Antibacterial activity of multidrug resistance Escherichia coli by some natural products

1. INTRODUCTION

Over the past decade, the intensive use of quinolones in human and veterinary medicines as well as in food animals has led to the rapid emergence of quinolones resistance(Haque et al., 2016). Fluoroquinolones (FQ) are effective antibacterial medications used to treat a wide range of nosocomial and community diseases. FQ resistance rates vary substantially by sex, age, type of urinary infection, and geographic region, although it has recently been revealed that E. coli isolated from community-acquired UTIs are becoming more resistant to FQ, with up to 29% of women carrying this strain (Hossain et al., 2016). Since the 1980s, when fluoroquinolones were first used in clinical treatments, this class of antibiotics has been frequently chosen to treat infections caused by Escherichia coli, Salmonella, and other Enterobacteriaceae in both humans and animals. This is due to the fluoroquinolones' oral administration benefit and bactericidal activity against Gram-negative bacteria(Kim et al., 2009). The rate of quinolone resistance differs widely from one country to another. According to the Center for Disease Dynamics, Economics and Policy the percentage of fluoroquinolone resistant E. coli among invasive isolates was 29% in United States of America, ranged from 7% to 46% in European countries, and reached>70% in some countries as India (78%) and Pakistan (72%) (Jeong et al., 2005). One of the most prevalent causes of various typical bacterial infections in both humans and animals continues to be Escherichia coli. Enteritis, uTIs, septicemia, and other clinical infections such infant meningitis are frequently brought on by E. coli(Briales et al., 2012). Chromosome mutations that change DNA gyrase, the medicines' biological target, or decrease intracellular drug concentration are two examples of fluoroquinolone resistance mechanisms in bacteria. Both chromosomal and plasmid-mediated mechanisms are involved in bacterial resistance to quinolones and fluoroquinolones (van der Zee et al., 2016).Escherichia coli are one of the microorganisms most frequently studied worldwide. they are Gram‐negative bacilli, facultative anaerobic, rod‐shaped bacterium, which are most often found in the gastrointestinal tract as a normal colonizer of warm blood organisms (mainly in mammals, but are also present in birds, reptiles and fish)(Ehwarieme et al., 2021).Escherichia coli can emerge from primary infection sites like percutaneous vascular systems asymptomatically. Bacteremia can develop into sepsis when bacteria in the blood result in a significant systemic response that leads to sepsis syndrome, severe sepsis (sepsis-induced malfunction of at least one organ or system), or septic shock. Septicemia accounted for 1.3% of all fatalities in the United States in 2000, making it the tenth most common cause of death(Aslam et al., 2020).Many societies have employed garlic (Allium sativum L.) to combat infectious diseases because it is effective against gram-positive bacteria. (Adler et al., 2002). It is advised to use garlic to cure bacteria that are resistant to many drugs. (Dini et al., 2011). The goals of this research are to evaluate the antibiotic resistance phenotype of E. coli that tested the effect of garlic and mint extract against multidrug-resistant E. coli.

 2. MATERIALS AND METHODS

  2.1. Population:

    Collection of samples

About 50 blood samples from patients (Approval No. H18RE34) who received care while hospitalised and whose data was thoroughly evaluated and subsequently studied were used for the testing. Samples were taken between October 2019 and February 2021 from Mansoura University Hospitals in Egypt. Blood was used to gather the samples. Samples were taken before the administration of antibiotics. Under sterile conditions, blood samples (5–10 ml) were drawn and inoculated into a blood culture container.

 2.2. Automated Broth Dilution AST Systems

 Vitek AST Automated System VITEK® 2 COMPACT (bioMérieux) Marcy l’Etoile, France Vitek 2 measures the turbidity changes over time (growth curve) and compare a growth control well with wells of other drug concentrations. MicroScan WalkAway, Vitek, and Vitek 2) is a broth microdilution method that utilizes a standard 96 microwell panel. These microwells contain serial dilutions of dehydrated antimicrobial agents. Results are obtained after about 18 hours by turbidimetric readings of overnight conventional panels or after about 7 hours by fluorometric readings of rapid panels. Fluorometric analysis depends on the degradation of fluorogenic substrates by viable bacteria. The phoenix system detects the susceptibility results by gravity-based inoculation process. Monitoring of the growth depends on a redox indicator system which provide results in 8 to 12 hours.

 2.3. Bacterial DNA extraction

 Rapid DNA extraction was performed using methods of (Salah et al., 2019). Where 10 samples were isolated as in Figure (2).

 2.4. Sequencing of the 16SrRNA gene of the isolates

 The thermal cycling conditions were 96 °C for 10 seconds, 50 °C for 5 seconds, and 60 °C for 4 seconds for the sequencing reactions in the 9700 thermal cycler with a volume of 20 L. (25 cycles). Afterwards, the cycle sequencing reaction was isolated from the extra dye terminators and primers using the DyeExTM 2.0 Spin Kit (Qiagen PN 63204). The resulting sequences were examined using the Finch TV (version 1.4.0) programme, and the phylogenetic tree was constructed using the closest published type strain sequences created using the Seaview programme.

 2.5.    Prepare aqueous garlic and mint extract

 Fresh garlic obtained from the local market were used to make aqueous garlic extract. Garlic weighing 100 grams have been cleaned. Then, a 60-sec immersion in 70% (volume/volume) ethanol was performed as a method to sterilize the surface (Kalyan, 2000). Garlic was homogenized using a sterile mortar and pestle after the ethanol had been vaporized in a sterile laminar flow chamber. The extract was filtered with 0.45-μm membrane, after centrifugation at 6000 rpm for 10 m. This extract contains 100% concentration of the original substance. Finally, 25, 50, 75 and 100% of the concentrated garlic extract was produced by diluting 100% of it with sterile distilled water (Durairaj et al., 2009). The mint from the local market was extracted in the same way as before.

