Efficacy of Acacia nilotica aqueous extract in treating biofilm-forming and multidrug resistant uropathogens isolated from patients with UTI syndrome

Escherichia coli is the dominant bacterial cause of UTI among the uropathogens in both developed and developing countries. This study is to investigate the effect of Acacia nilotica aqueous extract on the survival and biofilm of isolated pathogens to reduce UTIs diseases. A total of 170 urine samples were collected from Luxor general hospital and private medical analysis laboratories in Luxor providence, Egypt. Samples were screened for the incidence of uropathogens by biochemical tests, antibiotics susceptibility, detection of virulence, and antibiotic-resistant genes by multiplex PCR, biofilm formation, and time-killing assay. Escherichia coli is by far the most prevalent causative agent with the percentage of 73.7% followed by Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeuroginosa, and Acinetobacter baumanii. Isolates were multidrug-resistant containing blaTEM, blaSHV, blaCTX, qnrs, and aac(3)-Ia resistant genes. All isolates were sensitive to 15–16.7 mg ml−1 of Acacia nilotica aqueous extract. Time killing assay confirmed the bactericidal effect of the extract over time (20–24 h). A high percentage of 3-Cyclohexane-1-Carboxaldehyde, 2,6,6-trimethyl (23.5%); á-Selinene (15.12%); Oleic Acid (14.52%); Globulol (11.35%) were detected among 19 bioactive phytochemical compounds in the aqueous extract of A. nilotica over the GC-mass spectra analysis. The plant extract reduced significantly the biofilm activity of E. coli, K. pneumoniae, P. mirabilis, and P. aeuroginosa by 62.6, 59. 03, 48.9 and 39.2%, respectively. The challenge to improve the production of A. nilotica phytochemicals is considered a very low price for the return.

GC-MS analysis. The analysis and extraction of plant material play a significant role in the progress, reconstruction, and quality control of herbal formulations. Hence one of the important aims in the present study was to find out the bioactive compounds present in the aqueous extract of A. nilotica by using Gas chromatography-Mass spectroscopy. This shows the presence of 19 bioactive phytochemical compounds in the aqueous extract of A. nilotica. The highest percentage content of the compounds are as follows: 3-Cyclohexane-1-Carboxaldehyde, 2,6,6-trimethyl (23.5%); á-Selinene (CAS) (15.12%); Oleic Acid (14.52%); Globulol (11.35%). Other active compounds with their peak number, concentration (peak area%), and retention time (RT) are presented in (Table 4; Fig. 3).

Efficacy of A. nilotica aqueous extract as an antimicrobial agent. Antibacterial activity of A. nilot-
ica aqueous extract against the isolated uropathogens was analyzed by minimal inhibitory concentrations (MIC) by determining the bacterial viability using a colorimetric INT-formazan assay. Thus, we additionally determined the minimal bactericidal concentrations (MBC) which confirmed the killing of the isolated uropathogens over time. The results showed a reproducible and effective antibacterial effect against all isolated uropathogens (Preventing INT color change). Where, the concentration of 11.7 mg ml −1 was enough as MIC for all tested Table 1. Incidence of isolated uropathogenic bacteria in urine samples. Total number of examined urine samples was 170 sample, they were 110 sample from females and 60 samples from male. Number of positive samples was 133 samples comprised from 101 sample from females and 32 sample from males. a Different isolated uropathogens from urine samples. b Number of positive isolates from total number of positive samples. c Percentage of positive isolates from total number of positive samples. d Colony forming unit per ml for each isolated bacterium.    (Fig. 5a, b). In the case of P. mirabilis, P. aeruginosa and A. baumannii killing curve were also recorded a steady decrease of OD 595 over time starting after 8 h exposition with no viable microorganism in the initial inoculums could be observed after 24 h. Significant reduction starting at 16 h of treatment for all isolates. Positive control had a continuous increase to 24 h (Fig. 5c-e). Based on these results, Time-kill kinetic profile indicates that A. nilotica extracts exhibited bactericidal actions against all uropathogenic isolates (Fig. 5).

