The Synergistic Effects of Plant Extracts and Silver Nanoparticles on Colistin-Resistant Pseudomonas aeruginosa Isolates

The Synergistic Effects of Plant Extracts and Silver Nanoparticles on Colistin-Resistant Pseudomonas aeruginosa Isolates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Synergistic Effects of Plant Extracts and Silver Nanoparticles on Colistin-Resistant Pseudomonas aeruginosa Isolates Ali Ahmadi, Jamshid Faghri, Mitra Rabiei This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4688621/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The pathogen of Pseudomonas aeruginosa is a significant nosocomial infection. Metal nanoparticles (NPs) and plant extracts have demonstrated antibacterial efficacy against multidrug-resistant bacteria. This study aimed to determine the synergistic antibacterial effect of silver nanoparticles and Urtica and Zataria multiflora (Shirazi thyme) extracts against Colistin-Resistant Pseudomonas aeruginosa isolated from hospital patients. Methods A total of 95 Pseudomonas aeruginosa isolates were collected from selected teaching hospitals in Isfahan province. The Kirby–Bauer disk diffusion method used for antibiotic susceptibility and determining the minimum inhibitory concentration of colistin was checked by microdilution broth, and the presence of the mcr-1 gene was detected by PCR method. In addition, the effect of synergism of Zataria multiflora (Shirazi thyme) and Urtica extracts with silver nanoparticles was investigated by checkerboard procedure. Results The screening for antibiotic resistance showed 99% resistance to Levofloxacin and 6% resistance to colistin antibiotic. One isolate is positive for the mcr-1 gene. Furthermore, combining Zataria multiflora (Shirazi thyme) and the silver nanoparticle is efficient against Colistin-Resistant Pseudomonas aeruginosa. Conclusion For the first time, the synergistic effect of Zataria multiflora (Shirazi thyme) and Urtica extracts and silver nanoparticle extracts against Colistin-Resistant Pseudomonas aeruginosa was examined in our research. So plant extracts and silver nanoparticles might assist with managing multidrug-resistant pathogenic bacteria and be a possible source of medicinal application due to their potential antibacterial effect. Pseudomonas aeruginosa Zataria multiflora extract Urtica extract silver nanoparticles Figures Figure 1 Figure 2 Figure 3 Introduction Pseudomonas aeruginosa ( P. aeruginosa ) is a major opportunistic pathogen that is known for causing nosocomial infections and invasive diseases, such as pneumonia, especially in patients who are critically ill, debilitated, or immunocompromised (1, 2). Its wide distribution and capacity to thrive in damp environments contribute to its prevalence within hospital settings (3). The emergence of multi-drug resistant (MDR) Gram-negative bacteria, particularly the global prevalence of MDR P. aeruginosa , is concerning due to its ability to form biofilms and cause various infections (4, 5). Biofilms created by bacterial species disrupt the effectiveness of most antibiotics. Minimal treatment options are available for multidrug-resistant P. aeruginosa . Typically, these strains resist fluoroquinolones and reduce sensitivity to beta-lactam antibiotics. Significant concerns exist regarding P. aeruginosa XDR and mainly high-risk P. aeruginosa clones (6, 7). The antibiotic of cationic lipid type known as colistin primarily targets the lipopolysaccharides (LPS) found in the outer membrane of gram-negative bacteria. By binding to lipid A, it disrupts the electrostatic balance, leading to the displacement of Mg 2+ and Ca 2+ cations. This process causes the outer membrane to open, resulting in osmotic changes within the periplasm and eventual bacterial lysis (8). Plasmid-mediated resistance to colistin, known as mcr-1 (mobile colistin resistance), was identified as a phosphoethanolamine transferase enzyme family member. This enzyme can modify the target site of colistin, reducing its affinity for lipid A by adding phosphoethanolamine. The discovery of mcr -1 was first reported in China in 2015, and that same year, Vietnam reported its first clinical case in a diarrheal sample that had been cryopreserved since 2008. In this case, a Shigella sonnei carrying the mcr -1 gene was isolated (9–11). One important medicinal plant from the Urtica genus is Diotica. This plant contains saponins, which have been reported to exhibit medicinal properties, including an antibiotic effect against both Gram-positive and Gram-negative bacteria (12, 13). Another noteworthy medicinal plant is Zataria Boiss, known as "multiflora Zataria" or "Shirazi thyme." It belongs to the Lamiaceae family and is a fragrant herbaceous shrub. Geographically, it grows in the central and southern regions of Iran, Afghanistan, and Pakistan. This medicinal plant, traditionally known as Satar or Zatar, is recognized for its potent and effective properties in treating various infectious diseases (14, 15). In addition to medicinal plants, nanotechnology has gained significant attention in the field of medicine due to the unique properties of metallic nanoparticles. Nanoparticles offer several advantages over antibiotics, such as their broad spectrum of activity against a wide range of microorganisms, the resistance of bacteria to nanoparticles, and their minimal adverse effects on human cells (16). The application of nanotechnology in medicine holds great promise and has attracted considerable interest. Among metallic nanoparticles, silver nanoparticles are expected to play a significant role in combating future drug-resistant microorganisms. The antimicrobial properties of silver nanoparticles are utilized to eliminate a wide range of bacteria, yeasts, fungi, and viruses. Research has shown that the bactericidal effect of silver is attributed to the damage it causes to the cellular membranes of bacteria (17, 18). Despite the clinical importance of colistin-resistant strains of P. aeruginosa , there is limited information regarding effective treatment options for these strains in Iran. This study aims to investigate the synergistic effects of plant extracts, specifically from Urtica and Zataria multiflora (Shirazi thyme), in combination with silver nanoparticles, on colistin-resistant P. aeruginosa isolates obtained from selected teaching hospitals in Isfahan province. The goal is to identify a suitable and effective treatment for these resistant strains. Methods Isolation and identification of bacteria P. aeruginosa was isolated from clinical specimens such as blood, urine, wounds, urinary catheters, and biopsy chips in hospitals and medical centers of Isfahan, Iran, from 2021 to 2022. At first, to ensure the purity of the isolates, we performed Gram staining. Then, the isolates were cultured on nutrient agar and Eosin Methylene Blue (EMB) agar. Afterward, to determine the definitive identity and confirm the final diagnosis of the isolates, Gram staining and different media, including Triple Sugar Iron (TSI), Sulfur Indole Motility (SIM), Voges-Proskauer/Methyl Red (VP/MR), OF medium, Simmons citrate agar, urea agar, and oxidase and glucose fermentation tests were used. Finally, the special Pseudomonas colonies were stored in a -20°C freezer after stock preparation. Antibiotic susceptibility testing The Kirby–Bauer disk diffusion method, as described by the Clinical and Laboratory Standards Institute (CLSI 2020) guidelines, was utilized to determine anti-microbial susceptibility. Bacterial suspensions in saline solution were standardized to a concentration of 0.5 McFarland standard and applied onto Mueller Hinton Agar plates using swabs. Small circular discs containing specific antibiotics such as Ciprofloxacin (5 µg), Imipenem (10 µg), Ceftazidime (30 µg), Aztreonam (30 µg), Amikacin (30 µg), Levofloxacin (30 µg), Cefepime (30 µg), Sulfamethoxazole (23.75 µg), Piperacillin / Tazobactam (10/100 µg), Ceftriaxone (30 µg) and Cefoxitin (30 µg) from Becton Dickinson were placed on the surface of the inoculated plates. After incubating the plates at 37°C for 24 hours, the diameter of the growth inhibition zones around the antibiotic discs was measured. Minimum inhibitory concentration (MIC) for antibiotic colistin The MIC was defined through the broth microdilution method. In this method, the MIC for colistin antibiotic on the strain containing the mcr -1 gene was determined. To perform the MIC for colistin antibiotic, a microbial suspension with turbidity equivalent to 0.5 McFarland standard was prepared from the 24-hour cultures of the mentioned strains in Muller-Hinton broth. A stock solution of the desired antibiotic at a concentration of 10 mg/ml was prepared, and consecutive dilutions of this stock were made to achieve a final concentration range of 0.256 µg/ml to 0.25 µg/ml after mixing. Preparation of silver nanoparticles and herbal extracts The silver nanoparticles were purchased in a ready-to-use from Yasatab Medical Company. The particle diameter was 20 nanometers, and they had a spherical shape. The plant extracts were also obtained commercially from Ibn Masawayh Pharmaceutical Company. The extracts were alcohol-based and used at concentrations ranging from 1 to 8 (1, 2, 4, 8) mg/ml for Urtica extract and from 0.5 to 4 (0.5, 1, 2, 4) mg/ml for Shirazi thyme extract. Survival assay (MTT assay) For cytotoxicity assay, the Human Dermal Fibroblasts (HDF s ) were seeded on 96 wells and then incubated for 24 hours at 37°C with CO 2 . The wells were washed with PBS twice. Different concentrations of Urtica and Zataria multiflora extracts and silver nanoparticles were added and incubated for 24 hours again. The wells were rinsed and washed with PBS and 10 µl of MTT solution with 100 µl DMEM were added in each well. Plate containing MTT incubated for 4h at 37°C. The media were gently removed, and 100 µl DMSO was added and incubated in darkness. The absorbance reading in 96-well plates was carried out in a chromate ELISA Reader at 570 nm. The following formula calculated the cell survival (19): Molecular characterization of the mcr -1 gene After examining the physical and biochemical characteristics of the bacteria, the genomic DNA of P. aeruginosa isolates was extracted by the phenol-chloroform method for molecular identification of the mcr -1 gene. PCR was conducted using the following primers: forward: 5'-CGGTCAGTCCGTTTGTTC-3' and reverse: 5'-CTTGGTCGGTCTGTAGGG-3'. The amplification process included an initial denaturation at 94°C for 5 minutes, followed by 35 cycles at 94°C for 1 minute, 51°C for 30 