Molecular detection of shigatoxin gene in escherichia coliisolated from patients in Thi-Qar province, Iraq

Escherichia coli is a bacterium that has a reputation that may be both advantageous and disadvantageous. Certain types are crucial for maintaining gut health, while others have the potential to cause severe illnesses. An alarming characteristic of these pathogenic Escherichia coli is their increasing resistance to antibiotics and their strong inclination to build biofilms. Escherichia coli bacteria have shown a growing resistance to frequently used antibiotics. The main cause of this issue is essentially the excessive and incorrect use of antibiotics in both people and animals. Escherichia coli can aggregate and create cohesive, mucous-like coatings known as biofilms. Biofilms serve as strongholds, impeding the access of antibiotics and the immune system to eradicate the germs. Biofilms exacerbate the problem of antibiotic resistance. The compact nature of a biofilm provides a conducive environment for the survival of bacteria that are resistant to antibiotics, shielding them from the impact of these drugs. Shiga toxin, which is synthesized by some strains of Escherichia coli bacteria, is a very powerful antagonist in the realm of foodborne diseases. The Shiga toxin family consists of two primary variants: Stx1 and Stx2, both of which have the potential to cause significant harm to the human body. Specific variants, known as Shiga toxin-producing Escherichia coli, can generate these detrimental toxins, such as O157. H7 is well known for its propensity to induce sickness. Some strains of Escherichia coli possess a harmful collection of virulence factors, proteins, and structures that aid in causing disease. These include flic genes that produce flagella, which are hair-like structures used for movement, evading the immune system, and contributing to the formation of biofilms. Biofilms can increase resistance to treatment. The hlyA gene is responsible for producing a toxin known as hemolysin A, which can break down red blood cells. This process leads to tissue damage and inflammation when an infection occurs. Additionally, it has the potential to impair the immunological response of the host. Moreover, the rfb-o157 genes are accountable for synthesizing a distinct form of lipopolysaccharide, which constitutes a component of the outer membrane and serves many roles, including engagement with the host immune system. During this cross-sectional study, 250 clinical samples from patients at Nasiriyah Teaching Hospital with diarrhea and urinary tract infections were collected using disposable sterile urine and stool containers. All samples were grown on iv rich media (Blood agar), then cultured on differential and selective media (MacConkey agar) to grow only Gram-negative bacteria, and then all Gram-negative positive cultures were evaluated by using a series of biochemical tests to isolate only E. coli isolates. Following confirmed identification and investigation of the antibiotic resistance profile, the automated VITEK compact-2 system was used. To differentiate the E. coli O157:H7 isolate from other E. coli isolates, all isolates were cultured on differential media (Sorbitol agar). The Saccharomyces bulradii were growing on sabouraud dextrose and then added to the bacterial broth in order to evaluate their effects on the expression of E. coli genes. The biofilm formation capacity of bacteria was determined using microtiter plate methods. All virulence genes were detected using a conventional polymerase chain reaction and then detected by gel electrophoresis. T The expression of Shiga toxins genes was estimated using a quantitative real-time polymerase chain reaction. The E. coli phylogeny was analyzed after extracting 16S rRNA and sent to a determined sequencing Microgene company in Korea. The highest number of E. coli O157:H7- infected patients 6 (46.20%) was associated with the age group 21–30 years, while both age groups (11–20 and 41–50 years) were associated with lower-infected patients (8.3% and 14.3%, respectively). In contrast to the non-O157:H7 patients, the highest number was 11 (91.7%) in the age group 11–20 years, while the age groups <=10 and 31–40 years were associated with a lower number of 4 (50% and 44.4%), respectively. The female numbers were higher 15 (40.5%) and 22 (59.5%) among E. coli H7-infected and E. coli NON-infected patients, respectively, compared to male numbers of 5 (25%) and 15 (75%), among the same groups. Among 250 urine and stool samples, only 29 (50.88%) urine and 28 (49.12%) stool samples were positive for E. coli, and only 37 (64.90%) had the ability to sorbitol ferment, while 20 (35.10%) did not. According to hemolysis ability, 19 (33.30%) and 11 (19.30%) had alpha and beta patterns of hemolysis ability, while 27 (47.40%) had a gamma pattern of hemolysis ability. The STX1, STX2, rfo157, and hylA gene frequencies were positive among 19 (33.30%) and negative among 38 (66.70%). While the fliC gene was positive among 37 (64.90%) and negative among 20 (35.10%), Most isolates, 48 (54.21%), had the ability to form biofilm, categorized as follows: 11 (19.30%) of isolates had weak ability, 16 (28.07%) had strong ability, and 21 (36.84%) had moderate ability to form biofilm. The highest resistance level was against amikacin, while the lowest resistance levels were against pipracillin, tazobactam, and tobramycin. The fold change of STX1 gene expression was lower (3.96) after adding saccharomyces bulradii, and there was a two-fold lower change in STX2 gene expression (0.11) after adding saccharomyces bulradii. According to phylogeny, all our E. coli 16S rRNA sequences were completely identical (100%). While, when compared with NCBI references, there was a high identical sequence rate ranging from 99.63% to 100%. In conclusion, E. coli showed high levels of virulence and antibiotic resistance; however, Saccharomyces bulradii has a crucial role in influencing Shiga-toxin gene expression, and this yeast may be incorporated in different chronic and acute E. coli infection diseases