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Bita Hatefizade, Farzaneh Hosseini, Soheila Moradi Bidhendi,
Volume 23, Issue 148 (10-2016)
Volume 23, Issue 148 (10-2016)
Abstract
Background: Excessive use of antibiotics and biocides has led to emergence of resistant Staphylococcal strains. The aim of this study was to examine resistance of Staphylococcal strains biofilm isolated to biocidal components.
Methods: 100 clinical isolates of Staphylococcus aureus were collected from hospitalized patients with infectious skins from two Shohadaye Tajrish and Valiasr hospitals for one year in Tehran City. Isolated strains were identified by standard biochemical tests and confirmed using polymerase chain reaction. The pattern of resistance of strains to antibiotics and biocides such as Savlon, Decosept and Deconex (Dermocept) were determined by disc diffusion method and their minimal inhibition and cidal concentrations were estimated using microdilution. The biofilm of MDR strains were formed on polystyrene microplates.
Results: Most of strains were resistant to penicillin, amoxicillin, ampicillin and methicillin and the most sensitivity was seen to clarythromicin. The phenotyping findings of biofilm formation show that 6%, 23.2% and 50.4% of isolates were able to biofilm formation as strong, intermediate and weak, repectively, and only 20.4% were unable to form biofilm. Biofilm had the lower and higher resistant to Savlon and Deconex, respectively.
Conclusion: The prevalence of hospital resistance strains with ability of biofilm formation can be serious danger for health of society in an extended spectrum of patients.
M.s Asma Teymuri, Ph.d Mohammad Bokaeian, M.s Seyed Amin Papoli Baravati,
Volume 23, Issue 152 (2-2017)
Volume 23, Issue 152 (2-2017)
Abstract
Background: Today with increasing use of antibiotics and prevalence of resistant strains, there is need for antimicrobial drugs that have fewer side effects than the present antibiotics. Satureja hortensis is a medicinal plant which has many uses in traditional medicine. In this study, antimicrobial effect of extracts Satureja hortensis biofilm on some important human bacterial pathogens was studied.
Methods: In this in vitro study Satureja hortensis was used to evaluate its antimicrobial effects. Extract Satureja hortensis with concentrations of 50, 25 and 12/5 ppm were prepared and antibacterial activities were evaluated by well diffusion method on strains of Staphylococcus aureus, Streptococcus pyogenes, Pneumococcus, Staphylococcus saprophyticus and Proteus mirabilis. Minimum inhibitory concentration and maximum inhibitory concentration test was determined by microtitre plate. Satureja hortensis extracts were also prepared by Maceration method.
Results: The levels of MIC ranged from 12.5 to 50ppm. The highest MIC value observed against Staphylococcus aureus and lowest MIC value of Satureja extract concentration in 12.5ppm that against P. mirabilis. In addition, the results of this study showed that the rate of absorption (OD) for biofilm formation of Staphylococcus aureus in concentrations 50ppm Satureja extract was zero value.
Conclusion: Results of this study suggest that the extract of Satureja hortensis can be useful in treatment of bacterial infections.
Kobra Salimiyan Rizi, Hadi Farsiani,
Volume 25, Issue 10 (1-2019)
Volume 25, Issue 10 (1-2019)
Abstract
Background: Oral and dental diseases such as dental caries and gingivitis are common problems in human societies. The formation of biofilms is considered as a very important mechanism of disease by these bacteria. The present study is an overview of these infectious diseases and their bacterial etiology, the mechanism of formation and role of dental plaque biofilm in the development of these infections and vaccination against dental caries is a new research field for dealing with these oral-dental diseases.
Methods: In the current review paper, relevant articles were collected at Science Direct, Google Scholar, and PubMed databases from 1995 to 2017 and reviewed over a period of 2 months. Articles related to the role of bacterial biofilms in oral and dental diseases were used to write this research.
Results: As a result of the review of articles, it was seen that bacteria are one of the main causes of oral and etiology infections. Although the formation of bacterial biofilm at the different dental and oral surfaces is not necessarily related to the disease, but the formation of biofilms is considered as a very important mechanism of disease by these bacteria.
Conclusion: The dental plaque biofilm in health condition often consists of non-pathogenic bacteria and humans. However, the in pathogenic biofilms, the levels of acidogenic and aciduric bacteria have increased dramatically and, by creating a stable acidic environment, demineralization occurs more rapidly.
