Antimicrobial efficacy of Mentha piperata-derived biogenic zinc oxide nanoparticles against UTI-resistant pathogens

Misuse of antibiotics leads to the worldwide spread of antibiotic resistance, which motivates scientists to create new antibiotics. The recurring UTI due to antibiotics-resistant microorganism’s challenges scientists globally. The biogenic nanoparticles have the potential to meet the escalating requirements of novel antimicrobial agents. The green synthesis of nanoparticles (NPs) gained more attention due to their reliable applications against resistant microbes. The current study evaluates the biogenic ZnO NPs of Mentha piperata extract against resistant pathogens of urinary tract infections by agar well diffusion assay. The biogenic ZnO NPs revealed comparatively maximum inhibition in comparison to synthetic antibiotics against two bacterial strains (Proteus mirabilis, Pseudomonas aeruginosa) and a fungal strain (Candida albicans).The synthesized biogenic ZnO NPs alone revealed maximum activities than the combination of plant extract (PE) and ZnO NPs, and PE alone. The physiochemical features of ZnO NPs characterized through UV–Vis spectroscopy, FTIR, XRD, SEM, and EDX. The UV–Vis spectroscopy revealed 281.85 nm wavelengths; the XRD pattern revealed the crystalline structure of ZnO NPs. The FTIR analysis revealed the presence of carboxylic and nitro groups, which could be attributed to plant extract. SEM analysis revealed spherical hollow symmetry due to electrostatic forces. The analysis via EDX confirmed the presence of Zn and oxygen in the sample. The physiochemical features of synthesized ZnO NPs provide pivotal information such as quality and effectiveness. The current study revealed excellent dose-dependent antimicrobial activity against the pathogenic isolates from UTI-resistant patients. The higher concentration of ZnONPs interacts with the cell membrane which triggers oxidative burst. They may bind with the enzymes and proteins and brings epigenetic alteration which leads to membrane disruption or cell death.

The clinical utilization of antibiotics such as; penicillin was presented in 1930 and sulfonamide in 1940 during World war-II in 1940.As a result, individuals of that era showed that infectious diseases were completely controlled by antibiotics.But recently, the uncontrolled use of antibiotics develops antibiotic resistance worldwide and nowadays resistance to antibiotics is a crucial public health problem 1 .The worldwide emergence of pathogenes of multi-resistant nature is due to the misuse and overuse of antibiotics in human, veterinary, and agricultural medicines around the world.Antmicrobial resistance (AMR) was noticed shortly afterdiscovery of antibiotics 2 .For instance, resistance to penicillin after its clinical debut in 1942 was noticed in 1947 by Staphylococcus strains 3 , The resistance case of streptomycin debuted in 1944 and vancomycin took 30 years after their clinical debut 4 .The debut history of vancomycin starts 65 years ago and remains standard against the infections of methicillin-resistant Staphylococcus aureus.Its long-time resistance is due to controlled utilization of an initial 25 years; therefore, the long delay creates a big opportunity to develop effective antibiotics during the www.nature.com/scientificreports/The features such as; the utilization of ZnO NPs as antibacterial agents, toxicity to microorganisms, and control of outstanding biocompatibility of the human cell make it unique 41 .
The different antibacterial properties of nano-materials reduce to their aspect ratio as high specific and display a unique physiochemical effect.Day by day the resistant bacterial infections are major health problem across the globe and a big challenge to social and economic complications.Outbreaks of infectious strains, resistance of antibiotic to bacteria, change in bacterial genome, absence of satisfactory vaccine in progressive nations, and hospital-associated infections are global health threat to population especially in youngster for instance yearly 1.5 million deaths occur from the infections of resistant Shigella flexneri while other microbes include Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Enterococcus faecalis and Salmonella typhi 42 .ZnONPs is reported as one of the important, functional, strategic, promising and versatile particles for multiple applications including antibacterial properties.In periodic table Zn and O are categorized into two and six group 43 .ZnO shows a light covalent character but exhibits strong ionic bonding.Further, the higher selectivity, longer durability and heat resistance of ZnO are presided than inorganic and organic materials 41 .The synthesis of small sized ZnO is used as new antibacterial molecule due to its specific antifungal and antibacterial applications.ZnO have highest photochemical and catalytic activities.In the UV ZnO have highest optical absorption (325-400 nm) and UVB (280-315) with outstanding antibacterial responses 44 .Nowadays ZnO NPs are used in biomedical and as antiviral agents, due to their potential biocompatibility on the other metal oxides.
Hence, the fundamental aim of this investigation encompassed the formulation of biologically derived Zinc Oxide Nanoparticles (ZnO NPs) sourced from M. piperata, primarily intended for employment as antimicrobial agents targeting antibiotic-resistant bacterial strains isolated from human urinary tract infections (UTI).This current study represents a distinctive and pioneering endeavor, marking its novel approach towards assessing the effectiveness of biogenic ZnO NPs in addressing UTI-causing resistant microorganisms, with potential implications for the biomedical industry.

