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2- Molecular Biology Research Center, Systems biology and poisonings institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
3- Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran ,
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Pseudomonas aeruginosa is a gram-negative, non-fermenting, opportunistic, and highly invasive bacillus with an increasing resistance to antimicrobials. It is the normal flora of the skin and intestines that causes respiratory, urinary tract, and gastrointestinal infections, keratitis, otitis, and bacteremia in patients with weakened immune systems (such as cancer, burns, AIDS, cystic fibrosis). These infections often cause death (1). The antibiotic resistance of this bacterium is a major health problem that has increased the cost of treating this disease. Therefore, the limitations of treatment and complications of the disease are also rising (2).
P. aeruginosa pathogens include exotoxin A, exoenzyme S, phospholipase C, and pyocyanin (3). Exotoxin A is one of the main components of the type 2 secretion system that causes tissue necrosis and has a function similar to diphtheria toxin. The toxin inhibits protein synthesis by ADP-ribosylation of elongation factor 2 (4). Inhibition of this toxin reduces cell damage. Nowadays, various antimicrobials are known to prevent the growth of resistant bacteria and to eliminate resistance to antibiotics. Nanoparticles are a broad class of substances with antimicrobial properties. Silver nanoparticle (AgNPs) are best known for their high index of antimicrobial effects.
AgNPs are used for various purposes in medicine (5). They are used as a new antimicrobial agent for sterilization (6). They have high antimicrobial effects on various organisms (1, 7). These nanoparticles accumulate in the bacterial membrane and cause cavities, resulting in unstable cell membrane arrangement and increased permeability (8).
Studies have shown that AgNPs in most cases inhibit the formation of biofilms in P. aeruginosa and Staphylococcus epidermidis. This property can be used in medicine in the future (9). There are also several studies on the synergistic effect of AgNPs and antibiotics. Mirnejad et al. (2013) showed that streptomycin and AgNPs have a synergistic effect with each other (10, 11). Ahmadi et al. (2017) also showed the synergistic effect of tetracycline and AgNPs. They reported a synergistic relationship for wound healing and microbial inhibition (12).
The production of this material is economically valuable. The methods of synthesis of AgNPs have been reviewed and described according to recent studies and approaches which include the physical, photochemical, biological, and chemical approaches. Some examples of chemical approach include the synthesis of nanoparticles using aqueous polymers, the synthesis of AgNPs using low molecular weight compounds as a double dispersing agent, and the method of one-step synthesis of AgNPs-C sintered at temperature (2).
Clindamycin, erythromycin, and imipenem antibiotics are used to treat pseudomonas infections, but the resistance to them is increasing. Therefore, the most effective way to prevent increased resistance is to use alternative antimicrobials and combine other antimicrobials with antibiotics. In this study, first, the antibiotic resistance of erythromycin and AgNPs were evaluated, and then a test was designed to evaluate the synergistic effect of these two substances on clinical samples in vitro.
The present descriptive cross-sectional study was conducted during December 2017 and June 2018. A total of 40 samples were extracted by simple random sampling from the wounds of burn patients admitted to Imam Musa Kazem Hospital in Isfahan, Iran. The samples were identified using standard biochemical and microbiological tests. Samples were first cultured on eosin methylene blue medium (Ibersco, Italy). Then, hot staining, catalase, oxidase, glucose, fermentation, oxidation, and TSI (Triple Sugar Iron) tests were used to diagnose P. aeruginosa. After identification, P. aeruginosa was stored in Tryptic Soy Broth medium (Merck, Germany) with 15% glycerol in a research laboratory at 4°C (13).
Preparation of nanoparticles
To prepare the nanoparticles, we added 640 µg of AgNPs into 10 mL of double distilled water, then used an ultrasonic device (Skymen/China) for two hours to homogenize the solution.
Determination of Minimum Inhibitory Concentration (MIC) of Erythromycin
The MIC of bacterial growth for erythromycin was determined by microdilution method. In this study, 100 µl of bacterial culture medium at a concentration of 0.5 McFarland was added in a ratio of one to ten to all wells except the first column. Then, in the first column, sterile culture medium and the highest inhibitory concentration without bacteria were used for the negative control group. In column 12, only the culture medium solution and 200 µl of bacteria without any inhibitors as a positive control were added.
