Effects of Imipenem-containing Niosome nanoparticles against high prevalence methicillin-resistant Staphylococcus Epidermidis biofilm formed

Ethics approval and consent to participate

The authors of this article state that all methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org). All protocols were performed in accordance with the Ethical Committee and Research Deputy of the Islamic Azad University of Shahrekord Branch, Iran for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee guidelines of Islamic Azad University, Shahrekord, Iran (IR.IAU.SHK.REC.1400.043). Ethics approval needed for microbial testing and isolation of pathogenic bacteria in Iran.

Preparation of Niosomes encapsulated Imipenem

Formulation and encapsulation of Imipenem in noisome

According to instructions provided by previous studies^20,21, Noisome encapsulation of imipenem was conducted through thin-film hydration, and 2 drug compounds were prepared as follows. The first compounds were prepared. The first compound contained Span 60 (CAS: 1338-41-6) (Henan Daken Chemical CO., LTD., China) and Tween 60 (CAS: 9005-67-8) (Career Henan Chemical Co, China) mixed with cholesterol (CAS: 57-88-5) (Capot Chemical Co., Ltd., China) at molar ratios of 3:3:4 and was dissolved in 10 ml of chloroform and methanol mixed at ratios of 2:1. The second compound contained Span 40 (CAS: 26266-57-9) (Henan Daken Chemical CO., LTD., China) and Tween 40 (CAS: 9005-66-7) (Shanghai Jizhi Biochemical Technology Co., Ltd, China) mixed with cholesterol (CAS: 57-88-5) (Capot Chemical Co., Ltd., China) with respective molar ratios of 3:3:4 and was dissolved in 10 ml of chloroform and methanol mixed with respective ratios of 2:1 (Table 1). After adding glass beads to all 2 compounds, drug compound solvents were evaporated using a rotary evaporator (Heidolph, Germany) for one hour at 60 °C and 120 rpm rotation. Afterward, dried thin films were hydrated for one hour using a solution of imipenem dissolved in 10 ml of PBS at 60 °C with a speed of 120 rpm to obtain different Niosome formulations. The minimum inhibitory concentration of imipenem against Staphylococcus (1 mg/ml) was used as the concentration of this drug for encapsulation. The resulting particles were sonicated using a probe sonicator in an ice bath using SONOPULS ultrasonic homogenizers (amplitude: 25%, 200 wt) for five minutes to reduce the size of Niosomes containing imipenem, and samples were stored at 4 °C for subsequent experiments.

Noisome-encapsulated imipenem morphological characteristics

The average size, polydispersity index, and zeta potential of imipenem loaded in noisome were obtained using dynamic light scattering (DLS) and ZetaPlas palladium electrodes (Brookhaven Instruments Corp., USA). The polydispersity index (PDI) is a size-based metric for assessing sample variability. Polydispersity can develop as a result of a pattern’s particles size or accumulation or aggregation during extraction or evaluation. PDI was measured by DLS (Brookhaven Instruments Corp., USA). Various newly prepared Niosome formulations were diluted two times using distilled water at a ratio of 20:1 to prevent multiple scattering as a result of interactions between particles, and analysis was conducted at 25 °C with a 90-degree light scattering angle. The average z diameter and Niosome multiple scattering index were determined, and their zeta potentials were measured. Niosome-encapsulated imipenem particles were coated with a gold layer to generate electrical conductivity and were examined using an electron microscope (SEM) device model MIRA3 (TESCAN, Czech Republic).

Entrapment efficiency (EE)

Encapsulation efficiency was obtained by determining the amount of noncapsulate imipenem (free imipenem) in the formed Niosomes. Noisome-encapsulated imipenem particles were isolated over an hour at 4 °C in a refrigerated centrifuge at 1400 rpm. The imipenem content of the supernatant was examined through ELISA Reader Stat Fax2100’s (Awareness Technology, Ukraine) light absorbance reading of the supernatant at 276 nm wavelength, and the EE percentage was obtained based on Formula (1).

Evaluation of noisome-encapsulated release and stability

Dialysis was employed to examine imipenem’s release of the noisome. The dialysis tube was soaked in distilled water for 24 h. Then, 0.5 ml (10 mg) of imipenem-loaded noisome was placed in a dialysis bag, and 0.5 ml imipenem antibiotic aqueous solution containing 10 mg imipenem was also used as a control sample. Dialysis bags were immersed in conical flasks in 75 ml distilled water and shaken at 50 rpm in a water bath at 37 °C. Five milliliters were withdrawn from the receptor medium at intervals of one, two, four, six, 12, and 24 h, Aliquots of samples were replaced with a new medium at 37 °C, and imipenem was measured by spectrophotometry at 281 nm. The diffusion profile was determined using various kinetic models. This method was used to monitor the stability of diffusion of various Niosome-encapsulated imipenem formulations at intervals of 7, 14, 21, 28, 35, 42, 49, and 56 days for a storage period of two months at 25 °C.