Discussion
UTIs are considered one of the most common groups on infections in humans worldwide that upset kidney, pyelonephritis, bladder, and cystitis 11 . As stated by the CDC, UTIs are the greatest common bacterial infection demanding medical care, resulting in 8.6 million ambulatory care visits in 2007 12 . It is an infection of the urinary tract with a pathogen causing inflammation and occasionally life-threatening 13 . In the current study, although all patients were showing some or all of UTIs symptoms like burning feeling during urination, frequent urge for  (Table 1). This is possible because UTI symptoms are not a dependable indicator of disease. So, urine culture is necessary for the diagnosis of UTI for confirming the presence of bacteriuria 14 .
The study also verified a lower UTI rate of 7.2% in males comparing with 92.8% in females In agreement with 15 , who stated that UTIs are common in women than men with a ratio of 8:1. A low rate of infection in males may be due to the occurrence of antimicrobial substances in prostatic fluid. Also, maybe a long urethra (20 cm) that provides a distance barrier that eliminates microorganisms from the bladder 16 . The principal step for effective treatment of UTIs is to classify the type of infection, such as acute uncomplicated cystitis or pyelonephritis, acute complicated cystitis or pyelonephritis, catheter associated-UTI, asymptomatic bacteriuria (ASB), or prostatitis depending on identification of causing organism for describing defective antibiotic 17,18 . About 95% of uncomplicated UTIs are mono-bacterial and E. coli is the major causing agent of uncomplicated UTI, which accounts for up to 75-90% of cases 19,20 . This study is in agreement with our study where E. coli isolated with a percentage of 73.7% from all isolated uropathogens. Klebsiella pneumoniae was the second isolated organism with a percentage of 13.5%, in agreement with 20,21 . Followed by P. mirabilis (6.7%), P. aeruginosa (4.5%), and A. baumanii (1.5%) ( Table 1). Also in agreement with 22 , who revealed that uropathogenic E. coli (UPEC) is the most common causative agent for both complicated and uncomplicated UTIs, other causative agents are involved like K. pneumoniae, P. mirabilis, P. aeruginosa and other types. Uropathogenic E. coli (UPEC) contains several virulence factors that facilitate its colonization and invasion of host cells 23,24 . Surface virulence factors (adhesions) are among the most important virulence factors 25,26 . As the main attachment factor. P fimbriae are particularly associated with pyelonephritis and cystitis which encoded by pap genes 27,28 . Other important virulence factors in UPEC are toxins (secretory virulence factors) 26 . The most important toxin is a-hemolysin (HlyA), which encoded by hly gene that has been detected among pyelonephritis and cystitis 28 . eaeA (intimin or E. coli attaching and effacing gene) 29 . Most bacteria regulate a multitude of fimbrial adhesions such as fimbriae 1 type which encoded by fimH gene that was first recognized in E. coli 30 . Our results confirmed the presence of papC, hly and fimH genes among E. coli isolates while, the eaeA gene was absent among E. coli isolates (Table 3; Fig. 1). Several factors contribute to the virulence of K. pneumoniae such as the capsular serotype, lipopolysaccharides, iron-scavenging system, and adhesions 31 . These genes include those encoding for regulators of mucoid phenotype A (rmpA) which is detected among local urinary isolates 32,33 . Other Klebsiella virulence genes such as type 1 (fimH), type 3 adhesions (mrkD), aerobactin (hydroxamate siderophore which is produced by some enterobacterial strains and TraT gene [34][35][36][37][38] . Our results confirmed the presence of TraT gene and the absence of aerobactin, fimH, and rmpA genes among K. pneumoniae isolates (Table 3; Fig. 1). Proteus mirabilis contains several virulence genes that contribute to its pathogenicity such as an atpD gene (ATP synthase beta chain) 39 . This gene detected with the percentage of 100% among P. mirabilis isolates in our study (Table 3; Fig. 1). Virulence genes in P. aeruginosa such as Pilli, exoenzyme S, endotoxin A, and phospholipase C are important for the acute phase of disease while, siderophores and pseudo-capsule of alginate are essential for chronic phase of infections 40 . Elastase (LasB gene), phospholipase C, toxin A (toxA), and exoenzyme S was assessed in P. aeruginosa isolates from UTI 41 (Table 3; Fig. 1). Our study confirmed the presence of pslA gene and www.nature.com/scientificreports/ absence of lasB, toxA, and fliC genes. Some of the most significant virulence genes of A. baumannii are colicin V production, curi fibers (csg), siderophores like aerobactin (iutA), and cytotoxic necrotizing factor (cnf) 42,43 . Acinetobacter baumannii in our study was free from these genes. However, there is always the possibility of mutation at the level of the corresponding gene, leading to the lack of its detection. Consequently, a positive PCR shows the occurrence of the virulence gene, but a negative PCR does not point to its absence [44][45][46] (Table 3).