Samaneh Alizadeh Sarvandani, Kumarss Amini, Parvaneh Saffarian,
Volume 26, Issue 9 (12-2019)
Volume 26, Issue 9 (12-2019)
Abstract
Background: Antibiotic-resistant forms of Enterococcus faecalis, the second leading cause of severe nosocomial infections, have begun to emerge worldwide. Evidences have shown that the Esp expression is related to the primary adherence and biofilm formation of E. faecalis. The present study investigated the effect of curcumin nanoparticles on the Enterococcal surface protein, Esp, involved in biofilm formation of antibiotic-resistant forms of enterococcus faecalis.
Methods: In this study, 60 clinical specimens collected from patients admitted to major hospitals of Tehran, Iran and all specimens were identified by standard bacteriological and biochemical methods. The strains were evaluated for the presence of Esp in E. faecalis by PCR method. After treatment, broth microdilution method and Real-time PCR were used to assess the inhibitory activity of curcumin nanoparticles on biofilm formation and the expression level of Esp gene, respectively.
Results: Twelve E. faecalis harboring Esp gene strains were included. The result of MIC testing and gene expression assay showed that curcumin nanoparticles did not show any inhibitory activity against biofilm formation in clinical isolates of E. faecalis and no significant changes in transcription were observed.
Conclusion: Considering the high prevalence of Esp gene among E. faecalis strains, molecular identification might serve as a potent drug-resistant marker of E. faecalis, as essential elements of E. faecalis for effective infection control program. No significant changes in transcription were detected when the minimal medium was supplemented with curcumin nanoparticles, suggesting that these nanoparticles contribute very little, if at all, to inhibition of the Esp operon.
Shirin Sayyahfar, Gholamreza Bayazian, Ehsan Tourchi,
Volume 27, Issue 4 (6-2020)
Volume 27, Issue 4 (6-2020)
Abstract
Background: Biofilm, is an organized complex of bacteria that aggregates in an extracellular matrix enriched of polysacchrids, acid nucleic and proteins. Due to this evolved structure, the bacteria in the biofilm becomes highly resistant to the host's defense system and can adhere to the mucosal surface, leading to defects in the host's immune response. At first, bacteria with or without movement, reversibly adhere to the surfaces and then with the multiplication of glycocalyx by bacteria, this connection becomes irreversible. Biofilm growth progresses with the proliferation of baseless bacteria and gradually increases with the addition of other bacteria in the environment. These accumulations act as a chronic bacterial reservoir resistant to common antibiotics, and its debridement appears to justify the patient's clinical symptoms.
Biofilm formations have been observed and reported on the surface of adenoids and tonsils, especially in children with recurrent infections. The adenoid contains a large number of pathogenic bacteria and is more commonly known as a source of bacteria in children with rhinosinusitis than as a cause of mechanical obstruction. Studies have shown that culture of the sample prepared with swab and adenoidectomy is closely related with aerobic and anaerobic flora have been observed in both samples. Most aerobic strains were Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, Moraxella catarrhalis, and most anaerobes were Staphylococcus aureus, Peptostreptococcus, Prevotella and Fusobacterium.
Our sudy aims to evaluate the association between adenoid tissue cultures and microorganisms with biofilms grades in children with upper airway obstruction.
Methods: This cross-sectional study was conducted among the children referred to the ENT clinics of Ali Asghar Children hospital. We included patients with symptoms of nasal obstruction who were candidates for adenoidectomy. Indication of adenoidectomy in this study (inclusion criteria) was existence of confirmed adenoid hypertrophy on examination and lateral neck X-ray (Adenoid view) with symptoms of chronic upper airway obstruction including obstruction sleep apnea, snoring, open mouth breathing, adenoid face, speech disorders and restlessness during sleep (if accompanied by night snoring). Exclusion criteria also included cystic fibrosis, immunodeficiency, respiratory disorders including asthma, and the use of antibiotics during the two weeks prior to surgery. Demografic data of patients were gathered on questionnaire. At this stage, according to the patient history accuracy based on the number of upper airway infections in the past year, patients were divided into five groups less than 5 infections, between 5-8, between 8-12, between 12-15 infections and more than 15 infections per year. After removing the adenoid tissue of the patients by the ENT surgeon in the operating room, a sample was sent to laboratory for culture in normal saline solution and the rest of the sample was cut from several places with a knife after washing with normal saline solution and placed in 2.5% glutaraldehyde solution for fixation. Samples were dried in vacuo, then, after coating the surface of the samples with a layer of gold with a thickness of 30-50 nm (voltage 800v, 100 mA), the samples were classified according to the presence and size of biofilm using Philips XL30 Environmental Scanning Electron Microscope (ESEM). Based on the degree of bacterial biofilm in the electron microscope samples , the amount less than 20% was named as Grade I, 20-40% as Grade II, 40-60% as Grade III, 60-80% as Grade IV and we considered more than 80% as Grade V. Statistical analysis was done by SPSS.