Investigation of antibacterial activity

Bacterial isolation and antibiotic susceptibility

This descriptive study collected a total of 300 clinical samples from Shariati (150 samples), Firoozgar (150 samples), in Tehran. The samples used are from hospitals bio-bank. Samples were cultivated in blood agar cultures using sterile swabs at 37 °C for 24 h. First, S. epidermidis isolates were detected using Gram staining, catalase, mannitol fermentation, and DNase tests. Then identification of the species was performed by analyzing the 16S rRNA gene. Strain sensitivity to various antibiotics was examined by disc diffusion according to the Clinical and Laboratory Standards Institute (CLSI, CLSI supplement M100 (ISBN 1-56238-804-5 (Print); ISBN 1-56238-805-3 (Electronic)) method after S. epidermidis strains were detected and confirmed. S. epidermidis isolates’ sensitivity to vancomycin (VA, 10 μg), cefoxitin (FOX, 10 μg), ciprofloxacin (CIP, 5 μg), clindamycin (CD, 2 μg), mupirocin (MU, 5 μg) and imipenem (IMI, 10 μg) (MAST, UK) discs was measured in Muller Hinton agar culture (Merck, Germany). It must be noted that cefoxitin antibiotic discs were used to detect MRSE methicillin resistance, and the S. epidermidis ATCC 14990 standard strain was used as the positive control. Multidrug-resistant (MDR) was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories.

DNA extraction process

The bacterial genomic DNA was extracted according to the directions of the Cinna Gene company’s extraction kit (Cinna Pure DNA KIT, Alborz, Iran). Briefly, the bacterial solution was centrifuged. After centrifuging the bacterium, the precipitate was mixed with 100 μl of proteinase K and protease buffer and heated at 55 °C for 30 min. Lysis buffer was introduced in 200 μl and vortexed for 15 s. Afterward, 300 μl of precipitation solution was added and inverted ten times before being subjected to – 20 °C for one hour. The supernatant was drained after 10-min centrifugation at 13,000 rpm, then 700 μl of wash buffer was added and centrifuged for 5 min at 13,000 rpm. The PCR test was used to identify 16S rRNA gene.

Molecular detection of icaD, FnbA, and EbpS biofilm genes and resistance to vancomycin VanB

The bacterial genomic DNA was extracted according to the directions of the Cinna Gene company’s extraction kit (Cinna Pure DNA KIT, Alborz, Iran), and the purity was verified using a spectrophotometer at 260 nm. The M-PCR test was used to identify biofilm decoding genes icaD, FnbA, EbpS and the vancomycin resistance gene VanB utilizing oligonucleotide sequences of particular primers listed in Table 2. Primer design for genes icaD (AAQ88121.1), FnbA (KAB2208440.1), EbpS (WP_032604310.1) and VanB (OLS07309.1) were performed with Oligo 7 software and the results were reviewed in the online blast tool in the NCBI database. The final reaction volume (50 µL) was considered to contain 30 µL PCR master mix (PCR buffer, MgCl2, dNTP, 0.2 units of Taq polymerase), 0.5 µL icaD reverse primer, 0.5 µL icaD forward primer, 0.5 µL FnbA reverse primer, 0.5 µL FnbA forward primer, 0.5 µL EbpS reverse primer, 0.5 µL EbpS forward primer, 2 µL template DNA, and 15 µL distilled water (Amplicon, Denmark). The PCR program was performed in the form of an initial denaturation at 95 °C for five minutes and 35 initial denaturation cycles for one minute at 94 °C, one minute of annealing at 58 °C, one minute of extension at 72 °C, and a final extension at 72 °C for five minutes. The amplified products were investigated through 1% agarose gel electrophoresis to detect the desired genes of vancomycin resistance. Enterococcus faecalis ATCC 51299 were used as Vancomycin-resistant control.