Materials and methods
Sample collection and processing.The present study was carried out at Centre for Biotechnology and Microbiology, University of Swat and Pakistan Council for Scientific and Industrial Research (PCSIR), Peshawar.The urine samples were collected from 100 patients at Miangul Abdul Haq Jahanzeb Kidney Hospital [MJKH] (https:// www.mjkhkp.gov.pk) formerly known as Nawaz Sharif Kidney Teaching Hospital, Manglawar Swat-Pakistan.The samples were collected with the permission of the administrative authority of the hospital following the ethical and standard procedure as given in 45 .The urine sample from the patients was collected in sterilized containers and stored in refrigerator at 4°Cfor bacterial culture.
Bacterial and fungal culturing procedure.Nutrient agar (NA) and blood agar (BA) media for culturing was prepared according to the protocol of 46 .A known amount media powdered was poured into boiling water and then autoclaved (121 °C; 20 min; 15 psi) and broad-spectrum antifungal antibiotics were added after autoclaving to inhibit the growth of unwanted fungi in culturing media.The media was allowed for cooling and then transferred to sterile petri dishes and incubated for further processing.Mac Conkey agar (MCA) media was used to isolate gram negative bacterial strains on the basis of lactose fermentation which differentiate bacterial species.The species which ferment lactose display pink color on MCA surrounded by salts precipitation.Due to the production of acid the MCA color changed to red from neutral.The bacteria lacking the ability to ferment lactose appear colorless colonies.The culturing media was placed in incubator (37 °C) for 24 h to check contamination.The urine samples were transfer onto culture media under sterile condition and subsequently the cultured plates were incubated for 24-48 h at 37 °C.After incubation the different colonies appeared on NA was marked using colony morphology.Furthermore, fresh mannitol salt agar media was applied for sub-culturing of marked colonies and again incubated for 24-48 h at 37 °C to isolate pure cultures of bacteria using colony morphology.Furthermore, for the isolation of C. albicans, the urine samples were inoculated onto PDA and incubated for 24 h at 37 °C.After inoculation and incubation various fungal colonies appear on PDA.During the procedures, penicillin, ampicillin and ciprofloxacin were added to the culture media to inhibit the growth of unwanted bacteria.

Identification of pure bacterial and fungal strains.
For the identification of pure cultures of Proteus mirabilis and Pseudomonas aeruginosa different tests were performed including gram staining (GS) and multiple biochemical tests using the procedure of 47 .Pure culture can be identified using the GS from the prepared subcultures.A smear was prepared for each pure culture by excising a small part of the colony using sterilized loop.The smear was placed on glass slide by adding sterile water and then heating it for fixation.Initially the slide was exposed to crystal violet dye for 60 s and then washed with autoclaved H 2 O. Secondly, the slide was exposed to iodine for 60 s and then washed again with autoclaved H 2 O.In the third step the slide containing pure culture was treated with 70% alcohol as decolorizing agent for 60 s and then washed with autoclaved H 2 O.The slide was dried carefully and placed under microscope (40x) using immersion oil.In case of fungal isolation and identification, the fungal colonies were detected through general morphological analysis on PDA media and then the gram staining was also performed and finally the germ tube formation lactophenol staining was done to confirm the target C. albicans.
Different biochemical tests were performed for further identification that includes catalase, oxidase, triple sugar iron test, indole test and urease test.For all these tests, the procedure of 48 was followed.Catalase test was performed to test the catalase enzyme production by the organism.The role of this enzyme is the release of oxygen and water from the H 2 O 2 .If the reaction release bubbles it means that the culture is catalase positive and the specie is gram negative.Coagulase is special test used for the identification of Pseudomonas aeruginosain in which tetra-methyl-p-phenyl diamine di-hydro-chloride reagent was used to identify the oxidase producing bacteria.The positive test revealed that the culture consists of Pseudomonas otherwise, negative results represents the presence of family Enterobacteriaceae.The TSI test media composed of glucose, sucrose, lactose, phenol red and iron sulphate as indicator.It also displays that the culture is able to release hydrogen sulphide (H 2 S) or not.Pure culture was streak on the surface of slant and stabbed on TSI agar Butt following incubation for 24 h at 37 °C.The tubes were examined in order to measure the change in slant or Butt.If the culture is able to use glucose, then the slant or Butt color changed into yellow due to the production of acid.The appearance of cracks in the media represents the release of bubbles or gas.If no fermentation occurs the slant and Butt color do not change from initial red color.If the culture produces hydrogen sulphide using ferrous sulphate which combined with iron again and form ferrous sulphide and produce black precipitation.Indole test was performed to check the bacterium ability to produce indole from the tryptophan amino acid.In this test the addition of Kovacs reagent changes the media color to cherry red from yellow color.Urease test was also performed in which the enzyme converts urea to ammonia and CO 2 in the presence of H 2 O.The three components react with each other and produce ammonium carbonate and convert phenol red which is an indicator from orange yellow to bright pink color.
In case of C. albicans slacto-phenol cotton blue staining (LPCB) is a specific technique commonly used for the preparation semi-permanent microscopic fungi.LPCB has three main parts that is phenol used as antiseptic to killed living entities, lactic acid used as preservative agent that preserve and maintain fungal structure and cotton blue strain that provide blue color to fungal cell wall components.Here a drop of 70% ethanol was poured on sterilized glass slide and the specimen was mixed with it, further, one or two drops of LPCB is added to the slide before the ethanol is dried.A cover slip was placed upon the smear carefully to avoid bubble formation.The slide was studied under microscope using higher power of 40X objective lense for detailed investigation of spores and other structures.

