GC-MS Profiling and In vitro Antibacterial, Anti-Biofilm and Anti-adhesive Activities of Tamarix ericoides Rottl. Leaf Extract Against Catheter-Associated Urinary Tract Infectious Agents

GC-MS Profiling and In vitro Antibacterial, Anti-Biofilm and Anti-adhesive Activities of Tamarix ericoides Rottl. Leaf Extract Against Catheter-Associated Urinary Tract Infectious Agents | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Confirmatory Results GC-MS Profiling and In vitro Antibacterial, Anti-Biofilm and Anti-adhesive Activities of Tamarix ericoides Rottl. Leaf Extract Against Catheter-Associated Urinary Tract Infectious Agents Muhammad Musthafa Poyil , View ORCID Profile Mohammed H Karrar Alsharif , Mahmoud H. El-Bidawy , Mohammed Saad Alqahtani , Tarig Gasim Mohamed Alarabi , Ahmed Abdullah Albadrani , Alaa Azhari Mohamed Hamid , Abdullah Mohammed Radwan Arafah , Ahmed Abdel Tawab , Saad Alqasem , Ali Al-Gonaim doi: https://doi.org/10.1101/2025.01.21.634068 Muhammad Musthafa Poyil 1 Department of Basic Medical Sciences, College of Medicine, Prince Sattam bin Abdulaziz University , Al-Kharj, 11942, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: m.poyil{at}psau.edu.sa Mohammed H Karrar Alsharif 1 Department of Basic Medical Sciences, College of Medicine, Prince Sattam bin Abdulaziz University , Al-Kharj, 11942, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mohammed H Karrar Alsharif Mahmoud H. El-Bidawy 1 Department of Basic Medical Sciences, College of Medicine, Prince Sattam bin Abdulaziz University , Al-Kharj, 11942, Saudi Arabia 2 Department of Physiology, Faculty of Medicine, Cairo University , Kasr Al-Aini, Cairo, Egypt Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mohammed Saad Alqahtani 3 Department of Internal Medicine, College of Medicine, Prince Sattam bin Abdulaziz University , Al-Kharj, 11942, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tarig Gasim Mohamed Alarabi 4 Department of Anatomy, College of Medicine, King Khalid University , Abha, 61421, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ahmed Abdullah Albadrani 3 Department of Internal Medicine, College of Medicine, Prince Sattam bin Abdulaziz University , Al-Kharj, 11942, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alaa Azhari Mohamed Hamid 5 College of Medicine, Khartoum University , Khartoum – 77121, Sudan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abdullah Mohammed Radwan Arafah 1 Department of Basic Medical Sciences, College of Medicine, Prince Sattam bin Abdulaziz University , Al-Kharj, 11942, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ahmed Abdel Tawab 6 Department of Microbiology & Immunology, Faculty of Medicine, Cairo University, Al-Azhar University , Cairo, 11884, Egypt Find this author on Google Scholar Find this author on PubMed Search for this author on this site Saad Alqasem 7 Department of Surgery, College of Medicine, Prince Sattam bin Abdulaziz University , Al-Kharj, 11942, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ali Al-Gonaim 7 Department of Surgery, College of Medicine, Prince Sattam bin Abdulaziz University , Al-Kharj, 11942, Saudi Arabia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract Catheter-associated urinary tract infection (CAUTI) is one of the most important nosocomial infections among hospitalized patients and causes serious complications due to the development of drug-resistant, biofilms forming strains of microorganisms resulting in treatment challenges. As the chemical catheter-coating agents often fail to prevent biofilm formation, the researchers are looking for compounds of natural origin, and the phytocompounds with multiple modes of action pose as a promising option. Thus, the present study investigated the antibacterial anti-biofilm potentials of the phytocompounds in one of the least explored medicinal plants - Tamarix ericoides Rottl. and its leaf methanolic extract was analyzed against Escherichia coli – one of the most notorious multidrug-resistant, biofilm- forming uropathogens in CAUTIs. The well-diffusion method showed the antibacterial activity of methanolic leaf extract against E. coli and using the microdilution method, the minimal inhibitory concentration (MIC) of the extract against E. coli was calculated as 1 mg/ml. GC-MS profiling of methanolic fractions of T. ericoides showed the presence of eight important phytochemicals such as diethyl phthalate, ethanol, 2-[2-[(2-ethylhexyl)oxy]ethoxy]-, n-hexadecanoic acid, 9-octadecenoic acid, (E)-, 9,12-octadecadien-1-ol, (Z,Z)-octadecanoic