رسائل دكتوراة
English
2023
Genes among Escherichia coli Isolated from Urinary Tract Infections Patients in Hilla City-Iraq
ايمان محمد جار الله, حيدر عبد الأمير عبد الرضا الاسدي
كلية العلوم-جامعة بابل · العراق
Urinary tract infections (UTIs) are one of the most widespread bacterial infections with a global extension. These infections are predominantly caused by uropathogenic Escherichia coli (UPEC). The relationship between multidrug resistance of Escherichia coli and resistance genes profiles among carbapenems resistance of uropathogenic Escherichia coli isolates that isolated from patients with urinary tract infection in Hilla City. Therefore, the current survey was carried out in order to determine the phenotypically, genotypically and determine genes expressions among multidrug resistance uropathogenic Escherichia coli isolates that isolated from patients with UTIs in Hillah City. The study included collection and analysis of 646 midstream urine specimens which is immediately examined by general urine examination (GUE) after collection to detecting physical, chemical and microscopical examinations to identifying presence of infections due to the urinary tract infection occurs without any symptoms observed in the patient, followed by phenotypically, genotypic and gene expression detecting methods. The valuable midstream urine specimens were carefully collected from February to July/2022 from different hospitals in Hilla City. The clinical specimens from individuals with UTI symptoms were obtained from the following hospitals which is including: Al-Imam-Alsadiq teaching hospital (377 urine clinical specimens), Marjan teaching hospital (175 urine clinical specimens), and Babylon for women's and children's hospital (94 urine clinical specimens). Subsequently, all midstream urine clinical specimens were examined by microscopical examination and cultured onto MacConkey agar, Eosin Methylene Blue (EMB) agar and blood agar plates followed by incubation for 24 hrs at 37°C to detect lactose and non-lactose fermenting isolates. Out of total 646 midstream urine specimens, only 320 (49.5%) isolates were successfully identified as Escherichia coli bacteria through its growth on the following selective and differential culture media which is including MacConkey and EMB agar media and the rest 326 (50.5%) isolates were belong to other gram-negative bacteria. Results indicated the presence of 320 clinical specimens, encompassing 273 (85.3%) females and 47 (14.7%) males. These Escherichia coli isolates were obtained from urinary tract infections (UTI) patients are subjected to antibiotics susceptibility testing (AST). The results of this study showed the highest incidence of UTIs was observed in the adult (30–60) years old age group among the rest ages groups. The biochemical results and Vitek2 System tests were also confirmed as confirmatory tests for these isolates, which showed that all the isolates were E. coli bacteria. Antibiotics susceptibility testing (AST) revealed notable resistance trends among E. coli isolates. Notably, β-lactam combinations, including Amoxicillin-clavulanate and Ampicillin, exhibited resistance rates of 87.1% and 75.9%, respectively. Aminoglycosides (e.g., Gentamicin) demonstrated resistance in 66.8% of cases, while Sulfonamides (e.g., Trimethoprim) and Nitrofurans (e.g., Nitrofurantoin) exhibited resistance rates of 49.6% and 43.1% respectively. The susceptibility of isolates varied, with Ciprofloxacin, Cefepime, Ceftriaxone, and Piperacillin showing sensitivities in the 34.3% to 52.1% range. Moreover, the results of AST showed only 20 Escherichia coli isolates (6.2%) were resistant to the meropenem and imipenem. The results of phenotypical examinations which is including 20 carbapenems resistant Escherichia coli isolates showed positive result for amoxiclav double disk synergy test and modified Hodge's test (MHT) which confirms that these isolates have the ability to produce beta-lactamase enzymes.
The molecular detection methods have been used to identify carbapenems resistance genes among 20 E. coli isolates. The molecular detection methods were used to detect the following genes which is including blaCTX-M-1 and blaDHA were most prevalent with percentage levels (no=19) (95%), followed by blaTEM (no=16) (80%), blaNDM (no=15) (75%), blaIMP (no=12) (60%), blaVIM (no=10) (50%), and others with varying frequencies. While each of the genes of blaGES and blaSME were not detected during this study. According to the study's findings, the majority of isolates included at least two genes. Isolate number E.co-9 revealed a high percentage level which carried 10 distinct genes, while other isolates which is including (E.co-2, E.co-3, E.co-11 and E.co-18) each carried nine different genes. According to Ambler classification, the results of molecular investigation of this study showed among 280 β-lactamase genes have been detected during this study that showed positive results for 126 (45%) genes whereas the rest of the genes totally 156 (55%) genes showed negative result. The molecular characteristics of 20 carbapenems resistant E. coli isolates showed all of the genes belong to class A within n=53 (42.1%) β-lactamase out of 126 positive genes followed by class B within n=37 (29.3%) β-lactamase genes, while class C was n=30 (23.8%) and finally class D showed the lowest percentage levels among β-lactamase genes that detected during this study it was n=6 (4.8%). Finally, this study assessed gene expression in the presence of Imipenem (IMP) inhibitors, revealing a 1.8 to 12.4 fold increase in blaIMP mRNA levels in response to exposure to Imipenem antibiotics. The results of gene expression showed varying levels in the presence of the imipenem inhibitor for all bacterial isolates. It is worth noting that isolate No. 13 showed the highest level of gene expression among the rest of the other isolates. It could be concluded that the investigation highlights the prominence of urinary tract infections, particularly among uropathogenic E. coli, and demonstrates the prevalence of multi-resistance gene profiles among urinary tract infections patients in Hillah City.