Study of Antibacterial Chemical Substances and Molecular Investigation among Sulfamethoxazole-trimethoprim (SXT)-Resistant Escherichia coli Isolates

Background: Escherichia coli ( E. coli ) remains one of the leading agents of urinary tract infection (UTIs), it has become resistant to many drugs. Current work aimed to evaluate some chemical substances as antibacterial agents and molecular study of virulence factors associated with UTIs. Methods: This work involved 133 urine specimens obtained from females’ patients suffering from UTIs, Methods of well diffusion and disk diffusion were achieved to assay the effect of some chemical substances and antibiogram profiles toward Sulfamethoxazole-trimethoprim (SXT)-resistant E. coli respectively. Virulence genes were done based on the technique of Polymerase Chain Reaction (PCR). Results: The results recorded 49/133 (36.84%) E. coli among women suffering UTIs, 28/49 (57.14%) were resistant to SXT drug. imipenem, meropenem, and nitrofurantoin were recorded more effectively. Chemicals substances at the concentration 0.3 (g/ml) recorded percentages of inhibition, reaching 9.143±1.442, 15.36±0.5914, and 21.82±0.8699 for NaHCO3, Ch4c, and Viroxide Super™ respectively. PCR demonstrated that 28/28 (100%) of SXT-resistant E. coli isolates were harbored Sul-2 , FeoB and PapC genes, while 14/28 (50%), 15/28 (53.57%), 19/28 (67.85%) and 26/28 (92.85%) in U250 ( pet ), FumC , Sul-1 and IutA genes, respectively. Sul-3 gene was not observed. Conclusions: Observed a high percentage of E. coli that were resistant to SXT drug, and having several virulence genes, poses a real threat, it requires a real pause to create substitutions to limit the spreading of this threat.


Introduction
Urinary tract infections (UTIs) represent a popular reason for morbidity in females and it considers a significant cause of hospital visitation worldwide. the most prevalent bacteria are Escherichia coli (1,2). Antibiotic medical care is vital within the UTI treatment however in recent years it's changing into more difficult thanks to the increasing resistance of UTIs to habitually applied antibiotics. Drug of Sulfamethoxazole-trimethoprim (SXT) is another vital and wide used first-line antibacterial agent within the treatment of uncomplicated urinary tract infections. Recently, resistance to the drug of SXT among clinical isolates of E. coli observed increased, as well as patients have been infected with an isolate of SXT-resistant E. coli and cured by this drug may they more effect into clinical failure (3)(4)(5).
There are several microorganisms have capable to create and release virulence factors, which are mainly proteins. In bacterial pathogens, these components are encoded by particular genes on the chromosome or mobile genetic elements (6). Escherichia coli (E. coli) can acquire an admixture of mobile genetic agents through a horizontal exchange in related bacteria or in bacteria from different families, which consist of genes that encode virulence factors allowing them to become an emergent human pathogen (7). Therefore, one of the objectives of current work was to detect levels of SXTresistant in E. coli isolates which were responsible for UTIs and to estimate evaluate three chemical substances as antibacterial agents as well as antimicrobial susceptibility patterns to determine the suitable treatment, also this work involved molecular detection for some important virulence factor in this pathogen.

Patients and E. coli Diagnosis
This study included 133 non-recurring women suffering from UTIs symptoms who visited some hospitals and laboratories in the City of Najaf, during the period from August to November 2021. Mild stream urine was taken from all patients and immediately cultured on Blood agar, MacConkey agar and chromogenic agar (Pronadisa) then overnight incubated aerobically at 37 °C under sterile conditions. Bacterial growth was diagnosed firstly through morphological character, as well as some biochemical tests (IMViC), (8) and finally identified according to the Vitek-2 system.

Antibacterial agents' susceptibility
Initially, all 49 isolates of E. coli were tested to screen its SXT susceptibility, then only SXT-resistant E. coli isolates were involved in this work. Briefly, the method of disk diffusion that described through Kirby-baur method were approval to determine antibiogram profile of SXT-resistant E. coli isolates to differs categories of antimicrobial agents as shown in table 2 (9). Under sterile condition, all isolates were lawn on Mueller-Hinton agar after adjacent turbidity with McFarland tube (0.5) and incubated at 37 °C aerobic overnight. Guide of CLSI (10) was employed to characterize the isolates sensitivity, intermediate, and resistance.

Chemical substances and measuring antibacterial activity
Present study involved tested the activity of three various chemical substances toward SXT-resistant E. coli isolates, these substances Sodium bicarbonate, Ch4c (chalcone derivative compound) that obtained it from Department of Chemistry, Faculty of Science, University of Kufa, this derivative compound prepared and creative base on Adam et al., (11), and current study involved using chemical disinfectant of Viroxide Super™ (Neogen®).
Based on the method of agar well diffusion The activity of three chemical substances by three concentrations for each substance (0.1,0.2, and 0.3 g/ml) as antibacterial agents were evaluated for 28 isolates of SXTresistant E. coli. All isolates were lawn on the medium of Mueller-Hinton agar after adjacent turbidity with McFarland tube (0.5), depending on the micropipette add 50 µl from each concentration for all chemical substances under the antiseptic condition within the well has a five-milliliter diameter which done on the medium and incubated at 37 °C aerobic overnight. Using a ruler, the zone of inhibition was measured.