Results: Fifty-one children with a mean age of 7.31 years were enrolled in the study. The average rate of upper airway infection during last year was 9.58. In terms of accompanying symptoms, noctornal snoring and open mouth breathing were seen in all cases. The mean duration of symptoms from onset was 2.5 years (ranging from 2 months to 7.5 years). 30 patients had Grade IV adenoid size in their graphy and noone were graded as grade 1. Biofilm structures in 100% of samples were observed.
According to the achived data, the highest frequency of the organisms was in the biofilm with the grade of 60-80%. Also, biofilm grade above 60% had the highest number of positive cultures. Alpha hemolytic Streptococcus viridans was resulted for 26 samples of adenoid tissue culture and just one sample did not show any bacterial growth. The mean number of infections in different culture groups did not differ significantly (p=0.985, Krusskal Wallis). The mean duration of symptoms did not differ significantly in different culture groups (p=0.159, Krusskal Wallis). There was no statistically significant difference between the gender and different culture groups (p=0.701, Chi2). There was a statistically significant difference between adenoid grade and various groups of bacterial culture (p=0.003, Chi2), the larger the size of the adenoids, the more likely the culture to be positive. As other studies have only examined the most common microorganisms, this study is in fact the first study to evaluate both types of organisms and its comparison with the degree of biofilm. Streptococcus alpha is a hemolytic microorganism that oxidizes RBC hemoglobin to cause a green color in the culture medium. Streptococcus alpha hemolytic viridans is the oral type of this organism, which is a type of normal flora. Studies have shown that the same microorganism can cause pathogenicity.
Disagreement for surgery by parents of some patients was one of the our study limitations. Another limitation of our current study was the difficulty of accurate history of the number of upper airway infections in the past year due to the lack of a recorded medical registry system.
Conclusion: In this study, as presented before, there was a significant relationship between the increase in the surface grade of adenoid biofilm and the number of upper airway infections. Also, the highest frequency of the organisms was observed in biofilms with higher grades, and biofilms above 60% had the highest amount of positive culture. Therefore, this study also confirms that the presence of biofilm in the adenoid as a reservoir of infection causes inflammation and can justify the effectiveness of adenoidectomy as an acceptable therapeutic approch for children with recurrent upper airway infections.
Seifi Atena, Amir Mirzaie, Ehsan Ehsani, Afshin Mahmoodzadeh,
Volume 28, Issue 7 (10-2021)
Volume 28, Issue 7 (10-2021)
Abstract
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.
Shamim Ashkezari, Sarvenaz Falsafi, Sohameh Mohebbi, Amir Mirzaie, Neda Moosavi Niri,
Volume 30, Issue 3 (5-2023)
Volume 30, Issue 3 (5-2023)
Abstract
Background & Aims: Staphylococcus aureus is one of the human pathogens that causes a wide range of diseases such as endocarditis, blood, bone, skin and soft tissue infections (1). One of the resistant strains of this bacterium is methicillin-resistant strains of Staphylococcus aureus (MRSA), which has been reported as a serious risk by the Centers for Disease Control and Prevention. One of the pathogenic mechanisms and antibiotic resistance of this bacterium is the formation of biofilm, which causes this bacterium to bind to different surfaces (2). Biofilm-forming strains have become resistant to many antibiotics, so that biofilm-forming strains form extracellular matrices that are immune to the immune system and antibiotics. Various genes are involved in biofilm production, one of which is the icaB gene, which plays a key role in the production of poly N-acetyl glucose amine and biofilm production. The design of new antimicrobial drugs for the treatment of this bacterium is important, so finding a suitable treatment option for the treatment of infections caused by this bacterium is one of the challenges of researchers (3). Recent advances in nanotechnology have suggested alternative solutions, such as drug delivery systems, that increase drug specificity and efficiency (4). One of these drug delivery systems is nanosystems, which are composed of bilayer nonionic surfactants. Due to the importance of nanosystems, the aim of this study was to synthesize nanoparticles containing naproxen, to investigate their physicochemical properties and their antimicrobial and antifouling effects against Staphylococcus aureus strains.
Methods: Nanosomes containing naproxen were synthesized by thin layer hydration method. The confinement efficiency is indicative of the drug encapsulated in the nanosystem structure relative to the drug used. For this purpose, the nanonosomal formulation was first centrifuged at 4 ° C at 14000 g for 45 minutes. The nanosystem containing the drug precipitates and the free drug remains in the supernatant. The absorbance of the supernatant sample at 270 nm was read by a spectrophotometer and the amount of free drug was calculated from the initial value.
