Abstract

This study was conducted for the purpose of detecting some virulence factor genes of the bacterial species isolated from the urine transfusion dependent beta Thalassemia TDT with UTI, 173 urine samples were collected from both study group  TDT patient with UTI and control group, Results showed that sample (n=38, 21.96%) of the total samples studied gave positive bacterial growth, while (n=135, 79.03%) gives negatives growth on media cultivation in control samples. In patient with beta Thalassemia have shown positive growth of 38 samples (21.96%) and samples (135) (79.03%) were not give any growth. the purpose of study is to detecting some virulence factors for gram negative studied isolates, DNA was extracted for all isolates. (n=17)  gram negative isolates were taken, represented by (n=9, 23.7%) isolates of E. coli and (n=3, 7.9%) isolates of E. cloacae, (n=5, 13.2%) of K. pneumonia their isolated from transfusion dependent thalassemia with   UTI with use of universal primers for each bacterium and partial 16s rRNA sequence for diagnosis of this isolated bacteria.

Highlights:

1. Detecting virulence factor genes in bacteria from transfusion-dependent beta thalassemia UTI patients.
2. 173 urine samples, 38 (21.96%) showed positive bacterial growth, 135 negative.
3. DNA extraction, 17 gram-negative isolates identified: E. coli, E. cloacae, K. pneumoniae.

Keywords: universal primers, Thalassemia, Urinary tract infection, Virulence Factors.

Introduction

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.

Methods

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.

PCR conditions

Results and Discussion

Results of Bacterial Culture on Artificial media.

The initial identification of bacterial isolations was conducted using bacteriological methods, such as the Gram stain. The characteristics of Gram stain isolations were subjected to biochemical tests for identification. A Gram stain was performed on all positive cultures, encompassing both Gram positive and Gram-negative bacteria. The identification of all bacterial species was achieved through biochemical tests, both manual and automated. Urine samples were cultured overnight on Blood agar, MacConkey agar, and CLED agar in an incubator set at 37°C, followed by microscopic examination at a magnification of 100X. Subcultures were performed on Blood and Mannitol salt agar, Eosin Methylene Blue (EMB), and HiCrome™ UTI Agar (Chromogenic Agar).

In this study It were observed only (21.96% n=38) were positive for bacterial culture, this result is similar to (Obed et al., 2024) they have same positive growth percentage of UTI sample was 20% n=12/50.

Gram positive bacteria represent 55.3% n=21/38 and gram negative bacteria represent 44.7% n=17/38 our results agreed with (Al Badry et al., 2016) since their result showed gram positive bacteria Staphylococcus genus has the highest prevalence 38.88% n=7/18 while other gram negative bacteria showed less prevalences that’s my due to the iron overload in TDT so the Gram-positive bacteria thrive in this patients and to their compromised immune system.

Molecular detection of 16s rRNA of pathogen isolates:

In this study depending on Vitek2 and MA120 techniques we used a specific primer (listed in Table (2-7) in chapter two) used to identify bacterial species that were diagnosed by previous mentioned techniques. Some of isolated bacteria didn’t show any band in agarose gel electrophoresis, so the idea took a shape of misdiagnosis of bacteria by early mentioned techniques. For that reason, we used universal 16s rRNA primer (Table (2-5) in chapter two), and confirmatory partial sequencing for certain diagnosis of the all isolates.

All isolates (thalassemic patient n=38 and control group n=20) showed positive band with size 372bp in 1.5% agarose gel as showed below in Fig (3-1) other isolates figures are in appendix.

Figure 1. PCR products of the amplification of partial region of gene 16s rRNA of Bacteria

The size of the PCR product is 372bp. The gel was 1.5% and the DNA dye is RedSafe (Intron, Korea). V: 90, Time: 45 minutes. M: DNA ladder

The frequency and distribution of bacterial species for the thalassemic patients according to the results of PCR and 16s rRNA partial sequencing as showed in the chart below in table (4) and Fig (2).

Figure 2. Bacterial distribution for thalassemic patients

Molecular detection of virulence factors genes:

All isolates of TDT and 20 isolates of predominant bacteria of control group were molecularly diagnosed for virulence factors of iron acquisition systems, the genes were salmochelin (iroN), aerobactin (iutA). The highest prevalent genes in gram negative bacteria in TDT were iutA 94.1% n=16/17. The lowest prevalence was for iroN gene 23.5% n=4/17.

The control group isolates were all gram-negative bacteria since it the predominant bacteria. The highest prevalence was also for iutA showed higher prevalence than TDT with 100% n=20/20. The lowest prevalence was also for iroN gene 35% n=7/20. The table (5) show the details of gene prevalence.

The statistical analysis in table (6) of studied genes shows non-significant correlation between bacterial infection and studied groups.