Biofilm is an accumulation of bacteria reserved within a microbial-derived matrix, which assists their persistence 47 . It contains water passages for transporting oxygen and essential nutrients for growth. Microcolony is the main structural unit of the biofilm it may be composed of 10-25% cells and 75-90% exopolysaccharide (EPS) matrix depending on the species complex 48 . They characterized by a high degree of resistance to antibiotics and host immune defense response substances 4,49 . It also plays an essential role in the pathogenicity of several chronic human infections 50 . In our study, all isolated uropathogens were biofilm former (Fig. 4). Interestingly, the detection of latent virulence genes in the clinical urine isolates also the ability for biofilm formation confirmers the pathogenicity of these isolated uropathogens in the current study. Also has some great epidemiological outcomes to control the dissemination of infectious disease caused by these pathogens. Increasing rates of antibiotic resistance and high repetition rates impend to greatly enhance the problem that these common infections place on society 22 . In a study by 51 , they revealed that antibiotics such as ciprofloxacin and ampicillin are the most commonly recommended therapeutics for UTIs. Interestingly, our study confirmed a great resistance for all isolated uropathogens to ampicillin and ciprofloxacin. In another study by Abuhandan et al. 52 , they reported that all of the isolated uropathogens were resistant to ampicillin-sulbactam, with, high resistance rates recorded for E. coli (64.1%) They also stated that the most effective antimicrobial agents were determined to be imipenem, quinolone, and aminoglycosides. It is worth saying that our study showed 100% resistance to ampicillin-sulbactam, 60% resistance to imipenem, 100% resistance to two members of aminoglycosides (Gentamicin and Amikacin) also 100% resistance observed for one member of quinolones (ciprofloxacin) Table 2. This confirms the seriousness of the wrong use of antibiotics over the years. In the current study, the presence of multidrug-resistant genes was determinant such as bla SHV , bla CTX , bla TEM as it was determined earlier by 53,54 . These genes were detected in our isolates with a percentage of 100, 60, and 60%, respectively (Table 2; Fig. 2). Quinolone resistance is usually resulting from mutations in genes coding for chromosomally-encoded type II topoisomerases, efflux pumps, or porn-related proteins, it also can be plasmid-mediated 55,56 . The plasmid resistance determinants are qnrA, qnrB, and qnrS 56,57 . The qnrS gene was detected with percentages of 100% among all isolated uropathogens (Table 2; Fig. 2). Multidrug resistance in P. aeruginosa can be caused by regulatory mutations nalB (mexR), nfxB or nfxC (mexT) leading to overexpression of three separate RND efflux systems which causing multiple antibiotic resistance profiles 58 . In our study mexR gene was detected with the percentage of 100% among P. aeruginosa isolates ( Table 2; Fig. 2). Aminoglycosides resistant genes such as aac and aad 59 . An example of Gm resistance (Gmr) genes was aac(3)-Ia 60 . Our results confirmed a 100% resistance to aminoglycosides through the detection of aac(3)-Ia gene ( Table 2; Fig. 2).
