Molecular identification, antibiotics resistance and detection of virulence factors in p. aeruginosa bacteria isolated from urinary tract infection patients in Tikrit /Iraq

Pseudomonas aeruginosa is one of the causes of urinary tract infections. UTIs are among the most frequent bacterial infections, affecting millions globally annually. UTIs are a major healthcare concern, especially as uropathogens become increasingly resistant to antibiotics. The study included collecting 175 urine samples from patients suffering from urinary tract infections of both sexes. The sample collection period was from February 2023 to June 2023 from Tikrit Teaching Hospital and Alaskari Hospital in Tikrit City. Thirty isolates (17.1%) of P. aeruginosa bacteria were obtained. The isolates were diagnosed using the traditional method of MacConkey agar, cetramide agar, and blood agar culture media, followed by catalase and oxidase tests and biochemical tests. The final diagnosis was made using the VITEK2 device. The probability was 97%, 98%, and 99%, respectively. Antibiotic sensitivity tests of the isolates for 14 antibiotics were tested by VITEK2. The results of the study showed a difference in antibiotic resistance. The highest resistance rate was 100% for Ticarcillin, aztreonam 93.3%, piperacillin 86.6%, gentamicin 83.3%, ticarcillin/clavulanate 76.6%, and tobramycin.73.3%. Then, the phenotypic virulence factors were detected using culture media such as hemolysin production, which was 100% for resistant and sensitive isolates, pyocyanin production was 80% for resistant and sensitive isolates, protease production was 93.3% for resistant isolates and 86.6% for sensitive isolates, lipase production was 80% for resistant isolates and 66.6% for sensitive isolates, phospholipase C production was 86.6% for resistant and sensitive isolates, and alkaline protease production was 66.6% for resistant strains and 53.3% for sensitive isolates. Then, DNA was extracted from 20 isolates, and the presence of virulence genes was detected using the PCR technique. The results showed that the presence of toxA and plcH gene was 100% in the resistant and sensitive isolates, the bands had a molecular weight (396 bp)and (307bp), respectively). The results showed that the presence of aprA and lasB genes was 80% of the sensitive isolates. It was found that bands had a molecular weight (450bp) and (482 bp)respectively. Determine the genetic sequence for 16 isolates registered in the National Center for Biotechnology Information (NCBI). toxA gene exhibited the highest level of genetic variation, with a total of eight mutations detected. Isolate no. 1 showed two transversion mutations; isolate no. 2 exhibited three transversion and one transition mutation; and isolates no. 3 and 4 each showed one transversion mutation. aprA gene ranked second in terms of variation, with five mutations observed. Isolates no. 1, 3, and 4 showed transition mutations, while isolate no. 2 exhibited both transversion and transition mutations. plcH gene showed two substitution mutations one transversion in isolate no. 1 and one transition in isolate no. 2. No mutations were observed in isolates no. 3 and 4, suggesting that the plcH gene may be more genetically conserved compared to toxA and aprA. Finally, no mutations were detected in the lasB gene across all four isolates . The phylogenetic tree of P.aeruginosa was drawn and analyzed for the toxA and aprA genes. When comparing local isolates with those from other countries, the identity percentage was 99%. The phylogenetic tree was also drawn for the plcH and lasB genes. When comparing local isolates with those from other countries, the identity percentage was 100%. Gene expression was analyzed in forty isolates (10 per gene): toxA, plcH, aprA, and lasB), which were divided into two groups: resistant isolates and sensitive isolates. Among the tested genes, toxA exhibited the highest fold change difference, with a (p.value P= 0.02) indicating a highly significant upregulation in resistant isolates compared to sensitive isolates. lasB gene followed, with a (p.value P= 0.020), also showing a statistically significant difference in expression levels between resistant and sensitive isolates. plcH gene displayed a significant expression difference as well, with a (p.value 0.013). Lastly, aprA gene showed a statistically significant difference in gene expression, with a (p.value 0.018), The sequences of the four genes toxA, plcH, lasB, and aprA have been successfully submitted and registered in the NCBI GenBank database under the accession numbers