acid, -hydroxy-3-(1,1-dimethylprop-2-enyl) coumarin and Cholestan-3,22,26-triol 16-[2- [formylthio]ethyl]- that are responsible for antibacterial activities. The killing kinetics of T. ericoides leaf extract against E. coli showed at 1 h. Further, the antibiofilm activity of T. ericoides leaf extract against E. coli on nonliving surfaces was analyzed and quantified by crystal violet assay. T. ericoides leaf extract reduced mature biofilms of E. coli by 81%, 85%, and 89 % after treatment with 1X MIC (1 mg/ml), 2X MIC (2 mg/ml), and 3X MIC (3mg/ml) concentrations of the extract respectively. This was further confirmed using SEM analysis wherein biofilm reduction was observed when compared to untreated. The catheter coating with T. ericoides leaf extract showed antibacterial activity in the in vitro bladder model and was quantified based on colony count. The CLSM reveals the anti-adhesive property of T. ericoides leaf extract on the catheter surface which reduced the biofilm formation and biofilm thickness when contacted with E. coli cells. Also, 82% of dead cells were observed in the FDA and PI combination. Further, SEM showed the impact of T. ericoides leaf extract on E. coli cell morphology as the cells displayed damage including cell shrinkage. Furthermore, the leaf extract was found to be non-toxic to normal cells. Based on the findings, the authors recommend further investigation to develop T. ericoides leaf extract as a potential catheter coating agent to manage CAUTIs. 1. Introduction Urinary tract infections are among the most commonly encountered nosocomial infectious conditions in hospitalized patients and these infections are mainly attributed to the presence of an indwelling urethral catheter [ 1 ], which also cause other complications like ascending UTIs that are vital in urology [ 2 , 3 ]. The catheters create serious complications like mechanical traumas including symptomatic bacterial infection, urinary leakage, perforation, partial urethral damage, catheter toxicity, hypersensitivity, and anaphylaxis leading to increased lengthy stays and high [ 4 , 5 ]. The urinary catheters are partially stretchy hollow tubes in structure that are intended to drain the liquid waste from the bladder. Unfortunately, catheters are susceptible to infection because they have direct contact with uropathogens and permit them from the outside environment to the urinary tract which is normally a sterile area damaging the bladder host defense mechanisms [ 6 ]. Among the whole device-associated infection, CAUTI represents the second most important infection which ranges up to 40% [ 7 , 8 , 9 ] in hospitalized patients. The significant occurrence of this infection originates from the urinary catheter which allows the opportunistic uropathogens entry through the lumen and makes bacterial adhesion and colonization lead to serious complications such as bladder stones, pyelonephritis, encrustation, bacteriuria, endotoxic shock, and septicemia [ 10 , 11 , 12 ]. Moreover, catheter usage is short or long-term, the patients are easily getting infected and ready to develop biofilms on the inner and outer catheter surfaces giving survival tactics to bacteria [ 13 , 14 ]. These biofilms are multifaceted distinguished groups encompassing many bacterial associations and produce extracellular polymeric substances that help bacteria to escape from antibiotics and continue to attach on biotic and abiotic surfaces resulting in treatment critical but also improving the development of resistant strains [ 15 , 16 ]. Several organisms including bacteria and fungi are commonly contaminating the catheters and also, responsible for biofilm development resulting in treatment challenges that lead to high morbidity and mortality. Amongst, one of the most important gram-negative bacteria, Escherichia coli is a frequently isolated organism with the ability to form biofilm on catheter surfaces [ 17 , 18 ]. Generally, CAUTIs are curable but in certain circumstances such as recurrent or inappropriate use of antibiotics increases infection severity [ 19 , 20 , 21 ]. This alarming situation stimulated the search for new antimicrobial drug development with potent antibiofilm as well as anti-adhesive properties to combat CAUTI-causing organisms. In the past several decades, natural resources have been the better choice for antimicrobial discovery owing to their contribution to pharmaceutics such as antimicrobial, anticancer anti-inflammatory, etc. [ 22 , 23 ]. The report from WHO says, that most of the global population are seeking plant-based traditional medicine for primary health care [ 24 ]. Tamarix ericoides is the least studied medicinal plant from the family of Tamaricaceae was commonly used to treat diabetes, gastrointestinal disorders, wounds, and dental problems and also, had anti-inflammatory properties [ 25 , 26 ]. Based on this indication, our study investigated the antibacterial, anti-biofilm activity of T. ericoides methanolic leaf extract against E. coli involved in CAUTI. 