Medical Laboratory Journal

Association of agr Gene Expression with Staphylococcus Aureus Virulence Genes in BHI Broth

Hadi Koohsari

Abstract

Background and Objective: Agr is the most important regulatory system for the expression of Staphylococcus aureus virulence factors in different conditions. Agr acts as a quorum sensing system in this bacterium which is activated by increased cell concentration during the transition from logarithmic growth phase to stationary phase. Its role is to upregulate the secretory virulence factors such as alpha-hemolysin and inhibit the transcription of surface proteins including protein A-encoding gene. The aim of this study was to assess the relationship between the agr system expression and some virulence factors of Staphylococcus aureus in Brain-heart infusion (BHI) culture medium.

Methods: The expression level of agrA and RNAIII genes from the agr locus along with the expression of hla, spa and mecA genes in BHI broth were assessed in different growth phases using Real time-PCR. Also, gyrB was used as an internal control in this study.

Results: The growth curve of the five tested isolates in BHI broth at 24 hours showed that all the isolates had relatively similar growth patterns. AgrA gene expression in the stationary phase was decreased by 0.89-fold compared with the logarithmic phase. Although the expression of RNAIII gene increased by 3-fold, hla expression decreased by 0.47-fold.

Conclusion: An inactive agr system is observed in the BHI broth medium. BHI broth medium contains high amounts of suitable nutrients for the growth of Staphylococcus aureus, thus the bacteria do not require the activity of the agr system for the regulation of the virulence genes in these conditions.

The Antibacterial Effect of Aqueous Extract of Garlic against Resistant Enterococci

Shaghayegh Nikpour Moghadam

Abstract

Background and Objective: Enterococci are relatively nonvirulent bacteria that rarely cause disease. Antimicrobial treatment of Enterococci is often challenging due to their antibiotic resistance. This study aimed to investigate the antibacterial activity of aqueous extract of garlic against Enterococcal isolates.

Methods: In this descriptive study, 120 Enterococcus isolates including 70 multidrug-resistant isolates were collected from hospitals of Babol, Iran. Isolates’ susceptibility to different antibiotics and the antibacterial activity of garlic extract were assessed using methods of minimum inhibitory concentration (MIC) measurement. The experiments were performed according to the Clinical & Laboratory Standards Institute guidelines, using Tryptic soy broth medium and disc diffusion method.

Results: Among the 120 Enterococcal isolates, 95 (79.2%) and 25 isolates (20.8%) were E. faecalis and E. faecium, respectively. Of the all Enterococcal isolates, the highest resistance was to erythromycin (95.8%), tetracycline (88.3%) and ampicillin (65.8%). While, the minimal level of resistance was to chloramphenicol (6.8%), vancomycin (20%) and ciprofloxacin (25%). Also, 53.3% of Enterococcal isolates showed simultaneous resistance to at least three antibiotics (tetracycline, erythromycin and ampicillin). Such resistance in E. faecium isolates was higher compared to E.faecalis (68% vs. 55.7%). The range of antibacterial activity of garlic extract against isolated Enterococci was determined by growth inhibition zone of 16.8 ± 1.8 mm and MIC of between 4 to 32 mg/ml.

Conclusion: This study indicates the clear anti-enterococcal effect of aqueous extract of garlic and confirms the use of garlic in treatments by medicinal plants.

Ochratoxin A in Cow’s Milk Collected from Cattle Farms of Golestan Province

Farhad Niknejad

ABSTRACT

Background and Objective: Ochratoxin is a fungal toxin produced by Penicillium verrucosum and some Aspergillus species. Ochratoxin is usually found in grains, cereal products and also animal feed of livestock. The aim of this study was to measure the level of Ochratoxin in pasteurized milk samples of Golestan Province, Iran.

Methods: Overall, 38 milk samples were collected from East and West of the Golestan province in accordance with standards 326 and 419 of the Institute of Standards and Industrial Research of Iran. The level of Ochratoxin was measured by ELISA method.

