Volume 27, Issue 8 (10-2020)                   RJMS 2020, 27(8): 54-64 | Back to browse issues page

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Ashrafi G, Barzegar Masoomi P, Parvini Kohnehshahri M. Determination of the prevalence of pathogenic genes in Pseudomonas aeruginosa isolated from Patients hospitalized in Urmia Hospitals. RJMS 2020; 27 (8) :54-64
URL: http://rjms.iums.ac.ir/article-1-6130-en.html
Urmia Branch, Islamic Azad University, Urmia ,Iran , m.parvini@iaurmia.ac.ir
Abstract:   (1283 Views)
Background: Pseudomonas aeruginosa is an opportunistic, gram-negative, non-fermented and catalase-positive pathogen that causes significant opportunistic infections such as urinary tract infection, pneumonia, and septicemia. P. aeruginosa is a unique bacteria that can make changes in the virulence genes expression and the rate of drug resistance in order to survive in different environments. Drug resistance resulting from beta-lactamases have been reported to be an important cause of various infections and are a critical therapeutic problem worldwide. Additionally, It has been reported that the clinical isolates of P. aeruginosa are challenging to treat because of their virulence factors and antibiotics resistances. This bacteria has a large number of pathogenic agents such as Exotoxin A, Exoenzyme S and Elastase, which causes resistance of bacteria against antibiotics. They are resistant to many antibiotics, including beta-lactams and third-generation cephalosporins. The objective of this study was to determine the prevalence of Exo A, and Exo S genes in clinical isolates of P. aeruginosa obtained from patients hospitalized in Urmia city hospitals. The other aim of this study was to evaluate the antibiotic resistance, and the prevalence of bla-SHV and CTX-M genes in clinical isolates of P. aeruginosa obtained from patients hospitalized in Urmia city hospitals.
Methods: Through this cross-sectional and experimental-applied study, one-hundred isolates of P. aeroginosa from diverse clinical sources, including blood, wound, trachea and urine samples, were collected from different Urmia city hospitals, Iran. The hospitals and private or public laboratories were included Motahari, Imam-Reza, Arian, Taleghani, Milani, Imam-Khomeini, and Arefian. Samples were incubated and cultured in a nutrient agar culture medium at 37°C for 24 hours, and then a slide of each sample was prepared for examination by Gram Staining. Biochemical tests were performed on the samples suspected of P. aeruginosa including catalase, oxidase, oxidation/ fermentation test, pigment production, arginine dehydrolase, and growth at 42°C. Approved samples were cultured on a Mueller-Hinton agar culture medium to evaluate drug resistance. After confirmation by biochemical tests, antibiotic susceptibility test was performed using the disk diffusion method. The disks were placed at regular distances on the plates' surface, and the size of the zone of inhibition was measured after 24 hours of incubation at 37 °C. CLSI reference tables were used to detect the rate of antibiotic resistance. The antibiotic disks used in this study were included: antibiotics from the family of aminoglycosides (gentamicin, ceftriaxone, imipenem, cefotaxime, colistin and amikacin) and the β-lactam family (ceftazidime), and ciprofloxacin. In order to determine the prevalence of Exo A, Exo S, bla-SHV and CTX –M genes, DNA of the samples was extracted using the boiling method and maintained in the TE buffer in 20°C for PCR reaction. The primer sequences of the desired genes were obtained from the valid articles to perform the PCR reaction. PCR cycles were optimized, and the prevalence of genes was determined using the standard PCR method and touchdown-PCR.  
PCR reaction was performed at a standard volume of 25 μl. In this volume, for each microtubule, a mixture of 1 μl of extracted DNA samples, one μl of each forward and reverse primers, 12.5 μl of Master Mix and 9.5 μl of deionized distilled water were mixed. Then the PCR reaction was performed in a thermocycler with a specific time and heat cycle for amplification of each gene as follows: For the Exo A gene, the first temperature range includes an initial cycle of denaturation at 95 °C for two minutes, repetition of 14 cycles for denaturation at 95 °C for 30 seconds, annealing temperature at 73/1 °C for 30 seconds, through which the temperature decreases half a degree at each cycle, the extension temperature at 72 °C for 40 seconds. The second temperature cycle was included the denaturation at 95 °C for 30 seconds that be repeated for 19 cycles, the annealing temperature at 66.1°C for 30 seconds and the extension temperature at 72°C for 40 seconds and a final expansion cycle at 72 ° C for 40 seconds. For Exo S, bla-SHV and CTX-M genes, the temperature program was one cycle of initial denaturation at 95 ° C for two minutes, denaturation temperature at 95 °C for 30 seconds was repeated for 25 cycles, the annealing temperatures were 64°C, 61 °C and 56.7 °C respectively for 30 seconds, the extension temperature at 72 °C for 50, 70 and 80 seconds, respectively, and a final extension cycle at 72 °C for five minutes for each gene. Finally, in order to qualify the PCR product, five microliters of each sample were transferred for electrophoresis on 2% agarose gel and finally stained with ethidium bromide. The obtained data were analyzed by the qualitative method as a percentage.
Results: In this descriptive cross-sectional study, from 100 samples collected from different hospitals, 24 samples were obtained from the male patients (24%), and 76 samples were obtained from the female patients (76%). Clinical samples prepared from patients included 10% wounds, 4% blood and 86% urine. The antibiotic susceptibility test results indicated about 63% of resistance to β‐lactamases and exotoxin genes. The highest resistance was to cefotaxime, 86%, and the lowest resistance rate was to colisitin, 3%. The other antibiotics' resistance rates were 28% for gentamicin, 61% for ceftriaxone, 28% for amikacin, 23% for ceftazidime, 24% for ciprofloxacin, and 30% for imipenem. PCR results showed 52/38% and 57/14% of the isolates contained exotoxin S and A genes, respectively. Positive genes of CTX-M and bla-SHV were detected in 7.93% and 4.76% of isolates, respectively.
Conclusion: The presence of genes encoding beta-lactamase and exotoxin enzymes and their transmission among gram-negative bacteria is a principal threat for consumers of broad-spectrum cephalosporins, especially when indiscriminate using these antibiotics. In recent decades, the emergence of resistant strains of this bacterium with multiple drug resistance has increased. Therefore, isolation and identification of P. aeruginosa from the clinical samples and reporting the results to health authorities is essential. The present study demonstrated a low occurrence of P. aeruginosa isolates carrying CTX-M and bla-SHV genes. Additionally, drug resistance and the presence of virulence factors such as exotoxins can be a severe warning for treatment centers in terms of control of disease caused by this bacterium.
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Type of Study: Research | Subject: Microbiology

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