2. Materials and methods 2.1. Chemicals and Inoculum Preparation The chemicals such as FDA, and PI were purchased from Sigma Aldrich, from Sigma Aldrich, Louis, MO, USA and the Mueller-Hinton (MH) broth and rifampicin were purchased from Hi Media, Mumbai, India. The culture Escherichia coli was obtained from the American Type Culture Collection (ATCC 25922). The overnight E. coli culture grown in MHB adjusted to 10 6 CFU/ml was used throughout the study and rifampicin and methanol were used as positive and vehicle control respectively. 2.2. Preparation of T. ericoides Leaf Methanolic Extract The methanolic extract of the collected, cleaned air-dried leaves of T. ericoides was prepared as per the standard protocol [ 27 ]. Briefly, in a new cellulose thimble, about 20 g of fine powder was filled and placed inside the Soxhlet apparatus. Once the methanol was added, the reaction continued for many hours until a clear solution was obtained. The solvent-evaporated product was used for further investigations. 2.3. GC-MS Profiling of T. ericoides Leaf Methanolic Extract To analyze the bioactive fraction from the methanolic extract of T. ericoides leaf, GC-MS profiling as per standardized procedure [ 28 ]. All the programs like temperature, gas, etc., were initiated, and using a micro syringe, the methanol extracts (1 µl) were injected into the GC-MS. The scanning was continued for 30 mins. Later, the compounds separated were eluted from the column, and with the help of a detector, it was detected using the detector. Each of the peaks in the chromatogram represents an individual molecule present in the extract and it then enters into the mass spectroscopy detector. The identification of compounds was completed by comparing the retention indices and the mass spectra patterns available in the computer library. 2.4. Antibacterial Activity of T. ericoides Leaf Methanolic Extract The methanolic leaf extract of T. ericoides antibacterial activity was determined against E. coli using the well-diffusion method [ 29 ]. Briefly, the well made on a sterile MHA plate swabbed with overnight E. coli culture was allowed to receive two different concentrations of methanolic leaf extract and incubated. The zone inhibition around the well indicates the antibacterial activity of T. ericoides leaf extract against E. coli . 2.5. MIC Determination for T. ericoides Leaf Methanolic Extract To calculate the MIC of methanolic T. ericoides leaf extract against E. coli , the well diffusion method [ 30 ]. Shortly, in MHB broth, 4 mg/ml of T. ericoides leaf extract was serially diluted until 0.03mg/ml was received overnight E. coli culture. The Optical density of turbidity was measured in all the wells at 600 nm after incubation. 2.6. Killing Kinetics of T. ericoides Leaf Methanolic Extract To determine the killing kinetics of T. ericoides leaf extract against E. coli , the killing assay was performed [ 30 ]. Briefly, E. coli (1x10 6 CFU) overnight culture treated with 1 mg/ml leaf extract was incubated at different time points including 0 h, 1 h, 2 h, 4 h, 6 h, and 12 h. Later, the samples (100 µl) collected from each time point were serially diluted (10-fold), and a spread plate was done for each sample to calculate viable cells by counting CFUs. 2.7. Effect of T. ericoides Leaf Extract on Biofilm Formation To study the influence of T. ericoides leaf extract on E. coli biofilm formation, the crystal violet assay was used [ 30 ]. Briefly, the E. coli biofilm formation on a polystyrene plate surface using MHB broth was allowed for 5 days in the presence of various concentrations (4 mg/ ml to 0.03 mg/ml) of T. ericoides leaf extract. Later, the formed biofilm washed with phosphate buffer saline (PBS) was fixed with methanol sometimes followed by crystal violet staining. The stained biofilm was detained using acetone and ethanol mixture and the end product was measured at 570 nm. 2.8. Effect of T. ericoides Leaf Extract on Mature Biofilm The qualitative and quantitative T. ericoides effect on mature E. coli biofilms were studied as per the reported procedure [ 30 ]. In brief, the effect of leaf extract