Calculation of the Zone of Inhibition and Sensitivity Pattern of Fenugreek Seeds Extract and Negila sativa Seeds Extract on ESBL E. coli Growth Plates

Background: The reason behind the poor outcomes in treating bacterial infections is mainly the limited treatment options. The strains of ESBL are generally resistant to broad-spectrum penicillin, monobactams, and third-generation cephalosporins. Literature is currently highlighting the efficacy of herbs against many resistant organisms. Hence, the study aims to identify the sensitivity pattern and zone of inhibition of Fenugreek seed extract and Nigella sativa Extract against ESBL E. Coli. Methodology: The calculated sample size was n = 40. The ESBL E. coli growth plate samples were recruited from an associated lab which were identified by performing biochemical tests on appropriate media i.e. Mueller-Hinton agar was used. Agar dilution methods were performed to examine the antibacterial effects of fenugreek seed extract (FGSE) and Nigella sativa seed extract (NGSE) against ESBL E. coli. wells of 8mm diameter were punched in the inoculated plates by using sterile cork borer, 100μl of different concentrations of Fenugreek seed extract (30, 40, 50, 60, Original Research Article Rehman et al.; JPRI, 33(60B): 1298-1303, 2021; Article no.JPRI.79986 1299 and 80mg/ml) and Nigella sativa extract (30, 40, 50, 60, and 80mg/ml) were added to wells and the plates and the plates were incubated for 24 to 48 hours at 37°C. After 48 hours presence or absence of zones of inhibition were investigated. Results: Total 40 samples were calculated for each concentration of the solution of extracts i.e. FGSE group and NGSE group. The effective concentration of FGSE was found to be 40mg/ml and for NGSE was found to be 60mg/ml that showed maximum. Both the extracts showed inhibition of growth of organism at different concentrations table 2 shows the zone of inhibition. Conclusion: The Fenugreek seed extract at 40mg/ml and Nigella sativa seed extract at 60mg/ml have highest sensitivity and are effective in inhibiting the growth of ESBL E. coli on growth plates.


INTRODUCTION
In the last two to three decades, infections caused by extended-spectrum β-lactamase (ESBL)-producing bacteria are significantly increasing over time. ESBLs can hydrolyze the oxyimino-β-lactam component of antibiotic drugs which is essential for their therapeutic effects in humans and animals, thus producing resistance [1]. Most ESBL infections spread through bodily fluids of an infected person, like blood, phlegm, or urine [2]. Contaminated surface contacts also contribute to the spread of ESBL-E. coli. The ESBL producing bacteria, especially E. coli, are frequently been reported in livestock, companion animals, and food chain [3]. The transmission of ESBLs through livestock animals to humans is now considered a potential threat. These ESBL-E. coli strains may cause various ailments among which the most common are communityacquired urinary tract infection, pneumonia, and septicemia [4]. The reason behind the poor outcomes is mainly the limited treatment options. The strains of ESBL are generally resistant to broad-spectrum penicillins, monobactams, and third-generation cephalosporins. Furthermore, a lot of ESBL-producing strains of E. coli show coresistance to the other antimicrobial agents such as tetracycline, aminoglycosides, and fluoroquinolones [5].
Drugs that are stable against hydrolysis by ESBL are required to treat such infections produced by ESBLs [6]. Due to resistance produced by many broad-spectrum antibiotics and cephalosporins, the choice of drugs for ESBLs are carbapenems, including ertapenem, meropenem, and imipenem [7]. These drugs are reserved for less-typical moderate to severe infections showing resistance to other common antibiotics [8]. The bactericidal activity of carbapenems is a result of its binding to penicillin-binding proteins that inhibits bacterial cell wall synthesis [9]. The limitations of carbapenems use include that these drugs usually produce common adverse effects caused by other antibiotics such as pruritus, rash, injection site reaction, and diarrhea [10]. Another potential weakness of carbapenems use is the high cost of these antibiotics that may lead to loss of compliance [11].
In the last few years, many strains of carbapenems-resistant bacteria have been diagnosed. In 2017, the World health organization (WHO) ranked carbapenemresistant Pseudomonas aeruginosa, carbapenem-resistant Enterobacteriaceae (CRE), and carbapenem-resistant Acinetobacter baumannii in the highest priority and critical category [12]. This urges the medical researchers to find out a novel, economical, and non-resistant alternative treatment for such infections. Trigonella foenum-graecum commonly known as Fenugreek is a traditional plant, is used in herbal medicines for a variety of ailments. Multiple studies have reported antiparasitic, anti-diabetic, and especially antimicrobial effects of fenugreek seed extract [13]. The second plant, Nigella sativa is a bioactive plant, which is also one of the most extensively used medicinal plants due to its diverse pharmacological actions [14]. Plenty of literature is available on the pharmacological and biological effects of Nigella sativa seeds extract on neurological illness, diabetes, cancer, infertility, and cardiovascular diseases. It has been also in use for many parasitic, viral, bacterial, and fungal infections [15]. Therefore, the study aims to identify the sensitivity pattern and zone of inhibition of Fenugreek seed extract and Nigella sativa Extract against ESBL E. Coli.

