Collection of samples

Fifty-two soil and sewage samples were collected for six months, from November 2022 to April 4, 2023, from various locations in Iraq. The samples were carefully transported to the laboratory for further analysis.

Laboratory examination of collected samples

The samples were cultured on a medium, such as nutrient medium and McConkey agar medium to isolate Pseudomonas aeruginosa. After that, the samples were stained with Gram stain, then biochemical tests were performed to identify the isolates. The identity of all the P. aeruginosa isolates was confirmed using VITEK 2 (BioMérieux).

Identification of Pseudomonas aeruginosa using the polymerase chain reaction

DNA was extracted from the P. aeruginosa isolates and subjected to PCR-amplification to detect the PA-SS (16SrRNA) gene to identify P.aeruginosa species.

The PCR mix was made in a total volume of 25 μl comprising oligonucleotide primers (forward and reverse) and Quick-Load® Taq 2X Master Mix, which was liquefied at room temperature. Table 1 shows the nucleotide sequences of the oligonucleotide primers used for amplification.

Detection of L-arginase gene

The design of primers (Table 1)to detect the gene encoding the L-arginase enzyme was carried out in a scientific laboratory at Al-Mustansiriyah University, Iraq.

The amplification conditions for the primers were optimized. The PCR to amplify the L-arginase gene was performed in a thermal cycler with the following conditions: 95^oC for 2 min (initial denaturation), followed by 35 cycles of 94^oC for 20 sec (denaturation), 58^oC for 20 sec (annealing), 72^oC for 40 sec (extension), and one cycle of 72^oC for 1 min (final extension). 

Results

Figures & Tables

Discussion

The present study's findings revealed no substantial differences in the number of isolates from soil and sewage. However, the number of isolates in the soil was more than in sewage water. This is because bacteria in the soil are more than other microorganisms due to their ability to replicate easily. The observations made in the present study are consistent with previous findings[8,9]. In addition, sewage water provides an ideal environment for the growth of pathogenic and non-pathogenic bacteria, because it contains many elements necessary for the growth and reproduction of these bacterial species, as reported by Mohammed [10].

In the present study, arginase produced a relatively high amount of urea (110 µg/ml). The high production of urea observed during the assay of the L-arginase enzyme by M9 enhanced the color change from yellow to pink (Figure 2). This is crucial in the conversion of ammonia to urea in the urea cycle. Furthermore, the activity of the enzyme can be stimulated by measuring the volume of the enzyme (ml) and the amount of urea released during the enzyme's action. This finding highlights the significance of arginase in maintaining ammonia homeostasis and suggests that enzymatic activity is functioning efficiently in the given context, which is in agreement with previous reports that have demonstrated the significance of arginase in the regulation of levels of urea production [11]. The measurement of urea concentration provides a valuable indicator of arginase activity and the proper functioning of the urea cycle. Also, Lee and coworkers  found that an increased concentration of urea was associated with increased metabolism of arginine [12].

The use of the PCR method in the identification of P.aeruginosa is a commonly employed molecular technique in research and clinical settings. The procedure allows for the amplification of specific regions of the bacterial DNA, enabling sensitive and specific detection of the test organism. This procedure was used by Li et al. (2011) whose study focused on the rapid detection of P. aeruginosa in medical samples utilizing real-time PCR. The researchers highlighted the benefits of the RT-PCR, including its sensitivity, specificity, and quick turnaround time for detecting P. aeruginosa infections in a clinical laboratory setting. As far as we are aware, the present study presents the first report on the extraction of the arginase gene from P.aeruginosa. This finding contributes to the understanding of the genetic repertoire of P.aeruginosa and expands our knowledge of the functional genes present in this bacterium.

The arginase gene is made up of a 300bp DNA fragment and encodes the enzyme arginase. The enzyme catalyzes the hydrolysis of arginine to ornithine and urea, contributing to various physiological processes and metabolic pathways in the cells [11]. Pseudomonas aeruginosa is a versatile and adaptable bacterium known for its metabolic diversity and ability to thrive in various environments [14]. The identification and characterization of the arginase gene in P.aeruginosa provides insights into its potential role in arginine metabolism and the adaptability of this bacterium to different ecological niches.

The discovery of the arginase gene in P.aeruginosa has significant implications. It opens avenues for further research into the functional properties of the enzyme and its potential applications. Understanding the regulation and expression of the arginase gene can provide insights into the metabolic pathways involved in arginine utilization by P.aeruginosa. Furthermore, the arginase gene may have implications for pathogenicity and virulence. Pseudomonas aeruginosa is an opportunistic pathogen associated with numerous infections, particularly in immune-compromised individuals [15]. The arginase enzyme can influence the availability of arginine, an essential amino acid for immune cells, and potentially impact host-pathogen interactions and disease progression.

The study’s findings revealed a novel pathway, presenting an opportunity for scientific exploration and the potential for leveraging this enzyme's capabilities in bacterial growth research. Also, the present study highlights the possibility of harnessing arginase from natural sources, offering a new approach to inquiring about its applications in scientific investigations. This research signifies a breakthrough in genetic manipulation and opens up promising prospects for future studies in microbiology and related disciplines. 

Acknowledgement

The authors declare that there is no conflict of interest.

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