on the mature biofilm of E. coli was studied qualitatively by allowing the E. coli cells to grow on Whatman No.1 filter paper strips for 5 days and treated with 1 mg/ml of leaf extract for 1 h. Later, the attached biofilm on the filter paper strip after washing was fixed with glutaraldehyde dehydrated with varying ethanol gradients and air dried. Then, the gold-coated air-dried filter paper strip was analyzed for SEM images using Supra 55, Carl Zeiss. Similarly, the extract effect on mature E. coli biofilm was studied quantitatively using the crystal violet staining method as performed before. In short, on polystyrene surfaces the E. coli was allowed to form biofilm for up to 5 days and treated with 1 mg/ml (1X MIC), 2 mg/ml (2X MIC), and 3 mg/ml (3X MIC) for 24 h. The PBS wash was given to remove non-adherent cells followed by crystal staining with methanol-fixed biofilm. The destained final purple color product was read at 570 nm. 2.9. Antibacterial Activity of T. ericoides Leaf Extract-Coated Catheters To investigate the antibacterial effect of T. ericoides leaf extractcoated catheter against E. coli , an in vitro bladder model [ 32 ] analysis was performed. The air-dried sterile small catheter tube coated with leaf extract was placed over the prepared MHA plates swabbed with overnight E. coli culture and incubated for clear zone inhibition around the tube which demonstrates the antibacterial activity of T. ericoides leaf extract coated catheter against E. coli . 2.10. Quantification of Bacterial Load on the Bladder Model To quantify the bacterial load from T. ericoides leaf extract-coated catheter against E. coli, the viable bacterial count method was followed as mentioned before [ 33 ]. In short, the extract-coated and non-coating small sterile catheter tube was allowed into contact with E. coli culture for 24 h. Later, the catheter tube was transferred to a fresh centrifuge tube and the tube was shaken vigorously to dislodge the attached cells followed by 10-fold serial dilution for both samples. Then, 100 µl of treated and untreated samples were used to calculate viable cell count based on CFU count, and another 100 µl samples from both samples were used to measure turbidity at 600 nm to calculate growth percentage. 2.11. Visualization of Bladder Model To visualize the non-adhesive property of T. ericoides leaf extract-coated catheters against E. coli, confocal microscopy was used [ 33 ]. Briefly, a small sterile catheter tube coated with leaf extract was permitted to form biofilm on the catheter surface by submerging the catheter tube into E. coli culture containing broth for five days followed by PBS wash. The washed tube was stained with fluorescein diacetate (FDA, 40 µl from 5 mg/ml) for10 mins followed by propidium iodide (PI, 20 µl from 1 mg/ml) for 5 mins and the images were observed using CLSM to calculate live/dead cell percentage. 2.12. Effect of T. ericoides Leaf Methanolic Extract on Cell Morphology To understand the effect of T. ericoides leaf extract on E. coli cell morphology, scanning electron microscopy analysis was employed [ 31 ]. Briefly, E. coli cells grown on Whatmann No.1 filter paper were treated with 1mg/ ml of leaf extract for 1h followed by PBS wash to remove unattached cells. The glutaraldehyde fixation was done for washed paper strips and dehydration with ethanol gradient. The strips were observed after gold coating for morphological changes and the changes were captured using SEM (Supra 55, Carl Zeiss, Wetzlar, Germany). 2.13. Cytotoxicity of T. ericoides Leaf Methanolic Extract To study the T. ericoides leaf extract’s cytotoxic effect on L 929 cells, the MTT assay [ 30 ] was conducted. In short, the cells grown in Dulbecco’s Modified Eagles Medium (DMEM) with 10% fetal bovine serum were reacted with various concentrations of T. ericoides leaf extract (0.5, 1, 2, 3, and 4 mg/ml) for 24 h followed by formazan formation was observed after the addition of MTT solution. Later, the formazan crystals dissolved were read at 570 nm after adding DMSO, and the cell viability percentage was calculated using the following formula. Cell viability percentage= [(Treated cells OD)/ (Untreated cells OD)] ×100 2.14. Statistical Analysis The mean and standard deviations were calculated for all the experiments like MIC determination, extract effect on biofilm formation, and biofilm eradication. Statistical significance was carried out for quantification of bacterial load and live dead assay using student t -test and also, the significance was predicted when p-value ≤0.05. 