اثرات نانوذرات کیتوزان بر موش های آلوده به لیستریا مونوسیتوژنز
Subject Areas : Biotechnological Journal of Environmental MicrobiologySara Kazemi Rad 1 , Mirsassan Mirpour 2 * , Mehdi Assmar 3 , Mohamad Reza Razavi 4
1 - گروه میکروبیولوژی، دانشکده علوم پایه، واحد لاهیجان، دانشگاه آزاد اسلامی، لاهیجان، ایران
2 - گروه میکروبیولوژی ، دانشکده علوم پایه، واحد لاهیجان ، دانشگاه آزاد اسلامی واحد لاهیجان ، لاهیجان ، ایران
3 - گروه میکروبیولوژی ، دانشکده علوم پایه، واحد لاهیجان ، دانشگاه آزاد اسلامی واحد لاهیجان ، لاهیجان ، ایران
4 - گروه انگل شناسی، انستیتو پاستور ایران، تهران، ایران
Keywords: نانوذرات, خواص ضد باکتریایی, موش Balb/c, لیستریوز,
Abstract :
لیستریا مونوسیتوژنز عامل بیماری لیستریوز است که عوارض زیادی به خصوص در زنان باردار دارد. با توجه به مقاومت آنتی بیوتیکی این باکتری، تلاش های زیادی برای معرفی ترکیبات دارویی مختلف از جمله نانوذرات مبتنی بر ترکیبات بیولوژیکی صورت گرفته است. این تحقیق با هدف بررسی مکانیسم اثر نانوذرات کیتوزان بر لیستریا مونوسایتوژنز در بدن یک موجود زنده انجام شد. سویه استاندارد لیستریا مونوسایتوژنز (ATCC 7644) در آزمایشگاه بیمارستان دی (ایران) تهیه و مورد تجزیه و تحلیل قرار گرفت. باکتری ها بر اساس آزمایش های بیوشیمیایی مورد بررسی قرار گرفتند. سپس فعالیت ضد باکتریایی غلظت‌های 4.88 تا 5000 میکروگرم بر میلی‌لیتر نانوذرات کیتوزان در برابر استاندارد لیستریا مونوسیتوژنز (ATCC 7644) با روش‌هایی مورد بررسی و کمترین غلظت‌های بازدارنده و باکتری‌کشی (به ترتیب MIC و MBC) محاسبه شد. اثرات غلظت‌های مختلف نانوذرات و آمپی‌سیلین در موش‌های آلوده به باکتری نیز مورد بررسی قرار گرفت. در موش‌های آلوده، اثر درمانی با افزایش غلظت نانوذرات افزایش یافت و غلظت 156.25 میکروگرم بر میلی‌لیتر در مقایسه با سایر تیمارها مؤثرترین بود. همچنین، آمپی سیلین (10 میکروگرم) و نانوذرات کیتوزان با غلظت 39.06 میکروگرم بر میلی‌لیتر تقریباً اثر درمانی مشابهی داشتند. با شناسایی به موقع آلودگی لیستریا در زنان باردار و استفاده مناسب از نانوذرات کیتوزان به جای داروهای رایج، می توان راه حل جدیدی برای درمان لیستریوز پیدا کرد.
Abstract
Listeria monocytogenes is the cause of listeriosis, which has many complications, especially in pregnant women. Due to the antibiotic resistance of this bacterium, many attempts have been made to introduce different medicinal compounds, including nanoparticles based on biological compounds. This research aimed to examine the mechanism of the effect of chitosan nanoparticles on L. monocytogenes in vivo (in the body of a living organism). The standard strain of L. monocytogenes (ATCC 7644) was prepared and analyzed in the Day Hospital Laboratory (Iran). The bacteria were examined based on biochemical tests. Then, the antibacterial activity of concentrations of 4.88 to 5000 µg/mL of chitosan nanoparticles against L. monocytogenes standard (ATCC 7644) was calculated with the investigated methods and the lowest inhibitory and bactericidal concentrations (MIC and MBC, respectively). The effects of different nanoparticle concentrations and ampicillin in mice infected with bacteria were also investigated. In infected mice, the therapeutic effect increased with increasing the nanoparticle concentration, and the concentration of 156.25 µg/mL was the most effective compared to other treatments. Also, ampicillin (10 µg) and chitosan nanoparticles with a concentration of 39.06 µg/mL had almost the same therapeutic effect. With the timely identification of listeria contamination in pregnant women and the proper use of chitosan nanoparticles instead of common drugs, a new solution can be found for the treatment of listeriosis.