Antibiotics susceptibility test.
The disk diffusion method as Kirby-Bauer method of antibiotics was performed for the microbial sensitivity.Through the help of sterilized swab, the targeted microbes were streak uniformly on Mullar Hinton Agar (MHA) plate surface.The cultured plates were kept for 5-6 min for drying and then the antibiotics (Ciprofloxacin, azithromycin and clotrimazole) discs were placed on the surface of the media and these plates were then incubated at 37 °C for 24 h.Here two cultured plates of MHA were used for testing of multiple concentrations.The results were collected as zone of inhibition around the disc and expressed in mm.The obtained results were interpreted as resistance or sensitive or intermediate 49 .
Preparation of plant extract.Fresh leaves of Mentha piperata were utilized for the synthesis of ZnO nanoparticles.The fresh plants were collected from the Medicinal section of the Botanical Garden of PCSIR Peshawar Pakistan.The procedure as adopted in 50 was followed for extract preparation with minor modifications.Fresh plant leaves of 20 gm were washed with sterile water and shade dried for the formation of dried powder.The powder was mixed with 50 ml distilled water and incubated for 24 h at room temperature.The extract was filtered and centrifuged at 4000 rpm for 40 min.The supernatant was utilized for the synthesis of Zinc Oxide NPs and stored at 5 °C for additional procedure.
Biosynthesis of zinc oxide nanoparticles.Zinc oxide NPs were synthesized according to the standard procedure 50 .The plant extract consists of different functional groups such as; phenolic, carboxyl, and amine which function to reduce the Zn ions.The plant extract of Mentha piperata was mixed with the solution of zinc oxide already taken in a flask.A solution of Zinc dioxide (3.5 mM) was prepared by dissolving Zinc dioxide salt in distilled water to reach a concentration of 0.64218 g/L.The solution was stirred for 4 h at 50 °C using a magnetic stirrer, and the solution was reduced by adding a plant extract drop-wise keeping the PH 7-8 and till the appearance of yellow to red-orange color.The solution was incubated at 30 °C for 24 h.Then the solution was centrifuged at 10,000 rpm for 10-15 min (Centrifuge: Model: Eppendorf 5424 R, Germany, with 24 × 2 ml A-45-24-11 Rotor).After that solution(extract + ZnO NPs) was poured into Petri dishes and dried overnight in the oven to make a powder of nanocrystals to use for additional processing.

UV -spectroscopy characterization of ZnO Nanoparticles.
The UV range of the combined NPs was acquired by scanning of ZnO NPs in between 200 and 600 nm utilizing UV-Vis Halo DB-20 spectrophotometer 51 and spectrum was captured against specimen with assistance of TU-1901 double-beam U-V Visible spectrophotometer which was dissipated in ethanol.

FTIR analysis.
The confirmation of organic functional group arranged in plant extract and NPs was made with FTIR by potassium bromide (KBr) pellet method with a settlement and examine range of 4 cm −1 , from 400 to 4000 cm −1 .The infrared (IR) ingestion spectra uncovered the presence of various functional groups on the particle's surface.