3. Results 3.1. GC-MS Profiling of T. ericoides Leaf Methanolic Extract The GC-MS profiling of extracted leaf extract of T. ericoides analyzed for the identification of various compounds present in the methanolic fraction is presented in Figure 1 . The compound structure was analyzed based on the fragmentation pattern of mass and the spectral data comparison with chemical profiles located in the National Institute of Standards and Technology (NIST) library. As seen in Figure, the chromatogram of eight important peaks represents eight individual compounds such as Diethyl Phthalate, Ethanol, 2-[2-[(2-ethylhexyl)oxy]ethoxy]-, n-Hexadecanoic acid, 9-Octadecenoic acid, (E)-, 9,12-Octadecadien-1-ol, (Z,Z)-Octadecanoic acid, - Hydroxy-3-(1,1-dimethylprop-2-enyl) coumarin and Cholestan-3,22,26-triol 16-[2- [formylthio]ethyl]- were observed in methanolic leaf extract of T. ericoides . The retention time, peak area, percentage peak area, height percentage, identified compound name, and structure are presented in Table 1 . Download figure Open in new tab Figure 1. GC-MS chromatogram of T. ericoides leaf methanolic extract. View this table: View inline View popup Table 1. Phytochemicals obtained from methanolic fractions of T. ericoides leaf using GC-MS. 3.2. Antibacterial Activity of T. ericoides Leaf Methanolic Extract The antibacterial activity of methanolic T. ericoides leaf extract was confirmed against E. coli and the observed growth inhibition around the well using the well- diffusion method is presented in Figure 2 . As seen in the figure, the increasing zone size was evidenced in dual concentrations (2 mg and 3 mg/ml) of leaf extract demonstrating the antibacterial activity was concentrations dependant. Download figure Open in new tab Figure 2. T. ericoides leaf extract antibacterial activity against E. coli. Note: V-vehicle control and P- positive control 3.3. MIC Determination for T. ericoides Leaf Methanolic Extract The lowest concentration required to inhibit E. coli growth calculated using the micro-dilution method is presented in Figure 3 . The MIC of T. ericoides leaf methanolic extract against E. coli growth was 1 mg/ml. Download figure Open in new tab Figure 3. MIC determination for T. ericoides leaf methanolic extract 3.4. Killing Kinetics of T. Ericoides Leaf Extract The investigated killing kinetics and calculated growth inhibitory effect of T. ericoides leaf extract against E. coli using a killing assay are represented in Figure 4 . The figure shows that the leaf extract-treated E. coli cells exhibited no life after 1 h but cells without treatment showed more viable cells. Download figure Open in new tab Figure 4. Killing kinetics of T. ericoides leaf extract showed no live E. coli cells after 1 h treatment. 3.5. Effect of T. ericoides Leaf Methanolic Extract on Biofilm Formation The effect of T. ericoides leaf extract on E. coli biofilm formation was studied quantitatively using crystal violet assay and the biofilm formation percentage after leaf extract treatment is displayed in Figure 5 . The Figure verified the biofilm inhibiting capacity of varying ranges of leaf extract concentrations against E. coli. Moreover, the biofilm formation was inhibited until the MIC of leaf extract and the gradual increase of biofilm formation was noted after its MIC represents the traces of extract can also able to slow down the biofilm formation. Download figure Open in new tab Figure 5. T. ericoides leaf extract influence on E. coli biofilm formation was investigated along with various leaf extract concentrations that showed biofilm inhibition until 1 mg/ml. 3.6. Effect of T. ericoides Leaf Extract on Mature Biofilm T. ericoides leaf extract effect on E. coli mature biofilm was studied qualitatively on cellulose matrices and the pictorial representation of biofilm eradication after leaf extract treatment is presented in Figure 6 . The Figure shows an SEM image of the minimal number of adherent cells on the cellulose matrix after treatment with leaf extract resulting in biofilm eradication when compared to the untreated matrix wherein abundant adherent cells were noticed on the matrix thus no biofilm eradication. In addition, three different T. ericoides leaf extract concentrations treated with E. coli mature biofilms quantified using the crystal violet method are presented in Figure 7 . The Figure shows the percentage of biofilm eradication after T. ericoides 1X, 2X, and 3X MIC concentrations treatment which reduced 81%, 85%, and 89 % of E. coli mature biofilms respectively. Download figure Open in new tab Figure 6. A qualitative study of T. ericoides leaf extract impact on biofilm eradication after treatment. A) SEM image of viable cells adherent on cellulose matrix B) Leaf extract treatment reduced the attached cells on the matrix was evidenced by SEM. Scale bar- 2µm. Download figure Open in new tab Figure 7. Quantitative representation of biofilm eradication after treatment with three concentrations of T. ericoides leaf extract. 