Key words: listeriosis , nanoparticles, antibacterial properties, Balb/c mice
1. Introduction
Listeria monocytogenes coccobacillus is Gram-positive, rod-shaped, short, regular, without spores, catalase-positive, and oxidase-negative; it can survive in the range of pH = 4.4-9.6 and is an opportunistic intracellular pathogen that can grow at 0 to 45 °C. Among Listeria species, L. monocytogenes is pathogenic for humans and is very important because of its role in human infection caused by food around the world (Manso, 2019, Mook, 2011). This bacterium causes listeriosis, which is an infectious disease. Listeriosis is one of the common but little-known diseases of the present era that affects humans and domestic and wild animals. It is mainly transmitted through food contaminated with L. monocytogenes (Zhang, 2021, Pizarro-Cerdá, 2019). L. monocytogenes can cause miscarriage, stillbirth, meningitis, and encephalitis in immunocompromised people and people with underlying diseases. In general, dairy products, unpasteurized milk, soft and semi-soft cheese, raw meat, vegetables, and fish are the main sources of listeriosis (Altuntas, 2012, Haubert, 2016 , Zarei, 2013). In 1932, the first study of the effect of listeriosis on pregnant animals was carried out, and during an epidemic, a Gram-positive bacillus was isolated, which resulted in abortion when entered into a vein or vagina; the microbe can be found in the fetal viscera, vaginal secretions, and uterine wall.. Listeria can stay in the reproductive organs for a relatively long time, cause frequent miscarriages, and even lead to infertility and keep its listeriosis hidden in all these stages. Resistance to human pathogens is a big challenge in the field of medicine. These antibiotic resistances and the continuous and indiscriminate use of chemical medicinal compounds cause the serious phenomenon of resistancein microorganisms (Mota, 2020, Caruso, 2020, Yang , 2020) , and for this reason, many studies have been done in connection with nanoparticles all over the world. One of these nanoparticles is called chitosan, which is one of the most important derivatives of chitin. Chitin is a mucopolysaccharide that has a structural role and is abundantly found in the exoskeletons of arthropods such as shrimp and crabs (Steckel , 2004). One of the most important derivatives of chitin is a compound called chitosan, which, unlike chemical polymer compounds, is nontoxic and decomposes in nature. Chitosan is a biopolymer derived from the removal of acetyl from chitin (Dutta ,2002, Dutta ,2004). The conversion of chitin to chitosan can be done using sodium or potassium hydroxide solutions (40-50%) at a temperature of 100 °C or higher by removing parts or all of the acetyl groups from the polymer. Ampicillin, in combination with an aminoglycoside such as gentamicin or streptomycin, is the first choice for the treatment of listeria. Ampicillin, penicillin, or rifampin alone or in combination with gentamicin are standard antibiotic treatments for listeriosis (Altuntas , 2012). The classification of the use of this antibiotic in pregnancy is group B, and it should also be used with caution during breastfeeding (Haginaka,1987 , Jung, 2006, Jung, 2012).
