder (Fermentas, Thermo) and gelpilot 100 bp plus ladder (Qiagen, gmbh, Germany) were used to determine the fragment size and photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.