3.7. Antibacterial Activity of T. ericoides Leaf Extract-Coated Catheters The catheter tube coated with T. ericoides leaf extract was investigated for antibacterial activity against E. coli using an in vitro bladder model and the zone formation around the catheter tube is displayed in Figure 8 . The zone formation indicated the antibacterial activity of the catheter tube coating with leaf extract. In addition, the bacterial load was quantified from a catheter tube coated with T. ericoides leaf extract using the colony counting method, and the calculated growth percentage of leaf extract is displayed in Figure 9 A-C which shows the least number of viable cells were counted in coated catheter tube when compared to uncoated catheter tube and also, the calculated growth percentage was 14% in coated catheter tube against E. coli represents the anti-adhesive property of T. ericoides leaf extract. Download figure Open in new tab Figure 8. The catheter coated with T. ericoides leaf extract antibacterial activity was investigated against E. coli and showed zone development around the catheter tube. Download figure Open in new tab Figure 9. Bacterial load quantified from coated and uncoated catheter tube against E. coli . A) More number of CFUs found in the uncoated catheter tube B) Leaf extract coated catheter tube revealed the less CFUs C) Graph denotes the growth percentage after coating the leaf extract. *** Highly significant 3.8. Visualization of Bladder Model The anti-adhesive property of T. ericoides leaf extract-coated catheter tube was visualized after five days of contact with E. coli cells through CLSM and the calculated percentage of live/dead cells is mentioned in Figure 10A-D . The catheter tube without coating was contacted with E. coli cells for 5 days after being stained with fluorescein diacetate (FDA, binds to live cells which emit green fluorescence) and propidium iodide (PI, binds to DNA of membrane damaged cells emit red fluorescence) mentioned in Figure 10A . Figure 10B denotes the three-dimensional view of E. coli biofilm thickness (25µm) on a catheter tube. As seen in Figure 10C , high red fluorescence was observed on the coated catheter which represents the leaf extract damaged the cell membrane and binds to DNA. As indicated in Figure 10D , catheters coated with leaf extract have the antiadhesive property against E. coli cells on the catheter surface which was evidenced through biofilm thickness reduction in three-dimensional structure (14 µm) when compared to uncoated catheters. Moreover, the FDA and PI combination provides a pictorial representation of live/ dead on the catheter surface, and also, the percentage of live/ dead cells calculated based on FDA and PI in combination is represented in Figure 11 . T. ericoides leaf extract-coated catheter displayed 82% of dead cells after treatment. Download figure Open in new tab Figure 10. Confocal Microscopy of the catheter tube. A) Uncoated catheter tube reveals biofilm formation on their surface after 5 days of contact with E. coli B) Three-dimensional view of biofilm formation on catheter surface which represents 25 µm thickness C) Catheter coated with T. ericoides showed biofilm eradication D) Three-dimensional structure revealed the reduction in biofilm thickness till 14 µm. Download figure Open in new tab Figure 11. Live and dead E. coli cells percentage from catheter tube coated with leaf extract and uncoated tube was calculated and showed 82% of dead cells after treatment. 3.9. Effect of T. ericoides Leaf Extract on Cell Morphology The SEM showed the changes that occurred in E. coli cell morphology during contact with the T. ericoides leaf extract for 1 h examined and the attained images are presented in Figure 12 . As monitored in Figure, the cell shrinkage was noted due to internal cell leakage when treated with leaf extract; whereas undamaged cell structure was observed on untreated E. coli cells. Download figure Open in new tab Figure 12. T. ericoides leaf extract effect on E. coli cell morphology. A) Untreated E. coli cells with undamaged morphology B) Red arrow indicates cell shrinkage after T. ericoides leaf extract. 