Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(1): 83-99, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2356 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Alghamdi, M.A., Al-Sarraj, F., Alamshani, W.H., Alotibi, I., Al- Zahrani, M., Albiheyri, R., Nass, N.M., Sajer, B.H., Bataweel, N.M., Al-Matary, M.A., Bouback, T., Attallah, D., & Alqahtani, T.M. (2024). Antibacterial power of Pomegranate extracts against Beta- Lactamase producing Escherichia coli. Caryologia 77(1): 83-99. doi: 10.36253/ caryologia-2356 Received: November 6, 2023 Accepted: April 30, 2024 Published: July 8, 2024 Copyright: © 2024 Alghamdi, M.A., Al- Sarraj, F., Alamshani, W.H., Alotibi, I., Al-Zahrani, M., Albiheyri, R., Nass, N.M., Sajer, B.H., Bataweel, N.M., Al- Matary, M.A., Bouback, T., Attallah, D., & Alqahtani, T.M. This is an open access, peer-reviewed article pub- lished by Firenze University Press (ht tps://www.fupress.com/caryolo- gia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Antibacterial power of Pomegranate extracts against Beta-Lactamase producing Escherichia coli Mashail A. Alghamdi1, Faisal Al-Sarraj1,*, Wafa H. Alamshani1, Ibra- him Alotibi2, Majid Al-Zahrani5, Raed Albiheyri1,4, Nada M. Nass1, Bayan H. Sajer1, Noor M. Bataweel1,6, Mohammed A. Al-Matary1,7, Thamer Bouback1,3, Dalya Attallah8, Tahani M. Alqahtani1 1 Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jed- dah, Saudi Arabia 2 Health Information Technology Department, Applied College, King Abdulaziz Univer- sity, Jeddah, Saudi Arabia 3 Princess Dr. Najla Bint Saud Al-Saud Center for Excellence Research in Biotechnology, King Abdulaziz University, Jeddah, 21589, Saudi Arabia 4 Centre of Excellence in Bio Nanoscience Research, King Abdulaziz University, Jeddah, Saudi Arabia 5 Biological Science Department, College of Science and Art, King Abdulaziz University, Rabigh, Saudi Arabia 6 King Fahd Medical Research Centre, King Abdulaziz University, Jeddah 21589, Saudi Arabia 7 Department of Animal Production, Faculty of Agriculture, Sana’a University, Yemen 8 Clinical and Molecular Microbiology Laboratory, Faculty of Medicine, King Abdulaziz University Hospital, Jeddah, Saudi Arabia *Corresponding author. Email: falsaraj@kau.edu.sa Abstract. Herbs, as the pomegranate (Punica granatum L.)(P. granatum), has significant chemical constituents with distinct pharmacological properties. These chemicals confer neuroprotective, antioxidant, anticancer, anti-inflammatory, and antibacterial proper- ties to the plant. Pomegranate has specific components that enable its pharmacologi- cal actions; one of the functions of pomegranate extracts is to deactivate what is called extended spectrum beta-lactamase (ESBL) that makes Escherichia coli (E. coli) resistant to standard antibiotics. Twenty E. coli strains identified as beta-lactamase producers, the strains isolated from urine samples collected from patients with symptomatic urinary tract infection (UTI) and molecularly characterised using 16S rDNA. The study evalu- ated the antibiotic sensitivity and antibacterial activities of acetone and ethanolic pome- granate leaf and peel extracts, assessing their antimicrobial susceptibility against nineteen antibiotics. The ehanolic peel (EP) and leave (EL) extracts showed inhibitory potential inhibition zones spanning (9.0-12.6-18.3 mm) against E. coli pathogen producing extend- ed-spectrum beta-lactamase as compared with (10.2-15.3 mm) inhibition scale exhibited by acetone peel (AP) and leave (AL) extracts treatment. Pomegranate leaves and peel extracts contain bioactive compounds with antioxidant, antimicrobial, and other biologi- cal effects, and can be fractionated for the identification of new antibacterial bioactive compounds for the development of drugs against ESBL- E. coli, in addition to their syn- ergy with antibiotics for combination therapy that may have effective management and treatment of multidrug-resistant infections such as urinary tract infection. Keywords: Punica granatum, E. coli, beta-lactamase enzyme, antibacterial activities, antimicrobial assay, DNA sequencing. http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2356 https://doi.org/10.36253/caryologia-2356 https://doi.org/10.36253/caryologia-2356 https://www.fupress.com/caryologia https://www.fupress.com/caryologia mailto:falsaraj@kau.edu.sa 84 Mashail A. Alghamdi et al. 1. INTRODUCTION In addition to use plants as food, shelter, and build- ing materials, plants were traditionally employed as medicinal herbs. Herbs were defined as those with medicinal properties (Kunle et al., 2012). The World Health Organization (WHO) defines herbal medicines as plants whose parts are used to treat and prevent ill- nesses in humans and animals (Msomi and Simelane 2019). They include active agents; either alone or in combination can be used in the treatment and manage- ment of chronic conditions, such as cancer and cardio- vascular difficulties. Researchers have resorted to herbal drugs as alternatives to standard therapy methods as no bad side effects (Sutan et al., 2023). These unique natu- ral constituents, herbal medication significantly contrib- uted to disease management (Sutan et al., 2023; Kam and Neergheen 2022; Facciola and Stephen 1990). The usage of herbal medicines has grown in popularity and awareness around the world because to their inexpensive cost, little to non-existent side effects, and lack of bacte- rial resistance. In addition, these herbs are easily acces- sible as they are readily available. The therapeutic prop- erties of P. granatum shrub are attributed to the extract from its numerous sections, which has been shown to be beneficial in treating and preventing the pathogenic E. coli strain (Stover and Mercure 2007). Pomegranate is a deciduous plant that grows between five and eight metres in height. It