Bacterial isolates
This work was established after the agreement of the research ethics committee of the Faculty of Pharmacy, Tanta University, Egypt (Research Ethics Committee Code: TP / RE /12-21-M-002). A total of 100 P. aeruginosa clinical isolates were collected from different clinical sources including 35 from urine (U), 17 from wounds (W), 15 from burns (B), 14 from sputum (S), 10 from ear (E) and 9 from eye (EY). These isolates were collected from patients in different Mansoura hospitals under medical attention with strict aseptic precautions in the period from April 2018 till April 2019. According to the standard microbiological techniques,³⁰ the isolates were biochemically identified as P. aeruginosa. Moreover, identity of the isolates was further confirmed by PCR amplification of their 16s rRNA as previously described.³¹ All cultures were grown at 37°C in Luria Bertani medium (LB broth; tryptone 1% w/v, yeast extract 0.5% w/v, and NaCl 1.0% w/v), otherwise specified, and stored in 80% glycerol/LB broth at -80°C.

Polymerase chain reaction
Chromosomal DNA was extracted from P. aeruginosa isolates by using QIA amp® DNA miniprep kit (Qiagen, Germany). All P. aeruginosa clinical isolates were screened for the presence of QS genes by PCR using the primers (lasR, lasI, rhlR and rhlI) shown in (Table 1) as previously reported.^32,33 The volume of PCR reactions was 25 μL, and each reaction composed of 12.5 μL Dream Taq PCR master mix 2x (Fermentas, USA), 1 μL forward primer (10 μM), 1 μL of reverse primer (10 μM) and 3 μL of template DNA, then the reaction was adjusted to total volume of 25 μL with nuclease free water. Negative control tubes were also performed without template DNA. The cycling conditions performed were; initial denaturing at 95°C for 5 min, then 35 cycles of (denaturation at 95°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 2 min), and final extension at 72°C for 10 min. Visualization of the amplified genes by electrophoresis was done using 1% agarose gel stained with ethidium bromide (MP biomedicals, France), and compared with a 100 base pair plus DNA ladder (Thermo Scientific, UK). The presence of the tested gene was indicated by the appearance of a single sharp band with the specific amplicon size for each gene (Table 1).

Table (1):
Specific amplification primer sets for the tested quorum sensing genes among P. aeruginosa isolates.

F: Forward; R: Reverse

Assay of P. aeruginosa virulence factors
Pyocyanin assay
Pyocyanin was extracted as previously described.³⁴ King’s A liquid medium (peptone 2% w/v, K₂SO₄ 1.0% w/v, and MgCl₂ 0.14% w/v) was used to cultivate the isolates for 48 hours with shaking at 150 rpm. Cultures were then centrifuged and pyocyanin was recovered from the supernatant using 3 mL chloroform. Thereafter, 1 mL of 0.2 N HCl was added to the pyocyanin (bottom layer) after it was transferred to a new clean tube. The extracted acidic form of pyocyanin, which has a pink colour, was spectrophotometrically measured at OD₅₂₀ nm. The optical density at 520 nm was multiplied by 17.072 to get the microgram amounts of pyocyanin.³⁵ All P. aeruginosa isolates were compared with the isolate with the highest amount of pyocyanin in percentage. In this experiment, King’s A liquid medium was used as a negative control.

Hemolysin assay
P. aeruginosa clinical isolates were cultured overnight in LB and incubated at 37°C with shaking at 150 rpm for an appropriate time. Thereafter, cultures were centrifuged at 10,000 rpm for 10 min at 4°C. The obtained supernatants were filtered through 0.2 µM Millipore filter and subsequently used in the assay of hemolysin activity. The assay was performed as previously described.³⁶ Prepared free supernatant of bacterial cultures (volume 600 µL) was mixed with equal volume of saline suspension of erythrocytes 2%, and incubated at 37°C for 2 hours. Then, centrifugation of the reaction mixtures was established at 10,000 rpm for 8 min. at 4°C, and the optical density at 540 nm was used to determine the degree of haemoglobin release. Control studies for spontaneous (negative control) and complete (positive control) lysis were conducted without hemolysin and with 0.2 % sodium dodecyl sulphate, respectively. The percentage (%) of cells lysed = [(X-B)/ (T-B)] × 100, where B (baseline) is a negative control and T is a positive control corresponding to the total lysis.

Protease assay
Assay of total proteases was established using skimmed milk assay as previously illustrated.³⁷ Volume of 200 µL of P. aeruginosa supernatant (prepared as described above in hemolysin assay) was mixed with 1 mL 1.25% skimmed milk and at 37°C incubation takes place for 15 min, and then, OD₆₀₀ was measured. Comparison of OD₆₀₀ of all P. aeruginosa isolates with OD₆₀₀ of skimmed milk in percentage was established to detect the protease activity of each isolate. Protease activity = (OD₆₀₀ of skimmed milk – OD₆₀₀ of the sample) X 100. In this experiment, LB was used as a negative control.

Lipase assay
Lipase assay was established as formerly outlined.³⁸ Culture supernatants of P. aeruginosa isolates were prepared as described above in hemolysin assay. A stock solution of p-nitrophenyl palmitate (p-NPP) (Sigma, USA) was prepared in HPLC grade of isopropanol. The reaction mixture contained 75 µL of p-NPP stock solution, 5 µL bacterial supernatant, and completed to a final volume of 3 mL with 0.1 M Tris buffer (pH 8.5). To stop the reaction, the reaction mixture was incubated at 37°C for 10 min before being refrigerated at -20°C for 8 min. At OD₄₁₀ nm, the optical density of emitted p-nitrophenol was measured. All P. aeruginosa isolates were compared with the isolate with the highest lipase activity in percentage. In this experiment, LB was used as a negative control.

Swimming motility assay
Swarming motility assay was performed as previously depicted.³⁹ Surface inoculation was used to measure the swimming ability of P. aeruginosa by using swimming agar plates (tryptone 1%, sodium chloride 0.5%, and agar 0.3%). The prepared plates were centrally stabbed with 5 μL of diluted overnight culture of P. aeruginosa isolates in tryptone broth. After 24 hours incubation period at 37°C, the swimming zones were measured. All P. aeruginosa isolates were compared with the isolate with the highest swimming activity in percentage. In this experiment, LB was used as a negative control.

Quantitative detection of biofilm using microtiter plate assay
By using the method described previously,^40,41 biofilm production was assayed. P. aeruginosa clinical isolates were cultured overnight in tryptic soy broth (TSB) (Oxoid, Thermo Fisher, UK) and diluted to produce a cell density of approximately 1 × 10⁶ CFU/mL. Using 96-well flat-bottomed polystyrene microtiter plate, 100 μL aliquots of each bacterial suspension was inoculated into the wells of at least in triplicate. Then, plates were incubated at 37°C for 24 hours. The contents of each well were aspirated to eliminate all non-adherent cells. Each well was rinsed three times with phosphate buffer saline (PBS, pH 7.4). The remaining adherent bacteria were fixed with 100% methanol per well. The bacterial cells adhered to the well walls were stained with 1% crystal violet for 20 min. Thereafter, the dye bound to the adhering cells was resolubilized using 33% (v/v) glacial acetic acid. The absorbance of solubilized stain was measured at OD₄₉₂ nm using Biotek spectrofluorimeter (Biotek, USA). All P. aeruginosa isolates were compared with the isolate with the highest biofilm formation in percentage. In this experiment, LB was used as a negative control.

Statistical analysis
Significance of difference was assessed in the results by repeating the experiments three independent times and comparing the data using Student’s t-test. P values lower than 0.05 were regarded significantly different.