Effect nanoparticles zirconium on bacteria growth multidrug resistance pseudomonas aerginosa isolated from burns patients

. A total of 153 samples (swabs) were taken from burned patients between the ages of 2 and 75, representing a variety of age groups and body regions. All samples were obtained from Baghdad's government hospitals. Beginning in early September 2022 and ending in early January 2023, (48/153) isolates (31.37%) were identified as P. aeruginosa Testing for antibiotic susceptibility It was conducted using various antibiotic classes ( 8 antibiotics), which were assessed using the VITEK 2 compact system. The results revealed that P. aeruginosa isolates were resistant to ceftazidime and cefepime (100%) in the same rate, the resistance to Tobramycin and Ciprofloxacin was (77.08), and the maximum sensitivity to Colistin was ) 79.1) and resistance to imipenem, meropenem, and Levofloxacin was (52.08%), (79.18%), and (72.92%), respectively. In this study used zirconium nanoparticles for inhibition growth bacteria. The efficacy of the synthetic nanoparticles against P. aeruginosa was tested as five different concentrations (1,2.5,5,10 and 15) mg/ml were adopted, and concentrations of (5,10 and 15) mg/ml showed efficacy in inhibiting bacterial growth while (1 and 2.5) mg/ml was not given any effectiveness, the results of minimum inhibitory concentration was (5) mg\ml.


Introduction
Burns were a widespread health issue around the world because they result in the rupturing of the skin, the body's first line of defense against infection. As a result, the patient is exposed to numerous microbes, including the bacterium Pseudomonas earuginosa, which is known to be antibiotic-resistan [1]. Antimicrobial resistance to several different drug families, including carbapenems, fluoroquinolones, cephalosporins, and aminoglycosides, has been created and spread by MDR P. aeruginosa. This resistance makes treatment more difficult, due to its ability to develop resistance through a number of mechanisms, including efflux pumps, the secretion of alginate during biofilm formation, the production of -lactamases and aminoglycoside modifying enzymes, and the production of penicillin-binding proteins, P. aeruginosa infections are difficult to treat [2]. Nanotechnology is a novel technique that provided multiple methods in treating chronic human diseases through directing the drug at a specific location to facilitate delivery of accurate medicines. The important applications of nanotechnology in this field are biosensing, anticancer therapy and drug delivery, nanoparticles are clusters of atoms, with sizes ranging between 1 and 100 nm, whereas a "nano" is used to indicate one billionth of a meter [3]. In the current study has concentrated on biogenic ZrO2NP synthesis and promoting their biological applications, with a focus on their antibacterial properties [4].

Collecation samples and identification
Samples (153) were collected. Under the supervision of the responsible doctor, samples were collected from burn patients using a transporter media cotton swab. The burned area was cleaned thoroughly with sterile normal saline, and a sterile cotton swab was gently passed over the burned area. The samples were then transferred to the lab and incubated at 37 °C for 24 hours before being cultured. Identified P. aeruginosa isolates by diagnostic tests including biochemical tests, culture media like (cetrimide agar) and VITEK-2 Compact System

Antibiotic Susceptibility Assays by Using VITEK2 Compact System
Based on a determination of the MIC approach utilizing AST cards, antibiogram testing was carried out using the automated VITEK-2 compact system. This card listed the antibiotics in tables (1) Most recent recommendations by consensus [5].  (2). The findings revealed that burns accounted for (48/153) (31.37%) of the P. aeruginosa isolates. This may be because this pathogen has a number of potentially virulent components that aid in its ability to colonize and infect mammalian tissues, including hemolysin, pyoverdine, and protease, which encourage adhesion to host cells, damage host tissue, and interfere with the immune system [10]. Clinical samples included (23\48) (47.9%) samples taken from male and (25\48) (52.1%) samples taken from females. This study's findings are consistent with those of Kirkuk University [11], who found that females were more than men were (52.9% vs. 47.1%) figure (1). The possible reasons for high percentage in curruent study to female may be due to the types of populations studied, females may have routine indoor work and are often at risk of infection from flame, oil and hot water, the age ranged between from (2 to 75 years). All samples were collected from Governmental hospitals in Baghdad. this study agree with [12], collected and diagnosed as P. aeruginosa (26\68) (38.23%) from burn patients. This study disagree with [13] showed higher rates of infection with P. aeruginosa (91.6%).

