Investigating the Presence and Expression of stx 1 Gene in Escherichia coli Isolated From Women With Urinary Tract Infection Using Real-Time PCR in Tabriz , Iran

© 2018 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Escherichia coli is the most common cause of urinary tract infections (UTIs), especially in young women.1 E. coli is responsible for more than 80%-90% of UTI among the community and 30%-50% in the hospitals. In addition, this bacterium is one of the main causes of hospitalization with significant complications and high health care costs.2 The verotoxigenic E. coli (VTEC) strains secrete a type of toxin which is called verotoxin due to its ability in killing Vero cells. Further, because of the similarity of verotoxin to the Shiga neurotoxin which is secreted by Shigella dysenteriae, type I is called Shigalike toxin and the strains which secrete it are referred to as Shiga toxin-producing E. coli (STEC).3 Furthermore, the verotoxins are categorized into stx1 and stx2 groups. These toxins inhibit the synthesis of proteins through affecting the ribosomal RNA, and the types of E. coli that produce these toxins are called STEC. The STEC strains are an important group of zoonotic and foodborne pathogens in human that can cause various diseases such as bloody diarrhea, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP).4 A number of current studies have shown that about 20 000 instances of infection and 250 cases of death are annually reported due to the consumption of contaminated foods by STEC in the United States.5 Today, it is well known that STEC is intermittently isolated from cattle and sheep stools, and as a result it can contaminate the meat of these animals.6


Background
Escherichia coli is the most common cause of urinary tract infections (UTIs), especially in young women. 1 E. coli is responsible for more than 80%-90% of UTI among the community and 30%-50% in the hospitals.In addition, this bacterium is one of the main causes of hospitalization with significant complications and high health care costs. 2 The verotoxigenic E. coli (VTEC) strains secrete a type of toxin which is called verotoxin due to its ability in killing Vero cells.Further, because of the similarity of verotoxin to the Shiga neurotoxin which is secreted by Shigella dysenteriae, type I is called Shigalike toxin and the strains which secrete it are referred to as Shiga toxin-producing E. coli (STEC). 3Furthermore, the verotoxins are categorized into stx1 and stx2 groups.These toxins inhibit the synthesis of proteins through affecting the ribosomal RNA, and the types of E. coli that produce these toxins are called STEC.The STEC strains are an important group of zoonotic and foodborne pathogens in human that can cause various diseases such as bloody diarrhea, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP). 4A number of current studies have shown that about 20 000 instances of infection and 250 cases of death are annually reported due to the consumption of contaminated foods by STEC in the United States. 5Today, it is well known that STEC is intermittently isolated from cattle and sheep stools, and as a result it can contaminate the meat of these animals. 6

Objectives
This study sought to examine the presence and expression of the stx1 gene in E. coli isolated from women with UTI in Tabriz.

Sample Collection
A total of 25 E. coli isolates were evaluated in this study, which were identified in our previous studies by the biochemical tests administered to women with UTI.The E. coli samples were isolated from women with UTI who referred to Asadabadi hospital in Tabriz.The collected urine samples were cultured in EMB (eosin methylene blue) agar (Merck, Germany) medium.Then, the obtained bacteria colonies were evaluated and biochemical tests including IMViC and triple sugar iron (TSI) agar were used to identify the E. coli strains.Next, the E. coli isolates were inoculated in tryptic soy broth (TSB) medium containing 40% glycerol and were kept at -20˚C until DNA and RNA were extracted.
Extraction of DNA Genomic DNA was extracted using the salting-out method.Brain heart infusion (BHI) medium (Merck, Germany) containing E. coli was transferred to 2 mL tubes and centrifuged at 10 000 g for 5 minutes.The supernatant was poured out and then 800 μL of lysis buffer solution was added to each of the tubes.The tubes were respectively placed in a bain-marie at 85°C (30 minutes), in a freezer at -70°C (10 minutes), and again in a bain-marie at 85°C (5-6 minutes).Then, 700 μL of chloroform-isoamyl alcohol solution containing chloroform (24 mL) and isoamyl alcohol (1 mL) was added to each of the tubes, and were stirred slowly.Next, the tubes were centrifuged at 12 000 g for 5 minutes and the supernatant containing DNA was transferred to the new tubes.Subsequently, cold isopropanol (1.5 times of the samples' volume) was added to the new tubes, and the tubes were stirred slowly and placed in a freezer at -70°C for the whole day.The next day, the samples were removed from the freezer and put at the room temperature for defreezing.Then, the tubes were centrifuged at 12 000 g for 5 minutes and the supernatant was poured out.Afterward, the tubes were thoroughly dried using a sterile napkin so that the tubes did not smell alcohol.Finally, 50 μL of deionized distilled water (DNase free) was added to each of the tubes 4 and then the optical density (OD) and concentration of the extracted DNA were measured using a NanoDrop instrument.

Primer Design
The sequence of the 16s rRNA gene and the related data were obtained from the National Center for Biotechnology Information (NCBI) database.The specific primers for amplification of the 16s rRNA gene were designed using Oligo software, version 5.Moreover, the primer sequence was synthesized in Takapo Zist Company, Iran.The primer sequences and their characteristics are provided in Table 1.