is a member of the Lythraceae fam- ily. Pomegranate was traditionally regarded as a sym- bol of life, fertility, health, wisdom, immortality, and longevity (Loizzo et al., 2019). Pomegranate is planted primarily in South Asia and the Middle East. Today, it is widely cultivated in dry and semiarid places due to its adaptability to harsh climates (Stover and Mercure 2007). Each anatomical component of the plant has a unique pharmacological and toxicological function. This includes the peel, seed, flower, juice, leaf, bark, and root. It is an edible fruit with a thick, reddish skin, a rounded hexagonal shape, and a 5 to 12 cm diameter. The fruit contains approximately 600 seeds, each wrapped in a water-filled aril (pulp) of varying hues ranging from white to deep purple to deep crimson. The fruit’s edible portion, the seeds, can be utilised in baking, cooking, and beverage preparation (Loizzo et al., 2019). However, the Pomegranate has been widely utilised by numerous nations and civilizations for the treatment of numerous ailments (Kim and Choi 2009). Previous research indi- cates that the chemical constituents contained in Pome- granate seeds, bark, flowers, pericarp, and roots confer the plant with a variety of therapeutic effects. In addi- tion, it has antioxidant, anti-atherosclerotic, anti-inflam- matory, neuroprotective, anticancer, antimicrobial, anti- hypertensive, anti-teratogenic, stimulant, antidiabetic, antiviral, antifungal, anthelmintic, nephron-protective, wound-healing, and antiulcer properties. In addition, its chemical ingredients help combat respiratory disorders and erectile dysfunction (Kim and Choi 2009). While Pomegranate seeds contain estro- genic chemicals, estradiol and oestrone, and the fruit has been widely used to treat microbial infections, aci- dosis, diarrhoea, haemorrhage, helminthic infection, res- piratory ailments, and dysentery, they do not have estro- genic properties themselves (Ricci et al., 2006). Juice and dried pericarp of the fruit have been used to treat colic, headache, colitis menorrhagia, piles, oxyuriasis, diuret- ics, allergic dermatitis, acne, and oral illnesses (Berthe et al., 2013). Escherichia coli (E. coli) is a bacteria found in the intestines of animals (Denamur et al., 2021). The majority of E. coli strains are digestively helpful and harmless, some are dangerous and pathogenic (Khalid and Andreoli 2019). Pathogenic strains of E. coli bacte- ria are spread mostly by contaminated human-to-human contact, contaminated food or water consumption, and contaminated surfaces (Denamur et al., 2021; Al-Sarraj et al., 2021a; Alotibi et al., 2022). Infections of the uri- nary tract, gastrointestinal tract, and respiratory sys- tem are caused mainly by pathogenic E. coli strains. E. coli infections are characterised by moderate diarrhoea, abdominal discomfort, bloody diarrhoea, and renal fail- ure (Qi et al., 2022). In order to treat or prevent E. coli infections, one must adhere to food safety and sanitary standards and practise proper hygiene. Beta-lactams are the most often used antibiotics for the treatment of urinary tract infections (Flores-Mire- les et al., 2015); nevertheless, the rise of multiple-drug resistant (MDR) bacteria, particularly beta-lactamase (ESBL) generating pathogens, is a huge concern to the global healthcare system (Shaikh et al., 2015; Hashem et al., 2017; Heidari et al., 2017; Houri et al., 2017; Mot- amedifar et al., 2015). The prevalence of E. coli bacte- ria that produce beta-lactamase enzymes has increased recently on a global scale (Pourakbari et al., 2012; Kazemian et al., 2016). It inhibits the effectiveness of certain beta-lactam antibiotics, such as penicillin and cephalosporins, by degrading them. By generating beta- lactamase enzymes, bacteria acquire antibiotic resist- ance. Enzymes degrade the structure of beta-lactam antibiotics, rendering their antibacterial effects inactive (Qi et al., 2022; Amanulla and Sundaram 2019). There- fore, treating bacterial infections caused by E. coli gener- ating beta-lactamase becomes problematic. The prolifera- tion of antibiotic-resistant bacteria has become a global health concern, compelling researchers to investigate 85Antibacterial power of Pomegranate extracts against Beta-Lactamase producing Escherichia coli alternate preventative and therapeutic measures (Huang et al., 2019; Chauhan et al., 2020). The strategies include the creation of novel antibiotics, the use of natural chemicals such as plant extracts, and the improvement of sanitation practises to avoid the spread of infectious diseases (Amanulla and Sundaram 2019; Hashim and Pharma 2013). Leaves of P. granatum contain chemical substances with the ability to suppress the actions of E. coli strains producing beta-lactamase. With the cor- rect concentration of the extract, pathogenic E. coli will eventually be incapable of degrading the structure of the antibiotics. Consequently, the molecules of the antibi- otic continue to combat bacterial infections. This study comprehensively evaluates the antibacterial impacts of P. granatum leaves and peel extracts on E. coli extended spectrum β-Lactamase isolate and the antibiotic suscep- tibility patterns of E. coli isolate. Additionally, it pro- vides a comparison between the concentrations of P. gra- natum extractions and seeks to find the most effective concentration and its synergy with antibiotics that may have potential for future treatment of multi-drug resist- ant infections. 2. MATERIALS AND METHOD All of the chemicals and antibiotics employed were of analytical quality, purchased from Sigma-Aldrich, and were utilised without further purification. 