Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacteria .That causes a range of healthcare illnesses, Includes ventilator-associated pneumonia (VAP), Infections at the critical care unit, surgical site infections, Infections in the circulation associated with central lines, urinary tract infections (UTI), keratitis, otitis media and Burn wound infections [1-2].

UTI is a bacterial infection of the urinary tract. When bacteria from the rectal region enter the urinary tract through the urethra and grow in the urine, an infection develops. In many situations, bacteria initially enter the urethra. When bacteria grow, an illness might develop. Urethritis is an illness that affects just the urethra. Cystitis occurs when bacteria enter the bladder and grow. If the infection is not treated immediately, bacteria may migrate farther up the ureters to grow and infect the kidneys, known as pyelonephritis. [3].

Quorum sensing (QS) was initially found in the marine bioluminescent bacteria Vibrio fisheri in the early 1970s. QS is extensively distributed in bacteria and regarded as a "speaking" system [4]. QS, also known as density sensing, governs a wide range of physiological functions in bacteria. Bacteria influence the behavior of the entire bacterial community by creating and secreting signal molecules known as autoinducers (AIs) [5]. The QS system modulates a range of cellular processes, including as bacterial virulence factors, luminescence, spore production, disinfectant tolerance, spore production, toxin synthesis, motility, biofilm formation, motility, and resistance for drug. When the amount of signal molecules rises above a certain threshold in relation to bacterial population density, the expression of certain genes can begin to regulate bacterial population adaptability [5]. In both acute and chronic infections, the behavior of P. aeruginosa is controlled by an advanced regulatory mechanism known as QS. Community-wide coordination of specific gene expression includes the interplay of distributed molecular signals. [6]. In general, QS is regulated by three systems in P. aeruginosa las, rhl, and pseudomonas quinolone signal (Pqs), which have been carefully reviewed [7]. To be a unique strategy to treating P. aeruginosa infection, all quorum sensing systems are interconnected and cannot exist on their own. These three systems constitute a complex hierarchical QS circuitry. Las is at the top of the QS hierarchy and is necessary for the proper activation of the PQS and rhl QS system. [8]

Las System regulates P. aeruginosa's QS network, and Las I produces the signaling (3-oxo-C12-HSL) chemical in nature N-(3-oxo-dodecanoyl)-L homoserine lactone. When the signal concentration is above the threshold, It interacts with the receptor protein LasR, creating a complex that promotes the expression of downstream target genes. [9]. Las signaling system control LasA staphylolytic protease, LasB elastase, Exotoxin A and Apr alkaline protease expression, as well as biofilm development, are all virulence factors. [10]. LasA and LasB are needed to modulate protease activity; they are responsible for the destruction of complement, elastin, interferon, fibrin, and collagen during infection. They may also impact toxicity levels. [11]. Rhl is another LuxR-type receptor family system found in P. aeruginosa's QS system that is similar to las. RhlI produces N-butanoyl-L-homoserine lactone (C4-HSL), the rhl AHL signaling molecule. The signal molecule interacts with its receptor, activating the transcriptional regulator RhlR. [12]. Rhl is involved in the generation of several virulence factors, including rhamnolipids, as well as the harmful exo-factors hydrogen cyanide and pyocyanin [13]. Rhamnolipids are biodegradable surfactants made up of rhamnose residues and two fatty acid molecules [14]. They serve an important role in protecting cells against oxidative stress, biofilm formation, and maturation. [15].

P. aeruginosa third QS system, known as pqs, is closely related to two AHL-based systems. It uses 2-heptyl-3-hydroxy-4-quinolone (PQS) as a signal molecule, and its precursor, 2-heptyl-4-hydroxyquinoline (HHQ), is frequently connected with QS and pqs. [16]. The transcriptional regulator PqsR (MvfR) and the pqs ABCDE operon are required for the synthesis of these two signal molecules. [17]. PqsR plays a function in pqs because it controle the synthesis of rhlI as well as several virulence activities such as biofilm formation, bacterial motility, pyocyanin, and hydrogen cyanide, which are triggered by LasR but inhibited by RhlR [18].

Detection and identification of quantitative and qualitative QS measurements may be accomplished using a number of strategies, which might be classed as either direct or indirect. The majority of them rely on the identification of signaling molecules with functional groups. Colorimetry, fluorescence, bioluminescence, chromatography-mass spectroscopy, chemiluminescence, and electrochemistry are currently standard approaches for both qualitative and quantitative investigation of QS compound [19].With the evolution of modern technology, such as high-performance liquid chromatography (HPLC), it is now able to provide thorough differentiation proof as well as an accurate, An objective analysis of the huge number of atoms detected in cell culture supernatants. [20]. Several methods are now being employed to demonstrate autoinducers and extracellular substances. Centrifuging bacterial cultures separates particles from supernatants for analysis. Dichloromethane, hexane, or acetic acetate are used as organic solvents in the extraction process. After extraction, the dissolved substance is dried to leave just active particles in methanol. [21].