Antibiotic resistance is one of the biggest problems that face the world. Scientists have begun to search for new safe antibiotic alternatives. Medicinal plants are a good substitute for antibiotics [61][62][63][64][65][66] . The pods of A. nilotica extract was good antibacterial agent against different bacterial pathogens 64 . Our study confirmed the greatest efficacy of Acacia nilotica extract against all isolated uropathogens with MBC of 15-16.7 mg ml −1 with the greatest MBC value obtained by P. mirabilis ( Table 3). The analysis of time killing data confirmed that A. nilotica aqueous extract kills E. coli and K. pneumoniae (within 20 h) faster than other uropathogens (Fig. 5). Acacia nilotica extract also reduces the biofilm of the tested pathogens (Fig. 4). This is could be due to the presence of some active phytochemicals such as 3-Cyclohexane-1-Carboxaldehyde, 2,6,6-trimethyl; á-Selinene; Oleic Acid; Globulol and Isochiapin that were detected in the GC-MS analysis (Table 4; Fig. 3). The antibacterial activity of crude extracts and different fractions could be largely due to the effect of the phytochemicals detected 67 . In study by 68 , the phytochemical analysis of A. nilotica pod extracts by LCMS, HPLC/DAD, and FTIR was confirmed as antibacterial agents against antibiotic-resistant strains of E. coli and Salmonella sp. Cyclohexane, for example, is considered the most potent antibacterial agent that had a reduced ability to inhibit solute transport in comparison with other active analogs 69 . The oleic acid produced by marine spp. also could be valuable as a biocontrol against gram-negative bacteria including Vibrio parahaemolyticus and might denote an influence in the clinical use 70 . Other important phytochemical components detected with a high percentage in A. nilotica aqueous extract such as á-Selinene; Globulol and Isochiapin were also recorded for their antibacterial activities [71][72][73] .
In conclusion, a new preventive measure against multidrug-resistant isolated uropathogens which confirmed by multiplex PCR, consists of the use of A. nilotica aqueous extract. Acacia nilotica considered a natural antimicrobial agent to prevent bacterial growth, biofilm formation, and decreases the dissemination of these multidrugresistant strains. However, optimizing the production of the active organic products of A. nilotica extract is a challenge that must be considered to use this compound to contrast the pathogenic action of UTIs. Also, it is recommended to make purification of A. nilotica extract to test one or more of the larger concentration of some compounds like 3-Cyclohexane-1-Carboxaldehyde, 2,6,6-trimethyl; á-Selinene (CAS); Oleic Acid; Globulol in the composition of the extract against uropathogens and performing in vivo experiments.

Ethical approval and informed consent. The study protocol was approved by the local Medical Ethics
Committees of the Medical University of Assiut, Egypt, which has been approved by the Egyptian Ministry of Higher Education and Scientific Research on 11/2009. General hospital and private medical analysis laboratories in Luxor province, Egypt ethically approved urine sampling and informed consent was obtained from all participants during the study work. The methods were carried out in accordance with the relevant guidelines and regulations, and the subjects gave written informed consent.
Scientific RepoRtS | (2020) 10:11125 | https://doi.org/10.1038/s41598-020-67732-w www.nature.com/scientificreports/ Sampling; isolation and identification of uropathogens. A total of one hundred and seventy urine samples were collected between January to June (2019) from the general hospital and private medical analysis laboratories in Luxor province, Egypt. Patients were between 8 and 86 years old, they were 110 females and 60 males. Urine samples were collected by clean catch mid-stream urine collection method into the sterile container from patients who had not received antimicrobials within the previous one week. Guidelines for proper specimen collection were given to all patients on a printed card 74 . All samples were subjected to COMISCREEN 10 SL urine test strips for a rapid-semi-quantitative determination of leucocyte (pyuria) using a leucocyte esterase test (LET) and nitrite to detect bacteriuria. Samples were examined using a light microscope/high-power (HPF) (LEICA DMLF2, China) for the presence of 10 or more white blood cells 75 . For isolation of uropathogens, samples were streaked onto MacConkey (OXOID), Eosin methylene blue (BIOWORLD, USA), and Tryptic soy agar (OXOID) plates then incubated at 37°C for 24h. Isolates were picked up and identified by standard biochemical methods [76][77][78] . CFU/ml (colony forming units) were also determined.