Results: The mean level of Ochratoxin A in 20 raw milk samples collected from the West of the Province was 3.32 ± 3.76 ng/ml. The mean level of Ochratoxin A in 18 raw milk samples collected from the East was 6.02 ± 4.42 ng/ml. Ochratoxin A levels in most samples were higher than the limits established by the European standards.

Conclusion: Based on the results of this study, Ochratoxin level of 84.2% and 52.6% of the samples from the West and East of the province are higher than the allowed limits (2 ng/ml), respectively.

The Effect of Citrus Aurantium, Foeniculum Vulgare and Rosmarinus Officinalis Essential Oils on Peroxidase Activity

Maryam Mohajerani

Abstract

Background and objective: Peroxidases catalyze protein oxidation and lipid peroxidation. The activity of these enzymes in nerve cells is involved in causing disorders such as Alzheimer's and Parkinson's disease. This study investigated the effect of Citrus aurantium, Foeniculum vulgare and Rosmarinus officinalis essential oils on activity of peroxidase enzyme.

Methods: All three medicinal plants were dried at room temperature. Their essential oil was extracted by steam distillation using a Clevenger apparatus. Optimal reaction conditions were determined in the presence of hydrogen peroxide and guaiacol as substrate and hydrogen donor, respectively. Enzyme kinetics of zucchini peroxidase were evaluated by increasing the amount of essential oils in optimal reaction conditions. Enzyme reaction rate for each of the essential oils and the K_m and V_max values were determined.

Results: The results indicated concentration-dependent effect of the extracted essential oils on enzyme kinetics at optimum temperature of 50 °C and optimal pH of 6.5. The essential oil of Citrus aurantium had non-competitive inhibitory effects on the enzyme with K_m of 6.25 mM, while the enzyme’s V_max significantly reduced by increasing the concentration. Foeniculum vulgare showed mixed inhibition effect with K_m of 7.14 mmol per 20 μl of the essential oil, but had a decreasing effect on the V_max in smaller amounts. Finally, Rosmarinus officinalis showed activating effects by reducing the K_m to 4-5.88 mM.

Conclusion: The essential oils of Citrus aurantium and Foeniculum vulgare are inhibitors of the peroxidase enzyme and can be further studied as natural herbal medicines.

Comparison of the Antimicrobial Effects of Silver Nanoparticles Alone and In Combination with Zataria Multiflora Extract On Some Gram-Positive and Gram-Negative Bacteria

Shirin Sheikholeslami

Abstract

Background and Objective: The spread of drug resistance in bacteria have prompted researchers to seek suitable alternative for antimicrobial drugs among various medicinal plants and nanoparticles. The aim of this study was to evaluate the effect of silver nanoparticles alone and in combination with methanol extract of Zataria multiflora on five Gram-positive and Gram-negative bacteria.

Methods: Different concentrations of the nanoparticles and extract alone or in combination with each other were tested against the bacteria, using well diffusion method. Three concentration levels (lowest, average and highest) were prepared form the nanoparticles and the extract for the combination, and finally nine different combinations were prepared.

Results: The extract and nanoparticles showed inhibitory effects against all the tested bacteria. The maximum diameter of growth inhibition zone in the presence of the extract and nanoparticles were observed in Streptococcus pyogenes (35.6mm) and methicillin-resistant Staphylococcus aureus (20.6mm), respectively. The maximum diameter of growth inhibition zone for the combination was measured in S. pyogenes (31mm).

Conclusion: The combination of low concentrations of the plant extract and nanoparticles are more effective against bacteria, but the combination of their high concentrations reduce the antibacterial effects in some cases.

Mutations at Nucleotide 1762, 1764 and 1766 of Hepatitis B Virus X Gene in Patients with Chronic Hepatitis B and Hepatitis B-Related Cirrhosis

Farzane Salarneia

Abstract

Background and objective: Hepatitis B virus (HBV) is a DNA virus with high tendency toward hepatic tissue. There are currently about 3 million HBV-infected people and 350 to 400 million chronic carriers of this virus in the world. X protein plays a role in the over-expression of oncogenes, carcinogenicity of liver cells and overlaps with the basal core promoter of the virus. Mutations at specific nucleotides of this region increase viral replication and liver disease progression. The aim of this study was to investigate the frequency of mutations at nucleotides 1762, 1764 and 1766 of HBV X gene in patients with chronic hepatitis B and hepatitis B-related cirrhosis.