seconds, and 72°C for 30 seconds. A final extension was performed at 72°C for 5 minutes. The size of the DNA fragments was confirmed using a 100 bp DNA ladder, with a target product size of 309 bp. Investigation of sequencing and registration in the NCBI database The purified PCR products were sent to the Gene Fanavaran Company (Tehran, Iran) for sequencing to confirm the presence of the antibiotic-resistant gene, mcr -1, in the resistant strain of Pseudomonas aeruginosa . The accuracy of the sequencing was examined using Chromas v 2.6.5 software. The obtained sequences were further analyzed using the online BLAST software in the NCBI database. Biofilm analysis The potential of the P. aeruginosa isolates to create biofilm was tested using a microtiter dish biofilm formation assay. In this procedure, the isolates were cultured overnight at 37˚C in tryptic soy broth (TSB) supplemented with 0.25% glucose. The cultures were then diluted 1:100 in the TSB medium. Flat-bottomed 96-well polystyrene microtiter plates were aseptically inoculated with 125 µL of the bacterial suspension and incubated for 24 hours at 37˚C without agitation. After incubation, the wells were washed three times with 300 µL of distilled water and air-dried in an inverted position at room temperature. Subsequently, the wells were stained with 125 µL of 0.1% crystal violet solution in water for approximately 10–15 minutes. Following staining, the wells were washed three times with distilled water. To destain the wells, 125 µL of 30% acetic acid in water was added to a new sterile microtiter plate, and the destaining solution was transferred to each well. The absorbance of the destaining solution was measured at 595 nm using an ELISA reader. Each experiment was performed in triplicate, and the uninoculated medium served as a control. Based on the optical density of the samples (ODi) and the average optical density of the negative control (ODc), the samples were categorized as strong biofilm producers (4xODc < ODi), moderate biofilm producers (2xODc < ODi ≤ 4xODc), weak biofilm producers (ODc < ODi ≤ 2xODc), or non-producers of biofilm (ODi < ODc). Checkerboard assay The checkerboard technique is used to evaluate the synergistic effects between two agents. In the present study, this method was employed to assess the efficacy of silver nanoparticles' minimum inhibitory concentration (MIC) along with the herbal extracts of thyme and Urtica on clinical isolates. Twofold dilutions were prepared for the herbal extracts and silver nanoparticles. In each well, a total of 50 µL of Mueller-Hinton broth was added to prepare the microdilution serial plate for the microdilution assay. Then, each desired herbal extract was serially distributed in the vertical wells, while the silver nanoparticles were distributed horizontally. Afterward, a standardized 0.5 McFarland bacterial suspension of the clinical isolate P. aeruginosa was prepared, and 10 µL of the suspension containing 5 × 10^5 CFU/mL of bacteria was added to each well. The plate was then incubated aerobically at 37°C for 24 hours. The checkerboard result represents a combination of two substances (herbal extract + nanoparticle) with the well that contains the lowest concentration of the herbal extract and nanoparticle, effectively inhibiting bacterial growth being selected. In the checkerboard experiments, the bactericidal effect of the two molecules was assessed using the formula ΣFIC = FIC A + FIC B, which is given as follows: $$\:\frac{\text{A}}{{\text{M}\text{I}\text{C}}_{\text{A}}}+\frac{\text{B}}{{\text{M}\text{I}\text{C}}_{\text{B}}}={\text{F}\text{I}\text{C}}_{\text{A}}+{\text{F}\text{I}\text{C}}_{\text{B}}=\text{F}\text{I}\text{C}\:\text{I}\text{n}\text{d}\text{e}\text{x}$$ In this formula, A represents the MIC value of drug A in combination with another drug, and B represents the MIC value of drug B when combined with drug A. MIC A corresponds to the MIC value of drug A alone, and MIC B is the MIC value of drug B alone. If the value of ΣFIC is lower than or equal to 0.5, it is defined as a synergistic effect. If it is between 0.5 and 1, it is considered ineffective, and if it is greater than one, it will exhibit an antagonistic effect. Agar well diffusion assay The antimicrobial activity of the herbal extracts against Pseudomonas aeruginosa was investigated via this method. A freshly cultured Pseudomonas aeruginosa suspension was prepared in 100 µL of PBS. Then, the suspension was streaked onto Mueller-Hinton agar medium using a swab. Approximately 6 mm of the medium surface was removed using a pipette tip, and 50 µL of the herbal extract dissolved in PBS at various concentrations was added to the wells. The plate was then incubated at 37°C for 24 hours. The formation of a growth inhibition zone around the well indicated the antimicrobial activity of the herbal extracts against Pseudomonas aeruginosa. Aztreonam disks were used as a positive control, and PBS was used as a negative control. Data analysis SPSS 22 (IBM Corp. 1989, 2013, New York, USA) was utilized to conduct the statistical analyses. The statistical significance of the data was assessed through one-way ANOVA (Tukey), and a p-value of 0.05 was deemed statistically significant. Results Isolation of strains of P. aeruginosa In the conducted study, 95 isolates of P. aeruginosa , which were obtained from severe infections in hospitalized individuals, were included in the study after species identification using Gram staining and tests including oxidase, glucose fermentation, citrate utilization, SIM, MR/VP, OF, and urea. Of these 95 people, 35 people (37%) were men and 60 people (63%) were women. Among the total isolated strains collected from patients in different units, the majority were associated with subjects in the Intensive Care Unit (ICU) and Neonatal Intensive Care Unit (NICU), accounting for 35 strains (36.84%) and 15 strains (15.78%), respectively. Additionally, 30 strains (31.57%) were derived from wound samples, and 15 strains (15.78%) were extracted from catheter samples. The age range of the patients varied from 12 days to 88 years. Antimicrobial susceptibility profiles The results of antibiotic susceptibility testing were performed on 95 confirmed clinical isolates using phenotypic tests. Among the isolates, 94 isolates were resistant to Levofloxacin, and 6 isolates were resistant to colistin antibiotic, and out of 6 isolates resistant to colistin, all of them were obtained from wounds. (Table 1 ). Table 1 Antibiotic resistance pattern of P. aeruginosa isolates Antibiotic class Antibiotic Resistant NO (%) Intermediate NO (%) Susceptible NO (%) Cephems Cefepime 66(70) 10(10) 19(20) Ceftazidime 49(51) 11(12) 35(37) Aminoglycosides Amikacin 82(86) 0 13(14) Penems Imipenem 72(76) 12(13) 11(11) β-lactam + Inhibitors Piperacillin/tazobactam 41(43) 15(16) 39(41) Quinolones Ciprofloxacin 87(92) 1(1) 7(7) Levofloxacin 94(99) 1(1) 0 Monobactam Aztreonam 45(47) 10(11) 40(42) Minimum inhibitory concentration According to the broth microdilution method results, The MIC values for 95 isolates were determined according to CLSI 2020 guidelines for the antibiotic colistin. Among the 95 isolates, 6 were found to be resistant to colistin. The MIC for silver nanoparticles was also determined to be below 8 micrograms per milliliter, specifically at a concentration of 2 micrograms per milliliter. Furthermore, 1 milligram per milliliter and 8 milligrams per milliliter were assigned for the extract of Shirazi thyme and Urtica, respectively. All experiments were performed in triplicate to ensure the reproducibility of the results. Survival assay (MTT assay) The surveyed Urtica extract and silver nanoparticle in lower concentrations showed no significant adverse effects on Human Fibroblast cell viability. The ordinary two-way ANOVA analysis of the data obtained from the MTT assay. Checkerboard assessment The checkerboard method was used to investigate the antimicrobial interaction of Urtica extract in combination with silver nanoparticles. In this method, 50 microliters of each antimicrobial compound were added to each well of the plate, and each horizontal row was assigned to a series of concentrations of one antimicrobial compound, while each vertical column was assigned to a series of concentrations of another antimicrobial compound. Subsequently, 100 microliters of microbial suspension were added to each well. Each plate included positive and negative controls. The positive control well contained bacterial suspension and growth medium, while the negative control well contained the antimicrobial combination and growth medium. After 24 hours of incubation at 37 degrees Celsius, the results were expressed using the fractional inhibitory concentration (FIC) formula and calculated inhibitory concentrations. We used the following formula to assess the synergistic effect of the two desired substances: The fractional inhibitory concentration (FIC) of the combination of silver nanoparticles and Shirazi thyme was calculated to be 0.37, which is less than 0.5. This indicates a synergistic effect between the two substances (Fig. 1 ). Biofilm assay Based on the microtiter dish biofilm formation assay, The measured OD values for 55 samples were higher than 0.2, indicating the presence of strong biofilm formation. All 6 resistant to colistin isolates (100%) showed strong biofilm formation. The measured OD values for 33 samples ranged from 0.1 to 0.2, indicating moderate biofilm formation. The measured OD values for 7 samples ranged from 0.1 to 0.07, indicating weak biofilm formation (Fig. 2 ). Agar Well Diffusion evaluation Agar well diffusion assay was performed using plant extracts of Urtica and "Shirazi Thyme." The inhibitory concentrations for Urtica and Shirazi Thyme were 8 and 1 milligrams per milliliter, respectively. Detection of mcr -1 gene by PCR and Sequencing PCR was performed on 95 isolates of P. aeruginosa , and one sample from the wound specimens was found to have the mcr -1 gene (Fig. 3 ). Discussion Colistin has gained renewed attention as an old antibiotic for treating severe infections, especially in carbapenem-resistant Enterobacteriaceae . The reported resistance rate to colistin in Pseudomonas aeruginosa ranges from 0–17.46% (20). In the present study, which was the first report from Isfahan (Iran), 95 strains of P. aeruginosa were isolated from severe infections in hospitalized people. Antibiotic susceptibility tests showed high resistance among P. aeruginosa isolates. The detailed pattern of antibiotic resistance, as presented in Table 