MIC of Nanoparticles
The concentration of AgNPs was based on one-half by microdilution method. Therefore, dilution series was prepared in 96-well plates from 125-250-500-1000-2000-4000-8000-16000-32000-64000 ng/mL. The concentration of erythromycin was also determined as a half with a dilution series of 7.81-15.62-31.25-62.5-125-250-500-1000-2000-4000 ng/mL. The plates were then placed in an incubator at 37°C for 24 h to grow. Then, 0.5 McFarland turbidity was prepared from bacteria according to standard buffer (106 C 6CFU/mL) (6).
Preparation of Nanoparticle Solution with Antibiotics
The solution of the purchased AgNPs was mixed with 40 µg of erythromycin; then the solution was homogenized using an ultrasonic device for two hours, and the solution was stored at ambient temperature.
MIC of Mixture of Nanoparticles and Antibiotics
Microdilution method was used to determine the MIC of bacterial growth against nanoparticles and
antibiotics. Mixture of 100 µl AgNPs was poured from the second column of the microplate into the third column, and then 100 µl of solution was poured from the second column into the fourth column, and this process continued until column eleven. Finally, dilution series was obtained from a mixture of AgNPs with antibiotics.
PCR Test
To test and confirm the samples, the samples were cultured on Müller-Hinton agar medium. After 24 hours of heating, the main bacterial genome was isolated using a gene extraction kit from Sinaclon Company (Sinagen, Iran). For PCR, pairs of ETA primers with Forward 5'-GACAACGCCCTCAGCATCACCAGC-3' and Reverse 5'-CGCTGGCCCATTCGCTCCAGCGCT-3' primer sequences with a product length of 396 bp were used (14). Also, from Mastermix containing 2.5 μL of PCR buffer, 1 μL of MgCl2, 0.5 μL of dNTPs, 0.5 μL of taq polymerase enzyme (Sinaclon, Iran), along with 1 μL of each primer and 3 μL of template DNA of 25 μL was prepared and placed in the device. The PCR steps are listed in Table 1 to identify the ETA gene in P. aeruginosa. These steps were repeated 34 times. The results of amplification of the fragments were electrophoresed on 1% agarose gel and was observed after staining the gel using DNA safe stain. The standard strain used in this study is P. aeruginosa ATCC 27853 bought from the Microbial Bank of Iran.
Table 1. Polymerase chain reaction conditions for exotoxin A gene amplification in Pseudomonas aeruginosa
| Time | Temperature | |
| First denaturation | 5 min | 95°C |
| Denaturation | 1 min | 95°C |
| Annealing | 1 min | 65.4°C |
| Extension | 1 min | 72°C |
| Final Extension | 1 min | 72°C |
The results of MIC examination showed complete resistance to erythromycin. In this study, the growth of P. aeruginosa was not stopped in any of the cells and all the cells belonging to the inhibitory concentrations (2-11) had turbidity due to the growth of P. aeruginosa.
Results of MIC of AgNPs
The minimum inhibitory concentration of bacterial growth was 2000 μg/mL. In this study, turbidity due to the growth of P. aeruginosa was observed in cells with a concentration of less than 2000 μg/mL.
Results of Synergy of AgNPs and Erythromycin
Lack of growth was observed for synergistic results in cell number 7 (2000) and turbidity due to growth of P. aeruginosa was still observed in lower concentrations.
Results of exotoxin A Gene Prevalence
Out of 26 P. aeruginosa samples, 24 had exotoxin A gene. The prevalence of P. aeruginosa exotoxin A gene in this study was 96.15% of the total samples (Figure 1).
Mechanism of Antimicrobial Effect of AgNPs and Erythromycin
In the introduction section, we discussed the mechanism of nanoparticles that act on cell membranes. Erythromycin, which has a known mechanism of action on protein synthesis, is described in detail in Ketolide section of the chapter 28 of the Jawetz Medical Microbiology; it binds to the S50 component of ribosomes on SrRNA23, interfering with the formation of the initial complex of peptide chain synthesis.
In this study, the prevalence of exotoxin A and antibiotic resistance by microdilution method and the synergistic effect between AgNPs and erythromycin on bacterial inhibition were investigated. According to the findings, the MIC for AgNPs with a diameter of 20-100 nm against P. aeruginosa in well 7 was reported 2 μg/mL.