2.1. Herbal material and microorganisms The pomegranate plant used in this investiga- tion was collected in Taif, Saudi Arabia. The Hema- tology department of the Faculty of Medicine at King Abdulaziz University in Jeddah, Saudi Arabia, reported urine samples from twenty E. coli isolates isolated from urine samples of twenty-one patients with urinary tract infections (UTIs) and one healthy control individual in June 2021. The bacteria were cultured in Luria-Bertani (LB) medium with 200 rpm shaking at 37 °C for twenty- four hours before being stored at 4 °C. Next, we culti- vated bacterial isolates on LB agar overnight at 37 °C for 24 hours. After incubation, the cultures were inspected to determine if considerable growth had occurred. All collected strains were sequenced for the 16S rDNA gene to validate the culture-based identification of bacteria. The optical density of bacterial cultures was set to 600 nm (OD600) with sterile 0.1 M saline solution for evalu- ating Disk Diffusion, Antibiotic Susceptibility Assay, and the antibacterial activity of pomegranate leaves and peel extracts. 2.2. Antibiotics Amikacin AK 30mcg, Ampicillin AMP 10mcg, Cefrtriaxone CTR 30 mcg, Amoxyclav AMC (Amoxycillin+Clavulinic Acid) (Augmentin) 30mcg, Ceftazidime CAZ 30 mcg, Cefoperazone CPZ 75 mcg, Netilmicin NET 30 mcg, Cefuroxime CX. 2.3. Antibiotics sensitivity assay for bacteria Twenty bacterial isolates were tested against eight distinct drugs to determine their susceptibility. The bacteria were cultured overnight in nutritional broth for activation. They were then determined by measur- ing optical density (OD) at 600 nm with a UV-visible spectrophotometer and subsequently standardised to 0.1 McFarland standards (3107 CFU/mL). Later, the bacterial suspension was dispersed on nutrient agar plates. The agar was then covered with antibiotic discs and incubat- ed at 37 °C for 18-20 hrs. Later, the sizes of the zones of inhibition were measured and reported. 2.4. Preparing pomegranate extracts The peel and leaves of the pomegranate were gath- ered by hand, separated, chopped into little pieces, then rinsed with tap water and then distilled water to elimi- nate any suspended soil and other particles. The samples were then air-dried in the shade at room temperature to preserve their freshness. An electric mill was used to grind the dried samples into a fine pomegranate powder. The powder was kept in airtight containers at 4 oC until usage. 50 g of the powder was soaked in 500 ml of etha- nol and 500 ml of acetone for 5 minutes, and then the mixture was swirled continuously at 200 rpm for 72 hrs to dissolve. After that, filtration was performed in three stages. Initially, the fluid was filtered through numer- ous layers of gauze to remove the suspended plant fibres and particles. Afterward, it was filtered with Whitman No. 1 paper. Using a rotary evaporator, the filtrate was evaporated. The storage solution was made by dissolving 500 mg/ml of the extracted powder in 40 ml of 2 percent diluted Dimethyl Sulfoxide (DMSO). 2.5. Antibacterial activity of pomegranate extracts Using the agar well diffusion method, the antibacte- rial activity of pomegranate leaf extract was determined. 20-25 ml of autoclaved Muller–Hinton agar media was poured into a sterile petri dish, which was then allowed to harden. Spread the bacterial suspensions on the plate 86 Mashail A. Alghamdi et al. using a sterile cotton swab. Each plate includes five holes that were drilled with a 6mm cork borer and filled with 100µl of extracts of varying concentrations. As a nega- tive control, 30 percent diluted DMSO without pome- granate leaf extract was used. The plate was left for 10 minutes to allow the chemicals to pre-diffuse into the agar. The plates were then incubated at 37 °C for 24 hours. The diameter of the inhibitory zone was then measured in millimetres. 2.6. Beta-Lactamase production assay In order to determine the beta-lactamase synthe- sis of all examined E. coli isolates, broth samples were point-injected onto Mueller-Hinton agar (MHA) con- taining 1 percent starch and incubated at 37 degrees Cel- sius. After 24 hours of incubation, the plates were refilled with PBS containing potassium iodide, iodine, and peni- cillin. The creation of distinct colorless zones around the E. coli growth indicates the production of -lactamase enzymes, which convert Penicillin to penicilloic acid and iodine to iodide, as shown by the decolorization of the starch iodine complexes. 2.7. Molecular characterization of bacteria DNA extraction and amplification of 16S rRNA genes Total genomic DNA was extracted from 20 urine samples collected from patients with symptomatic uri- nary tract infection using a slightly modified version of the Azcárat-Peril and Raya (2001) protocol, in which 1 ml of pure bacteria culture grown overnight in NB was transferred to a 1.5 ml tube, and the tube was centri- fuged at 10000 rpm, 4°C for 5 minutes. After discard- ing the supernatant, 200 µl of TES buffer is added and vortexed thoroughly. Then, 20µl of lysozyme (10 mg/ ml) was added and thoroughly mixed using a vortex. Two hours were spent incubating the mixture at 37°C in a water bath. Each sample received 20µl of protein- ase K (10 mg/ml), which was then vortexed and incu- bated at 37°C for two hours. The liquid was chilled for five minutes, then 250 µl of sodium acetate was added. After 5 minutes of centrifugation at 8000 rpm and 4°C, the top zone was carefully transferred to a new, clean Eppendorf tube. 250 µl of chloroform: isoa- myl (24:1) was added to the mixture, which was then mixed by hand and centrifuged at 8000 rpm, 4°C for five minutes. The aqueous phase was then transferred to a fresh Eppendorf. Equal volume of isopropanol was added to the mixture, which was then stored overnight at -20 degrees Celsius. The following day, the solu- tion was centrifuged at 10000 rpm for 5 minutes, after which the liquid zone was discarded and the pellet was allowed to dry at ambient temperature