1 , shows significant resistance to several antibiotics, including cefepime, ceftazidime, amikacin, imipenem, piperacillin/tazobactam, ciprofloxacin, levofloxacin, and azetronam. It is worth mentioning that 94 isolates were resistant to levofloxacin and 6 isolates showed resistance to colistin, which included 31.6% of the strains. This rate is comparable to the resistance reported in previous studies conducted in Iran (Iran). The majority of the isolates were from male patients (63%), and the samples were obtained from various intensive care units (ICU) and neonatal intensive care units (NICU). In a 2022 study by Shahri et al. in Gorgan (Iran), a colistin resistance rate of 9.27% in P. aeruginosa isolates was reported, aligning with our study (21). Balkhair et al.'s study in Oman in 2019 reported a colistin resistance rate of 2.7% in P. aeruginosa isolates. A 2023 study by Soni et al. in India, focusing on A. baumannii and P. aeruginosa isolates, documented colistin resistance rates of 2.2% and 5.3%, respectively (22, 23). The results of our present study deviate from some studies on colistin resistance, such as Akar et al.'s 2019 research in Turkey, which indicated a colistin resistance rate of 2.2% in P. aeruginosa isolates (24). In a 2020 study by Santimalee Woragun et al. in Thailand, the rate of colistin resistance in P. aeruginosa isolates was reported to be 1.6%, contradicting our findings (25). Araaf et al.'s 2022 study in Pakistan reported colistin resistance rates of 19.9%, 16.1%, and 13.1% in Escherichia coli, Klebsiella pneumoniae, and P. aeruginosa , respectively (26). In 2022, in Mashhad (Iran), Zamani and colleagues reported a 4% resistance rate to colistin in P. aeruginosa isolates, with the presence of the mcr-2 gene responsible for their resistance (27). These variations among different reports may be attributed to geographical differences, variations in resistance testing methods, the diversity of sample types, sample sizes, the general condition of patients, different antibiotic prescription policies, and adherence to infection control measures. Today, plasmid-mediated mcr genes are rapidly spreading among bacteria, increasing the potential for colistin resistance in bacterial populations. Previous studies have reported the presence of the mcr-1 gene in clinical isolates from more than 50 countries and regions, including 21 countries in Asia, 14 countries in Europe, 3 countries in North America, 8 countries in Latin America, and 3 countries in Africa (28). In our study, an isolate carrying the mcr-1 plasmid gene was obtained from a wound sample, emphasizing the importance of alternative therapeutic strategies. This finding is crucial for understanding the mechanisms contributing to colistin resistance. Talebi et al.'s report in Tehran on clinical isolates of P. aeruginosa obtained from burn units showed that no isolate carrying the mcr-1 gene was found (29). In the study by Moradi et al. in 2022 in Isfahan, 2% of P. aeruginosa strains had the mcr-1 gene (30). These differences in findings may be attributed to the increase in antibiotic use in recent years compared to the past, geographical changes, and the type and number of samples. The presence of the mcr-1 gene in clinical isolates is increasing in Iran. In general, bacteria carrying the mcr-1 gene are considered resistant to colistin. However, a 2017 study by Wang et al. in China reported that 3% of E. coli isolates carrying the mcr-1 gene remained susceptible to colistin (31). The first report of the mcr-1 gene in a colistin-susceptible strain of Shigella sonnei in Vietnam was made in 2016 by Fam et al. This finding suggests that silent release of this gene may occur or another gene may be inserted into mcr-1, leading to its inactivation. However, further large-scale studies are necessary to clarify this issue (10). In the present study, silver nanoparticles and thyme extract showed inhibitory effects individually and in combination against colistin-resistant P. aeruginosa . However, ginger extract did not exhibit any therapeutic inhibitory effect. In the study by Safari et al. in 2019 in Tehran, the inhibitory effect of thyme extract alone and in combination with gentamicin and chloramphenicol antibiotics on gram-positive and gram-negative bacteria was investigated, and it was found that the inhibitory effect was greater on gram-negative bacteria (32). In the study by Zinali Aghdam et al. in 2019 in Tehran, the inhibitory effects of ginger extract and silver nanoparticles alone and in combination against Acinetobacter baumannii were investigated, and it was found that they exhibited a synergistic strengthening effect against A. baumannii (33). In a study by Jyoti et al. in 2016 in India, the synergistic effect of silver nanoparticles and ginger extract on common pathogens was investigated, and silver nanoparticles showed a very good inhibitory effect, while ginger extract had a relatively weaker inhibitory effect (34). In the study by Guntherip et al. in 2022 in Germany, the effects of mint, thyme, and green tea extracts on Escherichia coli, Staphylococcus epidermidis, and P. aeruginosa were investigated. Thyme extract partially reversed the resistance of Escherichia coli to bacitracin and penicillin (35). In a study conducted by Mohammad et al. in 2022 in Egypt on multidrug-resistant P. aeruginosa isolated from dental implants, silver nanoparticles showed inhibitory effects on all isolates and had significant anti-biofilm activity (36). In a study conducted by Arshad and colleagues in 2021 in Pakistan, the effect of silver nanoparticles on multidrug-resistant microorganisms was investigated. It was shown that silver nanoparticles have a significant inhibitory effect against Gram-positive bacteria such as Staphylococcus aureus and Gram-negative bacteria such as Escherichia coli , Acinetobacter baumannii , and P. aeruginosa . Additionally, inhibitory activity against Candida albicans, a fungal pathogen, was demonstrated. These findings indicate that silver nanoparticles have significant inhibitory effects against a wide range of microorganisms, including bacteria and fungi (37). According to the results of the MTT test, nettle extract and silver nanoparticles at lower concentrations did not show any significant adverse effects on the viability of human skin fibroblast cells. This is an important observation that suggests the possible safety of using these substances. Biofilm formation was common among P. aeruginosa isolates. This emphasizes the role of biofilm in antibiotic resistance and challenges related to the treatment of biofilm-forming strains. In this study, a comprehensive review was conducted on the challenges posed by colistin-resistant P. aeruginosa isolates, and alternative strategies for their treatment were explored. These findings shed light on critical aspects of antibiotic resistance, antimicrobial interactions, and potential therapeutic roles of plant extracts and silver nanoparticles. The high prevalence of antibiotic resistance among P. aeruginosa isolates, especially resistance to colistin, emphasizes the urgent need for effective treatment options. Significantly, the emergence of resistance to last-resort antibiotics such as colistin raises concerns, and there is an urgent need for innovative approaches to address this issue. Our findings on the synergistic effects between nettle extract and silver nanoparticles showed a promising way to overcome antibiotic resistance. The observed synergistic effect, as evidenced by the fractional inhibitory concentration (FIC: 0.37), suggests that the combination of these substances may increase their antimicrobial potency. Detection of the mcr-1 gene in an isolate indicates the presence of plasmid-mediated colistin resistance and adds a layer of complexity to existing resistance mechanisms. Understanding such genetic factors is crucial for devising targeted interventions and regulatory strategies to manage and prevent the spread of antibiotic resistance. In summary, this study provides valuable information on the prevalence of colistin-resistant P. aeruginosa isolates, their antibiotic susceptibility patterns, and the potential synergistic effects of nettle extract and silver nanoparticles. The observed biofilm formation emphasizes the complexity of treating these strains, and the detection of the mcr-1 gene highlights the importance of monitoring plasmid-mediated resistance mechanisms. These findings help to understand alternative strategies to deal with antibiotic resistance in P. aeruginosa infections. Conclusion The emergence of multidrug-resistant (MDR) isolates and resistance to colistin poses a serious global problem. When new and effective antibiotics are unavailable, colistin becomes the last resort for treating Gram-negative bacteria. The prevalence of colistin resistance varies in different geographical areas due to distinct strategies in infection control and treatment across different hospitals. Therefore, obtaining information on colistin resistance and continuous monitoring to determine the exact frequency of the mcr-1 gene among Gram-negative bacteria in human and veterinary medicine worldwide is of great importance. Our study provides valuable information on the multifaceted challenge of colistin-resistant P. aeruginosa infections. The identified synergistic effects of nettle extract and silver nanoparticles, coupled with a comprehensive understanding of antibiotic resistance patterns and biofilm formation, lay the foundation for further research and the development of innovative therapeutic approaches. Implementing new methods to treat these resistant strains is highly practical. Strategies in this field include controlling antibiotic consumption, understanding the mechanisms of antibiotic resistance, and developing appropriate treatment methods with therapeutic effects and minimal side effects, such as plant extracts and nanoparticles, that can be used as alternative antibiotics. Declarations Funding “The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.” Competing Interests “The authors have no relevant financial or non-financial interests to disclose.” Author Contributions “All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ali Ahmadi and Mitra Rabiei . The first draft of the manuscript was written by Ali Ahmadi and Mitra Rabiei and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.” Ethics approval “This study was conducted in the in vitro phase and was registered in the Ethical Committee of Isfahan University of Medical Sciences with the ethical code "IR.MUI.MED.REC.1400-509" on 2021/09/26.” Consent to participate "This study was conducted in the in vitro phase, and due to the lack of human studies, there was no need to obtain consent from individuals." Consent to publish "This study was conducted in the in vitro phase, and due to the lack of human studies, there was no need to obtain consent from individuals." 