The Physico-chemical characteristics of prepared nanoniosome encapsulated naproxen was determined using scanning electron microscopy (SEM), Dynamic light scattering (DLS). The in vitro drug release study was done using dialysis bag (6). The Staphylococcus aureus strains were recovered from 100 clinical samples and their antibiotic resistance patterns were studied using disk diffusion method. The antibacterial activity of nanoniosome loaded naproxen and free naproxen were investigated using well diffusion and micro-dilution methods (7). The icaB biofilm gene expression analysis in S. aureus isolates which are treated with nanoniosome loaded naproxen and free naproxen were examined using Real Time PCR methods (8).
Draw diagrams and Statistical analysis was performed by GraphPad Prism software version 7 and SPSS version 23, and one-way analysis of variance was used for statistical analysis and p <0.05 was considered significant.
Results: In this study, using different molar ratios of surfactant, cholesterol and drug, different formulations of nanosystems containing naproxen were synthesized. The optimal niosome size synthesis was measured by DLS method. The results of electron microscopy (SEM) show that the synthesized nanonosomes have a spherical structure. In this study, the dialysis bag method was used to evaluate the drug release pattern. Figure 2 shows the pattern of naproxen release from nanosystems and free naproxen over 72 hours.
Out of 100 clinical specimens, 15 specimens of Staphylococcus aureus were isolated and identified using microbiological methods. The results of antibiotic resistance profile test showed that out of 15 strains, 10 strains were methicillin resistant (MRSA).
The antibacterial activity of nanoniosome encapsulated naproxen and free naproxen showed that the MIC was reduced by 2 to 4 times. The results of the well diffusion method showed that nanoniosomes containing naproxen had more significant antimicrobial power than free naproxen, so that the diameter of the growth inhibition zone increased with increasing nanosystem concentration. Real-Time PCR was used to evaluate the effect of nanoniosoms containing naproxen and free naproxen on icaB biofilm gene expression in Staphylococcus aureus strains. Also, after treating Staphylococcus aureus strains with naproxen-containing nanosystem sub-inhibitory concentration, it was shown that the expression of icaB gene was significantly reduced compared to the 16S rRNA reference gene (p <0.05).
Conclusion: In this study, naproxen was encapsulated as a drug compound in the nanoniosome structure and its physical and chemical properties including size, morphology, drug enclosure percentage and drug release were studied. The results of this study showed that the synthesized nanoniosome in the optimal formulation had a spherical shape, the average size was 125.3 nm with a confinement percentage of 66.84%. Drug release results also showed that naproxen in the formulated form in nanonosomes has a much slower release pattern than free naproxen, which is a suitable feature of a drug delivery system. The results of the antimicrobial test showed that naproxen-containing nanoniosome had more significant antimicrobial effects than free naproxen compared to free naproxen, reducing the MIC by 2 to 4 times. One of the antimicrobial mechanisms of nanoniosome containing naproxen is the fusion of nanoniosome with bacterial cell membranes, which can deliberately release the drug into the cytoplasm of the cell and cause bacterial cell death (10). The results of this study showed that nanoparticles containing naproxen have more significant anti-biofilm effects than free naproxen compared to free naproxen and can significantly reduce the expression of biofilm gene (11). One of the reasons for the anti-biofilm effects of naproxen-containing nanosystems is the greater penetration of naproxen-containing nanosystems into the biofilm structure, which can cause the death of bacteria, a decrease in the number of bacteria, and the conversion of biofilms into planktonic cells (12).
Methods: Nanosomes containing naproxen were synthesized by thin layer hydration method. The confinement efficiency is indicative of the drug encapsulated in the nanosystem structure relative to the drug used. For this purpose, the nanonosomal formulation was first centrifuged at 4 ° C at 14000 g for 45 minutes. The nanosystem containing the drug precipitates and the free drug remains in the supernatant. The absorbance of the supernatant sample at 270 nm was read by a spectrophotometer and the amount of free drug was calculated from the initial value.
The Physico-chemical characteristics of prepared nanoniosome encapsulated naproxen was determined using scanning electron microscopy (SEM), Dynamic light scattering (DLS). The in vitro drug release study was done using dialysis bag (6). The Staphylococcus aureus strains were recovered from 100 clinical samples and their antibiotic resistance patterns were studied using disk diffusion method. The antibacterial activity of nanoniosome loaded naproxen and free naproxen were investigated using well diffusion and micro-dilution methods (7). The icaB biofilm gene expression analysis in S. aureus isolates which are treated with nanoniosome loaded naproxen and free naproxen were examined using Real Time PCR methods (8).
