Volume 28, Issue 9 (12-2021)
RJMS 2021, 28(9): 12-20 |
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Jamshidian-Mojaver M, Amiri M, Farzin H. Prevalence of virulence genes in Escherichia coli isolates of patients with UTI. RJMS 2021; 28 (9) :12-20
URL: http://rjms.iums.ac.ir/article-1-5917-en.html
URL: http://rjms.iums.ac.ir/article-1-5917-en.html
Mashhad Branch, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran. , hrfarzin@yahoo.com
Abstract: (1343 Views)
Background & Aims: Escherichia coli, which is naturally isolated from the digestive tract of humans and animals, is one of the most important members of the microbiota of this system and its pathogenic form causes very important complications in humans and animals. Escherichia coli is often harmless or low in severity and its presence as a common organism in the lower parts of the intestine is essential to maintain its ecosystem and health. Escherichia coli may cause opportunistic infections in extrauterine areas such as the mammary glands and urinary tract. Also, some strains are considered as the main pathogens by having or acquiring genetic factors of virulence. Strains that cause gastrointestinal diseases are usually not part of the normal human and animal flora, and infection results from the consumption of food, contaminated water, or direct contact with humans and animals that are clinically and subclinically infected with acute strains. It happens. Pathogenic strains have factors that allow bacteria to colonize mucosal surfaces and then cause damage. Predisposing factors involved in bacterial colonization include age, immune status, nutrition, and severe exposure to pathogenic strains. The pathogenic potential of strains is determined by the virulence genes in the bacterium. The expression of virulence genes leads to certain types of injuries and clinical syndromes. Pathogenic strains of Escherichia coli can be classified into two main groups based on the factors of severity, location and type of damage: intestinal pathogenic strains and extraintestinal pathogenic strains.
In pathogenic strains, genes encoding virulence factors are often found in pathogenic islets, while commensal strains lack these pathogenic islets in their genomes. Many pathogens are also encoded by acquired genes in mobile genetic components such as plasmids, bacteriophages, and transposons.
Extraintestinal pathogens, most commonly meningitis-causing strains (MAEC) and urinary tract infections (UPEC), have been isolated from patients with pneumonia, cholecystitis, peritonitis, and other infections. Among UPEC strains, some bind diffusely to tissue culture cells. In addition, diarrhea of some Escherichia coli strains (DAEC) has been isolated from urinary tract infections in addition to diarrhea.
In the present study, 50 samples of positive cultures with urinary tract infections were referred to the laboratory located in Imam Reza Hospital located in Bojnourd city. Eosin methylene blue agar and McConkey agar plates containing bacteria with specific bacterial conditions (with green gloss in eosin methylene blue agar medium and in McConkey agar medium with smooth pink colonies) as suspected isolates of Escherichia coli Were selected and cultured in TSI, SIM, MRVP and citrate media to confirm these selected isolates after hot staining.Finally, gram-negative isolates whose biochemical tests were acid / acid in TSI , The manufacturer of indole, without producing hydrogen sulfide, with or without immobilization in SIM, MR-positive, VP-negative and without citratease, was approved as Escherichia coli.
Methods: Boiling method was used to extract DNA in this study. In this method, the bacteria stored on the medium of Luria Bertani Agar were cultured and exposed to 37 ° C for 16-18 hours. After the incubation period, DNA was extracted from single colonies. Used. First, 350 μl of sterile distilled water was transferred into sterile microtubes and then a colony was dissolved in it. The microtubes were placed in a heating block device at 98 ° C for 11 minutes. The microtubes were then cooled in the freezer at -20 ° C for 5 minutes. Finally, the microtubes were centrifuged for 2 minutes at a speed of 12,000 rpm and 250 μl of the supernatant was removed and placed in the freezer at -20 ° C for subsequent steps.
Identification of virulence genes was performed using specific primers from the study by Ewers et al. To prepare Mastermix polymerase chain reaction from ready mix Master in the amount of 12.5 microliters, 0.1 microliters (100 Picomoles per liter) of each of the primers, sample DNA 5 microliters and sterile distilled water up to the reaction volume (25 microliters) were used.