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Chemical composition and antimicrobial effect of the essential oil of Zataria multiflora Boiss endemic in Khorasan-Iran. Asian Pacific Journal of Tropical Disease. 2015;5(3):181-5. Mahboubi M, HeidaryTabar R, Mahdizadeh E. The anti-dermatophyte activity of Zataria multiflora essential oils. Journal de mycologie medicale. 2017;27(2):232-7. Mirzajani F, Ghassempour A, Aliahmadi A, Esmaeili MA. Antibacterial effect of silver nanoparticles on Staphylococcus aureus. Research in microbiology. 2011;162(5):542-9. Kumar S, Singh M, Halder D, Mitra A. Mechanistic study of antibacterial activity of biologically synthesized silver nanocolloids. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 2014;449:82-6. Marambio-Jones C, Hoek EM. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. Journal of nanoparticle research. 2010;12:1531-51. Mirzaei A, Nasr Esfahani B, Ghanadian M, Moghim S. Alhagi maurorum extract modulates quorum sensing genes and biofilm formation in Proteus mirabilis. Scientific Reports. 2022;12(1):13992. Dadashi M, Sameni F, Bostanshirin N, Yaslianifard S, Khosravi-Dehaghi N, Nasiri MJ, et al. Global prevalence and molecular epidemiology of mcr-mediated colistin resistance in Escherichia coli clinical isolates: A systematic review. Journal of global antimicrobial resistance. 2022;29:444-61. Shahri FN, Izanloo A, Goharrizi MASB, Jamali A, Bagheri H, Hjimohammadi A, et al. Antimicrobial resistance, virulence factors, and genotypes of Pseudomonas aeruginosa clinical isolates from Gorgan, northern Iran. International Microbiology. 2022;25(4):709-21. Balkhair A, Al-Muharrmi Z, Al’Adawi B, Al Busaidi I, Taher H, Al-Siyabi T, et al. Prevalence and 30-day all-cause mortality of carbapenem-and colistin-resistant bacteraemia caused by Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae: description of a decade-long trend. International Journal of Infectious Diseases. 2019;85:10-5. Soni M, Kapoor G, Perumal N, Chaurasia D, Soni Jr M. Emergence of Multidrug-Resistant Non-Fermenting Gram-Negative Bacilli in a Tertiary Care Teaching Hospital of Central India: Is Colistin Resistance Still a Distant Threat? Cureus. 2023;15(5). Acar A, Karaahmetoğlu G, Akalın H, Altay AF. Pooled prevalence and trends of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates over the past 10 years in Turkey: A meta-analysis. Journal of global antimicrobial resistance. 2019;18:64-70. Santimaleeworagun W, Thunyaharn S, Juntanawiwat P, Thongnoy N, Harindhanavudhi S, Nakeesathit S, et al. The prevalence of colistin-resistant Gram-negative bacteria isolated from hospitalized patients with bacteremia. Journal of Applied Pharmaceutical Science. 2020;10(2):056-9. Arif A, Ullah I, Ullah O, Zaman R. Identification of colistin resistance and its bactericidal activity against uropathogenic gram negative bacteria from Hayatabad Medical Complex Peshawar. Pakistan Journal of Medical Sciences. 2022;38(4Part-II):981. Zomorodi AR, Mohseni N, Hafiz M, Nikoueian H, Hashemitabar G, Salimizand H, et al. Investigation of mobile colistin resistance (mcr) genes among carbapenem resistance Pseudomonas aeruginosa isolates from bovine mastitis in Mashhad, Iran. Gene Reports. 2022;29:101695. Xiaomin S, Yiming L, Yuying Y, Zhangqi S, Yongning W, Shaolin W. Global impact of mcr-1-positive Enterobacteriaceae bacteria on “one health”. Critical reviews in microbiology. 2020;46(5):565-77. Talebi G, Hakemi-Vala M. Survey on some carbapenems and colistin resistance genes among Pseudomonas aeruginosa isolates from burn and cystic fibrosis patients, Tehran, Iran. Archives of Clinical Infectious Diseases. 2019;14(5). Moarefian M, Poursina F, Narimani T. Resistance to Colistin and Detection of mcr-1 Gene in Multidrug-Resistant Pseudomonas Aeruginosa Isolated from Wounds of the Hospitalized Burned Patients. Journal of Isfahan Medical School. 2022;40(686):678-84. Wang Y, Tian G-B, Zhang R, Shen Y, Tyrrell JM, Huang X, et al. Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study. The Lancet Infectious Diseases. 2017;17(4):390-9. Saffari Samani E, Jooyandeh H, Alizadeh Behbahani B. Evaluation of reciprocal pharmaceutical effect and antimicrobial activity of Shirazi thyme essential oil against some Gram-positive and Gram-negative bacteria. Journal of food science and technology (Iran). 2020;17(104):1-11. Zeinali Aghdam S, Minaeian S, Sadeghpour Karimi M, Tabatabaee Bafroee AS. The antibacterial effects of the mixture of silver nanoparticles with the shallot and nettle alcoholic extracts. Journal of Applied Biotechnology Reports. 2019;6(4):158-64. Jyoti K, Baunthiyal M, Singh A. Characterization of silver nanoparticles synthesized using Urtica dioica Linn. leaves and their synergistic effects with antibiotics. Journal of Radiation Research and Applied Sciences. 2016;9(3):217-27. Guntrip RB, Luce MJ. Peppermint, thyme, and green tea extracts modulate antibiotic sensitivity. Bios. 2022;92(3):104-10. El-Telbany M, El-Sharaki A. Antibacterial and anti-biofilm activity of silver nanoparticles on multi-drug resistance pseudomonas aeruginosa isolated from dental-implant. Journal of Oral Biology and Craniofacial Research. 2022;12(1):199-203. Arshad H, Saleem M, Pasha U, Sadaf S. Synthesis of Aloe vera-conjugated silver nanoparticles for use against multidrug-resistant microorganisms. Electronic Journal of Biotechnology. 2022;55:55-64. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4688621","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345642919,"identity":"485f7adb-f000-42d2-b996-1ef5ab5c574a","order_by":0,"name":"Ali Ahmadi","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Ahmadi","suffix":""},{"id":345642920,"identity":"1d0dc1c1-231b-49a0-84e2-d103c69cc205","order_by":1,"name":"Jamshid Faghri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBAC9gYGNgYGAxsgMwGIDUBijA14tfAcAGtJI1kLw2GoFmIAD/vxa48rCs5H87MnsG7mKWCQ529gbvuAVwtPTrnhGYPbuTN7HrDd5jFgMJxxgLF5Bj4t9gw5aZINQC0bbiSAtTBuYGBsxu8w/jcgLedy90O12BPWIpF+DKjlQO4GCYiWRCK0vGEDaknOnXHmYdvNOQYSyTMOE3RY+jPJhj92uf3tycduvPljY9vf3v4YrxagJgMoAxyDEgwMzAQ0AJPMA4JKRsEoGAWjYIQDAJcRRXOwtwq5AAAAAElFTkSuQmCC","orcid":"","institution":"Associate Professor of Microbiology, University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Jamshid","middleName":"","lastName":"Faghri","suffix":""},{"id":345642921,"identity":"aed73b64-a4f8-4f51-8d98-662de6e45476","order_by":2,"name":"Mitra Rabiei","email":"","orcid":"","institution":"Isfahan University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mitra","middleName":"","lastName":"Rabiei","suffix":""}],"badges":[],"createdAt":"2024-07-04 21:53:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4688621/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4688621/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64009528,"identity":"893a42ce-3e90-44e4-be02-b46262467499","added_by":"auto","created_at":"2024-09-04 23:17:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":292946,"visible":true,"origin":"","legend":"\u003cp\u003eCheckerboard test results to investigate the combined effect of Shirazi thyme extract and silver nanoparticles\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4688621/v1/10a0223b725d39ed88d9756e.png"},{"id":64009530,"identity":"1a81d112-e2a4-46ca-b311-0b0f75319017","added_by":"auto","created_at":"2024-09-04 23:17:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":330720,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of the micro titer plate test\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4688621/v1/7b43b915bee0201796f1c58a.png"},{"id":64009529,"identity":"6b83f093-163f-4786-9db5-8897feb420ba","added_by":"auto","created_at":"2024-09-04 23:17:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":128449,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophoresis pattern of mcr-1 gene (bp309). Well M: 100 bp marker, 3: positive sample, 8: positive control, 12: negative control (Escherichia coli without mcr-1 gene), 1, 2, 4, 5, 6, 7, 9, 10, 11: negative samples (lacking the mcr-1 gene)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4688621/v1/2104677f04aabf04989eff8c.png"},{"id":69260191,"identity":"11611853-eb37-4ff2-8d7e-60b0ac9c0c84","added_by":"auto","created_at":"2024-11-18 13:32:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1545353,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4688621/v1/46f844e1-2184-474d-a514-d5651f2f35cf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Synergistic Effects of Plant Extracts and Silver Nanoparticles on Colistin-Resistant Pseudomonas aeruginosa Isolates","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cem\u003eP. aeruginosa\u003c/em\u003e) is a major opportunistic pathogen that is known for causing nosocomial infections and invasive diseases, such as pneumonia, especially in patients who are critically ill, debilitated, or immunocompromised (1, 2). Its wide distribution and capacity to thrive in damp environments contribute to its prevalence within hospital settings (3). The emergence of multi-drug resistant (MDR) Gram-negative bacteria, particularly the global prevalence of MDR \u003cem\u003eP. aeruginosa\u003c/em\u003e, is concerning due to its ability to form biofilms and cause various infections (4, 5). Biofilms created by bacterial species disrupt the effectiveness of most antibiotics. Minimal treatment options are available for multidrug-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e. Typically, these strains resist fluoroquinolones and reduce sensitivity to beta-lactam antibiotics. Significant concerns exist regarding \u003cem\u003eP. aeruginosa\u003c/em\u003e XDR and mainly high-risk \u003cem\u003eP. aeruginosa\u003c/em\u003e clones (6, 7). The antibiotic of cationic lipid type known as colistin primarily targets the lipopolysaccharides (LPS) found in the outer membrane of gram-negative bacteria. By binding to lipid A, it disrupts the electrostatic balance, leading to the displacement of Mg\u003csup\u003e2+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e cations. This process causes the outer membrane to open, resulting in osmotic changes within the periplasm and eventual bacterial lysis (8). Plasmid-mediated resistance to colistin, known as \u003cem\u003emcr-1\u003c/em\u003e (mobile colistin resistance), was identified as a phosphoethanolamine transferase enzyme family member. This enzyme can modify the target site of colistin, reducing its affinity for lipid A by adding phosphoethanolamine. The discovery of \u003cem\u003emcr\u003c/em\u003e-1 was first reported in China in 2015, and that same year, Vietnam reported its first clinical case in a diarrheal sample that had been cryopreserved since 2008. In this case, a \u003cem\u003eShigella sonnei\u003c/em\u003e carrying the \u003cem\u003emcr\u003c/em\u003e-1 gene was isolated (9\u0026ndash;11).