XRD characterization of ZnO Nanoparticles.
The biogenic synthesized nanoparticles were characterized with different techniques to identify the desired nanoparticles.The ZnO nanoparticles were subjected to XRD.The XRD observations of ZnO NPs were recorded on X-ray diffractometer at the energy range of 20 from 10 to 80 with a scan rate of 20/min utilizing Cu Alpha (lamda = 1.5406A) radiation with an accelerating voltage of 40 kV.Different stages present in the samples were assessed by mean of X-pert high score programming having search match office.Energy dispersive X-ray spectroscopy analysis (EDX).Energy dispersive x-ray (EDX) examination joined with SEM decided the presence of various elements in the NPs sample.The specimen was resolved through EDX programming by applying ZAF correction whereas; Z = atomic number, A = absorbance, and F = fluorescence.The samples data were taken from the ideally shaped particles exposed on the top of the plate to get the last measurement of the element in the sample.

Antimicrobial activities.
In this study two different bacterial strains and one fungal strain were used from the isolated samples of from UTI patients.These bacterial strains include; Proteus mirabilis and Pseudomonas aeruginosa and fungal strain include; Candida albicans.All these microbes are pathogenic and Multi-drug resistant (MDR).Zinc oxide nanoparticle (20 mg) was dissolved in 20 ml distilled water.After that Well diffusion method was utilized for the antimicrobial activity of nanoparticle as described by 52 .The zone of inhibition was measured in mm as diameter around the well.The microbes (Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans) were streaked in each plate and three well were bored in each plate.After that, each well of the plate was poured with nanoparticle.Exactly, a 100 µL nanoparticle extracts (100 ug/ml) was introduced in first well, then 50 micro liter nanoparticle extracts (25 ug/ml) was introduced into the second well and 25 µL nanoparticle extracts (25 ug/ml) was introduced into the third well to checked the antimicrobial activity of nanoparticle and kept it on 37 °C in incubator for one day.After that Zone of inhibition were recorded in millimeter (mm).For positive control the broad-spectrum antibiotics was utilized.

Data analysis.
The antimicrobial activities of differential treatments were carried in triplicates.For descriptive statistics (mean values ± SE determination) excel sheet was used.For significance and least significant differences of data one-way analysis of variance (ANOVA) Statistics software (v.8.1; USA) was used.The graphical designing of thee data was carried out by Origin Lab software (v.8.5; USA).

Methods/experiments.
We confirmed that all methods/experiments were performed following the relevant guidelines and regulations.Further, the protocols used in this study were according to the approved guidelines of the institution.

Ethics committee approval. All experimental protocols were approved by the ethical committee of
Center for Biotechnology and Microbiology, University of Swat, UOS/EC/22-423.
Informed consent.Informed consent was obtained from all patients participating in the study.

Research involving plants.
We confirmed that all methods (field studies/collection) involving Mentha piperita (L.) used for the greens synthesis of NPs in the current study were in accordance with relevant guidelines/regulations/legislation.

Results and discussion
UV-visible spectrometry.In the initial stage, we confirmed the ZnO nanoparticle with the help of UVspectroscopy (Fig. 1).The absorption spectrum of the zinc nanoparticles synthesized from the Mentha piperata plant specified falls at 281.5 nm, which showed the presence of ZnO.A higher absorption peak at 385 nm of ZnO nanoparticles was also observed 53 .Similar results for AgNPs were also recorded by 54 , confirming the nanoparticles with broad peaks in the 420 to 425 nm range.A slight difference in data may be due to the differences in plant species used for the green biosynthesis of ZnO NPs.In the present study, the ZnO NPs were observed in the range of 200 and 600 nm.These findings are supported by previous studies such as; synthesized Ag NPs using Boswellia ovalifoliolata stem bark extract fall in 200 to 800 nm 55 .According to 56 , synthesized zinc oxide nanocomposite particle occurs in the range of 200 and 400 nm, and from 57 , prepared ZnO NPs fall in the 190 to 204 nm range.These findings show a strong correlation with our current study.Furthermore, the report of 58 indicated that 370 nm absorption of wavelength is the best range for the visibility of ZnO and ZnO hybrid nanostructures.These reports and the current results suggest that the visible appearance range of ZnO NPs falls within the 200 to 800 nm range.Halo compounds respectively as shown in (Fig. 3).These results revealed that the various groups represent the presence of various polyphenolics.In contrast, 59 reported that the FTIR results at 3314 cm −1 of n-type ZnO nanoparticles prepared from ginger and garlic roots represent the OH functional group.The differences in data no doubt are due to the selection of different plant extracts used for particle preparation.However, our findings are in agreement with the reported data 60 .In the present study, the peaks range from 744.24 to 686.54 cm −1 representing the presence of O-H, and C-O of carboxylic anions, while the findings of 61 reported that the peaks range from 1640-3313 cm −1 represent phenols (O-H  FTIR was performed by potassium bromide (KBr) pellet method with a settlement and examine range of 4 cm −1 , from 400 to 4000 cm −1 .The infrared (IR) ingestion spectra uncovered the presence of various functional groups on the particle's surface.bond) and primary amines (C=O) in CuO NPs using the plant extract of Syzygium alternifolium this difference of opinion is due to the differences of plant material and salt used in the preparation of NPs.A similar result of various functional groups falling within the same range was observed by 55 in ZnO NPs prepared from Boswellia ovalifoliolata stem bark.The data presented by 62 are in agreement with the current study in which novel cafestolloaded ZnO NPs were prepared from the diterpene in coffee (Cafestol).Ahmad et al. 63 also observed a similar C-N group in AgNPs prepared from the leaves of Catharanthus roseus.The comparative evaluation of extract and biogenic ZnONPs revealed uniform functional group region which confirmed the presence of important phytochemicals such as; flavonoids, alkaloids and polyphenols.The current FTIR results suggest that these biogenic ZnO NPs of M. piperita are a rich source of phytochemicals.

Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM), Selected Area Electron Diffraction (SAED) and Zeta potential.
The SEM analysis confirmed the morphological shape of the synthesized ZnO nanoparticle.The result investigated that ZnO nanoparticles have pyramidal symmetry of particles approximately ranges between 20-30 nm (Fig. 5).The transmission electron microscopy (TEM) was carried out for determination of shape and size of synthesized ZnNPs of Mentha piperita extract.TEM results showed the globular and oblong shaped nanoparticles having sizes ranging from 15 to 27 nm.The mean particle size calculated was 18 nm as per TEM micrographs.The size and form of biosynthesized silver nanoparticles revealed even distribution and capping of the biomolecules present in the extract.Besides, the Brags reflection rings were observed during selected area electronic diffraction (SAED) indicating the crystalline    www.nature.com/scientificreports/nature of nanoparticles (Fig. 6).TEM analysis verified the efficiency of Mentha piperita extract for the synthesis of ZnNPs.Zeta potential identifies the charge difference between the loop of ions (which surrounds the surface of opposite charge NPs) and bulk fluid (in which NPs suspended).It is often the binding of negative charge ions with positive charge particles therefore; the stability of NPs is directly proportional in the medium to higher electrostatic repulsion.The Zeta potential window given in Fig. 7-revealed 50 to 100 nm which confirms excellent sedimentation and well dispersed nature of the synthesized NPs.Characterization of zinc nanoparticles unveiled their spherical morphology, exhibiting a size spectrum of 7-9 nm, as investigated 66 .Correspondingly, a notably analogous outcome was documented by 62 in the context of ZnO nanoparticles, wherein particle dimensions ranged from 45 to 60 nm, and hexagonal structure was ascertained through Zeta Sizer analysis.In congruence with these findings, 67 reported similar outcomes, where the size of magnesium oxide nanoparticles, derived from Dalbergia sissoo extract, registered at less than 50 nm.In contrast, biogenic silver nanoparticles from Catharanthus roseus displayed a size range of 40 to 60 nm 63 .This difference in data may be due to the difference in plant material and metallic salts used for biogenic particle preparation.The current study's particle size (20 to 30 nm) was in good agreement with 65 , who reported particle size (30.87-47.89nm) using extract of hairy root cultures of Phoenix dactylifera for ZnO NPs synthesis.Using HR-TEM images, 59  www.nature.com/scientificreports/found that the n type ZnO nanoparticles prepared from ginger and garlic roots were less than 50 nm in size.
The current study's findings, as well as those reported in the literature, supported the size of ZnO NPs ranging from 20 to 60 nm.The minor differences in size and multiple symmetries are most likely due to differences in adapting different methodologies, plant species, and metallic salt selection used for nanostructure synthesis.