2. Materials and Methods
2.1. Bacterial Strain and Culture Conditions
The standard strain of L. monocytogenes (7644 ATCC) was purchased in the lyophilized form from the microbial collection of Pars Bioproduct Research and Production Center, and the vial was broken under sterile conditions. For activation of strains, the contents of the vial were placed in screw-capped tubes, each containing 10 mL. A liter of TSB medium was transferred to activate the bacteria, and the bacteria were incubated for 24 hours at a temperature of 37 °C. After the mentioned period, the activated bacteria were transferred to the brain heart infusion (BHI) culture medium and kept in a greenhouse for 24 hours at a temperature of 37 °C and then at 4 °C .Finally, diagnostic biochemical tests were performed to confirm the strains, which include: Gram staining, catalase, oxidase, Sulfur, Indole, Motility (SIM ), hemolysis, Christie-Atkins-Munch-Petersen(CAMP) , methyl red-Voges Proskauer (MR-VP), acid production from rhamnose, dextrose, xylose, triple sugar iron (TSI), and hydrolysis of bile esculin. A suspension of the standard strain was prepared in tryptic soy broth (TSB) medium, mixed with sterile glycerin at a ratio of 5 to 1, poured into sterile microtubes, and stored at -20 °C.
2.2. Chitosan Nanoparticle Preparation and Characterization
In this study, chitosan nanoparticles (CNPs) produced by Aleboyeh et al. were utilized (Alebouyeh, 2020). The physicochemical characteristics of CNPs were measured by zeta potential , particle size and particle dispersity index (PDI ) by dynamic light scattering method using Zetasizer (Malvern, Nano ZS, England). The CNPs had a diameter ranging from 22 to 80 nm and a purity of 99%. The polydispersity index (PDI) was determined to be 0.229. Furthermore, the zeta potential of the CNPs was measured to be -19.5 mV.
2.3. Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the CNPs were determined by the macrodilution method. The concentrations of 4.88 to 5000 μg/mL of CNPs were prepared. Then, 100 microliters of each of the microbial suspensions with a turbidity equal to half McFarland was added to each of the tubes. Next to the test tubes, a positive control tube containing bacterial suspension without CNPs and a negative control containing the TSB culture medium without bacteria were considered. The prepared suspensions were incubated next to the control samples at 37 °C for 24 hours in a shaker incubator at 2000 rpm.
The turbidity of the contents of each test tube was checked. The lowest concentration of the nanoparticle suspension that did not show turbidity in the tube was determined as the MIC of the nanoparticle. Next, 100 microliters of the Mueller-Hinton agar culture medium were cultured in tubes that had no growth and no turbidity and were incubated for 24 hours at 37 °C. After this time, each plate in which the bacteria did not grow was declared as MBC. All the experiments were performed in triplicate (Katabchi, 2017, Kazemi Rad , 2022).
2.4. The antibiotic effect of ampicillin on the standard strain
We performed all the steps mentioned for CNPs for ampicillin to compare the performance of the nanoparticle and antibiotic. Dilution of ampicillin was done with distilled water with pH=5.
2.5. Mice
Seventy female and 14 male BALB/c mice were purchased from the Razi Institute (Karaj, Iran). All the mice were 21 days old, and their weight was between 18 and 22 grams. The mice were kept in a room with a temperature of 24 ± 2 °C, a 12-hour light/dark cycle, and relative humidity of 40% according to the instructions of the Ethics Committee.
2.6. The side effects of the substances used on mice
Four groups of 5 healthy female mice were examined in separate cages. The first group was given 0.2 mL of the TSB medium. The second group was given CNPs with the obtained MIC concentration. The third group was given distilled water with pH=5, which was injected subcutaneously. The fourth group was not injected with any substance and was considered the control group. Then, 1 male mouse was placed inside each cage, and the results were analyzed 10 to 14 days later.