\u003c/p\u003e \u003cp\u003eOne important medicinal plant from the Urtica genus is Diotica. This plant contains saponins, which have been reported to exhibit medicinal properties, including an antibiotic effect against both Gram-positive and Gram-negative bacteria (12, 13). Another noteworthy medicinal plant is Zataria Boiss, known as \"multiflora Zataria\" or \"Shirazi thyme.\" It belongs to the Lamiaceae family and is a fragrant herbaceous shrub. Geographically, it grows in the central and southern regions of Iran, Afghanistan, and Pakistan. This medicinal plant, traditionally known as Satar or Zatar, is recognized for its potent and effective properties in treating various infectious diseases (14, 15). In addition to medicinal plants, nanotechnology has gained significant attention in the field of medicine due to the unique properties of metallic nanoparticles. Nanoparticles offer several advantages over antibiotics, such as their broad spectrum of activity against a wide range of microorganisms, the resistance of bacteria to nanoparticles, and their minimal adverse effects on human cells (16). The application of nanotechnology in medicine holds great promise and has attracted considerable interest. Among metallic nanoparticles, silver nanoparticles are expected to play a significant role in combating future drug-resistant microorganisms. The antimicrobial properties of silver nanoparticles are utilized to eliminate a wide range of bacteria, yeasts, fungi, and viruses. Research has shown that the bactericidal effect of silver is attributed to the damage it causes to the cellular membranes of bacteria (17, 18).\u003c/p\u003e \u003cp\u003eDespite the clinical importance of colistin-resistant strains of \u003cem\u003eP. aeruginosa\u003c/em\u003e, there is limited information regarding effective treatment options for these strains in Iran. This study aims to investigate the synergistic effects of plant extracts, specifically from Urtica and Zataria multiflora (Shirazi thyme), in combination with silver nanoparticles, on colistin-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates obtained from selected teaching hospitals in Isfahan province. The goal is to identify a suitable and effective treatment for these resistant strains.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and identification of bacteria\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e was isolated from clinical specimens such as blood, urine, wounds, urinary catheters, and biopsy chips in hospitals and medical centers of Isfahan, Iran, from 2021 to 2022. At first, to ensure the purity of the isolates, we performed Gram staining. Then, the isolates were cultured on nutrient agar and Eosin Methylene Blue (EMB) agar. Afterward, to determine the definitive identity and confirm the final diagnosis of the isolates, Gram staining and different media, including Triple Sugar Iron (TSI), Sulfur Indole Motility (SIM), Voges-Proskauer/Methyl Red (VP/MR), OF medium, Simmons citrate agar, urea agar, and oxidase and glucose fermentation tests were used. Finally, the special \u003cem\u003ePseudomonas\u003c/em\u003e colonies were stored in a -20\u0026deg;C freezer after stock preparation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotic susceptibility testing\u003c/h2\u003e \u003cp\u003e The Kirby\u0026ndash;Bauer disk diffusion method, as described by the Clinical and Laboratory Standards Institute (CLSI 2020) guidelines, was utilized to determine anti-microbial susceptibility. Bacterial suspensions in saline solution were standardized to a concentration of 0.5 McFarland standard and applied onto Mueller Hinton Agar plates using swabs. Small circular discs containing specific antibiotics such as Ciprofloxacin (5 \u0026micro;g), Imipenem (10 \u0026micro;g), Ceftazidime (30 \u0026micro;g), Aztreonam (30 \u0026micro;g), Amikacin (30 \u0026micro;g), Levofloxacin (30 \u0026micro;g), Cefepime (30 \u0026micro;g), Sulfamethoxazole (23.75 \u0026micro;g), Piperacillin / Tazobactam (10/100 \u0026micro;g), Ceftriaxone (30 \u0026micro;g) and Cefoxitin (30 \u0026micro;g) from Becton Dickinson were placed on the surface of the inoculated plates. After incubating the plates at 37\u0026deg;C for 24 hours, the diameter of the growth inhibition zones around the antibiotic discs was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMinimum inhibitory concentration (MIC) for antibiotic colistin\u003c/h2\u003e \u003cp\u003eThe MIC was defined through the broth microdilution method. In this method, the MIC for colistin antibiotic on the strain containing the \u003cem\u003emcr\u003c/em\u003e-1 gene was determined. To perform the MIC for colistin antibiotic, a microbial suspension with turbidity equivalent to 0.5 McFarland standard was prepared from the 24-hour cultures of the mentioned strains in Muller-Hinton broth. A stock solution of the desired antibiotic at a concentration of 10 mg/ml was prepared, and consecutive dilutions of this stock were made to achieve a final concentration range of 0.256 \u0026micro;g/ml to 0.25 \u0026micro;g/ml after mixing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of silver nanoparticles and herbal extracts\u003c/h2\u003e \u003cp\u003eThe silver nanoparticles were purchased in a ready-to-use from Yasatab Medical Company. The particle diameter was 20 nanometers, and they had a spherical shape. The plant extracts were also obtained commercially from Ibn Masawayh Pharmaceutical Company. The extracts were alcohol-based and used at concentrations ranging from 1 to 8 (1, 2, 4, 8) mg/ml for Urtica extract and from 0.5 to 4 (0.5, 1, 2, 4) mg/ml for Shirazi thyme extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSurvival assay (MTT assay)\u003c/h2\u003e \u003cp\u003eFor cytotoxicity assay, the Human Dermal Fibroblasts (HDF\u003csub\u003es\u003c/sub\u003e) were seeded on 96 wells and then incubated for 24 hours at 37\u0026deg;C with CO\u003csub\u003e2\u003c/sub\u003e. The wells were washed with PBS twice. Different concentrations of Urtica and Zataria multiflora extracts and silver nanoparticles were added and incubated for 24 hours again. The wells were rinsed and washed with PBS and 10 \u0026micro;l of MTT solution with 100 \u0026micro;l DMEM were added in each well. Plate containing MTT incubated for 4h at 37\u0026deg;C. The media were gently removed, and 100 \u0026micro;l DMSO was added and incubated in darkness. The absorbance reading in 96-well plates was carried out in a chromate ELISA Reader at 570 nm.\u003c/p\u003e \u003cp\u003eThe following formula calculated the cell survival (19):\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"321\" height=\"56\"\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular characterization of the\u003c/b\u003e \u003cb\u003emcr\u003c/b\u003e\u003cb\u003e-1 gene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter examining the physical and biochemical characteristics of the bacteria, the genomic DNA of \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates was extracted by the phenol-chloroform method for molecular identification of the \u003cem\u003emcr\u003c/em\u003e-1 gene. PCR was conducted using the following primers: forward: 5'-CGGTCAGTCCGTTTGTTC-3' and reverse: 5'-CTTGGTCGGTCTGTAGGG-3'. The amplification process included an initial denaturation at 94\u0026deg;C for 5 minutes, followed by 35 cycles at 94\u0026deg;C for 1 minute, 51\u0026deg;C for 30 seconds, and 72\u0026deg;C for 30 seconds. A final extension was performed at 72\u0026deg;C for 5 minutes. The size of the DNA fragments was confirmed using a 100 bp DNA ladder, with a target product size of 309 bp.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInvestigation of sequencing and registration in the NCBI database\u003c/h2\u003e \u003cp\u003eThe purified PCR products were sent to the Gene Fanavaran Company (Tehran, Iran) for sequencing to confirm the presence of the antibiotic-resistant gene, \u003cem\u003emcr\u003c/em\u003e-1, in the resistant strain of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. The accuracy of the sequencing was examined using Chromas v 2.6.5 software. The obtained sequences were further analyzed using the online BLAST software in the NCBI database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm analysis\u003c/h2\u003e \u003cp\u003eThe potential of the \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates to create biofilm was tested using a microtiter dish biofilm formation assay. In this procedure, the isolates were cultured overnight at 37˚C in tryptic soy broth (TSB) supplemented with 0.25% glucose. The cultures were then diluted 1:100 in the TSB medium. Flat-bottomed 96-well polystyrene microtiter plates were aseptically inoculated with 125 \u0026micro;L of the bacterial suspension and incubated for 24 hours at 37˚C without agitation. After incubation, the wells were washed three times with 300 \u0026micro;L of distilled water and air-dried in an inverted position at room temperature. Subsequently, the wells were stained with 125 \u0026micro;L of 0.1% crystal violet solution in water for approximately 10\u0026ndash;15 minutes. Following staining, the wells were washed three times with distilled water. To destain the wells, 125 \u0026micro;L of 30% acetic acid in water was added to a new sterile microtiter plate, and the destaining solution was transferred to each well. The absorbance of the destaining solution was measured at 595 nm using an ELISA reader. Each experiment was performed in triplicate, and the uninoculated medium served as a control. Based on the optical density of the samples (ODi) and the average optical density of the negative control (ODc), the samples were categorized as strong biofilm producers (4xODc\u0026thinsp;\u0026lt;\u0026thinsp;ODi), moderate biofilm producers (2xODc\u0026thinsp;\u0026lt;\u0026thinsp;ODi\u0026thinsp;\u0026le;\u0026thinsp;4xODc), weak biofilm producers (ODc\u0026thinsp;\u0026lt;\u0026thinsp;ODi\u0026thinsp;\u0026le;\u0026thinsp;2xODc), or non-producers of biofilm (ODi\u0026thinsp;\u0026lt;\u0026thinsp;ODc).