المستخلص
Urinary tract infections (UTIs) are among the most common infections worldwide. Uropathogenic Escherichia coli (UPECs) are the main causative agents of UTIs and are extremely resistant to antibiotics because of their several virulence factors, which allow them to produce UTIs and even severe chronic cases in women. A total of 250 clinical urine samples were collected from women suffering from UTI symptoms from Iraqi women ages ranging from 18 to 65 years old. These specimens were obtained from the Infertility Unit in Al-Batool Teaching Hospital, Women’s medical clinics in Baqubah city, and Baqubah Teaching Hospital during the period from December 2023 to the end of February 2024.
All the samples were cultured on blood agar, MacConkey agar, and Eosin-Methylene blue agar (EMB). Diagnoses were then confirmed using biochemical tests and the VITEK-2 system The results showed that bacterial isolates appeared in 223 of these samples. The results of the conducted study showed that most participants had E. coli growth 53 (21%), other bacterial growth 170 (68%), and no bacterial growth 27 (11%). The outcomes of the current study showed microbial growth in UTI-infected women was 223 (89.2%), of which UTI-infected pregnant women without PCOS comprised 36.4%, non-pregnant UTI-infected women without PCOS comprised 34.4%, while UTI-infected women with PCOS comprised 18.4%. A total of 53 E. coli isolates were obtained: 21(39.6 %) from pregnant women with UTI without PCOS, 17(32.07%) from non-pregnant women with UTI without PCOS, and 15(28.3%) from women with UTI and PCOS.
Susceptibility testing was done to test the isolates resistance, and it was found that the isolates had varying levels of resistance to different antibiotics. The results of the current study showed that the E. coli isolates exhibited the highest resistance to Ampicillin (94.3%), Amoxicillin-Clavulanic acid (92.5%), Ceftazidime (92.5%),Cefotaxime (90.6%),PiperacillinTazobactam(79.2%), Ceftriaxone(75.5), Piperacillin(73.6%),Resistance patterns for the isolates were also calculated, and 22 (41.5%) isolates were multidrug-resistant (MDR), 29 (54.7%) were extensively drug-resistant (XDR), and the remaining 2 (3.8%) were multidrug-sensitive (MDS).
The study also included phenotypic detection of some virulence factors. Biofilm production was tested using the microtiter plate (MTP) method. The findings showed that most E. coli isolates produced moderate biofilm (27, 50.9%), and the lowest was strong biofilm production (21,39.6%). The remaining 5 isolates, which accounted for (9.4%) of the total isolates, were weak biofilm producers. Results of the present study showed the positivity of E. coli isolates for enzyme production as follows: MBL 12 (22.6%), ESBL9 (17.0%), and AMPC 14 (26.4%), compared to the negativity of 41(77.4%),44(83.0%), and 39 (73.6%), respectively.
Molecular detection was carried out on 20 isolates (10 MDR and 10 XDR) out of the 53 isolates for virulence genes. Multiplex PCR was used to detect the adhesion genes (fimH, papGII), which play an important role in biofilm production. Results from the multiplex PCR technique showed that all 20 isolates (100%) had the fimH gene, followed by papGII, which was found in 55% (11/20) of the isolates. This study was conducted to detect ESBL genes in 20 extensively drug-resistant and multidrug-resistant E. coli isolates. ESBL genes (SHV, TEM, and blaOXA) were screened by the multiplex PCR technique. The results showed that 19 (95%) isolates were positive for the TEM gene. These results revealed that the TEM gene was the most widespread. Additionally, the results of the current study showed that 7 (35%) isolates were positive for the blaOXA gene. Noteworthy, none of the 20 extensively drug-resistant and multidrug-resistant E. coli isolates had the SHV ESBL gene.
The result of the biofilm gene (ndvB) for 20 selected extensively drug-resistant and multidrug-resistant E. coli isolates, using the uniplex PCR technique, showed that 13 (65%) of the isolates were positive for the ndvB gene. Among the 20 extensively drug-resistant and multidrug-resistant E. coli isolates, the results achieved using PCR revealed that 4 (20%) isolates had the VIM gene, while 2 (10%) isolates carried the NDM gene.
The conducted sequencing reactions indicated the accurate identity of the investigated samples, which were found to be attributed to E. coli. The alignment of nucleic acid sequences of the five isolates with the reference sequences of E.coli revealed the presence of fifteen nucleic acid variants distributed in E3, E11, E12, E14, and E19 isolates. Ten of these variants were given silent effects. Whereas five of them were given missense impact on the fimbrial protein. The tree suggested that the samples under investigation were located in four separate and neighboring phylogenetic locations within a single, different phylogenetic tree. As a result, several of the detected variations significantly altered the studied samples’ phylogenetic position within the included clades of E. coli. All the investigated genomic sequences were deposited in the NCBI web server, and unique accession numbers were obtained for all analyzed sequences. GenBank PQ273408, PQ273409, PQ273410, PQ273411, and PQ273412 were deposited in NBCI to represent the E3, E11, E12, E14, and E19 isolates, respectively.