Draw diagrams and Statistical analysis was performed by GraphPad Prism software version 7 and SPSS version 23, and one-way analysis of variance was used for statistical analysis and p <0.05 was considered significant.
Results: In this study, using different molar ratios of surfactant, cholesterol and drug, different formulations of nanosystems containing naproxen were synthesized. The optimal niosome size synthesis was measured by DLS method. The results of electron microscopy (SEM) show that the synthesized nanonosomes have a spherical structure. In this study, the dialysis bag method was used to evaluate the drug release pattern. Figure 2 shows the pattern of naproxen release from nanosystems and free naproxen over 72 hours.
Out of 100 clinical specimens, 15 specimens of Staphylococcus aureus were isolated and identified using microbiological methods. The results of antibiotic resistance profile test showed that out of 15 strains, 10 strains were methicillin resistant (MRSA).
The antibacterial activity of nanoniosome encapsulated naproxen and free naproxen showed that the MIC was reduced by 2 to 4 times. The results of the well diffusion method showed that nanoniosomes containing naproxen had more significant antimicrobial power than free naproxen, so that the diameter of the growth inhibition zone increased with increasing nanosystem concentration. Real-Time PCR was used to evaluate the effect of nanoniosoms containing naproxen and free naproxen on icaB biofilm gene expression in Staphylococcus aureus strains. Also, after treating Staphylococcus aureus strains with naproxen-containing nanosystem sub-inhibitory concentration, it was shown that the expression of icaB gene was significantly reduced compared to the 16S rRNA reference gene (p <0.05).
Conclusion: In this study, naproxen was encapsulated as a drug compound in the nanoniosome structure and its physical and chemical properties including size, morphology, drug enclosure percentage and drug release were studied. The results of this study showed that the synthesized nanoniosome in the optimal formulation had a spherical shape, the average size was 125.3 nm with a confinement percentage of 66.84%. Drug release results also showed that naproxen in the formulated form in nanonosomes has a much slower release pattern than free naproxen, which is a suitable feature of a drug delivery system. The results of the antimicrobial test showed that naproxen-containing nanoniosome had more significant antimicrobial effects than free naproxen compared to free naproxen, reducing the MIC by 2 to 4 times. One of the antimicrobial mechanisms of nanoniosome containing naproxen is the fusion of nanoniosome with bacterial cell membranes, which can deliberately release the drug into the cytoplasm of the cell and cause bacterial cell death (10). The results of this study showed that nanoparticles containing naproxen have more significant anti-biofilm effects than free naproxen compared to free naproxen and can significantly reduce the expression of biofilm gene (11). One of the reasons for the anti-biofilm effects of naproxen-containing nanosystems is the greater penetration of naproxen-containing nanosystems into the biofilm structure, which can cause the death of bacteria, a decrease in the number of bacteria, and the conversion of biofilms into planktonic cells (12).
Fatemeh Tavassolian, Zahra Chegini, Amin Khoshbayan, Abbas Farahani, Dr Aref Shariati,
Volume 31, Issue 1 (3-2024)
Volume 31, Issue 1 (3-2024)
Abstract
Nowadays, the most important problem in the treatment of bacterial infections is the appearance of drug-resistant bacteria and the scarce prospects of producing new antibiotics. In this regard, methicillin-resistant Staphylococcus aureus (MRSA) is an antibiotic-resistant agent that poses
a remarkable threat to health care by causing 19,000 deaths and a cost of $3–4 billion annually in the US. The number of cases influenced by Multidrug-resistant (MDR), Extensively drug resistant (XDR), and Pandrug-resistant (PDR) Gram-negative bacteria, such as Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, as well as MDR or XDR isolates of Mycobacterium tuberculosis, has been growing continuously in recent years. The limitation of current clinical options for confronting threats of infections caused by intricate pathogens has led to a critical problem that encourages researchers to discover new approaches to face the growing problem of drug-resistant bacteria. To this end, scientists are using different antibacterial agents, such as bacteriophages (phages), for the inhibition of bacterial infection and the improvement of antibiotic efficacy. Phages are viruses that infect bacteria, and due to the huge increase in antibiotic resistance in recent years, there is renewed interest in revisiting the use of phages to treat bacterial infections. The practice of phage therapy, the application of phages to treat bacterial infections, has been around for approximately a century. Phage therapy relies on using lytic phages and purified phage lytic proteins for the treatment and lysis of bacteria at the site of infection.
