One of the most prevalent bacterial illnesses in the world, urinary tract infections (UTIs) affect millions of people annually and significantly burden healthcare systems (Behshad et al., 2023). The most prevalent gram-negative urinary tract pathogen is Escherichia coli, which is followed by Proteus species, Klebsiella species, and other Enterobacteriaceae. Urine cultures frequently include gram-positive organisms such Staphylococcus species and Enterococci (Rané & Dasgupta, 2013).

The most common hereditary illness worldwide is β-thalassemia. B-thalassemia is mostly treated with iron chelation and frequent blood transfusions, while some individuals may be cured via bone marrow transplantation. However, gene therapy is not yet a common medical procedure (Zafari et al., 2021). Since iron is a cofactor for metabolic enzymes and elements of the electron transport chain, it is a necessary element for practically all bacteria (Liao et al., 2024). Iron is necessary for the growth and development of bacteria, but at concentrations six to ten times that needed for consternation. In fact, higher iron concentrations can be detrimental to bacteria and can have antibacterial or bactericidal effects on the microbes (Al-Zerfi & Al-Hilu, 2024). Both humans and animals store iron in proteins such as ferritin, lactoferrin, and transferrin, mostly to protect themselves against microbial diseases. In contrast, microorganisms produce and release siderophores, which are tiny molecules that actively extract iron from eukaryotic binding proteins. Lastly, certain infections do not develop siderophores; instead, they extract iron straight from transferrin, lactoferrin, and ferritin to persist in the host (Klebba et al., 2021). The energy-transducing TonB-ExbB-ExbD system mediates the cytosolic membrane (CM) potential-driven uptake of ferri-siderophore complexes by gram-negative bacteria through species outer membrane (OM) receptors. Ferri-siderophores are transported from the OM receptors to the CM ATP-binding cassette (ABC) transporters via periplasmic binding proteins, which then transfer them to the cytosol. where reduction most likely dissociates the complexes. Gram-negative bacteria, however, produce a different type of OM protein called ferric siderophore receptors, which specifically recognise individual ferric siderophores, bind them with high affinity, and transport them into the periplasm because ferric siderophore molecules are typically larger than 600 D and cannot therefore pass through the OM channels of general or specific porins (Runci et al., 2019).

The aim of this study

Is to identify the specific genes (iutA and iroN) that are responsible for the expression of siderophores in Gram negative bacteria E. coli, K. pneumoniae and Enterobacter cloacae.

Study design

This study was structured as a case-control investigation involving a minimum of 100 samples, which were gathered from November 2023 to April 2024. Participants ranged from 6 months to 50 years, with mean age among the patients (1.473+-0.687) In the Alkut hospitals, specifically the Thalassemia Center in Wasit and Al-Numaniyah General Hospital located in Wasit province, Iraq.

Inclusion Criteria of the Patients.

Patients of both genders, ranging from 6 months to 50 years, participated in the study. They were diagnosed with urinary tract infections based on clinical signs and confirmed through urine sample analysis. All the patients were transfusion dependent thalassemic patients and control group was UTI patient free of chronic illness. Verbal consent was obtained directly from the patients or patient's relative for participation in the study.

Exclusion Criteria of the Patients.

Patients under 6 months and over 50 years, those who were not dependent on transfusions for thalassemia, individuals with other significant chronic medical conditions, and patients with other hemoglobinopathy excluded from the study.

Inclusion Criteria of the Control Group.

Participants included individuals aged 6 months to 50 years, all of whom were free from any acute or chronic illnesses. There is no known family history of thalassemia. Verbal consent was obtained for the collection of urine samples and participation in the study.

Exclusion Criteria of Control Group.

Individuals who are younger than 6 months and older than 50 years. Individuals with recent acute and chronic medical conditions, as well as a family history of chronic medical issues.

The Specimens Collection.

The Urine Specimens Collection

A sterile container was used to collect approximately 15 mL of midstream urine. Utilizing a sterile standard loop (0.001ml) for the culturing of urine samples. All urine samples were cultured using MacConkey agar, Blood Agar, CHROMagar medium and Cysteine–Lactose–Electrolyte-Deficient (C.E.L.D) agar. The culture media were incubated under aerobic conditions at 37°C for a duration of 24 hours. Conventional biochemical assays, the MA120 Automated ID&AST System, and the VITEK2 system (ID-GN, ID-GP) were employed to validate the diagnosis, along with Determination (AST-GN, and AST-GP). via the clean catch method for microscopic examination and bacterial culture. The isolations were transported in a container with ice to the laboratory at the College of Medicine in Wasit University for confirmation of all the isolations through 16s rRNA analysis and partial 16s rRNA sequencing, as well as for the detection of the bacteria's ability to produce certain virulence factors using PCR.

Molecular Study kits

PCR Bacterial DNA Extraction

The PCR diagnostic kits utilized in this study Presto™ Mini gDNA Bacteria Kit (gram positive and gram-negative bacteria) Taiwan

Polymerase Chain Reaction kit

GoTaq® Green PCR master Mix Promega USA is a pre-prepared formulation including premium Taq DNA Polymerase, deoxynucleotides, and reaction buffer at a 2X concentration. It includes all essential reagents for DNA amplification. The GoTaq® G2 Green Master Mix includes an inactive green dye and a stabilizer, facilitating the direct loading of the end product onto a gel for examination.

Polymerase Chain Reaction Primers:

In the following tables (1) and (2) the lists of genes and primers sequence that used in this study.