for 10 minutes before being suspended in 50 µl of distilled water. DNA from each bacterial isolate was served as template for amplification of the 16S rRNA genes using the follow- ing universal primers: 27F (5’- AGAGTTTGATCCTG- GCTCAG-3’) and 1492R (5’-TACGGYTACCTTGTTAC- GACTT-3’). For the PCR, a total volume of 50µl was utilized, along with 2µlof 10 pmol of each appropriate primer and 25µl of Master Mix (GoTaq® Green Master Mix, 2X, Promega). Add 2l of DNA and use DEPEC-treated water to adjust the volume of the final PCR mixture to 50 µl. The 16S rRNA gene was amplified using a thermal cycler (applied biosystems TM Veriti TM 96-Well Ther- mal Cycler) under the following conditions: initial dena- turation at 95 °C for 4 minutes, followed by 30 cycles of denaturation at 95 °C for 30 seconds, annealing for 45 seconds, and extension at 72 °C for 60 seconds, and a final extension at 72 °C for 10 minutes. 2.8. Sequence analysis and phylogeny of E. coli strains The consensus 16S rDNA amplicon sequences of E. coli isolates were modified and subjected to a BLAST search using the NCBI database http://blast.ncbi.nlm. nih.gov/Blast.cgi to assign presumptive identity with similar sequences. On the basis of sequence similar- ity measurements and inferences of phylogenetic trees, each bacterial isolate was assigned to its operational taxonomic unit (OTU). Where necessary, alignments were edited by hand. The pathogenic E. coli nucleotide sequences were submitted to GenBank for assignment of accession numbers. The acquired sequences were sepa- rated into distinct datasets in order to examine the evo- lutionary relationships between species and families. The NJ technique was employed to create phylogenetic trees, whereas MEGA software, version 10.0 (Tamura et al., 2011) was utilised to undertake molecular evolutionary studies. 2.9. Statistical analysis This study’s statistical analysis was conducted using the SPSS software (Version 26.0). All analyses were conducted in triplicate using a one-way ANOVA analysis of variance with a significance level of p 0.05, and the results were represented as the mean standard deviation. http://blast.ncbi.nlm.nih.gov/Blast.cgi http://blast.ncbi.nlm.nih.gov/Blast.cgi 87Antibacterial power of Pomegranate extracts against Beta-Lactamase producing Escherichia coli 3. RESULTS 3.1. Molecular identification of the E. coli strains All 20 bacterial isolates were identified molecularly by amplification of the 16S rRNA gene using universal primers. According to the PCR results, the 16S rRNA gene sequences of the selected isolates were ampli- fied successfully from extracted template DNA. Using gel electrophoresis, the PCR products of the 16S rRNA genes of these isolates yielded bands with around 1500 bp (Figure 1). DNA sequencing was used to identify the 20 exam- ined pathogenic E. coli strains at the molecular level by analysing the 16S rRNA gene. The GenBank was accessed to obtain the accession numbers for all of the bacterium isolates listed in Table 1, and MEGA software was used to optimise the sequences that were obtained. Using the NCBI public database, nucleotide similari- ties between 16S rRNA gene sequences and those of GenBank-recognized species were analysed. The results indicated that the isolated microorganisms were E. coli. The sequences of the acquired E. coli strains were 99.2– 100% comparable to those of their closely related iso- lates previously deposited in GenBank. The homologous sequences from the BLAST search were utilised to con- struct a phylogenetic tree, demonstrating their relation- ship to E. coli strains, as demonstrated in the Figure 2. This Original Tree’s evolutionary history was inferred using the UPGMA method (Sneath and Sokal 1973), and the 500-replicate bootstrap consensus tree (Felsenstein 1985) is considered to represent the evo- lutionary history of the species examined (Felsenstein 1985). The collapsed branches correspond to partitions that are replicated in less than half of bootstrap repli- cates. Next to each branch is the percentage of 500 rep- licate trees in which the relevant taxa clustered togeth- er in the bootstrap test (Felsenstein 1985). Using the Maximum Composite Likelihood approach (Tamura et al., 2004) and the amount of base substitutions per site, the evolutionary distances were estimated. This analy- sis used 40 nucleotide sequences, and for each sequence pair, all ambiguous locations were eliminated (pairwise deletion option). The final dataset contained 1030 loca- tions in total. In MEGA X, evolutionary analyses were undertaken (Kumar et al. 2018). 3.2. Antibiotic sensitivity assay of ESBL E. coli strains In the present investigation, the beta-lactamase enzyme assay results indicated that all twenty studied E. coli strains produced beta-lactamase, and the posi- tive result was observed as an inhibitory zone surround- ing the bacterial colonies. The disc diffusion suscepti- bility test was carried out to determine the sensitivity or resistance of pathogenic microorganisms to various antimicrobial agents. On Mueller-Hinton agar, the path- ogenic bacteria were cultivated in the presence of anti- microbial-impregnated filter paper discs. The presence or absence of bacterial growth around the discs is regarded an indirect sign of the compound’s ability to inhibit the organism. Table 2 illustrates the antibiotic resistance characteristics of the pathogenic bacterial strains (Fig- ure 3A, 3B). In order to evaluate the antibiotic resistance patterns of the examined bacterial strains, the Antibiot- 1kb → 2kb→ M 1 2 3 4 5 6 78 9 10 11 1415 16 18 19 20 21 22E ←1.5kb Figure 1. Agarose gel electrophoresis of PCR products for 16s rDNA gene. Lane M: 2kb DNA size marker, Lanes 1-E: 16S gene amplicon of 20 E. coli isolates from clinical samples. Table 1. GenBank accession numbers and β-cell