Results: Based on the results of the above study, out of 50 isolates studied, 32 isolates (64%) had irp2 gene, 18 isolates (36%) had cvi / cvaC gene, 15 isolates had papC gene (30%), 15 isolates ( 30% had iucD gene, 8 isolates (16%) had astA gene, 8 isolates (16%) had iss gene and 3 isolates (6%) had tsh gene and 2 isolates had vat gene (4%). In this study, there were 17 profiles for the studied genes.
Conclusion: Given the prevalence and presence of the studied genes, in the present study and other studies, it can be said that these genes play an important role in causing and exacerbating infections caused by extraintestinal strains. These genes are also present in some isolates at the same time, which can prolong the treatment period of the infection.
In pathogenic strains, genes encoding virulence factors are often found in pathogenic islets, while commensal strains lack these pathogenic islets in their genomes. Many pathogens are also encoded by acquired genes in mobile genetic components such as plasmids, bacteriophages, and transposons.
Extraintestinal pathogens, most commonly meningitis-causing strains (MAEC) and urinary tract infections (UPEC), have been isolated from patients with pneumonia, cholecystitis, peritonitis, and other infections. Among UPEC strains, some bind diffusely to tissue culture cells. In addition, diarrhea of some Escherichia coli strains (DAEC) has been isolated from urinary tract infections in addition to diarrhea.
In the present study, 50 samples of positive cultures with urinary tract infections were referred to the laboratory located in Imam Reza Hospital located in Bojnourd city. Eosin methylene blue agar and McConkey agar plates containing bacteria with specific bacterial conditions (with green gloss in eosin methylene blue agar medium and in McConkey agar medium with smooth pink colonies) as suspected isolates of Escherichia coli Were selected and cultured in TSI, SIM, MRVP and citrate media to confirm these selected isolates after hot staining.Finally, gram-negative isolates whose biochemical tests were acid / acid in TSI , The manufacturer of indole, without producing hydrogen sulfide, with or without immobilization in SIM, MR-positive, VP-negative and without citratease, was approved as Escherichia coli.
Methods: Boiling method was used to extract DNA in this study. In this method, the bacteria stored on the medium of Luria Bertani Agar were cultured and exposed to 37 ° C for 16-18 hours. After the incubation period, DNA was extracted from single colonies. Used. First, 350 μl of sterile distilled water was transferred into sterile microtubes and then a colony was dissolved in it. The microtubes were placed in a heating block device at 98 ° C for 11 minutes. The microtubes were then cooled in the freezer at -20 ° C for 5 minutes. Finally, the microtubes were centrifuged for 2 minutes at a speed of 12,000 rpm and 250 μl of the supernatant was removed and placed in the freezer at -20 ° C for subsequent steps.
Identification of virulence genes was performed using specific primers from the study by Ewers et al. To prepare Mastermix polymerase chain reaction from ready mix Master in the amount of 12.5 microliters, 0.1 microliters (100 Picomoles per liter) of each of the primers, sample DNA 5 microliters and sterile distilled water up to the reaction volume (25 microliters) were used.
Results: Based on the results of the above study, out of 50 isolates studied, 32 isolates (64%) had irp2 gene, 18 isolates (36%) had cvi / cvaC gene, 15 isolates had papC gene (30%), 15 isolates ( 30% had iucD gene, 8 isolates (16%) had astA gene, 8 isolates (16%) had iss gene and 3 isolates (6%) had tsh gene and 2 isolates had vat gene (4%). In this study, there were 17 profiles for the studied genes.
Conclusion: Given the prevalence and presence of the studied genes, in the present study and other studies, it can be said that these genes play an important role in causing and exacerbating infections caused by extraintestinal strains. These genes are also present in some isolates at the same time, which can prolong the treatment period of the infection.
Type of Study: Research |
Subject:
Microbiology
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