Evaluation of the Prevalence of blaSHV, blaTEM, and blaCTX Genes in Escherichia coli Isolated From Urinary Tract Infections

Escherichia coli is the most important agent of urinary tract infections (UTIs) which accounts for nearly 75% of the isolates (1, 2). Annually, about 150 million women suffer from UTIs worldwide (3). Uropathogenic E. coli (UPEC) strains often produce α-hemolysin which can cause an inflammatory response by the lysis of cells and the release of cytokine (4). β-lactamases are the enzymes that disrupt beta-lactam antibiotics and secret into the periplasmic space in gram-negative bacteria or out of the cell by Gram-positive organisms (5,6). Based on the protein sequence homology (the Ambler classification scheme), beta-lactamases are divided into A, B, C, and D types. According to Tooke et al study (7), the A-, C-, and D-type β-lactamases have serine at their active site (seineβ-lactamases) whereas B-type enzymes are considered as zinc-containing metalloenzymes (metallo-β-lactamases). The extended-spectrum β-lactamases (ESBLs) were first recognized in Klebsiella pneumoniae and Serratia marcescens in the mid-1980s. These enzymes are produced by gram-negative bacilli and are often classified into temoneira (blaTEM β-lactamase), sulfhydryl variable active site (blaSHV β-lactamase), and the cefotaxime degrading enzyme (blaCTX-M β-lactamase) classes (8). The high prevalence of ESBLs in E. coli has raised concerns about the treatment of the infection caused by this bacterium (9). The ESBLs represent resistance to almost all β-lactam antibiotics except for carbapenems and cefamycins and are mainly produced by E. coli and K. pneumoniae (10). TEM-1, TEM-2, and SHV-1 are named narrow-spectrum β-lactamases because they hydrolyze penicillins and are considered as the first generation of cephalosporins such as cephalothin, cephaloridine, or cefazolin (11). The ESBLs are emanated from the narrow-spectrum Avicenna Journal of Clinical Microbiology and Infection

beta-lactamase genes (i.e., TEM-1, TEM-2, or SHV-1) by mutations that change the amino acid sequence at their active site (12). The genes encoding ESBLs are located on the bacterial chromosome or plasmid (13). The bla SHV , bla TEM , and bla CTX-M types are regarded as the most common ESBLs (14). The UPEC strains contain bla SHV and bla TEM genes which cause resistance to betalactam antibiotics and the bacterium also possesses a bla CTX-M gene that hydrolyzes cefotaxime and ceftazidime (15). Given the importance of resistance to penicillins, cephalosporins, and carbapenems, mediated by extendedspectrum β-lactamases and carbapenemases, this study was performed to investigate the prevalence of ESBL enzymes in the UPEC strains isolated from patients with UTIs.

Isolation and Detection of Escherichia coli
This study was conducted on 3192 patients who referred to the therapeutic centers of Babol and Qaemshahr, Mazandaran Province, Iran between March and December 2017. The urine samples were streaked on eosin methylene blue and blood agars (Merk Company, Germany) and placed at 37°C for 24 hours. UPEC strains were identified using the gram-stain, the IMViC test, β-hemolytic activity, and the other conventional biochemical tests. These strains were then stored in Luria-Bertani (LB) broth (Merk Company, Germany) including 20% glycerol at -20°C for future studies (34).
Combined Disc Test for the Phenotypic Detection of Extended-Spectrum β-Lactamases One hundred and ninety-two E. coli isolates were studied to detect the presence of ESBLs by the combined disc method. A 0.5 McFarland suspension of E. coli was spread on the Mueller-Hinton agar. In this study, ceftazidime (30 μg) disks (MAST, UK) alone and in the combination of ceftazidime and clavulanic acid (30/10 μg) disks (MAST, UK) were placed on the Mueller-Hinton agar at a distance of 20 mm from each other and were placed at 3 °C for 24 hours. The isolates that displayed an increase of ≥5 mm in the inhibition halo of the combined disk (ceftazidime plus clavulanic acid) were considered as an ESBL producer compared to the ceftazidim disc alone (16,17). The polymerase chain reaction (PCR) detection was carried out on all positive ESBLs. The isolates were confirmed by the PCR and the Basic Local Alignment Search Tool (BLAST) sequence analysis was used as a positive control.
Extraction of DNA by Boiling Lysis Method A single colony was used for inoculating 5 mL of LB broth and then was incubated at 37°C for 24 hours. Next, 1 mL of bacterial suspension was transferred to a 1.5 mL microcentrifuge tube and was centrifuged at 6000 rounds per minute (rpm) for 5 minutes. The supernatant fluid was discarded as well. The pellet was resuspended in 200 μL nuclease-free distilled water and boiled for 10 minutes and chilled immediately on the ice for 5 minutes. After the ice incubation, the tubes were centrifuged at 10 000 rpm for 5 minutes at 4°C and the supernatant was transferred into a new tube. An aliquot of 3 μL of the supernatant was used in the PCR mixes (18). This method of DNA purification was selected to harvest both plasmid and chromosomal DNA.