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eCheckerboard assay\u003c/h2\u003e \u003cp\u003eThe checkerboard technique is used to evaluate the synergistic effects between two agents. In the present study, this method was employed to assess the efficacy of silver nanoparticles' minimum inhibitory concentration (MIC) along with the herbal extracts of thyme and Urtica on clinical isolates. Twofold dilutions were prepared for the herbal extracts and silver nanoparticles. In each well, a total of 50 \u0026micro;L of Mueller-Hinton broth was added to prepare the microdilution serial plate for the microdilution assay. Then, each desired herbal extract was serially distributed in the vertical wells, while the silver nanoparticles were distributed horizontally. Afterward, a standardized 0.5 McFarland bacterial suspension of the clinical isolate \u003cem\u003eP. aeruginosa\u003c/em\u003e was prepared, and 10 \u0026micro;L of the suspension containing 5 \u0026times; 10^5 CFU/mL of bacteria was added to each well. The plate was then incubated aerobically at 37\u0026deg;C for 24 hours. The checkerboard result represents a combination of two substances (herbal extract\u0026thinsp;+\u0026thinsp;nanoparticle) with the well that contains the lowest concentration of the herbal extract and nanoparticle, effectively inhibiting bacterial growth being selected.\u003c/p\u003e \u003cp\u003eIn the checkerboard experiments, the bactericidal effect of the two molecules was assessed using the formula ΣFIC\u0026thinsp;=\u0026thinsp;FIC A\u0026thinsp;+\u0026thinsp;FIC B, which is given as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\frac{\\text{A}}{{\\text{M}\\text{I}\\text{C}}_{\\text{A}}}+\\frac{\\text{B}}{{\\text{M}\\text{I}\\text{C}}_{\\text{B}}}={\\text{F}\\text{I}\\text{C}}_{\\text{A}}+{\\text{F}\\text{I}\\text{C}}_{\\text{B}}=\\text{F}\\text{I}\\text{C}\\:\\text{I}\\text{n}\\text{d}\\text{e}\\text{x}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn this formula, A represents the MIC value of drug A in combination with another drug, and B represents the MIC value of drug B when combined with drug A. MIC A corresponds to the MIC value of drug A alone, and MIC B is the MIC value of drug B alone. If the value of ΣFIC is lower than or equal to 0.5, it is defined as a synergistic effect. If it is between 0.5 and 1, it is considered ineffective, and if it is greater than one, it will exhibit an antagonistic effect.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAgar well diffusion assay\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of the herbal extracts against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e was investigated via this method. A freshly cultured Pseudomonas aeruginosa suspension was prepared in 100 \u0026micro;L of PBS. Then, the suspension was streaked onto Mueller-Hinton agar medium using a swab. Approximately 6 mm of the medium surface was removed using a pipette tip, and 50 \u0026micro;L of the herbal extract dissolved in PBS at various concentrations was added to the wells. The plate was then incubated at 37\u0026deg;C for 24 hours. The formation of a growth inhibition zone around the well indicated the antimicrobial activity of the herbal extracts against Pseudomonas aeruginosa. Aztreonam disks were used as a positive control, and PBS was used as a negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eSPSS 22 (IBM Corp. 1989, 2013, New York, USA) was utilized to conduct the statistical analyses. The statistical significance of the data was assessed through one-way ANOVA (Tukey), and a p-value of 0.05 was deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIsolation of strains of\u003c/b\u003e \u003cb\u003eP. aeruginosa\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the conducted study, 95 isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e, which were obtained from severe infections in hospitalized individuals, were included in the study after species identification using Gram staining and tests including oxidase, glucose fermentation, citrate utilization, SIM, MR/VP, OF, and urea. Of these 95 people, 35 people (37%) were men and 60 people (63%) were women. Among the total isolated strains collected from patients in different units, the majority were associated with subjects in the Intensive Care Unit (ICU) and Neonatal Intensive Care Unit (NICU), accounting for 35 strains (36.84%) and 15 strains (15.78%), respectively. Additionally, 30 strains (31.57%) were derived from wound samples, and 15 strains (15.78%) were extracted from catheter samples. The age range of the patients varied from 12 days to 88 years.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAntimicrobial susceptibility profiles\u003c/h2\u003e \u003cp\u003eThe results of antibiotic susceptibility testing were performed on 95 confirmed clinical isolates using phenotypic tests. Among the isolates, 94 isolates were resistant to Levofloxacin, and 6 isolates were resistant to colistin antibiotic, and out of 6 isolates resistant to colistin, all of them were obtained from wounds. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibiotic resistance pattern of \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibiotic class\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntibiotic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eResistant\u003c/p\u003e \u003cp\u003eNO (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIntermediate\u003c/p\u003e \u003cp\u003eNO (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSusceptible\u003c/p\u003e \u003cp\u003eNO (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCephems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCefepime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66(70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10(10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e19(20)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCeftazidime\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49(51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11(12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e35(37)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAminoglycosides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmikacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e82(86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e13(14)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePenems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eImipenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72(76)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12(13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e11(11)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-lactam\u0026thinsp;+\u0026thinsp;Inhibitors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePiperacillin/tazobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41(43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15(16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e39(41)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuinolones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87(92)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e7(7)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLevofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94(99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1(1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonobactam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAztreonam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45(47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10(11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e40(42)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMinimum inhibitory concentration\u003c/h2\u003e \u003cp\u003e According to the broth microdilution method results, The MIC values for 95 isolates were determined according to CLSI 2020 guidelines for the antibiotic colistin. Among the 95 isolates, 6 were found to be resistant to colistin. The MIC for silver nanoparticles was also determined to be below 8 micrograms per milliliter, specifically at a concentration of 2 micrograms per milliliter. Furthermore, 1 milligram per milliliter and 8 milligrams per milliliter were assigned for the extract of Shirazi thyme and Urtica, respectively. All experiments were performed in triplicate to ensure the reproducibility of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSurvival assay (MTT assay)\u003c/h2\u003e \u003cp\u003eThe surveyed Urtica extract and silver nanoparticle in lower concentrations showed no significant adverse effects on Human Fibroblast cell viability. The ordinary two-way ANOVA analysis of the data obtained from the MTT assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCheckerboard assessment\u003c/h2\u003e \u003cp\u003eThe checkerboard method was used to investigate the antimicrobial interaction of Urtica extract in combination with silver nanoparticles. In this method, 50 microliters of each antimicrobial compound were added to each well of the plate, and each horizontal row was assigned to a series of concentrations of one antimicrobial compound, while each vertical column was assigned to a series of concentrations of another antimicrobial compound. Subsequently, 100 microliters of microbial suspension were added to each well. Each plate included positive and negative controls. The positive control well contained bacterial suspension and growth medium, while the negative control well contained the antimicrobial combination and growth medium. After 24 hours of incubation at 37 degrees Celsius, the results were expressed using the fractional inhibitory concentration (FIC) formula and calculated inhibitory concentrations.\u003c/p\u003e \u003cp\u003eWe used the following formula to assess the synergistic effect of the two desired substances:\u003c/p\u003e \u003cp\u003e\u003cimg 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003e\u003cp\u003eThe fractional inhibitory concentration (FIC) of the combination of silver nanoparticles and Shirazi thyme was calculated to be 0.37, which is less than 0.5. This indicates a synergistic effect between the two substances (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm assay\u003c/h2\u003e \u003cp\u003eBased on the microtiter dish biofilm formation assay, The measured OD values for 55 samples were higher than 0.2, indicating the presence of strong biofilm formation. All 6 resistant to colistin isolates (100%) showed strong biofilm formation. The measured OD values for 33 samples ranged from 0.1 to 0.2, indicating moderate biofilm formation. The measured OD values for 7 samples ranged from 0.1 to 0.07, indicating weak biofilm formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAgar Well Diffusion evaluation\u003c/h2\u003e \u003cp\u003eAgar well diffusion assay was performed using plant extracts of Urtica and \"Shirazi Thyme.