lactamase assay of different E. coli strains under study. Ser. Bacterial strains β-cell lactamase Accession No. 1 E. coli strain WFGM S1 Positive OR472881 2 E. coli strain WFGM S2 Positive OR472882 3 E. coli strain WFGM S3 Positive OR472883 4 E. coli strain WFGM S4 Positive OR472884 5 E. coli strain WFGM S5 Positive OR472885 6 E. coli strain WFGM S6 Positive OR472886 7 E. coli strain WFGM S7 Positive OR472887 8 E. coli strain WFGM S8 Positive OR472888 9 E. coli strain WFGM S9 Positive OR472889 10 E. coli strain WFGM S10 Positive OR472890 11 E. coli strain WFGM S11 Positive OR472891 12 E. coli strain WFGM S14 Positive OR472892 13 E. coli strain WFGM S15 Positive OR472893 14 E. coli strain WFGM S16 Positive OR472894 15 E. coli strain WFGM S18 Positive OR472895 16 E. coli strain WFGM S19 Positive OR472896 1 E. coli strain WFGM S20 Positive OR472897 18 E. coli strain WFGM S21 Positive OR472898 19 E. coli strain WFGM S22 Positive OR472899 Control E. coli strain WFGM SE Positive OR472900 88 Mashail A. Alghamdi et al. ic-disk assay was developed utilising various antibiotics. The majority of the bacteria exhibited both antibiotic resistance and sensitivity, according to the results. Also, antibiotics susceptibility testing data revealed that the highest resistant pattern ranged between (44-61 percent) was found in 8 isolates (S11, S18, S3, S22, S7, S15, S16 and S20) from 19 (44 percent) examined bacteria, which were resistant to (9-11) antibiotics, followed by resistant pat- tern ranged between (39-44 percent) was observed in 7 isolates (S4, S5, S8, S14, S6, S14, S21, S6, S The resistance patterns of E. coli strains S9 and SE (control) are mod- est (5 percent and 6 percent, respectively). All examined E. coli isolates exhibited resistance to at least one of the antimicrobials examined. In particular, 95% of E. coli was resistant to CAZ, whereas 90% of same bacteria were resistant to AMP and CN. And 85% showed resist- ance to CXM and CFM. Eighty percent of the strains were resistant to AMC, and with a moderate suscep- tibility of 55%, 50% were resistant to CTX and COT, respectively. Additionally, 30% of the studied E. coli iso- lates were resistant to CIP, whereas 15% were resistant to CPZ. Only 10% of the examined E. coli bacteria were resistant to CTR and K, while 5% were resistant to GEN. None of the tested isolates were resistant to AK, NET, OF, or LE. 3.3. Antibacterial activity of pomegranate leaves and peels extracts The antibacterial activity of pomegranate leaves and peels was evaluated against 20 E. coli isolates using the well diffusion technique. The results revealed sig- nificant heterogeneity in the zone of inhibition’s size. To extract the active compounds from the powdered peel and leaves of pomegranate used in this investigation, two organic solvents (80% ethanol and 80% acetone) were employed. The antibacterial activity of the exam- ined extracts in 2 percent DMSO was dose-dependent, ranging from 100 mg/ml to 500 mg/ml, and the inhibi- tory zone scale expanded as the concentration of the test extract increased. The data in (Table 3) and (Figure 4) indicate that the ethanolic extracts 100-500mg/ml of pomegranate peels (EP) extract. Most the investigated strains showed resistance with absence of inhibition zone at 100mg/ml, compared with a strong antibacterial activity against the tested clinical E. coli strains, and showed maxi- mum inhibition scale (9.0- 18.3 mm) as the EP extract concentration increased from 200 mg/ml to 500 mg/ ml, followed by the inhibitory zone range (8.3-15.9 mm) at a dosage of 400 mg/ml EP extract, and inhibitory zone range (8.1-15.8 mm) at a dosage of 300 mg/ml EP extract. The more susceptible clinical E. coli strains to the (9.0- 18.3 mm) as the concentration increased from 200 mg/ml to 500 mg/ml EP extract were strains S16, S20, S3, S4, S6, S11, and S12 strains. By regarding the antibacterial activity of acetone extraction of pomegranate peels (AP) at a dosage of 100- 500 mg/ml as data represented in (Table 4) and (Figure 5). Most the investigated strains showed resistance with absence of inhibition zone at 100mg/ml and 200 mg/ml AP extract, and by increasing AP extract concentration from 200 mg/ml to 500 mg/ml demonstrated a potent antibacterial activity with a (9.07-15.3 mm) inhibitory zone ranges between tested clinical E. coli strains, fol- lowed by the inhibitory zone range (9.04-14.4 mm) at a dosage of 400 mg/ml AP extract, and inhibitory zone range (9.07-14.0 mm) at a dosage of 300 mg/ml AP extract. The more susceptible clinical E. coli strains to the (9.0-15.3 mm) as the concentration increased from 200 mg/ml to 500 mg/ml EP extract were strains S9, S20, S7, S8, and S10 strains. E. coli strain WFGM S14 (OR472892) E. coli strain WFGM S4 (OR472884) E. coli strain WFGM S2 (OR472882) E. coli strain WFGM S5 (OR472885) E. coli strain WFGM S7 (OR472887) E. coli strain WFGM S8 (OR472888) E. coli strain WFGM S11 (OR472891) E. coli strain WFGM S15 (OR472893) E. coli strain WFGM S19 (OR472896) E. coli strain WFGM S20 (OR472897) E. coli strain WFGM S21 (OR472898) E. coli strain WFGM S22 (OR472899) E. coli strain WFGM SE (OR472900) E. coli strain WFGM S18 (OR472895) E. coli strain WFGM S16 (OR472894) E. coli strain WFGM S6 (OR472886) E. coli strain WFGM S3 (OR472883) E. coli strain WFGM S1 (OR472881) E. coli strain WFGM S10 (OR472890) E. coli strain WFGM S9 (OR472889) Figure 2. Phylogeny analysis of twenty E. coli strains from 16S region sequences compared with different related bacterial isolates obtained from GenBank. 