3.10. Cytotoxicity of T. ericoides Leaf Extract T. ericoides leaf extract cytotoxicity studied on L 929 cells after treatment with varying concentrations is presented in Figure 13 . The cell viability percentage after treatment was calculated for all the concentrations and the cell viability observed at 1 mg/ml was 91% proving that the leaf extract was not toxic to L 929 cells compared to untreated cells. Download figure Open in new tab Figure 13. T. ericoides leaf extract cytotoxicity was studied on L 929 cells A) Untreated cells B) Cells treated with Extract C) The cell viability percentage calculated after varying concentrations denotes leaf extract was not cytotoxic to normal cells. 4. Discussion Device-associated infections, particularly CAUTIs are a significant medical complication that occurs in hospitalized patients for various reasons. Generally, CAUTI is curable but sometimes the treatment process is ineffective due to biofilm-forming organisms such as E. coli an important uropathogen, and also, overuse of antibiotics makes treatment challenges as well as resistant strains development. This dreadful situation prompted us to find an alternative antibacterial agent immediately to fight against CAUTI-causing organisms. Hence, our study investigated the antibacterial activity of methanolic T. ericoides leaf extract against E. coli and found antibacterial activity using very low concentrations. Our study was supported by a recent work wherein Tamarix ericoides Rottler, an unfamiliar medicinal plant was extracted by various solvents like methanol, ethanol, aqueous solutions petroleum ether, etc., and studied the antimicrobial activity against Bacillus subtilis, Salmonella typhi, E. coli, and C. albicans and found the potent antimicrobial activity in methanol extract when compared to other solvents [ 34 ]. Similarly, the antimicrobial activity of T. nilotica (Ehrenb) Bunge from the Tamaricaceae family was investigated against Klebsiella pneumoniae, and strong antibacterial activity was observed in n-butanol fractions [ 35 ]. Likewise, numerous reports investigated T. aphylla from Saudi Arabia’s medicinal plant’s antimicrobial activity against various human pathogens. The antimicrobial activity of both methanolic and ethanolic leaf extract against E. coli , B. subtilis , S. typhi , S. aureus , Aspergillus flavus , and C. albicans showed potent activity and suggested that the solvents had no differences in their MICs against test pathogens [ 36 , 37 , 38 ]. In addition, different species such as T. gallica and T. ramosissima from the Tamaricaceae family were investigated against human pathogens and found that they had strong activity against gram-negative organisms rather than gram-positive and fungi owing to the biomolecules present in the extracts [ 39 , 40 , 41 , 42 ]. Similarly, the antimicrobial activity was further investigated by killing kinetics against E. coli to determine the dose of leaf extract for various studies. The phytochemicals present in the methanolic fraction of T. ericoides leaf extract were investigated to find out the phytochemicals such as diethyl Phthalate, Ethanol, 2-[2-[(2-ethylhexyl)oxy]ethoxy]-, n-Hexadecanoic acid, 9- Octadecenoic acid, (E)-, 9,12-Octadecadien-1-ol, (Z,Z)- Octadecanoic acid, -Hydroxy- 3-(1,1-dimethylprop-2-enyl) coumarin and Cholestan-3,22,26-triol 16-[2- [formylthio]ethyl]- in methanolic leaf extract of T. ericoides and these are responsible for antibacterial activity against E. coli . Many reports say diethyl Phthalate, 9- Octadecenoic acid, and coumarin showed antimicrobial activity against many important including Staphylococcus aureus, Pseudomonas aeruginosa , Candida albicans, [ 43 , 44 , 45 , 46 , 47 , 48 , 49 ]. The phytochemicals profiling of 6,10,14-trimethyl-2- pentadecanone, dodecanoic acid, and octadecane are major compounds along with many minor compounds were reported in hexane fractions of T. aphylla [ 50 ]. The three major compounds such as hispidulin, isorhamnetin, and cirsimaritin were identified in Tamarix ramosissima bark extract and they were found to inhibit the formation of 2-amino-1-methyl-6-phenylimidazo[4,5- b ] pyridine suggesting their great potential beneficial effects on human health [ 51 ]. All the results represent different species that have different phytochemicals for different applications. In addition, T. ericoides leaf extract antibiofilm activity was investigated against E. coli an important uropathogen that can able to form biofilm formation on the catheter surface and