\" The inhibitory concentrations for Urtica and Shirazi Thyme were 8 and 1 milligrams per milliliter, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003emcr\u003c/b\u003e\u003cb\u003e-1 gene by PCR and Sequencing\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePCR was performed on 95 isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e, and one sample from the wound specimens was found to have the \u003cem\u003emcr\u003c/em\u003e-1 gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eColistin has gained renewed attention as an old antibiotic for treating severe infections, especially in carbapenem-resistant \u003cem\u003eEnterobacteriaceae\u003c/em\u003e. The reported resistance rate to colistin in Pseudomonas aeruginosa ranges from 0\u0026ndash;17.46% (20). In the present study, which was the first report from Isfahan (Iran), 95 strains of \u003cem\u003eP. aeruginosa\u003c/em\u003e were isolated from severe infections in hospitalized people. Antibiotic susceptibility tests showed high resistance among \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates. The detailed pattern of antibiotic resistance, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, shows significant resistance to several antibiotics, including cefepime, ceftazidime, amikacin, imipenem, piperacillin/tazobactam, ciprofloxacin, levofloxacin, and azetronam. It is worth mentioning that 94 isolates were resistant to levofloxacin and 6 isolates showed resistance to colistin, which included 31.6% of the strains. This rate is comparable to the resistance reported in previous studies conducted in Iran (Iran). The majority of the isolates were from male patients (63%), and the samples were obtained from various intensive care units (ICU) and neonatal intensive care units (NICU). In a 2022 study by Shahri et al. in Gorgan (Iran), a colistin resistance rate of 9.27% in P. aeruginosa isolates was reported, aligning with our study (21). Balkhair et al.'s study in Oman in 2019 reported a colistin resistance rate of 2.7% in \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates. A 2023 study by Soni et al. in India, focusing on A. baumannii and \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates, documented colistin resistance rates of 2.2% and 5.3%, respectively (22, 23). The results of our present study deviate from some studies on colistin resistance, such as Akar et al.'s 2019 research in Turkey, which indicated a colistin resistance rate of 2.2% in \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates (24). In a 2020 study by Santimalee Woragun et al. in Thailand, the rate of colistin resistance in \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates was reported to be 1.6%, contradicting our findings (25). Araaf et al.'s 2022 study in Pakistan reported colistin resistance rates of 19.9%, 16.1%, and 13.1% in Escherichia coli, Klebsiella pneumoniae, and \u003cem\u003eP. aeruginosa\u003c/em\u003e, respectively (26). In 2022, in Mashhad (Iran), Zamani and colleagues reported a 4% resistance rate to colistin in \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates, with the presence of the mcr-2 gene responsible for their resistance (27). These variations among different reports may be attributed to geographical differences, variations in resistance testing methods, the diversity of sample types, sample sizes, the general condition of patients, different antibiotic prescription policies, and adherence to infection control measures.\u003c/p\u003e \u003cp\u003eToday, plasmid-mediated mcr genes are rapidly spreading among bacteria, increasing the potential for colistin resistance in bacterial populations. Previous studies have reported the presence of the mcr-1 gene in clinical isolates from more than 50 countries and regions, including 21 countries in Asia, 14 countries in Europe, 3 countries in North America, 8 countries in Latin America, and 3 countries in Africa (28). In our study, an isolate carrying the mcr-1 plasmid gene was obtained from a wound sample, emphasizing the importance of alternative therapeutic strategies. This finding is crucial for understanding the mechanisms contributing to colistin resistance. Talebi et al.'s report in Tehran on clinical isolates of \u003cem\u003eP. aeruginosa\u003c/em\u003e obtained from burn units showed that no isolate carrying the mcr-1 gene was found (29). In the study by Moradi et al. in 2022 in Isfahan, 2% of \u003cem\u003eP. aeruginosa\u003c/em\u003e strains had the mcr-1 gene (30). These differences in findings may be attributed to the increase in antibiotic use in recent years compared to the past, geographical changes, and the type and number of samples. The presence of the mcr-1 gene in clinical isolates is increasing in Iran.\u003c/p\u003e \u003cp\u003eIn general, bacteria carrying the mcr-1 gene are considered resistant to colistin. However, a 2017 study by Wang et al. in China reported that 3% of E. coli isolates carrying the mcr-1 gene remained susceptible to colistin (31). The first report of the mcr-1 gene in a colistin-susceptible strain of Shigella sonnei in Vietnam was made in 2016 by Fam et al. This finding suggests that silent release of this gene may occur or another gene may be inserted into mcr-1, leading to its inactivation. However, further large-scale studies are necessary to clarify this issue (10).\u003c/p\u003e \u003cp\u003eIn the present study, silver nanoparticles and thyme extract showed inhibitory effects individually and in combination against colistin-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e. However, ginger extract did not exhibit any therapeutic inhibitory effect. In the study by Safari et al. in 2019 in Tehran, the inhibitory effect of thyme extract alone and in combination with gentamicin and chloramphenicol antibiotics on gram-positive and gram-negative bacteria was investigated, and it was found that the inhibitory effect was greater on gram-negative bacteria (32). In the study by Zinali Aghdam et al. in 2019 in Tehran, the inhibitory effects of ginger extract and silver nanoparticles alone and in combination against \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e were investigated, and it was found that they exhibited a synergistic strengthening effect against \u003cem\u003eA. baumannii\u003c/em\u003e (33). In a study by Jyoti et al. in 2016 in India, the synergistic effect of silver nanoparticles and ginger extract on common pathogens was investigated, and silver nanoparticles showed a very good inhibitory effect, while ginger extract had a relatively weaker inhibitory effect (34). In the study by Guntherip et al. in 2022 in Germany, the effects of mint, thyme, and green tea extracts on Escherichia coli, Staphylococcus epidermidis, and \u003cem\u003eP. aeruginosa\u003c/em\u003e were investigated. Thyme extract partially reversed the resistance of Escherichia coli to bacitracin and penicillin (35). In a study conducted by Mohammad et al. in 2022 in Egypt on multidrug-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e isolated from dental implants, silver nanoparticles showed inhibitory effects on all isolates and had significant anti-biofilm activity (36). In a study conducted by Arshad and colleagues in 2021 in Pakistan, the effect of silver nanoparticles on multidrug-resistant microorganisms was investigated. It was shown that silver nanoparticles have a significant inhibitory effect against Gram-positive bacteria such as \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and Gram-negative bacteria such as \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e, and \u003cem\u003eP. aeruginosa\u003c/em\u003e. Additionally, inhibitory activity against Candida albicans, a fungal pathogen, was demonstrated. These findings indicate that silver nanoparticles have significant inhibitory effects against a wide range of microorganisms, including bacteria and fungi (37).\u003c/p\u003e \u003cp\u003eAccording to the results of the MTT test, nettle extract and silver nanoparticles at lower concentrations did not show any significant adverse effects on the viability of human skin fibroblast cells. This is an important observation that suggests the possible safety of using these substances.\u003c/p\u003e \u003cp\u003eBiofilm formation was common among \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates. This emphasizes the role of biofilm in antibiotic resistance and challenges related to the treatment of biofilm-forming strains.\u003c/p\u003e \u003cp\u003eIn this study, a comprehensive review was conducted on the challenges posed by colistin-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates, and alternative strategies for their treatment were explored. These findings shed light on critical aspects of antibiotic resistance, antimicrobial interactions, and potential therapeutic roles of plant extracts and silver nanoparticles.\u003c/p\u003e \u003cp\u003eThe high prevalence of antibiotic resistance among \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates, especially resistance to colistin, emphasizes the urgent need for effective treatment options. Significantly, the emergence of resistance to last-resort antibiotics such as colistin raises concerns, and there is an urgent need for innovative approaches to address this issue. Our findings on the synergistic effects between nettle extract and silver nanoparticles showed a promising way to overcome antibiotic resistance. The observed synergistic effect, as evidenced by the fractional inhibitory concentration (FIC: 0.37), suggests that the combination of these substances may increase their antimicrobial potency. Detection of the mcr-1 gene in an isolate indicates the presence of plasmid-mediated colistin resistance and adds a layer of complexity to existing resistance mechanisms. Understanding such genetic factors is crucial for devising targeted interventions and regulatory strategies to manage and prevent the spread of antibiotic resistance.