89Antibacterial power of Pomegranate extracts against Beta-Lactamase producing Escherichia coli The antibacterial activity of ethanolic extraction of pomegranate leaves (EL) at a dosage of 100-500 mg/ml, and the bioactivity data against clinical E. coli strains represented in (Table 5) and (Figure 6). Most the inves- tigated strains showed resistance, and no antibacterial activity was identified at 100mg/ml and 200 mg/ml EL extract, and by increasing EL extract concentration from 200 mg/ml to 500 mg/ml, the antibacterial activity sig- nificantly increased (8.5-12.6 mm) inhibitory zone rang- es between tested clinical E. coli strains, followed by the inhibitory zone range (8.1-10.0 mm) at a dosage of 400 mg/ml EL extract, and inhibitory zone range (7.6-9.2mm) at a dosage of 300 mg/ml EL extract. The more suscep- tible clinical E. coli strains to the (8.0- 12.6 mm) as the concentration increased from 200 mg/ml to 500 mg/ml EL extract were strains S5, S2, S4, S7, S8, and S9 strains. The antibacterial activity of 100-500 mg/ml acetone extraction of pomegranate leaves (AL) against clinical E. coli strains as demonstrated in (Table 6) and (Figure 7). The smallest concentrations (100mg/ml, 200mg/ml, 300 mg/ml AL extract showed high antibacterial resistance or a little antibacterial activity was identified, increasing AL extract concentration from 300 mg/ml to 500 mg/ml exhibited inhibitory zones spanning (7.8-10.2 mm) rang- es between tested clinical E. coli strains, followed with inhibitory scale (8.0-9.5 mm) at a dosage of 400 mg/ml AL extract, and smallest inhibitory scale (7.8-8.1 mm) at a dosage of 300 mg/ml AL extract. The more susceptible clinical E. coli strains to the (8.7.8- 110.2mm) as the con- centration increased from 200 mg/ml to 500 mg/ml AL extract were strains S6, S7, S8, S2, S6, and S17 strains. 4. DISCUSSION Due to its numerous health benefits, P. granatum is not only a tasty fruit but also a therapeutic herb. Its leaves are abundant in phytochemicals with antibacterial characteristics (Stover and Mercure 2007; Fernández- Mazarrasa et al. 2009; Al-Sarraj 2021b). In this work, the researcher examined the antibacterial activity of pomegranate leaf extract against E. coli strains resistant to antibiotics. According to the data the inhibitory zone in different investigated bacterial strains were ranged Table 2. Resistance (%) or susceptibility of the ESBL E. coli strains used in the present study to different antibiotics Strains Resistance or inhibitory zone (mm) by antibiotics Antibiotic resistance (%)AMP AK AMC CTX CIP CXM CPZ CAZ GEN NET OF NX CFM CTR CN K LE COT S1 7 21 R 11 25 22 10 9 17 23 23 20 R 12 R 18 26 29 22 S2 R 26 8* 30* R 14* 27* R 25* 16 14* R 15* 28* R 22* 22* 30* 28 S3 R 53 R R 9 R 8 R 12 21 13 R R 8 R R 18 R 56 S4 R 37 R 10 R R 14 R 11 21 11 14 R 16 R 13 16 31 39 S5 R 42 R 9 R R 13 R 20* 19 13 R R 13 R 14 18 25* 44 S6 R 42 R R 25 R 11 R 19 23 25 20 R 9 R 19 27 R 44 S7 R 47 R R 16* R 9 R 17* 15 25 12* R 8 R R 30 R 50 S8 R 42 R 8 8 R 12 R 20 23 13 R R 12 R 19 10 8 44 S9 11* 5 15* 29* 32 17* 28* R 19* 22 32* 27* 16* 29* 17* 19* 30 31 6 S10 R 26 9 11 25 R 12 R 22* 20 24 19 R 15 R 18* 22 22 28 S11 R 58 R R R R 8 R 12 20 8 R R R R 15 8 R 61 S14 R 42 R 9 R R 11 R 18 25 10 R R 11 R 18 10 26 44 S15 R 47 R R 20 R R R 17 21 21 18 R 9 R 17 25 R 50 S16 R 47 R R 10 R 8 R 17 20 12 R R 8 R 16 12 R 50 S18 R 58 R R R R R R 19* 21 10 7 R R R 11 11 R 61 S19 R 37 R R 25 R 8* R 18 21 28 20 R 16 R 18 22 25 39 S20 R 47 R R 22 R R R 17 21 25 24 R 8 R 17 25 R 50 S21 R 37 R R 22 R 8 R 18 21 23 19 R 12 R 18 22 24 39 S22 R 53 R R 9 R 9 R R 17 8 R R 9 R 15 11 R 56 SE R 23 8 29 34 9 25 R 18 23 31 26 10 27 9 18 31 25 11 *R: Resistance. Antibiotic compounds used; AMP: Ampicillin, AK: Amikacin, AMC: Amoxyclav (Amoxycillin+Clavulinic Acid) (Augmen- tin), CTX: Cephotaxime, CIP: Ciprofloxacin, CXM: Cefuroxime, CPZ: Cefoperazone, CAZ: Ceftazidime, GEN: Gentamicin, NET: Netilm- icin, OF: Ofloxacin, NX: Norfloxacin, CFM: Cefixime, CTR: Cefrtriaxone, CN: Cephalexin, K: Kanamycin, LE: Levofloxacin, COT: Co- Trimoxazole. 90 Mashail A. Alghamdi et al. between (7.6-18.3 mm). Concentration 200 mg/ml of ethanolic and acetone-extracted leaves resulted in for- mation a small scale of inhibition zone 7.6-8.5 mm) The 500 mg/ml concentration produced the greatest inhibi- tion zone scale in ethanolic and acetone extraction of peels ranging between (9.0-18.3 mm), overall, at different concentrations (100 g/l - 500 mg/ml) pomegranate peel and leaves, the ethanol extracts of peel and leaves were more active than the acetone peel and leave extracts. The specifics are as follows. The leaf extract inhibited the proliferation and growth of E. coli that was resistant to antibiotics by breaking the cell membrane of the bac- teria, so allowing antibiotics to enter the bacteria. The extract contains bioactive chemicals, including as tan- nins, alkaloids, and flavonoids, which generate antibac- terial substances (Di Sotto et al., 2019; Alamshani et al., 2023; Zam and Khaddour 2017). The chemicals damage the bacterial cell membranes, leading in the release of the cellular contents and the eventual death of the bacte- rium. According to mass spectrometry research, P. gra- natum includes oligomeric ellagitannins consisting of up to 5 core glucose units, which are the most potent anti- bacterial chemicals in pomegranate. In addition, multiple prior researches have demon- strated that the aqueous extract of pomegranate leaves inhibits the growth of uropathogenic E. coli. In addition, the extract exhibited a minimum inhibitory concentra- tion (MIC) value of 0.6 mg/ml, resulting in an eighty percent reduction in the E. coli adhesion index. Addi- tionally, the extract exhibited a minimum bactericidal concentration (MBC) of 1.2 mg/ml (Jam et al., 2022; Church et al., 2007). Thus, the peel extract inhibited E. coli biofilm formation and decreased bacterial adhesion capacity (Alamshani et al., 2023). Solvent extraction is a method used to extract antioxidant chemicals from plants. The research outcome is determined by the type and amount of solvent utilised during the extraction