Methods: In this study, 102 patients including 68 chronic hepatitis patients and 34 patients with hepatitis B-related cirrhosis were enrolled. After DNA extraction, HBV X gene was amplified and sequenced using Semi Nested-PCR. Obtained gene sequences were compared with the standard sequence of HBV virus X gene available in the gene bank (Okamoto AB033559). Then, the mutations in the gene X of HBV were identified.

Results: Comparison of the standard sequence with sequences obtained from patients showed the presence of A1762T / G1764A mutation in 12 chronic (17.64%) and 13 cirrhotic (38.23%) patients. Also, C1766G / G1764T mutations were found in 8.23% of chronic patients and 17.64% of cirrhotic patients.

Conclusion: A1762T / G1764A mutations in the overlapping region of the basal core promoter with gene X C-terminal may lead to liver disease progression from chronic hepatitis to cirrhosis, by changing the amino acid sequence of the X protein.

Investigating the Phytochemical, Antibacterial and Antifungal Effects of Thymus Vulgaris and Cuminum Cyminum Essential Oils

Soghra Valizadeh

Abstract

Background and Objective: The aim of this study was to determine the chemical composition, antibacterial and antifungal effects of Thymus vulgaris and Cuminum Cyminum essential oils against foodborne pathogens and Candida species in vitro.

Methods: The essential oils were extracted from the aerial parts of Thymus vulgaris and dried Cuminum Cyminum seeds using a Clevenger apparatus for 3 hours. Analysis of the essential oils’ constituents was performed using gas chromatography-mass spectrophotometry. The antibacterial activity of Cuminum Cyminum essential oil and essential oil of Thymus vulgaris against Bacillus cereus, Listeria monocytogenes, Escherichia coli and Salmonella typhimurium were evaluated in agar culture medium. The minimum inhibitory concentration (MIC) of these essential oils against fungal strains of Candida albicans, C. tropicalis, C. parapsilosis and C. dubliniensis was measured.

Results: Thymol (64.45%) and cuminaldehyde (29.02%) were the main components of the essential oil of Thymus vulgaris and Cuminum Cyminum, respectively. The largest inhibition zone diameter in the essential oils of Thymus vulgaris and Cuminum Cyminum in the agar disk diffusion method was related to B. cereus with 30 and 21 mm diameter, respectively. The largest growth inhibition zone diameter by the essential oil of Thymus vulgaris in the well diffusion method was 21 mm and against B. cereus. The MIC of essential oil of Thymus vulgaris in the microdilution method was 0.09% against all the four Candida strains. The MIC of Cuminum Cyminum essential oil against strains of C. albicans and C. tropicalis was 0.39%, while it was found as 0.19% against C. parapsilosis and C. dubliniensis.

Conclusion: In this study, Cuminum Cyminum essential oil and essential oil of Thymus vulgaris show suitable inhibitory effects against the growth of bacteria using well and disk diffusion methods. Regarding the antifungal effects, the MIC of essential oil of Thymus vulgaris is lower than the Cuminum Cyminum essential oil, which indicates the higher antifungal activity of the essential oil of Thymus vulgaris. This study has raised the possibility of using these essential oils as suitable antimicrobial compounds and alternatives for chemical preservatives in the food industry.

Comparison of Disk Diffusion and E-Test with the Reference Method of Microbroth Dilution for Susceptibility Testing of Acinetobacter baumannii Isolates to Tetracyclines

Abdollah Ardebili

Abstract

Background and Objective: Due to the continuous increase of multidrug-resistant Acinetobacter baumannii strains around the world, decision making for an effective treatment of infections caused by this organism depends on the results of antimicrobial susceptibility tests. In this study, the validity of disk diffusion and E-test methods was assessed by their comparison with the reference method of microbroth dilution for three antibiotics of tetracycline, doxycycline and minocycline.