2.7. The method of contamination and treatment of mice
The rest of the female mice were divided into 5 groups of 10, and each group was placed in separate cages. Then, 0.2 mL of L. monocytogenes bacterium suspension with a concentration of 0.5 McFarland was injected subcutaneously. After 48-72 h, to make sure that female mice were infected, we cut their tail area randomly with scissors in sterile conditions and cultured a drop of blood from the tail area; we also prepared a slide, cultured it in blood agar culture medium, and used it to check the presence of L. monocytogenes. After making sure that the mice were infected, we placed 2 male mice in each cage and waited 10 days for mating to take place (the male mice remained inside the cages until the end of the research); then, we started treatment. To the first group, which is the control group for therapeutic effects, we injected distilled water with pH = 5; to the second group, ampicillin was injected with determined MIC concentration; to the third group, we injected the nanoparticle with pre-MIC concentration; to the fourth group, the nanoparticle MIC concentration was injected; and to the fifth group, post-MIC concentration (MBC) of CNPs was injected every 8 hours at 0.2 mL. We recorded the results of this treatment process, including fertility or infertility, abortion, physical disabilities in newborns, and maternal and neonatal deaths after 3 weeks. It should be noted that ketamine (100 mg/kg) and xylazine (10 mg/kg) (intraperitoneal injection) were used as sedatives, and 2% lidocaine (spray at the injection site) was used as an analgesic.
Daily care and observations were performed to record the clinical signs and the mortality rate of the mice during the entire treatment period.
2.8. Statistical analysis
Data analysis was performed using SPSS v. 26 (IBM Corp., Armonk, NY, USA). P-values less than 0.05 were considered statistically significant.
3. Results
3.1. Antimicrobial activity
The findings of this research show that CNPs and ampicillin have a concentration of 5000 µg/mL compared to the standard strain. In the case of the standard strain, CNPs at concentrations of 78.12 to 4.88 µg/mL did not have any inhibitory effect on the tested bacterial strains. Also, the antibiotic ampicillin had no inhibitory effect at the concentrations of 156.25 to 4.88 µg/mL for the standard strain. Distilled water with pH=5 as a control had no growth halo.
The MIC and MBC values of CNPs were 156.25 and 312.5 µg/mL, respectively, for the standard strain and 312.5 and 625 µg/mL for the antibiotic ampicillin, respectively.
3.2. Effects of CNPs on infected mice
Control mice had normal reproduction and did not show any problems. In the study conducted on healthy female BALB/C mice, opposite results were obtained. Injection of TSB culture medium, CNPs with MIC concentration (78.12 µg/mL), and distilled water with pH = 5 in groups of 5 female BALB/c mice separately had no side effects compared to the control group in biological activities (fever, decreased feeding, cornering, body hair changes, including color change and spiking, pregnancy, abortion, infant disability, infant mortality, and maternal mortality), and all the biological activities of the mice were completely normal.
In this research, the injection of distilled water with pH = 5 to a group of 10 BALB/c mice infected with L. monocytogenes bacteria (group A) was considered as a control, and the results were obtained as follows: Out of 10 mice studied in this group, 70% had fever, 80% were withdrawn, 80% had changes in body hair, and 20% became pregnant; among these pregnant mice, 50% suffered abortion, and maternal mortality was not reported.
In groups of 10 infected female mice treated with ampicillin (156/25 µg/mL) (group B), 20% of the mice had a fever, 30% had decreased feeding, 30% were withdrawn, and 30% showed changes in the appearance of body hair, and 70% of them became pregnant. Among the pregnant mice, 25.8% had abortions, but the others were without clinical symptoms (P-value <0.05 and significant).
In this study, different concentrations of CNPs (39.02-12.78-156.78-25.02 µg/mL) were injected into 3 groups of 10 infected mice separately. The effect of the concentration of 39.02 µg/mL nanoparticles on infected mice (group C) is reported: 20% of the mice had a fever, 30% were isolated, 30% had reduced feeding, 30% showed changes in body hair, and 70% were pregnant. Among the pregnant mice of this group, a 14.3% abortion was seen, and the rest of the pregnant mice had no special complications (P-value<0.05).
The effect of the injection of 12.78 µg/mL nanoparticles on infected mice (group D) was observed as follows: 10% of mice had a fever, 20% had decreased feeding, 20% became withdrawn, 20% showed changes in body hair, and 70% reported pregnancy. Among the pregnant mice of this group, a 14.3% abortion was observed, and other pregnant mice had no symptoms (P-value <0.05 and statistically significant).