METHODOLOGY
It was a preclinical experimental study conducted at Baqai Medical and Dental College Karachi., from July -November 2021. The calculated sample size was n = 40. The ESBL E. coli growth plate samples were recruited from an associated lab which were identified by performing biochemical tests on appropriate media i.e. Mueller-Hinton agar was used. Fenugreek seed and Nigella sativa seeds were purchased from the local market and authentication numbers i.e. Specimen vouchers 53 and 96 were allotted. 1000-gram of both the seeds were soaked in 2500ml of 90% ethanol for 30 days after washing and grinding to powder. The filtrate was then filtered with Whitman filter paper that was further processed at 60 o C by using a water bath. The mixture was then dried at 50 o C until a wellconcentrated extract was produced. The extract was kept in an airtight bottle and stored in a refrigerator till usage. The extracts were diluted in different concentration i.e. 20, 30, 40, 50 and 60 mg/ml of DMSO. Agar dilution methods were performed to examine the antibacterial effects of fenugreek seed extract (FGSE) and Nigella sativa seed extract (NGSE) against ESBL E. coli. wells of 8mm diameter were punched in the inoculated plates by using sterile cork borer, 100μl of different concentrations of FGSE (30, 40, 50, 60, and 80mg/ml) and NGSE (30, 40, 50, 60, and 80mg/ml) were added to wells and the plates and the plates were incubated for 48 hours at 37°C. After 48 hours presence or absence of zones of inhibition were investigated. A diameter scale was used to measure and compare the zones of inhibition. ANOVA followed by post hoc Tukey was applied as a test of significance, <0.05 p-value was considered as significant at 95% confidence interval.

RESULTS
Total 40 samples were calculated for each concentration of the solution of extracts i.e. FGSE group and NGSE group. The effective concentration of FGSE was found to be 40mg/ml and for NGSE it was found to be 60mg/ml table 1 shows the mean inhibitory concentration of both the seeds. Both the extracts showed inhibition of growth of organism at different concentrations table 2 shows the zone of inhibition.

DISCUSSION
Misuse of conventional antibiotics is an issue of serious concern these days. To tackle the rise of new infectious diseases, there is a need to find new antibacterial agents having novel properties. As plants are the major contributors to traditional medicines since ancient times, around the globe. That shows that these plants provide us with a wide range of chemical compounds having multiple biological activities [16]. But, in the last few decades, extensive work has been done on the specific antibacterial role of various plant compounds [17]. That is why there is a significant improvement in the extraction of novel plant chemicals that contribute to the elimination of various diseases caused by pathogenic bacteria.
Seeds, leaves, and roots of both the plants of our study, that are Fenugreek and Nigella sativa, have proven their medical importance in various studies as potential anti-inflammatory, antibacterial and anticancer agents [18]. Infections caused by extended-spectrum-βlactamase producing E. coli is a serious issue of concern for many years, especially in immunecompromised patients [19]. The severe morbidity and increased mortality rate are directly connected to the delay in the treatment and depending upon already-resistant conventional antibiotics due to the presence of drug-resistant strains of E. coli [20]. In infections caused by ESBL-E. coli, the somehow effective antibiotics like carbapenems have a major issue of being uneconomical and are also associated with the major adverse effects of standard antibiotics [21].

CONCLUSION
The Fenugreek seed extract at 40mg/ml and Nigella sativa seed extract at 60mg/ml have highest sensitivity and are effective in inhibiting the growth of ESBL E. coli on growth plates. Furthermore, the phytoconstituents of FGSE and NGSE should be evaluated for possible antibacterial properties against resistant bacteria.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

COMPETING INTERESTS
Authors have declared that no competing interests exist.