DNA Sequences of qacE∆1 Gene in Pseudomonas Aeruginosa Isolated from Wounds and Burns Infections

A total of 69 isolates of Pseudomonas aeruginosa were obtained from different clinical samples including wounds 24 (35%) and burns 67 (65%)، the genes that responsible for Pseudomonas aeruginosa resistance isolates was detected, the results showed that quaternary ammonium compound delta 1 (qacE∆1) gene was 97.1% in Pseudomonas aeruginosa isolation. 24 (35%) in the wounds and 63 (62.31%) in burns, the size of the gene showed 285 bp, the results of the qacE∆1 analysis showed a single mutation of the mutation type. The results of genetic analysis tree were showed after comparing presence of genetic variations of the qacE∆1 gene with their relatives from the nucleotide sequences as containing variations occurring at one distance and equally in Pseudomonas aeruginosa and other bacterial species.


Introduction:
Pseudomonas aeruginosa is a major hospital-acquired pathogen and is an opportunistic pathogen. Its morbidity increased for people having cancer, immunodeficiency diseases and people with Acquired Immune Deficiency Syndrome AIDS [1]. As Pseudomonas aeruginosa is considered the first pathogen which causes many inflammation of the burns, wounds, bacteremia in serious injuries and urinary tract infection, Otitis media, eye infection, inflammation of the brain membranes (Meningitis), endocarditic, respiratory treat infection such as Pneumonia especially in people with cystic fibrosis patients and joints infection, gastrointestinal infection and skin and soft tissue infection [2,3].
Pseudomonas aeruginosa has many mechanisms for resistance to both antibiotic and antiseptics, making them widely spread and difficult to treat it owns the structural changes in the membrane a of bacteria that causes absence of outside membrane porins, as well as the presence of ESBLs β-lactamase, efflux system pumps and plasmid resistance, it also owns Pseudomonas aeruginosa the genes of resistance to disinfectants, including the qacE∆1, represent a mutation of the qac gene, which acts as a multidrug transfer gene. This gene is located in the integron class 1 which allows it to transport between the plasmid and the chromosome and make of Pseudomonas aeruginosa resistance to disinfectants that used in hospitals [4].
The aim of this study was isolate and diagnose Pseudomonas aeruginosa from wounds and burns infection with detection of qacE∆1 gene which is responsible for the resistance of these bacteria to disinfectants. DNA sequencing analysis of qacE∆1gene and detection the mutation and protein translate.

Isolation and diagnosis:
The samples were cultured on the Cetrimide agar medium, MacConkey agar, and blood agar. Biochemical tests (oxidase and catalase) were performed for the final detection of isolates using API20E system according to the instruction by BioMerieux Company [5].

Genomic DNA Extraction:
A Bioneer extraction kit was used for DNA Bacteria Kit extraction according to manufacture instruction Presto TM Minig the supplier of the company (Bioneer, Korea) to extract DNA from the isolates P. aeruginosa according to the instructions of the company.

DNA purity:
The purity of DNA output in the Nano-drop device was measured the purity of obtained was between (1.7-2).
The optimum conditions for the detection of this gene were one cycle for 5 minutes at 95° C for initiation 30 cycles for 30 seconds at 95 ° C for DNA denaturation, 30 seconds at 55.5° C for annealing to DNA, 45 seconds at 72° C to elongate and then only one cycle for 5 minutes and at 72° C for final elongation.

Electrophoresis:
5μl of DNA product was transferred to the gel electrophoresis system using 2% of agaros gel strength 12 cm with 100 volts for 60minutes. Then imaging by using ultraviolet radiation at a wavelength of 260 nanometers and then photographed with a high-resolution camera.

DNA Sequencing analysis :
After initial amplification of P. aeruginosa qacE∆1 gene, the PCR product of (20μl) DNA of each F primer and R primer was sent to US NICEM Company for sequencing by Genetic analyzer. DNA sequence data were analyzed using NCBI (National Center for Biotechnology Information) database and using BioEdit program (V.7.2.5) [6] 3. Results and discussion: After the laboratory diagnosis, 69 isolates of P. aeruginosa including 24 wounds isolates (35%) and 45 burns isolates (65%). P. aeurginosa is most bacteria causes burn infections because it lives in the humid environment and hospitals and is a major cause of both burns and wound infections [7], the present study is similar to that of the researchers [8,9]. These results was agreed with Kucken et al. [8] who determined the prevalence of the gene 81% in P. aeruginosa isolated from different clinical sources which encodes quaternary ammonium compound delta qacE∆1 which responsible for the resistance to disinfectants, that including quaternary ammonium compound and cetramide, as well as with the researcher Helal and Khan [9], who determined the prevalence of the gene 79% in P.aeruginosa, while the agreement with Dabiri et al. [10] who showed that 64.4% gene prevalence in P.
aeruginosa isolated from different hospitals in China, while Kazama et al. [11] showed the prevalence of the gene was 68%, while the Mahzounieh et al [12]determined the prevalence of the gene 91% in P. aeruginosa which is isolated from burns in Tahran and Isfahan hospitals, the difference in the proportion of the qacE∆1 gene in P. aeruginosa is due to the number of samples and the concentration of the disinfectant [13,14]. Fig 1 and 2. The sequencing analysis of the qacE∆1 gene was performed the results showed that isolation Ps-42 which is a single point mutation of type shown in Table 2 (1, 2,3 ,4, 5,6 ,7,8 ,9,10 ,11, 12, 13, 14,15,16 ,17 ,18,19,20

Conclusion:
the qacE∆1gene is responsible for resistance P. aeruginosa isolated from burns and wounds for disinfectant.