Extraction of RNA
The positive samples related to the presence of stx1 gene were cultured in Mueller Hinton broth medium and centrifuged.First, 500 μL of Trizol solution was added to each of the bacterial sediment and kept at 45°C for 30 minutes.Then, 200 μL of chloroform was added to each of the tubes and stirred slowly and placed at room temperature for 5 minutes.The tubes were then centrifuged at 12 000 g for 10 minutes and the supernatant (without interrupting the middle phase) was carefully transferred to the new tubes.Next, cold isopropanol (2.5 times of the samples' volume) was added to the new tubes, and the tubes were stirred slowly and placed in a freezer at -70°C for the whole day.The next day, the samples were removed from the freezer and brought to the room temperature for defreezing.Subsequently, the tubes were centrifuged at 12 000 g for 10 minutes and supernatant was poured out.Then, the tubes were thoroughly dried using a sterile napkin so that the tubes did not smell alcohol.Next, 20 μL of diethyl pyrocarbonate (DEPC) was added to each of the tubes and they were placed at room temperature for 10 minutes. 9Eventually, the OD and concentration of extracted RNA were measured applying a NanoDrop instrument.
cDNA Synthesis and Real-Time Quantitative PCR The real-time quantitative PCR method was implemented to evaluate the expression of the stx1 gene in E. coli isolated from UTI.Additionally, the fold changes were normalized to 16s rRNA.The cDNA was synthesized at 20 μL volume as follows: total RNA (500 ng), universal hexamer primer 0.2 Μm (1 μL), dNTP 10 mM (1 μL),  To obtain further details about the primer sequences and their characteristics refer to Table 1.

Analysis of Gene Expression Information
The threshold cycle (CT) was determined for each sample.
The expression of the stx1 gene was compared to that of the control group and the obtained data were normalized by 16s rRNA housekeeping gene.The expression ratio and production of Shiga toxin were calculated using the ratio of Michael Pfaffl's mathematical method:

Presence of stx1 Gene
The obtained results demonstrated that only 6 out of 25 E. coli isolates (24%) harbored the stx1 gene (Figure 1).

Expression of stx1 Gene
The expression of the stx1 gene in the studied E. coli isolates varied from 0.125 to 4.99.In addition, stx1 gene expression in 2 of the E. coli isolates was three times higher than the standard strain (Figure 2).

Discussion
The results indicated that 6 (24%) E. coli isolates contained the stx1 genes.Adeli et al reported that 1% of E. coli isolates from patients with UTI harbored the stx1 gene. 11Further, Mansouri et al and Mahdavi found that the prevalence of stx1 gene in 146 samples of E. coli isolated from patients with UTI and E. coli isolates from the local cheese was 0%. 12,14However, According to Nazemi et al, the prevalence of stx1 gene in E. coli bacteria isolated from patients with UTI was 10%.Similarly, base on a report by Beni et al., 11.1% of E. coli isolates from the lambs' meat contained stx1 gene. 15Except for E. coli O157, other Shiga toxin producing E. coli serotypes are the cause of 60% of the STEC infections in many parts of the world such as Argentina, Australia, Spain, Denmark, Chile, and Germany. 16The results of this study compared to those of previous studies revealed the difference in dispersion of the virulence genes of E. coli.This may be due to geographical differences as well as differences in the ecological origin of the isolated strains (e.g., milk, human, and animals).In the present study, we may have lost the stx1 gene in some E. coli samples due to repeated passages of the cultures or mutation.In a similar study, strains with three-fold increase in gene expression were considered positive while those with 2 or fewer times of increase were regarded as negative compared to the standard strain.Furthermore, strains with 2-3-fold increase in gene expression are called borderline. 17In this study, the expression range of the stx1 gene in E. coli isolates was between 0.125 and 4.99, and the average increase in gene expression was 1.79 times higher than that of the E. coli O157 (H7 PTCC43889 as the standard strain).Moreover, the expression of the stx1 gene in 2 E. coli isolates was three times more than that of the standard strain.It was revealed that Shiga toxin can be coded by phage, chromosome, or plasmid.If Shiga toxin-encoding gene was located on a phage or plasmid, it could be distributed between different strains of a bacterium or among different bacteria strains. 18Shiga toxin is found to have apoptotic effects on some of the cancer cells, and the clinical use of this toxin in some  cancers is at its early stages. 19,20If the aim is to use Shiga toxin for therapeutic purposes, it is advisable to select the strain that has the highest production of Shiga toxin.Additionally, as there may be a difference between the expression of phage, chromosomal, or plasmid genes, it is necessary to determine the genetic origin of Shiga toxin in a variety of clinical isolates.In addition, if the Shiga toxin-encoding gene is located on the phage or plasmid, it is possible to transfer it between the strains.Therefore, having knowledge regarding the genetic origin of clinical isolates is essential for providing basic information to counteract the release of toxin genes among the strains.

Conclusion
Generally, the presence of the stx1 gene in E. coli isolates from women with UTI was 24% which is considerably high.Further, all the isolates containing this gene could express it.Accordingly, the PCR method can be used as a routine test in reference laboratories due to its sensitivity and specificity.Furthermore, by employing this method, verotoxigenic strains can quickly and appropriately be detected in order to determine the type of secreted toxin by each strain and to increase the possibility of direct application on clinical samples.

Figure 2 .
Figure 2. The stx1 Gene Expression of the Isolated Escherichia coli Samples.

Table 1 .
The Sequences and Characteristics of the Primers * The primer design for 16s rRNA gene was performed using Oligo software.