Genotypic Detection of ESBL Genes
All the ESBL-producing E. coli strains confirmed by the phenotypic assay were screened using the uniplex PCR for the detection of bla TEM , bla SHV, and bla CTX-M genes. The PCRs were carried out in a final volume of 25 μL. The oligonucleotide primers used in the investigation are listed in Table 1.
The PCRs were performed with a 25 L reaction mixture containing 3 L of the solution containing DNA, 12.5 L of super PCR master mix 2X (Yekta Tajhiz Azma), 1 L of each primer (20 pmol), and 7.5 L of distilled water. In addition, the PCR was perfor.for 5 minutes. The BLAST analysis revealed that the PCR product sequences all 3 genes show high similarity with the corresponding genes in the GenBank database thus confirming the genes. A clinical isolate containing bla TEM , bla SHV, and bla CTX genes confirmed by the PCR and sequencing techniques were used as positive control and sterile distilled water was utilized as a negative control. The PCR products were identified on 2% agarose gel electrophoresis and the

Data Analysis
The data were analyzed by SPSS software, version 16. The prevalence of resistance genes was calculated using χ 2 and fisher extract tests for each gene. Statistical significance was considered at the P < 0.05.

Results
The urine samples were collected from hospitalized patients (inpatients) and outpatients. Of the 3192 urine samples cultured on the eosin methylene blue and blood agars, 192 isolates were identified as E. coli by IMViC and biochemical tests. Among these 192 E. coli strains, the ESBL producers were detected in 45 cases (28/12%) by the combined disc test. Figures 1, 2, and 3 illustrate the PCR product bands of bla TEM , bla SHV, and bla CTX-M genes. The amplified PCR products for bla TEM , bla SHV, and bla CTX-M genes were 500, 392, and 585 base pairs (bp), respectively. All the 45 E. coli isolates confirmed by the phenotypic methods were also positive for at least one ESBL gene by the molecular technique (P < 0.001), the details of which are provided in Table 2.
The uniplex PCR of the 45 ESBL-producing E. coli strains indicated that the bla TEM was the most abundant gene (89%), followed by CTX-M (27%) and SHV (20%). The co-existence of the bla CTX-M and bla TEM , as well as bla SHV and bla TEM genes was detected in 9 (20%) and 5 (12%) isolates, respectively. The co-existence of all 3 genes (i.e., bla SHV , bla CTX-M , and bla TEM ) were observed in 3 isolates (7%).

Discussion
Cephalexin or amoxicillin/clavulanate is considered as one of the first options in the treatment of acute uncomplicated cystitis and can be prescribed in the treatment of cystitis caused by gram-positive cocci because it is effective against enterococci and staphylococci (22). The ESBLproducing bacteria, particularly E. coli and K. pneumoniae has emerged as a significant problem in the treatment of bacterial infections worldwide (23). The value of the current study was 0.001 thus the results demonstrated that there is a significant relationship between phenotypic and genotypic methods for the detection of the ESBLs. In our study, the prevalence of ESBL-producing E. coli strains was 28.12%. Further, Yılmaz et al. found that the prevalence of ESBL-producing E. coli strains was 24% (24). In another study, Jena et al reported that the prevalence of ESBL-producing E. coli strains isolated from patients with UTIs was 59.74% (25). Although mutations can cause antibiotic-resistant, the overuse of antibiotics increases the selection and emergence of resistant bacteria strains. Therefore, the prevalence of ESBL-producing UPEC varies in different regions. In the present study, 40 (89%) isolates were positive for the bla TEM gene, which is consistent with the results of several studies. For example, Liu et al reported a prevalence of    (31) reported the co-existence of different ESBL genes within the same isolate. Based on previous evidence, the coexistence of ESBL genes (i.e., bla SHV , bla CTX-M, and bla TEM ) is because they are frequently located on the plasmid and can be transferred to other bacteria (32).

Conclusions
Due to the high prevalence of the ESBL uropathogenic E. coli, especially bla TEM (89%), it is thought that ESBLs should be detected by phenotypic or genotypic methods for selecting the appropriate antibiotics regarding treating the patients with UTIs. Thus, future studies are suggested to determine the prevalence of the metallo-β-lactamases of the Verona Integron-encoded metallo-β-lactamase, imipenemases, and New Delhi metallo-β-lactamase (33,34).

Ethical Approval
There are no ethical issues for this article.

Conflict of Interests
Authors declare no conflict of interests associated with this study.