DNA extraction and PCR assay
An extraction kit for whole-genome DNA (Favogen, Korea) was used to extract the total DNA from 28 isolates of SXT-resistant E. coli. the extraction has been accomplished as claimed by instructions of the manufacturer's company. All DNA was stored at -20 °C using a deep-freezing device, the PCR apparatus has applied to expose all the genes enrolled in Table 1. Gel document devices (Cleaver, United Kingdom) were employed to screen and analysis of positive bands of genes after migration through agarose (1%) (iNtRoN, Korea). Table 1. Oligo-sequences of primer applied in the current work.

Statistical analysis
Based on the program of GraphPad prism V.7, the data was expressed as (Mean±standard division) as well as (P-value) and it applied for comparison among diameters of inhibition zone.

Specimens and bacterial growth
Data of present work reported among 133 urine specimens observed that 99 (74.43%) were bacterial growth involved 26 (19.54%) and 73 (54.88%) returned to gram-positive and gram-negative respectively, while 34 (25.56%) of urine specimens were no growth.

Evaluation of some chemical substance as antibacterial against SXT-resistance E. coli isolates
Results of this method recorded different inhibition zone among SXT-resistance E. coli isolates based on the type of chemical substance and base on the concentration of the chemical substances. However, all three chemical substances gave a good effect against 28 isolates of SXT-resistance E. coli at a concentration of 0.03 (g/ml) involved 9.143± 1.442, 15.36±0.5914 and 21.82±0.8699 for NaHCO3, Ch4c and Viroxide Super™ (Neogen®) respectively. All isolates were resistant to Sodium bicarbonate substance at 0.01 g/ml and do not have any inhibition zone. Disinfectant of Viroxide Super™ was more effect on the growth of bacteria at all three concentration (0.01, 0.02 and 0.03 g/ml) while Ch4c gave good effect in both 0.02 g/ml and 0.03 g/ml concentrations (Table 3).

Discussion
Data of current work was close to another local study done by Abbas and Al-Mathkhury (19), they recorded that rate of E. coli isolate was a prevalent reach 42.5% among other genus of gram negative and gram-positive isolates which recovered from women suffering UTIs. At same respect, several previous studied were observed that E. coli was the most pathogen caused UTIs among patient (20,21). The data of current work was consistent with findings of several previous studies, they observed the rate of E. coli isolate was highest among bacterial agents which caused UTIs (22,23). This may be for several reasons especially it's formed a part of microbiota in the urinary tract as well as have many virulence factors that permit it to cause a huge range of infection.
Patients of UTIs require to be diagnosed early and treated with antimicrobial agents' therapy as soon as possible to reduce the damage of the kidney. In the past few decades, resistance to antimicrobial has increased considerably (24,25). Present work recorded a rising rate of SXT-resistant among E. coli isolates, in fact, drug of SXT has been widely used as an antimicrobial agent in UTIs treatment, which may demonstrate many reported' recorded resistance elevation among isolates of E. coli to this agent that led to complicated treatment options (26). Results in Table 2 were revealed different spectrum of resistance for antimicrobial agents, however There were a high rate of antibiotic resistance among SXT-resistant E. coli isolates for piperacillin, amoxicillinclavulanic acid, cefixime, cefotaxime, aztreonam and tetracycline. Locally, there are confronting diverse challenges that be able to promote the development or expansion of drugs resistance this may be repeated or misemployment of drugs in humans, animals, as well as administration of an antibacterial agent without recipe have contributed to increased resistance to these drugs. While the drugs of piperacillin/tazobactam, imipenem, meropenem and nitrofurantoin were more effect against isolates of SXT-resistant E. coli and the findings in this study were in accord with those reports in other parts of the worldwide, indicating the agents have good effective against E. coli isolates (27).
High concentration of all three chemical substances recorded a good effect on SXTresistant E. coli isolates, However, previous works were done by Gutiérrez-Huante et al., (28) and Dobay et al., (29) indicated that NaHCO3 reduces the time generation of bacteria growth due to its effect on the pH and this harmony with the results of current work. In the same respect, present data agree with the results of Adam et al., (11) they recorded a positive effect of the Ch4c derivative compound against bacterial growth. PCR data demonstrated the existence of sul-1 and Sul-2 genes correlated positively with SXT resistance. However, previous work done by Abbassi,et al.,(30) observed that 46.2%, 23.8, and 8.9% of E. coli isolates were harbored Sul-1, Sul-2, and Sul-3 respectively. Another study was done by Tabidehchi and Amini, (31) recorded that 76.6% and 61.6% of E. coli isolates were harbored Sul-1 and Sul-2, respectively.
Genes associated with main virulence factors such as adhesins, toxins, as well as systems of iron acquisition among isolates of E. coli usually have been associated with increased pathogenicity of this pathogen and caused human infection especially UTIs (32,33). Genes of U250 (pet toxin) and fum C were detected in the present study and this was conducted with previous reports that indicated that these genes are important virulence factors associated with UTIs (34,35). At same respect, current work observed that PapC gene was exist in all isolates of SXT-resistant E. coli, however, this result could be related to pathogeneses and confirms its crucial role during colonization of the urinary tract. This finding was conducting with previous works achieved by López-Banda et al., (36)  Possession of iron has a necessity for E. coli existence in an environment that is as iron bounded as the urinary tract (38). Metabolism of Iron in several microorganisms are dependent on a ferrous iron transporter (FeoB) (39). Current data was agreement with a result of a recent work done by Kocúreková et al., (14) recorded that genes of ironuptake FeoB and iutA were 94% and 58% respectively, among E. coli isolates. While Rocha et al. observed that iutA gene was (45.9%) among E. coli isolates (40).