Detection of Quorum Sensing Genes of Pseudomonas aeruginosa Isolated from Different Areas in Iraq

Pseudomonas aeruginosa is an opportunistic pathogen. Quorum sensing (QS) is one of processes that are responsible for biofilm formation. P. aeruginosa can live in different environments, some of which are pathogenic (clinical isolates) and some that are found outside the body (environmental isolates). The present study aimed to determine the presence of a number of genes responsible for QS in clinical and environmental isolates of P. aeruginosa . In the present study full DNA was separated from all environmental and clinical isolates that contained seven genes ( rhlA , rhlR , rhlI , lasR , lasI , lasB , phzA1 ) associated with QS occurrence. The total DNA was separated from all environmental and clinical isolates (PAE1, PAE2, PAE3, PAE4, PAE5, PAE6, PAE7, PAE8, PAE10, PAE11, PAE12, PAE14, PAC1, PAC2, PAC5, PAC7, PAC8, PAC9 and PAC10). This study found that all studied environmental and clinical isolates contained the seven genes rhlA , rhlR , rhlI , lasR , lasI , lasB , phzA1 , which was associated with QS occurrence.


Introduction
P. aeruginosa is a stick-shaped gram-negative pathogen that is associated with serious illnesses and infections such as pneumonia and various sepsis syndromes in humans, as well as it causes lung cancer. In addition, P. aeruginosa isolates infect immune compromised people, burns patients and individuals suffering from cystic fibrosis [1]. Its infection treatment is a big challenge as this bacteria is highly resistant to a wide spectrum of antibiotics [2]. P. aeruginosa swarming requires releasing of two external factors, rhamnolipids and 3-(3hydroxyalkanoyloxy alkanoic acids) that play role as wetting factors [3]. However, a tiny chemical molecule called auto-inducer has the main role in connecting and leading the bacterial cell to combine and form a biofilm [4]. There is a correlation between the ability of bacteria to form biofilm and its resistance to antibiotics [2]. Previous studies have showed that environmental isolates of P. aeruginosa have an ability to form biofilm [5]. Quorum sensing is a process of gene organizing that uses the chemical indicators by many hosts-related bacteria for observing accumulation of population of bacteria in a certain area [6]. It is well known that the quorum sensing style differs from bacterium to bacterium, i.e.,gram negative bacteria use acyl-homoserine lactone (AHL) [7]. The adhesion part of biofilm formation and quorum sensing regulation to synthesis the signal such as lasI in Pseudomonas bacteria which have role to synthesis quorum sensing signal regulates by many genes [8]. In biofilms, the gene expression depends on bacterial response to the native ecological conditions. Over the last decade, several researchers studied AHL indicators that are used by gram negative bacteria in the quorum sensing. The results clarified that P. aeruginosa genome expression of extracellular virulence agents created by P. aeruginosa is organized through quorum sensing [9]. Other studies found that quorum sensing has two specific patterns in P. aeruginosa of the lasI gene in the las quorum sensing patterns that are responsible for the diffusible extracellular indicator, N-(3-oxododecanoyl)-L-HSL.The first pattern is a las system, while the second pattern is rhl system that reacts together with LasR [10] to operate virulence genes including lasB, lasA, apr, toxA and lasI [11]. Composition of the siderophore pyoverdine also is stimulate by the las system [12],The 3OC12-AHL seems to division into cell membranes, and P. aeruginosa efflux pumps support divide the movement of this signal to the external environment although 3OC12-HSL is diffusible . The synthesis of N-butyryl-L-HSL (C4-AHL) catalyzes by rhlI product [13].Pyocyanin,Cyanide,and Chitinase are virulence factors, they are positively organized by the rhl system [14].
Previous studies highlighted the role of several genes in QS directly and indirectly such as las and rhlR [15]. These systems are coiled in a hierarchical manner, las system controls rhl system together, the transcriptional and posttranslational fields, It also has a role in the production and development of biofilms, In addition to antibiotic resistance [16] .A complicate phenazin biosynthetic lane found in P. aeruginosa, that consist of two symmetric core loci included phzA1B1C1D1E1F1G1, phzA2B2C2D2E2F2G2, they are responsible for the production of phenazine-1-carboxylic acid in addition to other three supplemental genes inclusive phzM, phzS and phzH encoding individual enzymes implicated in the transformation of phenazine-1-carboxylic acid to pyocyanin,1-hydroxyphenazine, in addition to phenazine-1carboxamide [17]. The genes that are responsible for QS had been covered by several studies but the question raised here is regarding the existence of QS genes in clinical isolates of P. aeruginosa in parallel with QS genes that are present in environmental isolates of P.
aeruginosa. The current study provided an answer to this question