 2.6. Evaluation of the antibacterial activity of garlic and mint extract

  Using a well-agar diffusion method two tested bacterial strains of E. coli were cultured on MacKonky Agar, then incubated at 37 °C for 8 h. The bacterial culture was spread on the surface of the nutrient agar plate, and a cork borer was used to puncture wells with a diameter of 6 mm. 100 μL of the following concentrations were added into each well: standard ciprofloxacin 5 mg/mL (a reference antibiotic) in sterile distilled water 25, 50, 75, and sterile 100% garlic and mint extract.

 2.7.MIC to garli

 Two strains of E. coli bacteria were tested for their response to various garlic doses. 100, 75, 50, 25, 12.5, 6.25 and 3.13.

 3. RESULTS AND DISCUSSION

 3.1. Identification of Escherichia coli by Vitek

 In the current investigation, 50 blood samples in total were gathered. Results of a Vitek sample test are displayed. All samples (n = 50) contained Escherichia coli. Furthermore, all samples (n = 50) had a probability of 93% to 99% for Escherichia coli identification by Vitek.

 3.4. DNA extraction

  All isolated samples identified MDR E. coli. Antibiotics are all over the place, which could explain the MDR. The causes that are most likely to contribute to the spread of antibiotic resistance include globalization, excessive antibiotic use in aquaculture and animal husbandry, the use of numerous broad-spectrum medications, and a lack of effective antimicrobial management (Vivas et al., 2019). The results showed that E. coli are resistant to a number of antibiotics, including ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, ceftazidime, ceftriaxone, cefepime, ciprofloxacin, gentamicin, moxifloxacin, nitrofurantoin, piperacillin/tazobactam, tobramycin, trimethoprim (Aabed et al., 2021). E. coli isolated from healthy children in China has extreme resistance to Ampicillin (54.0%), Arimethoprim/Sulphurmethoxazole (47.5%) and Tetracycline (58.9%) (Zhao et al., 2021). This research results showed that E. coli bacteria are resistant to a number of antibiotics, including ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, ceftazidime, ceftriaxone, cefepime, ciprofloxacin, gentamicin, moxifloxacin, nitrofurantoin, beprazobactamide. 100% of the evaluated ESBL-E. coli bacteria were ampicillin resistant. (Abalkhail et al., 2022). In this research 100% of the ESBL-E rating. coli resistant to ampicillin I agree with this. Major antibiotic-resistant E. coli strains are becoming more prevalent in almost all of Europe. (Chang et al., 2015). This research agreement with (Abalkhail et al., 2022), all E. coli isolates are MDR and that 78% of E. coli samples are ESBL E. coli. 15 antimicrobial medications, including Ceftriaxone, Cefotaxime, Ceftazidime, Cefpime, Cefuroxime, Cephalothin, Cefoxitin, Ampicillin, Amoxicillin-clavulanate, Aztreonam, Trimethoprim-sulfamethoxazole, Gentamicin, and Tigecycline, showed high resistance in ESBL-E. coli. E. coli with ESBLs present in Iran, Pakistan, and 40% (Hashemizadeh et al., 2018; Ali et al., 2016). This study proved aqueous garlic extract has antibacterial properties in different doses against E. coli MW719074 and MW719253. Garlic and ginger are natural herbs that have powerful antibacterial properties against pathogens that are resistant to several drugs and can be utilised to prevent the development of drug-resistant microbial illnesses (Karuppiah and Rajaram, et al., 2012). It was agreed that garlic affected all its concentrations, and the bacteria were affected by mint extract, and the results showed that mint did not affect all its concentrations at all. Many researches on suggested that allicin in garlic is the most vital compounds that makes antibacterial properties and restrictions the speed of RNA synthesis; the preliminary task of allicin is tricking RNA (Deresse, 2010). Allicin containing garlic extracts dominate inhibitory and bactericidal activities against the Burkholderia cepacia (Wallock-Richards et al., 2014). Ethyl acetate, Ethanol, Chloroform, Hexane, Petroleum ether and Methanol, extracts of the Garlic leaves and bulbs were possessing highest antibacterial activity against pathogenic bacteria (Prasad et al., 2018). Ultrasonicated garlic extract (garlic nanoparticles) displayed highest effective against Escherichia coli, Streptococcus mutans, Staphylococcus aureus, and Poryphyromonas gingivalis (Gabriel et al., 2022).Garlic aqueous extract was to damage bacteria and inhibit from working of the protein generating RNA synthesis (Mozaffari et al., 2014). The high antimicrobial activities of many bacterial types by garlic are related to the garlic contents such as allicin, ajoenes, and allyl sulfides (Bhatwalkar et al., 2021).

The possibility of interbreeding different species of the genus Mentha results in the fact that their taxonomy is complicated, and thus their chemical composition is heterogeneous. Therefore, the chemical composition and biological properties of individual hybrids can significantly vary from one another, depending on many factors, both environmental and genetic. The heterogeneous botanical systematics of the constantly emerging new mint cultivars creates problems with their proper identification, and thus with the appropriate determination of their chemical profile and possible use for therapeutic purposes. The active constituents of Mentha species leaves include flavonoids (eriocitrin, hesperidin, diosmin, luteolin and their glycosides), phenolic acids (derivatives of caffeic acid, e.g. rosmarinic acid), terpenoids, and volatile compounds (Fecka and Turek, 2007; Mahendran and Rahman, 2020; Nilo et al., 2017). 

4. Conclusion