In this research, injecting a concentration of 156.25 µg/mL into infected female rats (group E) resulted in the following results: 10% reduction in feeding, 10% withdrawal, 10% changes in appearance (body hair), and 80% pregnancy, but the mice did not develop fever. Among the pregnant rats examined in this group, no complications were reported (P-value<0.05) (Fig1).
4. Discussion
L. monocytogenes is an opportunistic pathogen and the cause of listeriosis with a wide clinical spectrum. Listeriosis is not a reportable disease in Iran's health and safety system, so the actual prevalence of L. monocytogenes in Iran is unknown. Listeriosis is a serious and invasive disease that enters the human body through inhalation, eating contaminated food, using animal products, and direct contact. Among Listeria species, only L. monocytogenes is pathogenic for humans. This bacterium is one of the few microorganisms that can pass through the placenta (Vazco-Boland, 2001).
It has been reported that chitosan generally has a stronger bactericidal effect on Gram-positive bacteria than Gram-negative bacteria (Jeon, 2001). This is probably due to the outer membrane barrier of Gram-negative bacteria (Zhang, 2021). In the research conducted, L. monocytogenes is a Gram-positive bacterium, and CNP has shown a significant inhibitory effect on this bacterium.
The low solubility of chitin is considered the most important factor limiting the use of this biopolymer. Despite this limitation, so far, many applications of chitin and its derivatives have been reported. When chitin is soluble in acidic aqueous solvents whose pH is less than 6, it is called chitosan.
Chitosan has important functional groups, such as amine groups, at the second carbon position and primary and secondary hydroxyl groups at the third and sixth carbon positions. Chitosan has antimicrobial properties that are created through interactions between the positive charge of the amine group and the negative charge on the cell wall of the microorganism. Among its prominent features are its biodegradability, high biocompatibility, and nontoxicity. According to the literature, antimicrobial activity is highly dependent on the degree of deacetylation and molecular weight, and it can also have different effects according to the bacterial strain (Aranaz, 2009 , Pillai, 2009 , Chen, 1998 , Feng, 2011, Chavez, 2011).
In fact, the antibacterial property of CNP depends on the protonation of amino groups, which can react with the negative charge of the cell surface and lead to the destruction of bacterial cells (Wei 2009). Perhaps in the present study, CNPs have a high positive charge density and have a good inhibitory effect on clinical and standard strains of L. monocytogenes; also, in this research, we dissolved chitosan at pH 5 and performed the other tests.
Radam et al. (2020) aimed to investigate the effect of dietary chitosan supplementation on the experimental infection of mice by L. monocytogenes. The results showed the isolation of bacteria mainly from the brain, spleen, and liver of positive control groups, while it showed slight growth in the other treated groups. In our research, symptoms decreased after treatment with chitosan nanoparticles (Radam , 2020).
In the research conducted by Ma et al., it was shown that the antimicrobial properties of CNPs increase with the increase in the concentration of these particles (Ma , 2010), which was also obtained in our research and the lack of growth halo. Bacteria, due to different concentrations of these nanoparticles, showed that the greatest inhibitory effect in chitosan with higher concentration is observed in the standard strain of L. monocytogenes. This indicates that the higher the nanoparticle concentration, the greater its inhibitory effect.
5. Conclusion
In this study, the antibacterial effect of CNPs on L. monocytogenes, the causative agent of listeriosis, was investigated. The results showed that the nanoparticle has beneficial effects on the treatment of this disease in the body of a living organism (laboratory white mouse), and the higher the concentration of this nanoparticle, the greater its therapeutic effect. Because it has a natural origin, it is a suitable alternative to antibiotics, such as ampicillin, used to treat listeriosis, especially in pregnant women.
6. Acknowledgements
The authors gratefully acknowledge the cooperation and support of Day Hospital Laboratory (Tehran, Iran).
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