\u003c/p\u003e \u003cp\u003eIn summary, this study provides valuable information on the prevalence of colistin-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e isolates, their antibiotic susceptibility patterns, and the potential synergistic effects of nettle extract and silver nanoparticles. The observed biofilm formation emphasizes the complexity of treating these strains, and the detection of the mcr-1 gene highlights the importance of monitoring plasmid-mediated resistance mechanisms. These findings help to understand alternative strategies to deal with antibiotic resistance in \u003cem\u003eP. aeruginosa\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe emergence of multidrug-resistant (MDR) isolates and resistance to colistin poses a serious global problem. When new and effective antibiotics are unavailable, colistin becomes the last resort for treating Gram-negative bacteria. The prevalence of colistin resistance varies in different geographical areas due to distinct strategies in infection control and treatment across different hospitals. Therefore, obtaining information on colistin resistance and continuous monitoring to determine the exact frequency of the mcr-1 gene among Gram-negative bacteria in human and veterinary medicine worldwide is of great importance. Our study provides valuable information on the multifaceted challenge of colistin-resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e infections. The identified synergistic effects of nettle extract and silver nanoparticles, coupled with a comprehensive understanding of antibiotic resistance patterns and biofilm formation, lay the foundation for further research and the development of innovative therapeutic approaches. Implementing new methods to treat these resistant strains is highly practical. Strategies in this field include controlling antibiotic consumption, understanding the mechanisms of antibiotic resistance, and developing appropriate treatment methods with therapeutic effects and minimal side effects, such as plant extracts and nanoparticles, that can be used as alternative antibiotics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e“The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e“The authors have no relevant financial or non-financial interests to disclose.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e“All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by \u003cem\u003eAli Ahmadi\u003c/em\u003e and \u003cem\u003eMitra Rabiei\u003c/em\u003e. The first draft of the manuscript was written by \u003cem\u003eAli Ahmadi\u003c/em\u003e and \u003cem\u003eMitra Rabiei\u003c/em\u003e and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e“This study was conducted in the in vitro phase and was registered in the Ethical Committee of Isfahan University of Medical Sciences with the ethical code \"IR.MUI.MED.REC.1400-509\" on 2021/09/26.”\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\"This study was conducted in the in vitro phase, and due to the lack of human studies, there was no need to obtain consent from individuals.\"\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent to publish\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\"This study was conducted in the in vitro phase, and due to the lack of human studies, there was no need to obtain consent from individuals.\"\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the information of this study is available to the authors of the article and according to the criteria of the journal of \"\u003cu\u003eInternational Microbiology\u003c/u\u003e\", it is provided to the researchers.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKern WV, Roth JA, Bertz H, G\u0026ouml;tting T, Dettenkofer M, Widmer AF, et al. 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Global prevalence and molecular epidemiology of mcr-mediated colistin resistance in Escherichia coli clinical isolates: A systematic review. Journal of global antimicrobial resistance. 2022;29:444-61.\u003c/li\u003e\n\u003cli\u003eShahri FN, Izanloo A, Goharrizi MASB, Jamali A, Bagheri H, Hjimohammadi A, et al. Antimicrobial resistance, virulence factors, and genotypes of Pseudomonas aeruginosa clinical isolates from Gorgan, northern Iran. International Microbiology. 2022;25(4):709-21.\u003c/li\u003e\n\u003cli\u003eBalkhair A, Al-Muharrmi Z, Al\u0026rsquo;Adawi B, Al Busaidi I, Taher H, Al-Siyabi T, et al. Prevalence and 30-day all-cause mortality of carbapenem-and colistin-resistant bacteraemia caused by Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae: description of a decade-long trend. International Journal of Infectious Diseases. 2019;85:10-5.\u003c/li\u003e\n\u003cli\u003eSoni M, Kapoor G, Perumal N, Chaurasia D, Soni Jr M. 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Identification of colistin resistance and its bactericidal activity against uropathogenic gram negative bacteria from Hayatabad Medical Complex Peshawar. Pakistan Journal of Medical Sciences. 2022;38(4Part-II):981.\u003c/li\u003e\n\u003cli\u003eZomorodi AR, Mohseni N, Hafiz M, Nikoueian H, Hashemitabar G, Salimizand H, et al. Investigation of mobile colistin resistance (mcr) genes among carbapenem resistance Pseudomonas aeruginosa isolates from bovine mastitis in Mashhad, Iran. Gene Reports. 2022;29:101695.\u003c/li\u003e\n\u003cli\u003eXiaomin S, Yiming L, Yuying Y, Zhangqi S, Yongning W, Shaolin W. Global impact of mcr-1-positive Enterobacteriaceae bacteria on \u0026ldquo;one health\u0026rdquo;. Critical reviews in microbiology. 2020;46(5):565-77.\u003c/li\u003e\n\u003cli\u003eTalebi G, Hakemi-Vala M. Survey on some carbapenems and colistin resistance genes among Pseudomonas aeruginosa isolates from burn and cystic fibrosis patients, Tehran, Iran. Archives of Clinical Infectious Diseases. 2019;14(5).\u003c/li\u003e\n\u003cli\u003eMoarefian M, Poursina F, Narimani T. Resistance to Colistin and Detection of mcr-1 Gene in Multidrug-Resistant Pseudomonas Aeruginosa Isolated from Wounds of the Hospitalized Burned Patients. Journal of Isfahan Medical School. 2022;40(686):678-84.\u003c/li\u003e\n\u003cli\u003eWang Y, Tian G-B, Zhang R, Shen Y, Tyrrell JM, Huang X, et al. Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study. The Lancet Infectious Diseases. 2017;17(4):390-9.\u003c/li\u003e\n\u003cli\u003eSaffari Samani E, Jooyandeh H, Alizadeh Behbahani B. Evaluation of reciprocal pharmaceutical effect and antimicrobial activity of Shirazi thyme essential oil against some Gram-positive and Gram-negative bacteria. Journal of food science and technology (Iran). 2020;17(104):1-11.\u003c/li\u003e\n\u003cli\u003eZeinali Aghdam S, Minaeian S, Sadeghpour Karimi M, Tabatabaee Bafroee AS. The antibacterial effects of the mixture of silver nanoparticles with the shallot and nettle alcoholic extracts. Journal of Applied Biotechnology Reports. 2019;6(4):158-64.\u003c/li\u003e\n\u003cli\u003eJyoti K, Baunthiyal M, Singh A. Characterization of silver nanoparticles synthesized using Urtica dioica Linn. leaves and their synergistic effects with antibiotics. Journal of Radiation Research and Applied Sciences. 2016;9(3):217-27.\u003c/li\u003e\n\u003cli\u003eGuntrip RB, Luce MJ. Peppermint, thyme, and green tea extracts modulate antibiotic sensitivity. Bios. 2022;92(3):104-10.\u003c/li\u003e\n\u003cli\u003eEl-Telbany M, El-Sharaki A. Antibacterial and anti-biofilm activity of silver nanoparticles on multi-drug resistance pseudomonas aeruginosa isolated from dental-implant. Journal of Oral Biology and Craniofacial Research. 2022;12(1):199-203.\u003c/li\u003e\n\u003cli\u003eArshad H, Saleem M, Pasha U, Sadaf S. Synthesis of Aloe vera-conjugated silver nanoparticles for use against multidrug-resistant microorganisms. Electronic Journal of Biotechnology. 2022;55:55-64.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pseudomonas aeruginosa, Zataria multiflora extract, Urtica extract, silver nanoparticles","lastPublishedDoi":"10.21203/rs.3.rs-4688621/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4688621/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe pathogen of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is a significant nosocomial infection. Metal nanoparticles (NPs) and plant extracts have demonstrated antibacterial efficacy against multidrug-resistant bacteria. This study aimed to determine the synergistic antibacterial effect of silver nanoparticles and Urtica and Zataria multiflora (Shirazi thyme) extracts against Colistin-Resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e isolated from hospital patients.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 95 \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e isolates were collected from selected teaching hospitals in Isfahan province. The Kirby\u0026ndash;Bauer disk diffusion method used for antibiotic susceptibility and determining the minimum inhibitory concentration of colistin was checked by microdilution broth, and the presence of the mcr-1 gene was detected by PCR method. In addition, the effect of synergism of Zataria multiflora (Shirazi thyme) and Urtica extracts with silver nanoparticles was investigated by checkerboard procedure.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe screening for antibiotic resistance showed 99% resistance to Levofloxacin and 6% resistance to colistin antibiotic. One isolate is positive for the mcr-1 gene. Furthermore, combining Zataria multiflora (Shirazi thyme) and the silver nanoparticle is efficient against Colistin-Resistant \u003cem\u003ePseudomonas aeruginosa.\u003c/em\u003e\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFor the first time, the synergistic effect of Zataria multiflora (Shirazi thyme) and Urtica extracts and silver nanoparticle extracts against Colistin-Resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e was examined in our research. So plant extracts and silver nanoparticles might assist with managing multidrug-resistant pathogenic bacteria and be a possible source of medicinal application due to their potential antibacterial effect.\u003c/p\u003e","manuscriptTitle":"The Synergistic Effects of Plant Extracts and Silver Nanoparticles on Colistin-Resistant Pseudomonas aeruginosa Isolates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-04 23:17:03","doi":"10.21203/rs.3.rs-4688621/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5aef3107-ecfd-40c0-bd01-cc1b69f0cf45","owner":[],"postedDate":"September 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-18T13:24:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-04 23:17:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4688621","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4688621","identity":"rs-4688621","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}