EDX Analysis.
The EDX analysis confirmed the presence of Zinc nanoparticles as spherical shaped.EDX analysis of Zinc nanoparticles revealed (Zn) Zinc, (C) Carbon, and (O) Oxygen which displayed the presence of metallic zinc acetate in the synthesis of Zinc oxide nanoparticles.EDX spectrum revealed three peaks viz., Zinc, Carbon, and Oxygen (Fig. 8), this confirmed the presence of Zinc with a strong prominent peak (85.71% weightage between the characteristic spectrum range of 3 to 4 keV).The findings of the current study are in agreement with the reported data 68 .In the present study, the strong peak of Carbon indicates that it is a capping and stabilizing agent for the formation of biogenic nanostructures of ZnO from the leaves of M. piperita.The work of 13 also reported similar results while using the extracts of leaf, stem, and callus cultures of Mussaenda frondosa for the biogenic synthesis of ZnO NPs.The work of 56 used the techniques of inductively coupled plasma optical emission spectroscopy for the presence of Zinc in graphene oxide/Zinc oxide and observed that the techniques demonstrated the quantity increases of Zinc in the nanocomposite.In this study, the EDX analysis confirmed the presence of Zn, C, and O in the biogenic ZnO nanostructures synthesized from M. piperita.
Antimicrobial potential of Biogenic ZnO NPs.The Zinc oxide nanoparticles were used against UTIresistant microbial species; Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans.Three different concentrations of ZnO nanoparticles (100, 50, and 25 ug/ml) were used against these microbes.While only the single higher concentration (100 ug/ml) of antibiotics was used.In the current study, the higher concentration (100 ug/ml) of zinc oxide NPs showed the maximum activity (34.16 ± 2.3 mm) against Pseudomonas aeruginosa, followed by 50 and 25 ug/ml and exhibited 24.0 ± 1.22 and 19.0 ± 3.5 mm zones of inhibitions against Pseudomonas aeruginosa (Figs.9a,10a).The ZnO nanoparticles were also used against Candida albicans.The highest concentration (100ug/mL) exhibited 20.83 ± 0.62 mm of activity while decreasing the nanoparticles concentrations, the activity also decreases (Fig. 9b).Here the highest activity (39.33 ± 3.29 mm) was observed against Proteus mirabilis using the higher concentration (100 ug/ml) of ZnO NPs (Fig. 9c) which was comparatively higher than Pseudomonas aeruginosa, Candida albicans, and antibiotics.As compared to ZnO NPs alone, the combination of ZnO NPs + PE (100 ug/ml) displayed 30 ± 0.9 mm, 36 ± 1.9 mm, and 17.2 ± 0.4 mm zones of inhibitions against Pseudomonas aeruginosa, Proteus mirabilis and Candida albicans (Fig. 10b).However, the lowest activities were displayed by applying different concentrations of PE alone (Fig. 10c).The antimicrobial potentials of ZnO NPs were compared with the antibiotics which are commonly used in UTI infections.The ciprofloxacin used against Pseudomonas aeruginosa displayed an 18 mm zone of inhibition (Fig. 9d).The clotrimazole exhibited a 17 mm zone of inhibition (Fig. 9e), while the azithromycin used against Proteus mirabilis showed a 16 mm zone of inhibition (Figs.9f, 10).These results suggest that ZnO NPs prepared from Mentha piperita strongly inhibited the growth of Proteus mirabilis, Pseudomonas aeroginosa, and Candida albicans.The overall findings revealed dose-dependent activities.The findings Due to the extensive use of antibiotics, the resistance in microbial cells also increases, like ciprofloxacinresistant P. aeruginosa is rapidly increasing.Ciprofloxacin resistance can arise through the acquisition of mutations in genes encoding the target proteins of ciprofloxacin and regulators of efflux pumps, which leads to the over-expression of these pumps.However, nanoparticles as alternative drugs fluctuate the gene expression level and efflux pumps in such a way that cause the death of microorganism 56,62,63,67 .Nanoparticle has the ability to disturb the metabolic activity of microbial cell which is more beneficial for the treatment of various infections caused by resistant microbes and also prevent the formation of biofilm 72 .When a nanoparticle crosses the cell membrane it must affect the shape and function of the cell membrane.After that NPs must contact with fundamental constituents of microbial cells such as DNA, ribosomes, and lysosomes, inhibition of enzymes, change in gene expression, deactivation of protein, heterogeneous alternation, electrolytic disturbance, variation of cell membrane permeability, and oxidative stress 35 .Metal oxides produce metal ions that dissolved through the cell membrane which affect proteins and nucleic acid functional groups such as carboxyl (-COOH), amino (-NH), and thiol/sulfhydryl/sulfanyl group (-SH) which cause different effects on physiological mechanisms, cell structure, enzymatic activity and finally killed the microbes that is why nanotechnology are currently utilized as a source of antimicrobial agents 35,73,74 .
From different sources, different microbes enter to host and cause numerous diseases [75][76][77] .These diseases can be controlled by various synthetic antibiotics.The frequent utilization lead to mutation and finally resistance in microorganisms 78 .Currently, multidrug-resistive strains of many microbes arises serious problems 59 .Such a problem needs alternative sources, such as the use of multiple nanoparticles prepared from medicinal plants which are natural, less toxic, and more effective against resistant pathogens.The mechanism (Fig. 11) showed how our formulated NPs get entered the cell by overcoming various barriers and how it disrupts the cell by interacting with various organelles and components of the cell.For instance: either interaction with the cell membrane may lead to distraction of the permeability of the membrane leading to cell disruption or by directly attacking the protein synthetic machinery and DNA component of the cell that may result in the loss of the cell integrity or simply to apoptosis or cell death.The aqueous extract of Leonotis nepetifolia flower bud was tested in different in-vitro activities suggesting its wide range of biomedical applications 79 .The modulated silver nanoparticles of Cucumus sativa (Cs-AgNPs) exhibited significant dose-dependent antimicrobial activity by agar well diffusion test against thetested pathogenic fungal and bacterial strains 80 .