Antimicrobial sensitivity testing. The antibiograms for all the recovered isolates were determined as described earlier according to the Kirby Bauer disk diffusion method 79 . The susceptibility of all isolates was tested for 12 antibiotics from different groups (BIOANALYZE). The used antibiotics were Imipenem (10 μg (3)-Ia, and qnrs. Besides, the mexR gene was performed for P. aeruginosa only. The encoding enterotoxins and antibioticresistant genes (twenty-one) were performed using (forty-two) primers sets including forward and reverse. All primer sequences with corresponding references are listed in Table 5  The reaction was performed in an applied biosystem 2,720 thermal cycler. All primers amplicon sizes and cycling conditions are summarized in Table 5 32,81-97 . The products of PCR were separated by electrophoresis on 1.5% agarose gel (APPLICHEM, Germany, GmbH) in 1xTBE buffer at room temperature using gradients of 5 V/ cm. For gel analysis, 15 μl of the products were loaded in each gel slot. Gelpilot 100 bp and 100 bp plus ladders (QIAGEN, Germany, GmbH) and GeneRuler 100 bp ladder (FERMENTAS, THERMO) was used as a marker for electrophoresis to determine the fragment sizes. The gel was photographed by a gel documentation system (ALPHA INNOTECH, BIOMETRA) and the data was analyzed through computer software (AUTOMATIC IMAGE CAPTURE, USA).

Gas chromatography-mass spectrometer (GC-MS) analysis. GC-MS technique was used in this
study as described earlier by Sadiq et al. 68 , to identify the Phyto-components present in the plant extract. For preparing A. nilotica aqueous extract, 20 gm of dry pods were ground into fine powder by using an electric grinder (SOGO, China). The powder was soaked in 100 ml of hot distilled water and then cooled down with continuous stirring at room temperature by using bigger bill shaker, USA, for extraction of active ingredients 98 . The mixture was filtered then sterilized using a syringe filter equipped with a 45μ membrane filter; then kept at 4 °C. Acacia nilotica material was subjected to gas chromatography-mass spectrometer technique (GC-MS) (THERMO SCIENTIFIC TECHNOLOGIES, TRACE 1,310) with capillary column TG-5 (30 m × 250 μm × 0.25 μm) system were used. The mass detector used in split mode and helium gas with a flow rate of 1.5 ml/min was used as a carrier. The injector was operated at 230 °C and the oven temperature for the initial setup was 60 °C for 2 min. ramp 10/min. to 300 °C for 8 min. Mass spectra were taken at 70 eV, total GC running time was 35 min.

Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) by INT reduction assay.
The determination of MIC and MBC were assayed as described by 99 Table 5. Primers sequences, target genes, amplicon sizes and cycling conditions. The specific sequences that were amplified for each of the used primers (Metabion, Germany). a fimH gene was also detected for these isolates.  Survival curve of the isolated uropathogens in the presence of A. nilotica aqueous extract. An increase, both in total cell mass and cell number can readily be estimated by measuring the turbidity of a cell suspension using an instrument such as a spectrophotometer 105 . So, the microbial population at the initial and completion of the experiment isolates were grown overnight on TSA plates, suspended in TSB to an OD 595 of 0.01 then incubated with the MBC value of A. nilotica aqueous extract for each isolate at 37 °C. Adjusted culture from each isolated uropathogens at OD 595 of 0.01 served as the positive control. Approximately, 1 ml aliquot was tested from the culture medium over time (0,4,8,12,16,20, and 24 h) for monitoring the optical density of all bacterial treatments at OD 595 nm using the ' 'SPECTRONIC GENESYS 2PC" Spectronic Instruments, USA. Readings were taken three times. Results were confirmed by taking 50 µl of each treatment at OD 595 of 0.0 (complete killing) onto fresh TSA and incubation at 37 °C for 24 h (Three plates were used for each isolate).  www.nature.com/scientificreports/ Statistical data analysis. Data were analyzed using the Mann-Whitney U test or a Kruskal-Wallis test followed by post hoc Dunn's multiple comparisons. Differences were considered significant at P values of ≤ 0.05. For all statistical analyses, GraphPad Prism version 5 was used.