Also, erythromycin could not be used against P. aeruginosa and all strains obtained were resistant to this antibiotic. The suitable concentration of AgNPs to prevent the growth of P. aeruginosa was 2 μg/mL. In testing the antimicrobial effect of a mixture of antibiotics erythromycin and AgNPs against the growth of P. aeruginosa, this bacterium did not show growth in the well 7 at a concentration of 2 μg/mL.
The prevalence of P. aeruginosa exotoxin A gene in this study was 96.15% of the total samples. This toxin is one of the pathogens of this bacterium. This study showed that the predominant population of P. aeruginosa produces the secretion of exotoxin A burn wound. One of the important factors in the pathogenicity of P. aeruginosa is the production of exotoxin A, which causes serious health problems, and therefore, by performing a test for the presence or absence of this factor, more effective diagnosis and treatment can be performed.
Shinashal et al. conducted a study on the effect of silver and gentamicin on P. aeruginosa by disk diffusion method and found that the antimicrobial effect of AgNPs was better than that of gentamicin; 250 mg of gentamicin and 10 mg of AgNPs were reported (7). In the present study, the antimicrobial effect of AgNPs was reported 2 μg, which is less than that reported by Shinashal et al. The size of the nanoparticles is probably the reason for their increased antimicrobial properties.
Marck et al. (2014) studied the synergy of AgNPs and azithromycin against P. aeruginosa PAO1 biofilm. In this study, AgNPs in the sizes of 10 nm, 20 nm, 40 nm, 60 nm, and 100 nm were used and each of these concentrations were mixed with 0.234 μg/mL, 0.625 μg/mL and 1.25 μg/mL, 2.5 μg/mL, and 7.5 μg/mL of aztreonam antibiotic, respectively; then the MIC was evaluated. The best concentration of AgNPs was reported to be 0.13 μg/mL for 10 μm and 0.500 μm/mL for 20 nm. AgNPs may also have a synergistic relationship with azithromycin against inhibition of P. aeruginosa (15). The results of this study were consistent with the present study.
He T. et al., in an experiment on the antibacterial effect and analysis of graphene-based AgNPs proteome on the pathogen P. aeruginosa, concluded that P. aeruginosa did not grow at 5 μg/mL in the first 7-9 hours but had a slight growth after 48 hours, which was considered as the MIC of bacterial growth inhibitor. It has also been reported that growth stopped at 20 μg/mL and this concentration was considered as the minimum lethal concentration (16). This concentration is consistent with the present study and shows that the antimicrobial effect of AgNPs on P. aeruginosa is positive.
Sondi et al. (2004) studied the effect of AgNPs as antimicrobial agents on Escherichia coli, as a gram-negative bacterial model. They found that AgNPs with a diameter of 12 nm and a concentration of 10 μg cm−3 were able to stop the growth of E. coli by 70% and they significantly reduced the growth at a concentration of 20 μg cm-3 and completely stopped the growth at a concentration of 50-60 μg cm−3 (8).
Kalishwaralal et al. (2010) showed that AgNPs inhibit biofilm formation by P. aeruginosa and S. epidermidis; AgNPs with a diameter of 50 nm were performed by well test method. The diameter of the inhibitory region of 100 nM AgNPs was reported 9.5±0.9 mm for P. aeruginosa and 12±1.2 mm for S. epidermidis; also, for blocking exopolysaccharide (Biofilm), 50 nM was reported for both bacteria (9). The results of the present study are also consistent with the results of the study by Kalishwaralal et al. Finally, it is recommended that this test be performed with other antibiotics.
Exotoxin A is one of the main pathogens of P. aeruginosa. The bacterium is sensitive to AgNPs that have effects on microbes. AgNPs can be used in a way that they are not harmful to host cells. P. aeruginosa is resistant to erythromycin and has no effect on it. Due to the fact that AgNPs and erythromycin were used together and there was no change in the inhibitory concentration, these two antimicrobials have no synergistic effect on this bacterium.
Thanks to guidance and advice from "Clinical Research Development Unit of Baqiyatallah Hospital".
Authors declared no conflict of interests.
Received: 2019/07/7 | Accepted: 2020/07/9 | ePublished: 2020/07/9
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