Two hundred forty five urine samples, clean catch midstream, were collected from urinary tract infections (UTIs) including both sexes with different ages; there are various teaching hospitals in Mosul. The research was carried out from March 2023 to September 2023. All specimens were obtained in suitable sterile containers. The specimens were inoculated on Blood agar media and MacConkey agar media, incubated at 37 °C. Overnight aerobically, (Growth which appears non lactose fermenter and oxidase positive). Subculture on selective Pseudomonas cetramide agar and incubated in the same condition. Isolates were identified on the basis of their colony morphology, cultural, Gram stain, and biochemical characteristics by using Analysis Profile Index (API) [22-23].  

Molecular Diagnosis

A molecular diagnosis was performed using 16SrRNA as a detecting gene. according to (Khaleel etal .,2023)  [24] and QS Genes (lasl , lasR ) as a detection the isolate strain of P.aeruginosa in present study produce QS according to [25] , Primers (Table 1) were designed by Primer3 program according to National Center for Biotechnology Information( NCBI).

The extraction of DNA 

The genomic DNA was extracted directly from pure bacterial growth utilizing Geneaid's genomic DNA isolation kit. The manufacturer's recommendations were followed. The concentration and purity of genomic DNA were determined; the DNA was stored at -20 °C until future usage.

The polymerase chain reaction (PCR)

Promega's GoTaq G2 Green Master Mix (USA) was used for a 20-μL PCR reaction. Table 1 shows the universal primers used to amplify the whole 16srRNA gene region [24]. Primers (1 μM each) and template DNA (100 ng) were added according to manufacturer recommendations. The 16SrRNA gene PCR procedure was as follows: initial denaturation at 95°C for 3 minutes, followed by 30 cycles of amplification that included a denaturation phase at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for one minute. The last extension stage was set to 72 °C. PCR products were separated on a 2% agarose gel and stained with Midori Green Advance DNA dye. 100-bp DNA marker (New England Biolabs, UK) is used to indicate molecular weight.

Detection of QS virulence factors genes using PCR

The Bacterial DNA was extracted using a Geneaid purification kit. The PCR reaction mixture consisted of 2μl purified DNA, 10μl master mix, 4μl primer mix, and 4μl nuclease-free water, totaling 20μl [24]. HELINI Biomolecules provides oligonucleotide primers for lasl and lasR [25] were used, as shown in Table (1). The PCR protocol included an initial denaturation at 95 °C for 5 minutes, followed by 35 cycles of denaturation, annealing, and extension at 94 °C for 30 seconds, 58 °C for 30 seconds, and 72 °C for 30 seconds. The last extension lasts 5 minutes at 72 °C. Genes were identified using a 2% agarose gel stained with Midori Green Advance DNA dye. The band pattern was discovered after 40 minutes of electrophoresis at 50 volts using a transilluminator.

Table 1: Sequence of primers and their size

F: Forward sequences, R: Reverse sequences

Extraction of (QS crude) From Culture Supernatants

Extraction was carried out using the method previously reported by (Vivero-Gomez et al., 2021) [26]. To separate each stationary phase culture, 10 mL broth was centrifuged at 10000 rpm for 10 minutes at 4°C. (cool centrifuge method). As previously described, QS molecules were recovered from the cell-free supernatant. Supernatants were filtered via sterile 0.22 mm filters (BHD, England) and placed in a 50-mL falcon tube. The filtered supernatant was then mixed with 10 mL of acidified Ethyl acetate (0.01% acetic acid) and was vortexed for 30 seconds to mix the aqueous and organic phases. The mixture was transferred to a separate funnel that had previously been cleaned with acetone and left alone until the two phases separated. The organic phase was then transferred to another falcon Tube [26]. The extraction procedure was done Twice or more until a Total volume of 30 mL was reached. The supernatant extracts were separated into 50-mL round-bottom flasks and rotated and evaporated until the liquid phase was entirely dry. Each extract was rinsed with methanol and put in two mL vials. [26]. The QS crude Confirm it using Fourier-transform infrared spectroscopy (FT-IR) based on identification of signaling molecules that possess functional groups such as (C=O, C-O, N=H, OH), functional groups were discovered by FT-IR as stated (Taghadosi et al., 2015) [27]. In summary, a drop of the QS extract was put on a KBr plate, followed by a second plate, and the samples were evaluated using IR (Bruker, Germany). Another validated experiment was performed using HPLC equipment (Sykam, Germany). The QS extract crude was placed into an HPLC 1200 infinite configuration, which pumps molecules by weight through a solvent (mobile phase) and a solid (stationary phase). Molecules collide and adsorb onto the strong substance at that place, eluting at variable rates [28]. Section dimensions were 2.1 × 100 mm, with an estimated molecular size of 1.8 mm. The stationary stage employed C18-reinforced silica, whereas the versatile stage used acetonitrile. Non-polar atoms were separated into pH groups ranging from (2 to 9). We changed the draw and discharge rates to 2000 mL/min^-1, the infused volume to 5 mL, the suction temperature to 30 °C, and the stream rate to( 0.4 )mL/min^-1. The low pressure limitation was set to zero, while the high pressure limit was set to 900 bars. At (1) minute, the solvent included (95%) soluble A (water, 5 mM ammonium formate, and 0.1% formic acid) and (5%) soluble B (ACN). After (10) minutes, the solvent gradient had changed to 5% solvent A and 95% solvent B. [28].