Antibacterial activity of noisome-encapsulated imipenem

The antibacterial activity of various noisome-encapsulated imipenem formulations against MRSE strains was examined through broth microdilution. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were then determined as follows. The MIC is the lowest concentration of a chemical, usually a drug, which prevents visible growth of a bacterium. The MBC is measured by sub-culturing the broths used for MIC determination onto fresh agar plates. MBC is the lowest concentration of a drug that results in killing 99.9% of the bacteria being tested. To determine the MBC, the dilution representing the MIC and at least two of the more concentrated test product dilutions are plated and enumerated to determine viable CFU/ml. The closer the MIC is to the MBC, the more bactericidal the compound. After culturing S. epidermidis in Müller-Hinton broth for 24 h, 5 × 10⁵ CFU/mL (5 μl) bacteria, 100 μl of various Niosome-encapsulated imipenem formulations, free imipenem and free Niosome (at a concentration ranging from 0.03 to 64 μg/ml), and 95 μl pure Hinton broth were poured into each well of a 96-well plate and incubated for 24 h at 37 °C. The sub-MIC values of negative control well (containing pure culture medium), and positive control well (containing culture medium plus standard S. epidermidis strain ATCC 14,990) were determined, and the test was replicated for a 3 time.

Inhibition of biofilm formation

Sub-MIC concentrations of free imipenem and Niosomes and various formulations of Niosome-encapsulated imipenem were used to determine the minimum biofilm inhibition concentration (MBIC) against the S. epidermidis strains. The wells of the 96-well plate were filled with 100 μl drug sample and 100 μl of the bacteria cultured in Müller Hinton Broth. The plates were incubated for 48 h at 37 °C and stained with crystal violet 1% after being washed. The tests were replicated two times, and the mean MBIC was determined to be OD630 < 0.1.

Analysis of biofilm gene expression

The expression of the biofilm genes icaD, FnbA, and EbpS was examined through polymerase chain reaction (qRT–PCR) using the specific primers indicated in Table 2. RNA was extracted from resistant MRSE bacteria using an RNX-Plus kit (Sina gene, Iran) after 24 h of exposure to sub-MIC concentrations of free imipenem and Niosome-encapsulated imipenem, and cDNA was fabricated based on the RNA extracted from treated and untreated bacteria according to the protocol of the YTA Kit (Yekta Tajhiz, Iran). Biofilm gene expression to the 16S rRNA gene (as the reference gene) was examined using a Corbett 5 Plex HRM real-time PCR device (Corbett, Australia). The final reaction volume was 15 μl, containing 1 μl of cDNA, 1 μl of the forward primer and 1 μl of reverse primer, 8 μl of master mix and 4 μl of deionized water (Merck, Germany), and the temperature cycle included initial denaturation for five minutes at 95 °C followed by 40 cycles of 20 s at 95 °C, 40 s at 58 °C, and 40 s at 72 °C. The final stage was selected to be at 53–95 °C to draw melting curves. Gene quantitative relative expression was examined using the ΔΔCt method.

Cytotoxicity study

According to standard ISO10993-5 and Li et al.²², the cytotoxicity test was performed by the MTT method on HDF normal cells. To check the cytotoxicity, an L929 confluence flask was placed under a hood, and the cells were cultured in fully sterile DMEM with high Glu (catalog number 11965118) (Thermo Fisher Scientific, United States) culture medium²³. The surface of the cells was washed with PBS, and trypsin was added to remove the cells from the fibroblastic state and make them spherical. Then, DMEM containing 10% FBS (bovine fetal serum) was poured onto the cells to neutralize trypsin, and the cells were poured into a Falcon tube and centrifuged at 1200 rpm for 5 min. The supernatant was discarded, DMEM containing 10% FBS was poured, and cell counts were performed on a Neobar slide. For each well, a 24-cell plate of 5 × 10⁴ cells were considered. Sterile samples of free imipenem and niosomal encapsulated imipenem were also placed in the center of each plate under sterile conditions, and the cell suspension was added and placed in a 37 °C incubator containing 5% CO₂ for 24 h. According to standard ISO10993-5, after 24 h, the medium was removed, the cells were washed with PBS, and then a sufficient amount of MTT (1 mg/ml) was poured onto the cell layer. The culture plate was incubated for 3 to 5 h, and the cells were then washed with PBS to remove unreacted MTT. The product of Formosan solvent by isopropanol and OD560 nm of each cell was evaluated. Cell viability was obtained from the absorption fraction of each sample on the control sample (polystyrene container). Additionally, the percentage of living cells or the rate of cell survival was calculated based on the following Eq. (2):

Statistical analysis

Statistical analysis in this study was calculated using SPSS software version 16, and the results were subjected to one-way analysis of variance (ANOVA). Additionally, the expression of target genes between the control and treated samples was calculated by Tukey’s HSD post statistical method. The confidence interval (CI) of the analysis was 95% (CI 2.23–4.23, P < 0.001).

Ethics approval and consent to participate

The study was approved by the Committee of Islamic Azad University of Shahrekord Branch in Iran (IR.IAU.SHK.REC.1400.043).