Salmochelin siderophore receptor ( iroN ):

The IroN protein has been identified as an outer membrane receptor responsible for the uptake of Fe3+-bound salmochelin, exhibiting similarities to the FepA protein(Mohsen et al., 2023). The outer membrane siderophore receptor (IroN) facilitates the transport of various catechol siderophores(Etefia, 2021). The iroN gene was found on a significant plasmid known as pLVPK, which has been shown to play a vital role in the pathogenicity of hypervirulent K. pneumoniae (hvkp)(Tang et al., 2020). The figure (3) showed the iroN bands in agarose gel detected by conventional PCR in some isolates. In this study iroN prevalence was 23.5% n=4/17 in TDT gram negative isolated bacteria Figure (4) showed the prevalence of iroN gene in TDT isolates). All isolates of K. pneumoniae were negative n=0/5 for iroN gene that’s results similar to (Lam et al., 2018) were the found low prevalence of iro locus about < 10% of 2503 genome analyzed, the low prevalence of iro locus in their study considering the great specimens number explaining the negative result in our study since our specimens number much lower n=38. Other studies disagreed with our results such as (Abdalhadi, 2024) in their study conducted in Baghdad Iraq showed iroN gene was found 63.3% n=38/60 isolates, since iroN carried on plasmid that’s may explaining the absence of it from our isolates of TDT and this categorized our TDT isolates as Classical K. pneumoniae (cKP) is frequently linked to nosocomial infections, including pneumonia, urinary tract infections (UTI), and bacteremia, particularly in individuals at the extremes of age or those with underlying immunodeficiencies. In contrast, hypervirulent K. pneumoniae (HvKP) tends to cause infections such as pyogenic liver abscess (PLA), lung abscess, and endophthalmitis in otherwise healthy individuals (Dai & Hu, 2022). E. cloacae isolates with iroN gene prevalence was 33.33% n=1/3 there is no previous study showed the presence of iroN in E. cloacae and we investigate about it because iroN could transmitted horizontally by plasmid. E. coli also exhibit same prevalence of iroN 33.33% n=3/9 this result almost agreed with (Al-Zerfi & Al-Hilu, 2024) were they found iroN represent 11.1% n=5/45 of E. coli isolates from UTI in Najaf-Iraq but our results showed an inconsistency with (Krawczyk et al., 2023) were they discovered a high prevalence of iroN in E. coli isolated from UTI in Poland 64% n=54/85 this contradiction in results explained by the environmental causes and also to the presents of plasmids that carried the iroN gene. The control group in this study represented by two predominant bacteria E. coli and K. pneumoniae, E. coli shows lower prevalence than study group for iroN was 25% n=3/12 and K. pneumonia showed higher prevalence than study group 50% n=4/8, from previously results the bacterial isolates of K. pneumoniae from control group were more virulent from TDT group. statistically non-significant differences were found in the distribution of iroN between TDT and control group (p= 0.447).

Figure 3. Bands of PCR products of the amplification of partial region of gene iroN The size of the PCR product is 553 bp. The gel was 1.5% and the DNA dye is RedSafe. Tracks from LH04 to LH30 for TDT isolates and from LH34 to LH42 for control group.

Figure 4. showed the prevalence of iroN gene in TDT isolates.

Aerobactin siderophore receptor ( iutA ):

Aerobactin is a receptors located on the outer membrane that interact with siderophores(Rahmani et al., 2020). Aerobactin iutA is encoded by the plasmid and plays a role in the transferring of iron across the membrane, participating in the tonB-dependent transport pathway for iron (R. et al., 2021). The figure (5) showed the iutA bands in agarose gel detected by conventional PCR in some isolates . In this study iutA prevalence was 94.1% n=16/17 in TDT gram negative isolated bacteria as showed in Figure (6) down there. The lowest prevalence of iutA in this study was in E. cloacae it was 66.67% n=2/3 this results almost similar to (Mosaffa et al., 2024) were their results was 80% n=12/15 from multiple clinical isolates of E. cloacae including UTI isolates in their study conducted in Kerman Iran. The iutA prevalence for E. coli isolates was 100% n=9/9 this result has exact match’s with a study done in Eastern Finland by (Kurittu et al., 2022) in UTI isolates n= 12/12. From other side our results disagreed with (Krawczyk et al., 2023) in their study conducted in Poland iutA gene represent 52% n=44/85, also two other studies showed low prevalences of iutA (Rezaei et al., 2023) in Iran find iutA prevalence was 20% n=8/40 and (Dossouvi et al., 2023) in Dakar Senegal their result was 34.6% n=27/78 this different results could be explained by the different isolated strains and horizontal gene transmission. Also K. pneumoniae exhibit 100% n=5/5 presence of iutA in TDT with UTI isolates this results agreed with study in China by (young Lee et al., 2022) were they found iutA represents 90.5% in hvkp group of K. pneumoniae from other side in same study our result disagreed with them result of ckp group which showed iutA in prevalence 41.3% their study links the high prevalence of sitA with hvkp group in UTI cases that could develop to bacteremia from results above we could considering our isolates as hvkp. Our result strongly disagreed with study conducted in Uganda and Kenya by (Decano et al., 2021) were they found iutA represent only 10% of K. pneumonia UTI isolates. statistically non-significant differences were found in the distribution of iutA between TDT and control group (p= 0.272). control group of this study showed similar results of iutA prevalence both E. coli and K. pneumoniae exhibit 100% presence of iutA in all isolates.

Figure 5. Bands of PCR products of the amplification of partial region of gene iutA the size of the PCR product is 302 bp. The gel was 1.5% and the DNA dye is RedSafe. Tracks from LH04 to LH30 for TDT isolates and from LH34 to LH42 for control group.

Figure 6. showed the prevalence of iutA gene in TDT isolates.

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