procedure (Ellatif et al., 2021; Ellatif et al., 2022a; Ellatif K N H M B L F A D I G E Q C J M R O P A E A B Q D J L M B C I F N O P R H G K O Q E D M K R P B C L A J H N B G I F N L O I P K D G L A B F C R J B E Q H L P K C O E M D A R F H J B Q N G I A C L K P Q N M R D G C J L A J H E F K Q B L M G I F R I O K A C H D P H A E F G J E O H C N B K B K K J N B R G L I Q F E K K K K I Q O K B N I O Q D R K K G L P O D J M H C E A P F B D R P M Figure 3A. Antibiotic resistance profiles of ten (1-10) E. coli pathogenic bacterial strains used in these studies. Antibiotic compounds used; Ampicillin (A), Amikacin (B), Amoxyclav (Amoxycillin+Clavulinic Acid) (Augmentin) (C), Cephotaxime (D), Ciprofloxacin (E), Cefuro- xime (F), Cefoperazone (G), Ceftazidime (H), Gentamicin (I), Netilmicin (J), Ofloxacin (K), Norfloxacin (L), Cefixime (M), Cefrtriaxone (N), Cephalexin (O), Kanamycin (P), Levofloxacin (Q), Co-Trimoxazole (R). 91Antibacterial power of Pomegranate extracts against Beta-Lactamase producing Escherichia coli et al., 2022b). For such experiments, aqueous solvents such as acetate, ethanol, ethyl acetate, and methanol are advised. Due to their polarity, ethanol and methanol have been widely utilised to extract antioxidant com- ponents from numerous plants and plant-based meals. Moreover, the two solvents are compatible with the human body (Sutan et al., 2023; Park et al., 2011). The plant was extracted using ethanol and acetone in this investigation. Mueller-Hinton agar is a regularly used solid culture medium for assessing the antibiotic suscep- tibility of microorganisms. Its composition (beef extract, casein hydrolysate, and starch) allows for precise testing of the susceptibility of microorganisms to antibiotics. Due to its low concentration of magnesium and calcium ions, Mueller-Hinton agar can be used for antimicrobial susceptibility testing. The low ion concentration inhibits the medium from reacting with certain antibiotics and guarantees that the medications’ effects on the bacteria are not obscured. In this work, Mueller-Hinton agar was utilised to reveal E. coli’s susceptibility to the employed antibiotics. McConkey Agar is a differential and selec- tive culture medium used to isolate and differentiate gram-negative bacteria, namely members of the Entero- bacteriaceae family. It is composed of bile salts, neutral red indicators, crystal violet dye, peptones, and lactose (Erylmaz et al., 2010; Ho et al., These components pro- mote the growth of Gram-negative bacteria while inhib- iting the growth of Gram-positive bacteria. Its unique- ness rests on the capacity of bacteria to ferment lactose, hence creating acid and lowering the pH of the medium. This often results in a shift in the colour of the colonies, from pink to red (Phillips and Garda et al. 019; George et al., 2008; Giri et al., 2021). Effective culture medi- um because E. coli is an example of an enteric bacteria (Alamshani et al., 2023). The results of the investigation reveal that the majority of strains demonstrated both antibiotic resist- ance and sensitivity. The most effective antibiotics against the pathogen E. coli are therefore Amikacin, Netilmicin, Ofloxacin, Cefrtriaxone, and Levofloxacin. E D G M P L O I A B K J Q C H F A R H Q C I N O Q N R E J L F O N I C R K L A D M P G N E R J B I K G F C A J F Q K B F C A L D M K E H A A D E O O J R I H A J P M L I F O K I A G O D K G R N G C P M H O E C F D L G N I F P B E J K R D N N H P B P M I L A K Q C Q G M R G C L B M H A K B P L F O Q Q D A B K E H C A B A J R N D G Q L I P H H M E C A J B J D E P A H A F R B N J Figure 3B. Antibiotic resistance profiles of ten (11, 14, 15, 16, 18- 22 and C) E. coli pathogenic bacterial strains used in these studies. Anti- biotic compounds used; Ampicillin (A), Amikacin (B), Amoxyclav (Amoxycillin+Clavulinic Acid) (Augmentin) (C), Cephotaxime (D), Cip- rofloxacin (E), Cefuroxime (F), Cefoperazone (G), Ceftazidime (H), Gentamicin (I), Netilmicin (J), Ofloxacin (K), Norfloxacin (L), Cefixi- me (M), Cefrtriaxone (N), Cephalexin (O), Kanamycin (P), Levofloxacin (Q), Co-Trimoxazole (R). 92 Mashail A. Alghamdi et al. These antibiotics were chosen because the drugs’ micro- bial membrane was protected from damage by the bacte- ria injected. According to the findings, the pomegranate component extract has the greatest ability to inhibit the growth of pathogenic microorganisms (Alamshani et al., 2023). Additionally, the higher the concentration of the leaf extract, the simpler it is to alter the structural mem- brane of the E. coli-causing pathogen. This extract can be used to replace or even enhance the performance of standard antibiotics. Antimicrobial activity was greatest in the ethanolic extract. This is in contrast to acetone. In conclusion, P. granatum leaves and peels have a substantial effect on E. coli that is resistant to antibiotics. The study indicated that the leaf extract had antibacte- rial properties that suppress the growth of E. coli strains resistant to antibiotics. The active chemicals in the extract eliminate antibiotic resistance by destroying the integrity of the bacteria. When P. granatum leaf extract is mixed with standard antibiotics, synergistic effects are produced. The combo eradicates antibiotic-resistant E. coli bacteria more effectively than conventional anti- biotics alone. These findings demonstrate the potential of P. granatum leaves as a natural alternative and treat- ment for conventional antibiotics in the management and treatment of antibiotic-resistant bacterial illnesses. To determine the precise mechanisms of action, optimise the extraction procedures, and assess the long-term effi- cacy and safety of P. granatum leaf extracts, additional research is required. However, the antibacterial proper- ties of P. granatum have permitted the development of