The use of two or more phage mixtures with different host ranges in a single suspension as a bacteriophage cocktail is usually more effective for inhibiting bacterial infections. Phage cocktail causes a better reduction of bacterial density and improves phages’ efficiency, and in vitro studies have also shown that phage cocktail results in a higher reduction in bacterial infection. Additionally, polysaccharide depolymerase, a polysaccharide hydrolase encoded by phages, can specifically degrade the macromolecule carbohydrates of the host bacterial envelope. This enzyme helps the phage adsorb, invade, and disintegrate the host bacteria. Furthermore, phages generate peptidoglycan hydrolase enzymes, called Endolysins, at the end of the lytic cycle. They decompose peptidoglycan from the inside and assist in forming new progeny phages to release from the cell. Endolysins are always proposed as antibacterial agents because of their high specific activity and unique mode of action against bacteria. The activity of Endolysins is independent of antibiotic susceptibility patterns.
The combined use of phage and antibiotics also indicated promising effects for inhibition of MDR bacteria. The sub-lethal concentrations of conventional antibiotics, for example, ciprofloxacin, could lead to an increase in progeny production. Antibiotic treatments could enhance the release of progeny phages by shortening the lytic cycle and latent period. Thus, sub-lethal concentrations of antibiotics combined with phages can be used for the management of bacterial infections with high antibiotic resistance. In addition, combination therapy exerts various selection pressures that can mutually decrease phage and antibiotic resistance.
It’s noteworthy to mention that bacterial biofilm was introduced for the first time in 1987 as a community of microorganisms capable of binding to surfaces and forming an exopolysaccharide and extracellular matrix. Biofilms are communities of bacteria that are surrounded by a complex polysaccharide matrix (glycocalyx). They are highly resistant to antibiotics, making them a major challenge. The antibiotics are unable to penetrate the matrix, which makes biofilms between 10 to 1000 times more resistant to antibiotics compared to individual planktonic cells. This has led to an increase in the prevalence of multi-drug resistant (MDR) strains of bacteria in recent years. Unfortunately, there are no fully effective antibiotics available to stop these bacteria. However, phages can be used to eradicate biofilms. They work by destroying the extracellular matrix of the biofilm, which increases the permeability of antibiotics into the inner layer of the biofilm. Phages also inhibit the formation of biofilm by stopping the quorum-sensing activity.
Furthermore, the combined use of bacteriophages and other compounds with anti-biofilm properties, such as nanoparticles, enzymes, and natural products, can be of more interest because they invade the biofilm by various mechanisms and can be more effective than the one used alone. On the other hand, using bacteriophages to destroy biofilm has some drawbacks, like a limited range of hosts, high-density biofilm, subpopulation phage resistance in biofilm, and quorum sensing in biofilm, which stops phage infection. Therefore, phages not only kill MDR bacteria but also destroy their biofilm structure. To this end, recently published studies used phage therapy for the inhibition of different bacterial infections, such as wounds, urinary tract infections, and chronic cystic fibrosis infections. Recently published studies have proposed phage therapy as a potential alternative against MDR urinary tract infections (UTI) because the resistance mechanism of phages differs from that of antibiotics and few side effects have been reported for them. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are the most common uropathogenic bacteria against which phage therapy has been used. Phages, in addition to lysing bacterial pathogens, can prevent the formation of biofilms. Besides, by inducing or producing polysaccharide depolymerase,
Phage can easily penetrate into the deeper layers of the biofilm and degrade it. Notably, phage therapy has shown good results in inhibiting multiple-species biofilm, and this may be an efficient weapon against catheter-associated UTI.
Additionally, wound infections kill a large number of patients worldwide each year. S. aureus, K. pneumoniae, A. baumannii, and P. aeruginosa are the most important colonizing pathogens of wounds that, with various virulence factors and an impaired immune system, cause extensive tissue damage and non-healing wounds. Furthermore, the septicemia caused by these pathogens increases the mortality rate due to wound infections. Because of the prevalence of antibiotic resistance in recent years, the use of antibiotics to inhibit these pathogens has been restricted, and the topical application of antibiotics to wound infections increases antibiotic resistance. The results of published studies showed that phages have an excellent ability to inhibit MDR bacterial pathogens and wound infections and accelerate wound healing.
Studies have demonstrated that phages can prevent septicemia, which arises due to wound colonization by different pathogens. Furthermore, phages have good stability in different environmental conditions. Also, based on the studies, they have negligible side effects, which may increase their potential to be used for patients with underlying diseases or unstable physiological conditions, because they are more tolerable for patients than the toxic antibiotics.
