Volume 28, Issue 7 (10-2021)
RJMS 2021, 28(7): 37-49 |
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Research code: 91486
Ethics code: IR.IAU. TMB.REC. 25.196
Clinical trials code: IR.IAU. TMB.REC. 22.168
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Atena S, Mirzaie A, Ehsani E, Mahmoodzadeh A. Evaluating the antibacterial and biofilm inhibitory activity of aluminium (Al2O3) nanoparticles against clinical isolates of Klebsiella pneumoniae. RJMS 2021; 28 (7) :37-49
URL: http://rjms.iums.ac.ir/article-1-6901-en.html
URL: http://rjms.iums.ac.ir/article-1-6901-en.html
Islamic Azad University , Amir_mirzaie92@yahoo.com
Abstract: (1947 Views)
Background & Aims: The genus Klebsiella belongs to the family Enterobacteriaceae, which consists of gram-negative and immobile species that have mucosal colonies and live in the gastrointestinal tract (1). Among Klebsiella species, Klebsiella pneumoniae is one of the most common opportunistic pathogens that cause serious diseases (3). These strains have become resistant to many antibiotics and one of the strategies of antibiotic resistance of K. pneumoniae strains is biofilm formation (4-6). One of the important genes in biofilm formation in this bacterium is mrkA gene and this gene encodes MrkA protein, which is one of the important proteins of type 3 fimbriae (7). Excessive use of antibiotics leads to increasing antibiotic resistance of these bacteria and therefore the use of alternatives to treatment other than antibiotics is important. One of these solutions is the use of nanotechnology (8). So far, few studies have been conducted to investigate the antimicrobial and antifouling effects of aluminum nanoparticles against bacterial clinical strains. The aim of this study was to investigate the antimicrobial and anti-biofilm effects of Al2O3 nanoparticles against clinical strains of K. pneumoniae.
Methods: In this experimental study, 100 clinical samples including wounds, blood, urine, and cerebrospinal fluid were collected from different hospitals in Tehran (Imam Khomeini Hospital and Pars Hospital). Subsequently, strains of K. pneumoniae were identified by growing one in McConkey agar medium, Gram staining, and biochemical tests (9). Antibiotic resistance pattern of K. pneumoniae strains was determined by disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) instruction. Briefly, adjusted cultures to give a 0.5 McFarland concentrations of the K. pneumoniae strains were inoculated into Muller-Hinton broth (Oxoid, UK) and the susceptibility of the strains to the antibiotics including ampicillin, gentamicin, amikacin, tetracycline, cefepime, ciprofloxacin, levofloxacin and trimethoprim sulfamethoxazol was evaluated (10). In order to investigate the presence of biofilm in K. pneumoniae strains, microtiter plate quantification method was used (11). In this study, Al2O3 nanoparticles were purchased from the American company US nano. In order to investigate the physicochemical properties of Al2O3 nanoparticles, FTIR, electron microscopy (SEM), XRD and DLS tests were used. The SEM, DLS, XRD and FTIR were used for study of morphology, size, nanomaterial characterization and analyze the structure of matter at the molecular scale based on the resonant vibration modes of various molecules on the aluminum nanoparticles, respectively. In order to investigate the antibacterial effects of Al2O3 nanoparticles, the microdilution method was used in a 96-well plate according to the CLSI standard (16). Briefly, various concentrations of Al2O3 nanoparticles including 15.62, 31.25, 62.5, 125, 250, and 500 µg/mL, were added into 96-well containing each K. pneumoniae strain (500 µl) with 5 × 105 concentration, and the 96-well plates were incubated 24 h at 37 0C. The next day, absorbance was taken using UV-visible spectrophotometer at 600 nm and the concentration giving the least optical density corresponds to minimum inhibitory concentration (MIC) of nanoparticles for that particular microorganism. In order to quantitatively study the anti-biofilm effects of aluminum nanoparticles, Crystal violet (CV) assay was used (12). A 96-well plate-based Crystal violet (CV) assay was used for evaluating of anti-biofilm efficacy of Al2O3 nanoparticles. The biofilm-forming K. pneumoniae strains were cultured into 96 well microtiter plates for 24 h at 37 0C. The K. pneumoniae strains were then treated with sub-MIC values of Al2O3 nanoparticles for 24 h incubation at 37 0C. All the wells were then again washed with PBS three times and fixed with methanol for 15 minutes. The plate was air dried for 30 minutes and 0.1% CV solution was added to each well and incubated at room temperature for 20 minutes. After washing with distilled water, 33% acetic acid was added to each well and absorbance was taken at 590 nm. Mean absorbance values of each sample was calculated and compared with the mean values of controls.