Conclusion
The phenomenon of heightened resistance exhibited by microorganisms, particularly bacteria, towards contemporary synthesized antibiotics, presents a pervasive and concerning global predicament.When subjected to adverse conditions, bacteria engage in the excretion of specific compounds or toxins as a countermeasure against the efficacy of antibiotics.Scientists endeavor to direct their investigations towards these stress-inducing molecules, devising strategies to hinder the defensive capabilities of these microorganisms.Nevertheless, the intricate landscape of bacterial mechanisms, encompassing both acquired and inherent traits, poses a substantial challenge in the meticulous design of compounds tailored to disrupt these targeted pathways.Moreover, the situations become worsened when misuse of the antibiotics particularly in case of recurring UTIs develops resistance in microorganisms.This phenomenon compels world researchers to develop cost-effective antibiotics with relatively no or few side effects.The rate of infectious diseases is increasing with the global increase in population.Therefore, the synthesis of new antimicrobial agents is the need of the day.The biogenic NPs can better fill this gap.We used M. piperata-derived Zinc NPs against pathogenic microbes which displayed a strong antimicrobial effect against these microbes as compared to synthetic antibiotics including ciprofloxacin, azithromycin, and clotrimazole and we further affirm that ZnO nanoparticles are more potent than medicinal plants as well as conventional and commercial drugs.As a result, we believe that Zinc Nanoparticles (NPs) derived from M. piperata could aid in the development of antibiotics for UTI strains that do not respond to standard treatments.However, more research is needed to determine how these naturally produced NPs work effectively.
FTIR investigates the presence of functional groups of various phytochemicals present in the biogenic nanoparticles.The spectrums showed the range in cm −1 for Mentha piperata.FTIR analysis of ZnO NPs shows that the Carboxylic functional group (COOH) was present at 3773.67 cm −1 .The peak at 2241.32 cm −1 indicates the presence of alcohol groups OH.Whereas; 1881.51 cm −1 , 1574.32, and 1451.68 cm −1 specify the presence of C-H and N-H stretching.Other peaks displayed at 744.24 cm −1 , 686.54 cm −1 , and 523.72 cm −1 show numerous functional groups such as O-H, C-O of carboxylic anions and C-N nitrite respectively as shown in (Fig.2).The FTIR spectrums for aqueous extracts of M. piperata showed the range in cm −1 with spectral wavelength between 400 and 4000 nm and investigate the presence of functional groups of various phytochemicals present in the aqueous extracts of M. piperata.The FTIR peaks and stretching bands commonly found in extracts, -CH (Alkyl) group at 3414.12 cm −1 and -CH (Alkene) group at 2918.40 cm −1 .The wavelength at 2850.88 cm −1 indicated the presence of alkene (C=C) group.The wavelength 1618.33 cm −1 indicated the presence of Alpha-beta unsaturated ketone (C=C) group, 1419.66 for R-NO2 group, 1305.85 cm −1 phenol (O-H) group and 1261.49cm −1 indicate Alkyl-aryl-ether (C-O) group, and 1084.03cm −1 depicted the presence of aliphatic (C-O) group.The wavelength 1037.74 cm −1 revealed the presence of Anhydride (CO-O-CO) group and the wavelength at 871.85 cm −1 indicated the occupation of Alkene (C=C) group.Other peaks displayed habitation of bending at 713.69 cm −1 , 669.32 cm −1 , 613.38 cm −1 , and 534.30cm −1 revealed the presence of numerous functional groups such as; O-H, C-O, C-N, and C-I of carboxylic anions, nitrite and

Figure 1 .
Figure 1.UV-Visible spectrometry of biogenic Zinc NPs prepared from Mentha piperata.The antimicrobial ZnO NPs absorption spectra appear in the range of 280 to 320 nm which represent the fine structure of NPs and high band gaps energy arising from strong electronic structures of defects and inner shell electron transitions and finally quantum confinement effect.