Finally, pulmonary infections involving P. aeruginosa are among the leading causes of the deterioration of the respiratory status of cystic fibrosis (CF) patients. The emergence of MDR strains in such populations, favored by iterative antibiotic cures, has led to an urgent need for new therapies. Among them, phage-based therapies deserve a focus.
In this regard, studies have shown that phages can be used as a preventive or curative treatment for P. aeruginosa lung infections. However, the preferred route of administration, the dose, the duration of treatment, co-treatment with antibiotics, and the choice of a single agent or of a host-adapted or preexisting cocktail are still unclear.
Therefore, as mentioned, phages have shown promising results for managing bacterial infections. However, phages have different host ranges in various studies, which limits their use because MDR bacteria are usually nosocomial bacteria that may have different origins, and the isolated phage may not be able to infect some of them. Also, isolation of specific phages may be time-consuming and unnecessary for the patient. Furthermore, phages have low stability in long-term storage, but it is possible to use them in different ways, such as liposomal capsules and lyophilization. Therefore, using phages along with antibiotics, natural substances that have antimicrobial properties, or biological bands that increase wound healing can increase the chances of successful treatment. It is noteworthy that, using phage cocktails and providing phage banks can also increase their chances of success, as this is less time-consuming to isolate them and covers a wider host range. However, determining the use of phages to do the least harm to humans, methods to boost their effect on bacterial pathogens, the best time for the treatment, and the route or dosage of the administration need further studies.
Collectively, recently published studies used phage therapy for the inhibition of different bacterial infections, such as wounds, urinary tract infections, and chronic cystic fibrosis infections. However, despite the positive use of phage therapy, various studies have reported phage-resistant strains, indicating that phage-host interactions are more complicated and need further research. Additionally, these investigations are limited, and further clinical trials are required to make this treatment widely available for human use.
a remarkable threat to health care by causing 19,000 deaths and a cost of $3–4 billion annually in the US. The number of cases influenced by Multidrug-resistant (MDR), Extensively drug resistant (XDR), and Pandrug-resistant (PDR) Gram-negative bacteria, such as Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, as well as MDR or XDR isolates of Mycobacterium tuberculosis, has been growing continuously in recent years. The limitation of current clinical options for confronting threats of infections caused by intricate pathogens has led to a critical problem that encourages researchers to discover new approaches to face the growing problem of drug-resistant bacteria. To this end, scientists are using different antibacterial agents, such as bacteriophages (phages), for the inhibition of bacterial infection and the improvement of antibiotic efficacy. Phages are viruses that infect bacteria, and due to the huge increase in antibiotic resistance in recent years, there is renewed interest in revisiting the use of phages to treat bacterial infections. The practice of phage therapy, the application of phages to treat bacterial infections, has been around for approximately a century. Phage therapy relies on using lytic phages and purified phage lytic proteins for the treatment and lysis of bacteria at the site of infection.
The use of two or more phage mixtures with different host ranges in a single suspension as a bacteriophage cocktail is usually more effective for inhibiting bacterial infections. Phage cocktail causes a better reduction of bacterial density and improves phages’ efficiency, and in vitro studies have also shown that phage cocktail results in a higher reduction in bacterial infection. Additionally, polysaccharide depolymerase, a polysaccharide hydrolase encoded by phages, can specifically degrade the macromolecule carbohydrates of the host bacterial envelope. This enzyme helps the phage adsorb, invade, and disintegrate the host bacteria. Furthermore, phages generate peptidoglycan hydrolase enzymes, called Endolysins, at the end of the lytic cycle. They decompose peptidoglycan from the inside and assist in forming new progeny phages to release from the cell. Endolysins are always proposed as antibacterial agents because of their high specific activity and unique mode of action against bacteria. The activity of Endolysins is independent of antibiotic susceptibility patterns.
The combined use of phage and antibiotics also indicated promising effects for inhibition of MDR bacteria. The sub-lethal concentrations of conventional antibiotics, for example, ciprofloxacin, could lead to an increase in progeny production. Antibiotic treatments could enhance the release of progeny phages by shortening the lytic cycle and latent period. Thus, sub-lethal concentrations of antibiotics combined with phages can be used for the management of bacterial infections with high antibiotic resistance. In addition, combination therapy exerts various selection pressures that can mutually decrease phage and antibiotic resistance.