Antibiotic Susceptibility of P. aeruginosa
The results of the present study shown indicated that from all P. aeruginosa (48) were tested for their antibiotic resistance to (8) antibiotics, using VITEK 2 compact system in recommendation with the clinical and laboratory standards institute [14], guidelines depending on the diameter of inhibition zone (mm). The antimicrobial potency of selected antibiotics against the P. aeruginosa summarized in Table (1). This study's percentage of imipenem users concur with [15], finding which resulted in resistance (53.3%) for Imipenem in study of P. aeruginosa isolated from contaminated burns and wounds in Baghdad, this study Disagree with the results obtained by [16] in Babylon who showed extremely low resistance rate to Imipenem (8%). In this research the percentage of resistance for meropenem agree with [17], who found resistance to meropenem was (73%) in Iran. While disagree with This study [18] in Baghdad, he found resistant rate Meropenem 35% respectively. High-level Carbapenem resistance include (Imipenem and Meropenem) reported in this study as shown in a table (1) may be multiple mechanisms including Carbapenemase production and effluxpump over-expression according [19]. The percentage of resistance for colistin agree with [20] findings about the degree of resistance to the antibiotic Lipopeptides (colistin). Rate resistance of colistin was (18.5%) in Saudi Arabia. Resistance to colistin is caused by the phosphoethanolamine transferase enzyme, its encoded gene is located on mobile genetic elements [21]. According to [22], who showed that the rate of tobramycin resistance was (79.6%), the proportion of resistance for aminoglycoside antibiotics (Tobramycin) is similar to that found in this study. This study contrast the findings of [23] in Saudi Arabia, he discovered that the rate of tobramycin resistance was (12%). Certain genetically encoded enzymes, such as N-acetyl-transferases, adenyl-transferases, and phosphosphor-transferases, may be the cause of the bacterial resistance to this family of antibiotics. These enzymes are recognized as particular AMEs (aminoglycoside-modifying enzymes) [24]. And alteration of the ribosomal binding site by mutation expression of 16S ribosomal RNA methylases [25].
The percentage of resistance for fluoroquinolone drugs (ciprofloxacin and levofloxacin). Levofloxacin antibiotic was agreement with [26] found rate resistance of levofloxacin was (72.83%) from isolated P. aeruginosa from clinical samples in diyala and while the result of [27] in duhok city, iraq disagree with current study score low resistance (13.3%). For the antibiotic ciprofloxacin, the percent resistance was identical to that shown by [28] found that almost all P. aeruginosa isolates were (75.5%). This study disagree with [29], the researcher found the resistance of P. aeruginosa isolates against Ciprofloxacin (26%). P. aeruginosa have two types essential mechanisms of resistance to fluoroquinolone; structural modification of target enzymes and efflux pump [30]. Targets of the fluoroquinolone drugs (levofloxacin and ciprofloxacin) include DNA gyrase (type II topoisomerase, topoisomerase IV), Fluoroquinolone resistance is mostly caused by mutations in the fluoroquinolone resistance-determining region (FRDR) of the DNA gyrase subunit-coding genes parC and gyrA or gyrB. The topoisomerase II (gyrA), topoisomerase IV (parC), and efflux regulatory (mexR) gene alterations are therefore the primary causes of fluoroquinolone resistance in gram-negative bacteria. In gram-negative bacteria, such as P. aeruginosa, amino acid changes discovered in the genes gyrA, gyrB, parC, and mexR are linked to high levels of fluoroquinolone [31].
In this study the percentage of resistance for Cephems (Cephalosporin IV) including (Cefepime and Ceftazidime) Cefepime agree with [32] rate resistance of cefepime was (100%) and In this study disagree with [33] the rate was (23.5%) resistance. In this study the percentage of resistance for Ceftazidime agree with the results of [34] in Basrah, that P. aeruginosa were resistant to ceftazidime, (100%).

X-Ray Diffraction Technique (XRD)
The prepared sample's XRD pattern was recorded in order to verify the phase development of ZrO2. When compared to Tetragonal JCPDS No. 70-1769, XRD reveals a single broad peak associated with tetragonal ZrO2 nanoparticles, 300 of which belong to the plan. Using Scherrer's equation, this broad peak figure (3), indicates a crystallite size of 2 nm. Additionally, there are no additional unidentified peaks, indicating that the prepared ZrO2 is single crystalline.

Energy-dispersive X-ray spectroscopy (EDX)
While the EDX spectra of pure ZrO2 show peaks for the elements Zr and O, the spectra of composites show peaks for the elements Zr, O, F, Ca, C, and Si. Due to the small peaks of C, Ca, and Si in the spectrum, negligible levels of contaminants have been discovered. As seen in (figure 4), the Zr/O weight ratio between the O and Zr peaks is approximately (40.5:30.5), and the intensities of the O and Zr peaks vary linearly with the concentrations of their respective predecessors of ZrO2.

FESEM characterization of zirconia nanoparticles
The form and microstructure of the material were examined with SEM. Particles in this zirconia sample range in size from 30 to 65 nm. The XRD results were discovered to be entirely consistent with the pictures of the zirconia samples. The size distribution of zirconia particles as seen through a SEM is shown in Figures (5). Zirconia particles, whose size distribution is roughly (45 nm), as previously mentioned, will fill the holes in the resin specimen

Determination the Minimum Inhibitory Concentration (MIC)and Minimum Bactericidal Concentration (MBC) of ZrO2NPs against P. aeruginosa
Multi-drug resistant microorganisms have emerged as a result of excessive or inappropriate usage of antimicrobials. Nanotechnology offers an alternate approach for creating alternative antimicrobial agents that can effectively destroy bacterial cells and show enormous potential for usage in both medical and veterinary applications. This will help to overcome the drawbacks of existing synthetic antimicrobial chemicals. The result in Figure