Figure 2 .
Figure 2. FTIR spectrum of ZnO NPs prepared from Mentha piperita.FTIR was performed by potassium bromide (KBr) pellet method with a settlement and examine range of 4 cm −1 , from 400 to 4000 cm −1 .The infrared (IR) ingestion spectra uncovered the presence of various functional groups on the particle's surface.

Figure 3 .
Figure 3. FTIR spectrum of Mentha piperita extract performed by potassium bromide (KBr) pellet method with a settlement and examines range of 4 cm −1 , from 400 to 4000 cm −1 .The infrared (IR) ingestion spectra uncovered the presence of various functional groups in the extract of Mentha piperita.

Figure 4 .
Figure 4. Peaks of biogenic ZnO NPs through XRD.The XRD pattern of ZnO NPs was recorded on an X-ray diffractometer at the range of 20 from 10 to 80 with a scan rate of 20/min utilizing Cu Alpha (lambda = 1.5406A) radiation with an accelerating voltage of 40 kV.Different stages present in the samples were assessed by means of X-pert high score programming having a search match office.

Figure 5 .
Figure 5. SEM of the prepared biogenic ZnO NPs.SEM (model Jeol JSM-6480LV) was utilized at 20 kV for 4 min.The ZnO NPs have pyramidal symmetry of the particle ranges 20-30 nm in size.

Figure 6 .
Figure 6.TEM micrographs along with SAED pattern of the prepared biogenic ZnO NPs.TEM (JEM-2100; manufacturer JEOL, Japan) was utilized at 200 kV.The synthesized ZnO NPs ranges 15 to 27 nm with 18 nm average in size.

Figure 7 .
Figure 7. Zeta potential measurement window revealed 50 to 100 nm of the effective electric charge on the synthesized ZnONPs surface and the electro mobility.This depicts the positive charge at ZnONPs.

Figure 8 .
Figure 8. EDX analysis of Zinc Oxide nanoparticles.The specimen was resolved through EDX programming that applies ZAF correction and carries out the incorporation of the top of the plate to get the last measurement of the element in the sample.

Figure 9 .
Figure 9. Pictorial presentation of bacterial and fungal growth inhibition by ZnO nanoparticles (a) effect of different concentrations of ZnO NPs against resistant Pseudomonas aeroginosa (b) Candida albicans (c) Proteus mirabilis (d) ciprofloxacin against Pseudomonas aeruginosa (e) clotrimazole against Candida albicans and (f) azithromycin against Proteus mirabilis.

Figure 10 .
Figure 10.Antimicrobial potential of different concentrations (25, 50, and 100 ug/ml) of biogenic zinc oxide nanoparticles, zinc oxide nanoparticles in combination with plant extracts and plant extracts alone against pathogenic microorganisms.All the values are mean ± SE.Bars labeled with different letters (LSD values) exhibited significant variation (α < 0.05).

Figure 11 .
Figure 11.Different mechanisms of action of NPs in bacterial cells.The biogenic ZnO NPs interact with MDR microbes and activate oxidative stress mechanisms.It also inhibits enzymes and proteins as well as epigenetic changes in DNA and ultimately causes cellular leakage of the membranes leading to the death of the microorganisms.

and Transmission electron microscopy (TEM), Selected Area Electron Diffraction (SAED) and Zeta potential.
The structural observations of ZnO NPs were carried out by SEM (model Jeol JSM-6480LV).The SEM was utilized at 20 kV for 4 min.The nanoparticles were settled on stub with the assistance of adhesives, covered with gold.The structural observations of ZnO NPs were also carried out by TEM (JEM-2100; manufacturer JEOL, Japan).The TEM was utilized at 200 kV voltage at a magnification of 1,500,000X with a resolving power of 1.4 Å.The charge measured on the surface of the NPs calculated through a test known as Zeta potential (Zetasizer Blue; Malvern Panalytical, UK).As the NPs carry and float a loop of opposite electric charge.So, the NPs surface net charge is screened out using the abundance of ions carrying the opposite charge near the body of NPs.The analysis of SEM, TEM, SAED and Zeta potential observations were carried out in the central resource's laboratory (CRL), University of Peshawar.