It’s noteworthy to mention that bacterial biofilm was introduced for the first time in 1987 as a community of microorganisms capable of binding to surfaces and forming an exopolysaccharide and extracellular matrix. Biofilms are communities of bacteria that are surrounded by a complex polysaccharide matrix (glycocalyx). They are highly resistant to antibiotics, making them a major challenge. The antibiotics are unable to penetrate the matrix, which makes biofilms between 10 to 1000 times more resistant to antibiotics compared to individual planktonic cells. This has led to an increase in the prevalence of multi-drug resistant (MDR) strains of bacteria in recent years. Unfortunately, there are no fully effective antibiotics available to stop these bacteria. However, phages can be used to eradicate biofilms. They work by destroying the extracellular matrix of the biofilm, which increases the permeability of antibiotics into the inner layer of the biofilm. Phages also inhibit the formation of biofilm by stopping the quorum-sensing activity.
Furthermore, the combined use of bacteriophages and other compounds with anti-biofilm properties, such as nanoparticles, enzymes, and natural products, can be of more interest because they invade the biofilm by various mechanisms and can be more effective than the one used alone. On the other hand, using bacteriophages to destroy biofilm has some drawbacks, like a limited range of hosts, high-density biofilm, subpopulation phage resistance in biofilm, and quorum sensing in biofilm, which stops phage infection. Therefore, phages not only kill MDR bacteria but also destroy their biofilm structure. To this end, recently published studies used phage therapy for the inhibition of different bacterial infections, such as wounds, urinary tract infections, and chronic cystic fibrosis infections. Recently published studies have proposed phage therapy as a potential alternative against MDR urinary tract infections (UTI) because the resistance mechanism of phages differs from that of antibiotics and few side effects have been reported for them. Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis are the most common uropathogenic bacteria against which phage therapy has been used. Phages, in addition to lysing bacterial pathogens, can prevent the formation of biofilms. Besides, by inducing or producing polysaccharide depolymerase,
Phage can easily penetrate into the deeper layers of the biofilm and degrade it. Notably, phage therapy has shown good results in inhibiting multiple-species biofilm, and this may be an efficient weapon against catheter-associated UTI.
Additionally, wound infections kill a large number of patients worldwide each year. S. aureus, K. pneumoniae, A. baumannii, and P. aeruginosa are the most important colonizing pathogens of wounds that, with various virulence factors and an impaired immune system, cause extensive tissue damage and non-healing wounds. Furthermore, the septicemia caused by these pathogens increases the mortality rate due to wound infections. Because of the prevalence of antibiotic resistance in recent years, the use of antibiotics to inhibit these pathogens has been restricted, and the topical application of antibiotics to wound infections increases antibiotic resistance. The results of published studies showed that phages have an excellent ability to inhibit MDR bacterial pathogens and wound infections and accelerate wound healing.
Studies have demonstrated that phages can prevent septicemia, which arises due to wound colonization by different pathogens. Furthermore, phages have good stability in different environmental conditions. Also, based on the studies, they have negligible side effects, which may increase their potential to be used for patients with underlying diseases or unstable physiological conditions, because they are more tolerable for patients than the toxic antibiotics.
Finally, pulmonary infections involving P. aeruginosa are among the leading causes of the deterioration of the respiratory status of cystic fibrosis (CF) patients. The emergence of MDR strains in such populations, favored by iterative antibiotic cures, has led to an urgent need for new therapies. Among them, phage-based therapies deserve a focus.
In this regard, studies have shown that phages can be used as a preventive or curative treatment for P. aeruginosa lung infections. However, the preferred route of administration, the dose, the duration of treatment, co-treatment with antibiotics, and the choice of a single agent or of a host-adapted or preexisting cocktail are still unclear.
Therefore, as mentioned, phages have shown promising results for managing bacterial infections. However, phages have different host ranges in various studies, which limits their use because MDR bacteria are usually nosocomial bacteria that may have different origins, and the isolated phage may not be able to infect some of them. Also, isolation of specific phages may be time-consuming and unnecessary for the patient. Furthermore, phages have low stability in long-term storage, but it is possible to use them in different ways, such as liposomal capsules and lyophilization. Therefore, using phages along with antibiotics, natural substances that have antimicrobial properties, or biological bands that increase wound healing can increase the chances of successful treatment. It is noteworthy that, using phage cocktails and providing phage banks can also increase their chances of success, as this is less time-consuming to isolate them and covers a wider host range. However, determining the use of phages to do the least harm to humans, methods to boost their effect on bacterial pathogens, the best time for the treatment, and the route or dosage of the administration need further studies.
Collectively, recently published studies used phage therapy for the inhibition of different bacterial infections, such as wounds, urinary tract infections, and chronic cystic fibrosis infections. However, despite the positive use of phage therapy, various studies have reported phage-resistant strains, indicating that phage-host interactions are more complicated and need further research. Additionally, these investigations are limited, and further clinical trials are required to make this treatment widely available for human use.
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