Expression analysis of mrkA gene in K. pneumoniae strains under control MIC of free curcumin and curcumin were investigated using the Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) method through specific primers. For this purpose, after treating strains, total RNA was extracted using a high purity RNA extraction kit (Qiagen, USA) according to the manufacturer protocol. Then, cDNA synthesis was performed through the Qiagen Press Kit based on the manufacturer protocol. The qRT-PCR method was performed using SYBER Green containing Master Mix (Ampriqon, Denmark). The 16S rRNA gene is used as your home gene and the relative expression of the gene was calculated by ΔΔCт method.
Statistical calculation of this study was performed using Graph pad prism software and the results were analyzed by one way ANOVA. The results are displayed as mean ± standard deviation (SD) and P<0.05 was considered significant.
Results: In this study, lactose positive and mucoid colonies were isolated from 100 clinical specimens suspected of K. pneumoniae and 15 strains of K. pneumoniae were isolated using microscopic and biochemical tests. The results of antibiotic resistance showed that 10 samples (66%) had a pattern of multidrug-resistant resistance (MDR). (MDR: strains resistant to one of the antibiotics from three different classes of antibiotics).
Microtiter plate test was used to evaluate the quantitative biofilm formation and the results showed that out of 15 strains of K. pneumoniae, all MDR strains (10 strains) formed biofilm, so that 5 strains formed strong biofilm (50%). 3 strains forming moderate biofilm (30%) and 2 strains (20%) forming weak biofilm.
The results of SEM and DLS showed that Al2O3 nanoparticles have a polyhedral structure and have an average size of 164.1 nm. The FTIR results also showed the chemical bonds associated with Al2O3 nanoparticles. XRD results showed the presence of peaks of 31.8, 35.5, 40.5, 46.7 and 47.98, which confirms the Al2O3 nanoparticles.
Antimicrobial and anti-biofilm effects of Al2O3 nanoparticles
The results showed that Al2O3 nanoparticles had antimicrobial properties at the lowest concentration of 500 μg/ml and the highest concentration of 2000 μg/ml. The results of this test showed that Al2O3 nanoparticles at sub-inhibitory concentration (sub-MIC) significantly reduced the biofilm formation (light absorption was significantly reduced compared to the control group) (P <0.05).
The results showed that the amplification of the gene was done correctly and the analysis of the melting curve shows the amplification of the target gene. Also, after treatment of biofilm-forming strains with sub-inhibitory concentrations of Al2O3 nanoparticles, the expression of mrkA gene in all biofilm-forming strains decreased significantly compared to the 16S rRNA reference gene (P <0.05), so that in some Strains expression was reduced 5 to 6-fold.
Conclusion: The results of this study showed that aluminum nanoparticles have antimicrobial and anti-biofilm effects against drug-resistant K. pneumoniae strains and can reduce the expression of biofilm gene. Therefore, this nanoparticle can be used as a candidate for antimicrobial and antifouling film for future studies.
Methods: In this experimental study, 100 clinical samples including wounds, blood, urine, and cerebrospinal fluid were collected from different hospitals in Tehran (Imam Khomeini Hospital and Pars Hospital). Subsequently, strains of K. pneumoniae were identified by growing one in McConkey agar medium, Gram staining, and biochemical tests (9). Antibiotic resistance pattern of K. pneumoniae strains was determined by disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) instruction. Briefly, adjusted cultures to give a 0.5 McFarland concentrations of the K. pneumoniae strains were inoculated into Muller-Hinton broth (Oxoid, UK) and the susceptibility of the strains to the antibiotics including ampicillin, gentamicin, amikacin, tetracycline, cefepime, ciprofloxacin, levofloxacin and trimethoprim sulfamethoxazol was evaluated (10). In order to investigate the presence of biofilm in K. pneumoniae strains, microtiter plate quantification method was used (11). In this study, Al2O3 nanoparticles were purchased from the American company US nano. In order to investigate the physicochemical properties of Al2O3 nanoparticles, FTIR, electron microscopy (SEM), XRD and DLS tests were used. The SEM, DLS, XRD and FTIR were used for study of morphology, size, nanomaterial characterization and analyze the structure of matter at the molecular scale based on the resonant vibration modes of various molecules on the aluminum nanoparticles, respectively. In order to investigate the antibacterial effects of Al2O3 nanoparticles, the microdilution method was used in a 96-well plate according to the CLSI standard (16). Briefly, various concentrations of Al2O3 nanoparticles including 15.62, 31.25, 62.5, 125, 250, and 500 µg/mL, were added into 96-well containing each K. pneumoniae strain (500 µl) with 5 × 105 concentration, and the 96-well plates were incubated 24 h at 37 0C. The next day, absorbance was taken using UV-visible spectrophotometer at 600 nm and the concentration giving the least optical density corresponds to minimum inhibitory concentration (MIC) of nanoparticles for that particular microorganism. In order to quantitatively study the anti-biofilm effects of aluminum nanoparticles, Crystal violet (CV) assay was used (12). A 96-well plate-based Crystal violet (CV) assay was used for evaluating of anti-biofilm efficacy of Al2O3 nanoparticles. The biofilm-forming K. pneumoniae strains were cultured into 96 well microtiter plates for 24 h at 37 0C. The K. pneumoniae strains were then treated with sub-MIC values of Al2O3 nanoparticles for 24 h incubation at 37 0C. All the wells were then again washed with PBS three times and fixed with methanol for 15 minutes. The plate was air dried for 30 minutes and 0.1% CV solution was added to each well and incubated at room temperature for 20 minutes. After washing with distilled water, 33% acetic acid was added to each well and absorbance was taken at 590 nm. Mean absorbance values of each sample was calculated and compared with the mean values of controls.
Expression analysis of mrkA gene in K. pneumoniae strains under control MIC of free curcumin and curcumin were investigated using the Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) method through specific primers. For this purpose, after treating strains, total RNA was extracted using a high purity RNA extraction kit (Qiagen, USA) according to the manufacturer protocol. Then, cDNA synthesis was performed through the Qiagen Press Kit based on the manufacturer protocol. The qRT-PCR method was performed using SYBER Green containing Master Mix (Ampriqon, Denmark). The 16S rRNA gene is used as your home gene and the relative expression of the gene was calculated by ΔΔCт method.
Statistical calculation of this study was performed using Graph pad prism software and the results were analyzed by one way ANOVA. The results are displayed as mean ± standard deviation (SD) and P<0.05 was considered significant.
Results: In this study, lactose positive and mucoid colonies were isolated from 100 clinical specimens suspected of K. pneumoniae and 15 strains of K. pneumoniae were isolated using microscopic and biochemical tests. The results of antibiotic resistance showed that 10 samples (66%) had a pattern of multidrug-resistant resistance (MDR). (MDR: strains resistant to one of the antibiotics from three different classes of antibiotics).
Microtiter plate test was used to evaluate the quantitative biofilm formation and the results showed that out of 15 strains of K. pneumoniae, all MDR strains (10 strains) formed biofilm, so that 5 strains formed strong biofilm (50%). 3 strains forming moderate biofilm (30%) and 2 strains (20%) forming weak biofilm.
The results of SEM and DLS showed that Al2O3 nanoparticles have a polyhedral structure and have an average size of 164.1 nm. The FTIR results also showed the chemical bonds associated with Al2O3 nanoparticles. XRD results showed the presence of peaks of 31.8, 35.5, 40.5, 46.7 and 47.98, which confirms the Al2O3 nanoparticles.
Antimicrobial and anti-biofilm effects of Al2O3 nanoparticles
The results showed that Al2O3 nanoparticles had antimicrobial properties at the lowest concentration of 500 μg/ml and the highest concentration of 2000 μg/ml. The results of this test showed that Al2O3 nanoparticles at sub-inhibitory concentration (sub-MIC) significantly reduced the biofilm formation (light absorption was significantly reduced compared to the control group) (P <0.05).
The results showed that the amplification of the gene was done correctly and the analysis of the melting curve shows the amplification of the target gene. Also, after treatment of biofilm-forming strains with sub-inhibitory concentrations of Al2O3 nanoparticles, the expression of mrkA gene in all biofilm-forming strains decreased significantly compared to the 16S rRNA reference gene (P <0.05), so that in some Strains expression was reduced 5 to 6-fold.
Conclusion: The results of this study showed that aluminum nanoparticles have antimicrobial and anti-biofilm effects against drug-resistant K. pneumoniae strains and can reduce the expression of biofilm gene. Therefore, this nanoparticle can be used as a candidate for antimicrobial and antifouling film for future studies.
Keywords: Klebsiella pneumoniae, aluminum nanoparticles, antimicrobial activity, anti-biofilm, gene expression
Type of Study: Research |
Subject:
Microbiology
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