makes treatment ineffective. Normally, the uropathogens enter into the catheter from the exterior environment resulting development of biofilm including many stages such as attachment, colony formation, and maturation which makes treatment less effective [ 52 , 53 ]. Hence, our study focused on each stage of biofilm formation to prevent biofilm formation, the T. ericoides inhibited the biofilm formation of E. coli on non-living surfaces the antibiofilm activity was proved. The antibiofilm activity was quantified by investigating the activity on mature biofilms and eradicating the biofilms after treatment. In support of this, electron microscopy revealed the T. ericoides effect on E. coli biofilms by eradicating or reducing biofilm after treatment. Similarly, a recent study evaluated dichloromethane (DCM) and ethyl acetate (EtOAc) fractions of T. nilotica antifungal activity against clinical isolates of Candida albicans and demonstrated antifungal activity with the least inhibitory concentration of 64-256 and 128-1024 µg/mL, respectively. The SEM examination revealed reduced or decreased biofilm formation DCM fraction treatment suggesting T. nilotica can be an important source antifungal agent against C. albicans [ 54 ]. Further, the formed biofilms can able to block the catheter and protect the bacteria from host defense and antibiotic treatment resulting in antibiotics failure to eliminate the bacteria present in the biofilms [ 55 , 56 ]. To prevent biofilm formation on the catheter’s inner and outer surfaces, the coating catheter with any antimicrobial agent is an excellent method [ 57 ]. Henceforth, our study proved the T. ericoides extract-coated catheter antimicrobial activity against test pathogens. It was further supported by the quantification of bacteria from a catheter coated with extract revealed minimal viable cells of E. coli when compared to an uncoated catheter which suggests that the catheter coated with T. ericoides extract prevents biofilm formation. To confirm the biofilm inhibition on the catheter surface, the coated and uncoated catheter contact with E. coli was visualized by staining with FDA and PI revealing the development of structured biofilms on the uncoated catheter surface and reduced biofilms on the coated catheter surface which strongly suggest that, the catheter coated with T. ericoides extract is efficient in preventing biofilm formation on the catheter surface. Our findings were supported by many groups wherein abundant antimicrobial agents including antibiotic combinations, fosfomycin, silver nanoparticles, polymer, and zinc oxide coated with catheter exposed the robust antimicrobial activity against S. aureus, E. faecalis , K. pneumoniae, and E. coli [ 58 , 59 , 60 , 61 , 62 ]. Apart from that, when E. coli contact with T. ericoides extract creates cell damage representing weakened cells and also osmotically unstable cells which creates cells more permeable to internal components resulting in leakage of inner components and finally cell death which was evidenced through SEM. Finally, our goal is intended for human use, hence, the T. ericoides extract toxicity was checked for safety purposes which showed no toxic effect on normal cells suggesting that, the methanolic T. ericoides extract can be a useful antibacterial agent for CAUTI infection. 5. Conclusion The methanolic leaf extract of T. ericoides was investigated against CAUTI-causing uropathogens , E. coli, and exhibited antibacterial activity at low concentrations due to the phytochemicals present in the extract which was evidenced in GC-MS analysis. The T. ericoides showed killing kinetics against E. coli and the antibiofilm activity was proved by inhibiting biofilm formation and also, eradicating mature biofilms after treatment on non-living surfaces and it was further confirmed by SEM analysis. The antiadhesive property of T. ericoides leaf extract was studied using an in vitro bladder model wherein the antibacterial activity was determined and it was further quantified based on colony count. The CLSM reveals the visual effect of T. ericoides leaf extract on the catheter surface which suggests the antiadhesive property of leaf extract against E. coli. T. ericoides leaf extract was not toxic to normal cells. Based on the above findings, the T. ericoides leaf extract can be a potent coating antibacterial agent against E. coli . Conflict of interest The authors declared that the present study was performed in the absence of any conflict of interest. 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