Methods: Total of 68 A. baumannii isolates were obtained from patients hospitalized in the burn center of Shahid Motahari Hospital in Tehran, Iran. Susceptibility of the Acinetobacter isolates was evaluated using the disk diffusion, E-test and microbroth dilution methods, according to the guidelines of Clinical and Laboratory Standards Institute.

Results: Among the isolates, 82.3% were tetracycline-resistant (with minimum inhibitory concentration 50 (MIC₅₀) and MIC₉₀ of 32 and more than 32 µg/ml, respectively and 41.2% were doxycycline-resistant (with MIC₅₀ and MIC₉₀ of 4 and more than 32 µg/ml, respectively). Minocycline, with resistance of up to 13.3% (MIC₅₀ and MIC₉₀ of 1 and 8 µg/ml, respectively) showed the highest antimicrobial activity against the A. baumannii isolates. Antimicrobial susceptibility of bacteria was different depending on the type of methods used. No very major error was observed in any of the methods of susceptibility testing. Overall, the level of major and minor errors in the E-test was lower than the disk diffusion method.

Conclusion: The results of this study indicate that minocycline has notably high antimicrobial activity against A. baumannii compared to other antibiotics of the tetracycline group.

Association of HDL/TG Ratio as an Insulin Resistance Marker with Various Levels of Fasting Blood Glucose

Maryam MoradiBinabaj

ABSTRACT

Background and Objective: The association of Triglyceride/High Density Lipoprotein-Cholesterol (TG/HDL-C) ratio with fasting serum insulin, which is an alternative method of insulin resistance (IR) measurement, is well-recognized. Thus, the measurement of TG/HDL-C ratio is useful to determine both IR and dyslipidemia, which itself is a characteristic of individuals with IR. Therefore, this study aimed to investigate the relationship between TG/HDL ratio as an indicator of IR, with different fasting blood glucose levels.

Methods: This case-control study was performed on 343 volunteers with no history of diabetes or use of blood glucose-lowering medications and fasting blood sugar (FBS) levels of less than 126 mg/dl. After sampling, the subjects were divided into three groups based on their FBS level. First group included healthy subjects with FBS of less than 100 mg/dl. Second group consisted of subjects with impaired fasting glucose (IFG) and FBS of 110-100 mg/dl and a third group including those with impaired glucose tolerance (IGT) and FBS of 110-125 mg/dl.

Results: The amount of TG/HDL-C ratio was 3.8 ± 2.8, 4.0 ± 2.1 and 5.4 ± 3.8 for the healthy group, individuals with IFG and IGT, respectively. The TG/HDL index was significantly different among the tested groups with no significant difference between healthy subjects and subjects with IFG. Moreover, there was a statistically significant difference between the IGT and IFG groups with healthy individuals.

Conclusion: Considering the significant increase of the TG/HDL ratio in groups with impaired glucose, using this index can be helpful in evaluation of glycemic disorder.

The Impact of High Intensity Interval Training On Lipid Profile, Inflammatory Markers and Anthropometric Parameters in Inactive Women

Nasrin Zaer Ghodsi

Abstract

Background and Objective: High-intensity interval training (HIIT) is a recently proposed exercise protocol, which is time-effective. The aim of this study was to evaluate the effect of HIIT for 8 weeks on the lipid profile, C-reactive protein (CRP), fasting blood sugar (FBS) and anthropometric parameters of young women who do not exercise.

Methods: In this study, 20 young physically inactive women performed HIIT workouts for 8 weeks and 3 sessions per week. The training protocol consisted of 10-times treadmill running for 15 seconds at maximum effort and then 30 seconds of resting. Blood samples were taken while fasting, a day before and after the training and then the considered parameters were measured. Wilcoxon test was used to compare the obtained data.

Results: HIIT significantly reduced FBS, cholesterol, low-density lipoprotein, high-density lipoprotein /cholesterol ratio and CRP while increasing the HDL levels. There was a significant difference in the weight, body fat percentage, waist circumference, abdominal circumference and chest circumference of the subjects before and after the training (p <0.05).

Conclusion: HIIT can improve lipid, inflammatory and anthropometric parameters, thus it can be considered as a suitable alternative to time-consuming exercises, especially for physically inactive women who like to spend less time to achieve optimal physical wellness and body fitness.