Materials and Methods Samples Collection
Fifty-three samples were collected from burn wound, sputum of patients suffering from respiratory tract infections and ear infections. Samples were collected from indoor patients after getting an ethical approval from the Ethical Committee in the Department of Biology, College of Science, University of Baghdad and signed consents from the patients. Samples were cultured onto nutrient agar under sterile conditions. Subsequently, seventy samples collected from Shatt Al-Hillah and the Al-Yahudia rivers, were placed in sterile glass containers and 0.1 ml from liquid specimens were cultured onto nutrient agar [18].

P. aeruginosaIsolation and Identification
Suspicious isolates of P. aeruginosa were cultured onto cetrimide agar. The isolated colonies of suspected P. aeruginosa appeared yellow-green and blue-green under fluorescent down ultra violet rays light. Standard method was followed to identify bacterial isolates. VITEK 2 DensiCheck tool (bioMe´rieux) (ID-GNB card) was used in the present study [19,20].

QS genes Detection
Nucleic acid was extracted to detect several genes (rhlA, rhlR, rhlI, lasR, lasI, lasB, and phzA1) that are responsible for QS and biofilm formation in the clinical and environmental isolates of P. aeruginosa. DNA was extracted according to the manufacturer's instructions of G-spin TM iNtRON kit. Primers were used for the detection of genes by polymerase chain reaction (PCR). Sequences of primers used in the current study are mentioned in Table 1.  Gel Electrophoresis 0.8 gram of the agarose was added to 80 ml of the Tris-borate-EDTA (TBE) buffer. The mixture was heated using microwave oven. Subsequently, the mixture temperature was decreased to 50-60°C. Finally, 4 μl of Red Safe stain (iNtRON, Korea) was added to agarose prior to solidifying. Comb was put at one end of the gel template and then the agarose mixture was poured into template. Next it was left at room temperature for 30 minutes. After that the tank tray was loaded with TBE buffer. Finally, after releasing the comb from the template, the template was placed into the tank tray. DNA samples were prepared by mixing 1 μl of bacterial DNA by blending 1 μl of dye. The samples and leader were loaded in the agarose wells and then the electrophoresis device (BIORAD, UK) was turned on at 75 volt/cm, 20 mA. Gel documentation system was used to get the pictures of bands [22].
In the current study, DNA was extracted from both clinical and environmental isolates of P. aeruginosa. PCR technique was applied to determine the presence of seven genes (rhlA, rhlR, rhlI, lasR, lasI, lasB, and phzA1) associated with the QS and biofilm phenomenon.The results showed that the studied genes were found in all clinical and environmental isolates of P. aeruginosa. Figures 1-7 show an electrophoresis image from PCR results that were taken by gel documentation system which showed the amplification genes by PCR technology. The figures show that all the identified genes had the number of base pairs (bp) around 100, except rhIR gene where the number of base pairs was 120.       Several previous results showed the ability of P. aeruginosa to form biofilm [23,24]. There are different factors that play an important role in its formation, QS phenomenon being one of them [25]. There are two pyramidal structures of the QS for P. aeruginosa, las and the rhl (26). The condensation of molecule signaling for 3-oxo-dodecanoyl of the homoserine lactone increased when the inhabitant's thickness grew in particular to form a complex with lasR. This complex operates the genes transcription as a lasI, rhlR and other genes [26]. rhl structure consists of the rhlR protein and the auto inducer synthesize RhlI which is incorporated to form auto inducer N-butyryl homoserine lactone. These structures are twisted in the manner of pyramidal structures. Indeed, the las structure is controlled by the rhl structure, in terms of transcriptional and also the next translational areas together. According to this fact, the condensation of marking molecule 3-oxo-dodecanoyl homoserine lactone which interacts with LasR at to activate a number of virulence genes such as lasB. These structures are wrapped up in the pyramidal manner.. Our results proved that proteases, exotoxin A, rhamnolipids and pyocyanin are considered as the virulence factors in the P. aeruginosa. This formation and organization is considered as an indicator of the QS, and also merge the formation and development of the biofilm. It also plays an important role in antibiotic resistance [16].
The results of the present study agree with recent research that indicates the selection genes chosen as targets, and also as one of the causes of epidemic, help spread of the P. aeruginosa bacteria. Further, the clinical isolates were able to form biofilm which had lasR, rhlI, and rhlR genes. As for the general, isolates included lasI. This finding also agrees with a previous study [27].