The results showed that Eight (4%) P. aeruginosa were identified from Urine specimens based on cultured and biochemical tests (API). These isolates did not ferment lactose and generated pale yellow colonies on MacConkey agar. They also caused β-hemolysis on blood agar. And pigments produced on the selective ceramide agar were more obvious the final results for cultures were shown in (Figure 1).

Figure 1: Culture results of P. aeruginosa. (A) Culture Results and percentage, (B) P. aeruginosa isolation percentage.

To confirm P. aeruginosa isolates, 16SrRNA gene detection was done by using PCR, The 16SrRNA genes were purified and delivered to Psomagene Sequencing Company (USA). Using the NCBI's BLAST program, the retrieved sequences were compared to published genes uploaded to GenBank. The 16SrRNA sequencing showed that all isolates (99)%  P. aeruginosa, were recorded as New four strains in NCBI, Coded (AhmHiy) ACCESSION NO. (OR186518, OR186519, OR186520 and OR186521).  Some of QS genes (lasl and lasR) were detected in Two Isolates, these isolates had lasl and lasR genes. The result of gel electrophoresis for amplification (Figure 2).

Figure 2: Gel PCR results. (A) Detection of P. aeruginosa 16SrRNA gene (1465 bp). Lane 1 shows the DNA ladder (1500bp) and lanes 2-9 represent positive results.(B) An electropherogram of the pathogenic gene Appoint with lane 1 displaying the DNA ladder (1500bp), lane 2,3 displaying the positive amplicons of lasR. (378 bp) and lane 4,5 lasI (311 bp).

After detecting QS genes from Two isolates, QS Crude was extracted according (Vivero-Gomez etal.,2021) [26]. The QS Crude has been confirmed to contain (HSL and pqs) by Fourier-transform infrared spectroscopy (FTIR) and High-pressure Liquid Chromatography (HPLC), As shown in the Figure 3. The current study appeared and confirmed found the AHLs in the QS crude especially 3oxoC12HSL and C4HSL (AHLs) while not found (PqS). 

Figure 3: (A) FT-IR Chart. (B) HPLC Chart.

Eight strains of p. aeruginosa were obtained from 200 positive bacterial cultures of urine samples for the period from March 2023 till September 2023, The percentage of p. aeruginosa was 2 (4%) compared to other bacterial isolates, These results were identical to what was obtained. Farhadi, Z., & Bahador, N. [29] and (AL-Khikani and Ayit, 2019) [30], where found to be the third most common causative agent of UTI. In P. aeruginosa, population-dependent QS is triggered by AHL secretion and binding to regulatory receptors, initially LasR and RhlA, which activate the production of additional LasR, RhlA, and PqsR receptors, which activate the entire QS system and activate biofilm formation and virulence factor production. [31]. Therefore, it was necessary to confirm the p. aeruginosa isolates 16SrRNA gene detection was done by using PCR according (Khaleel et al .,2023) [24], and then also to verify that they possess some of the genes responsible for the production of QS according (Shravani etal.,2023) [25] before proceeding with the extraction of the QS crude as in the study conducted by (Vivero-Gomez et al.,2021) [26], and then to ensure that this crude contains QS compounds, which was done using FT-IR to identify Functional groups are found in the mid-IR range (4000 - 400 cm^-1), hence the development of an absorption band in this area may be utilized to assess whether or not particular functional groups are present in the molecule. The IR spectra of AHLs show unique absorption peaks at 1650, 1710, and1780 cm^-1 due to the presence of the amide carbonyl, 3-OX0, and lactone ring respectively [32], [27]. It is similar to what we obtained in this study, Figure (5 A). Another confirmed done by used HPLC, which determine (Retention Time) for each types of QS, then comparative HPLC results with several studies like (Teiber and Draganov, 2011; Laj etal., 2022) [33],  [28] which appear that QS crude component from Qs types (C4HSL and 3oxoC12HSL) in the retention time 3.55, 5.9 respectively, while did not appeared the third types of QS in p. aeruginosa which is PQS when comparative HPLC results with results of (Vivero-Gomez et al.,2021) [26].

QS Genes were found in the majority of P. aeruginosa, and they play an important role in the pathogenesis of P. aeruginosa infection; however, not all virulence factors are regulated by the QS system. The lasI and lasR genes play significant roles in the generation of virulence factors. AHL has been discovered and validated using FT-IR and HPLC. P. aeruginosa contained two distinct AHL molecules linked to its quorum sensing system. This is the first comprehensive research on detecting and identifying AHLs in P. aeruginosa isolates acquired from urine specimens.

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