innovative, safe, and successful therapeutic techniques against E. coli resistant to antibiotics. tannins and alka- loids metabolites in the leaves and peels of pomegran- ate are considered antimicrobial (Joshi et al., 2019; Shaygannia et al. 2016; Wu and Tian 2017; Warsi and Sulistyani 2018; Joshi and Nair 1960). Alkaline groups in alkaloids interact with amino acid groups in cells, result- ing in structural and chemical changes that are damag- ing to the cell (Johan et al., 2020; Seeram et al., 2005; Nurdin et al., 2019). Antibacterial flavonoids in pome- granate inhibit bacterial growth by inhibiting the DNA gyrase mechanism, resulting in intracellular leakage (Shaygannia et al., 2016). Queercetin, a flavonoid found in pomegranate, may kill microorganisms by increasing membrane permeability and hurting the potential of bac- teria within membranes (Khan et al. 2012). Table 3. The inhibition zone diameters (mm) of different concen- trations (100-500 mg) of Pomegranate peels ethanol extracts against E. coli strains. Data are expressed as the mean ± SD Strains Pomegranate peels ethanol extracts (mg/ml) 100 200 300 400 500 1 R R 8.6±0.01e 14.6±0.12c 16±0.24b 2 R R 14.3±0.21c 15±0.14b 15.8±0.13b 3 R 9.1±0.12e 14.1±0.12c 15.90.12b 17.2±0.21a 4 R R 13.2±0.11c 15.7±0.14b 17.1±0.15a 5 R 8.2±0.14e 9.6±0.04e 10.3±0.1d 11.6±0.02 6 R R 9.3±0.025e 10.2±0.012d 11.1±0.11 7 R R 8.1±0.014e 10.3±0.14d 12.3±0.14 8 9.0±0.02e 11.3±0.13d 13.3±0.11c 14.6±0.012c 15.6±0.12b 9 R R R 8.4±0.02e 10.0±0.01d 10 R 10.6±0.11d 12.1±0.07d 13.6±0.04c 14.6±0.1 11 R R 12.2±0.10d 14.05±0.12c 15.2±0.09b 12 R 8.0±0.01e 10.1±0.011d 14.3±0.13c 15.6±0.14b 13 R R R R R 14 R R R R R 15 R R R R 9.0±0.05e 16 R R 15.8±0.12b 14.2±0.011c 18.3±0.24a 17 R R R R 9.2±0.10e 18 R 10.1±0.05d 11.2±0.1d 12.02±0.10d 13.3±0.12c 19 R R R 8.3±0.04e 9.1±0.01 20 R R 9.1±0.05e 12.3±0.013d 16.1±0.13b *R: Resistance. Table 4. The inhibition zone diameters (mm) of different concen- trations (100-500 mg) of Pomegranate peels acetone extracts against E. coli strains. Data are expressed as the mean ± SD. Strains Pomegranate peels acetone extracts (mg/ml) 100 200 300 400 500 1 R 8.0±0.021d 9.1±0.014d 10.2±0.015c 13.2±0.02b 2 R R R 11.1±0.012c 14.3±0.2a 3 R R 11.2±0.01c 12.6±0.014b 13.4±0.14b 4 R R R 9.04±0.016d 9.2±0.025d 5 R 9.3±0.04d 9.07±0.015d 10.2±0.017c 11.1±0.1c 6 R R R R R 7 9.1±0.14d 10.20.25c 12.2±0.03b 14.0±0.23a 15.1±0.023a 8 R 9.1±0.18d 10.4±0.02c 14.0±0.018a 15.0±0.17a 9 9.2±0.21d 12.3±0.16b 14.0±0.021a 14.4±0.17a 15.3±0.014a 10 R 12.2±0.12b 13.2±0.22b 14.1±0.3a 14.6±0.1a 11 R R R R R 12 R 10.1±0.13c 11.8±0.16c 12.2±0.21b 13.4±0.012 13 R R R 12.1±0.16b 11.12±0.014c 14 R R 11.1±0.015c 13.2±0.24b 14.11±0.13a 15 R R R R 8.1±0.01d 16 R R 11.7±0.02c 12.4±0.12b 15.1±0.024a 17 R 8.0±0.02d 9.1±0.04d 11.05±0.02c 12.2±0.016b 18 8.1±0.012d 9.1±0.012d 10.2±0.02c 10.7±0.011c 3±0.02b 19 R R R R R 20 R 8.0±0.02d 12.1±0.12b 14.3±0.15a 15.2±0.17a *R: Resistance. 93Antibacterial power of Pomegranate extracts against Beta-Lactamase producing Escherichia coli 5. CONCLUSION As a result of the global problem with MDR bacteria, scientists have been attempting to develop novel bioactive compounds derived from natural resources that can be exploited as safe phytotherapy. According to the findings of this study, pomegranate peels and leaf extract can sup- press the growth of Extended-Spectrum Beta-Lactamase -E. coli pathogenic strains at doses 100 and 200 mg/ml with highest significant antibacterial activity. According- ly, the ethanolic and acetone extracts exhibited the best antibacterial activity and may be used to enhance or even replace antibiotics with less cytotoxic effect. ACKNOWLEDGMENTS This research work was funded by institutional fund projects under grant no. (IFPIP:971-247-1443 ). The authors gratefully acknowledge technical and financial support provided by the Ministry of Education and King Abdulaziz University, DSR, Jeddah, Saudi Arabia. FUNDS This research work was funded by institutional fund projects under grant no.( IFPIP:971-247-1443 ). The authors gratefully acknowledge technical and financial support provided by the Ministry of Education and King Abdulaziz University, DSR, Jeddah, Saudi Arabia. Figure 4. The inhibition zone diameters of different concentrations (100mg-500mg) of Pomegranate peels ethanol extracts against E. coli strains. 94 Mashail A. Alghamdi et al. AUTHOR CONTRIBUTIONS All the authors contributed to the study’s concep- tion and design. The Faisal Al-Sarraj, Raed Albiheyri and Noor M. Bataweel authors were Identify ultimate objective of study and designed the required analysis and bio-assays. Wafa H. Alamshani, Majid Al-Zahrani, Tahani M. Alqahtani, Mashail A. Alghamdi, Nada Nass and Thamer Bouback authors were involved in funding acquisition, prepare the chemical, kits and performed the practical part of this work. Ibrahim Alotibi, Moham- med A. Al-Matary and Bayan H. Sajer authors were par- ticipate in study analysis, obtained raw data collection, supervision, and perform the statistical analysis. Faisal Al-Sarraj, Raed Albi, M. Attallah and Wafa H. Alamsha- ni authors were shared in paper writing the manuscript body, create a strong conclusion and language editing REFERENCES Alamshani HW, Al-Sarraj FM, Algamdi M. 2023. The inhibitory effect of Punica granatum on Escherichia coli and Klebsiella pneumonia extended spectrum β-lactamase strains. Novel Research in Microbiology Journal, 7(1):1836-56. Alotibi I, Al-Sarraj FM, Albiheyri R, Alghamdi MA, Nass N, Bouback T, Sajer BH, Al-Zahrani M, Basingab F, Alharbi M. 2022. Study the apoptosis and necro- sis inducing of fosfomycin into associated infected urothelial tissue by extended spectrum beta lac- tamase positive of E. coli. Microbial Pathogenesis. 173:105838. Al-Sarraj FM. 2021a. 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