بررسی مولکولی ژنهای slyA، stn، sopB، Phop/Q و spvc در سالمونلا تیفیموریوم جدا شده از نمونههای بالینی با روش Multiplex PCR
محورهای موضوعی : میکروب شناسیزهرا معصومعلی نژاد 1 , بابک خیرخواه 2 , فهیمه میرزاده 3
1 - دانشجوی دکتری، گروه میکروبیولوژی، دانشگاه آزاد اسلامی، واحد سیرجان، سیرجان، ایران
2 - استادیار، گروه میکروب‌شناسی، دانشگاه آزاد اسلامی، واحد کرمان، کرمان، ایران.
3 - دانشجوی دکتری، گروه میکروبیولوژی، دانشگاه آزاد اسلامی، واحد سیرجان، سیرجان، ایران
کلید واژه: سالمونلا تیفیموریوم, MultiplexPCR, ژنهای ویرولانس,
چکیده مقاله :
سابقه و هدف: سالمونلا یک ارگانیسم روده‌‌ای گرم‌‌منفی و عامل بروز مسمومیت‌‌های غذایی در انسان میباشد. جنس سالمونلا دارای پنج ژن ویرولانس stn،Phop/Q ،spvc ، slyAو sopB میباشد. این ژن‌‌ها پروتئین‌‌هایی را در قسمت‌‌های مختلف باکتری کد می‌‌نمایند که مقابله با سیستم ایمنی، کمپلمان و مرگ داخل سلول را باعث می‌شوند. هدف از مطالعه حاضر شناسایی ژن‌‌هایویرولانسدر سویه‌‌های سالمونلاتیفی‌‌موریوم جدا شده از نمونههای بالینی به روش Multiplex PCR و تعیین الگوی مقاومت آنتی‌‌بیوتیکی میباشد. مواد و روش‌ها: در این مطالعه توصیفی- مقطعی طی سال 1396 تعداد 60 نمونه مدفوع به‌‌منظور شناسایی سالمونلا تیفیموریوم از بیمارستان البرز شهر کرج جمع‌‌آوری شد. آزمون حساسیت آنتی‌‌بیوتیکی روی محیط مولر هینتون آگار و بر اساس استاندارد (CLSI) انجام گردید. آزمون Multiplex PCR جهت تشخیص ژنهای ویرولانس با استفاده از آغازگرهای اختصاصی انجام شد. نتایج: نتایج آزمون حساسیت آنتی‌‌بیوتیکی نشان داد تمامی ایزوله‌‌ها دارای حساسیت به ایمی‌‌پنم، جنتامایسین و آمیکاسین بودند. همچنین، فراوانی ژنهای Phop/Q، slyAوstn به‌‌ترتیب برابر 100، 3/98 و 6/91 درصد بود و ژنهای sopB و Spvc در جدایه‌‌های سالمونلا تیفیموریوم مشاهده نگردید. نتیجه‌گیری: نتایج مطالعه حاضر حاکی از شیوع ژنهای ویرولانس در سویه‌‌های بالینی سالمونلاتیفی‌‌موریوم میباشد که می‌‌تواند به‌‌عنوان زنگ خطری برای انتشار این ژن‌‌ها به دیگر سروتیپ‌‌های سالمونلا باشد.
Background: Salmonella is a Gram-negative intestinal organism and causes food poisoning in human. Salmonella has five virulence genes, stn, Phop/Q, spvc, slyA and sopB. These genes encode proteins in different parts of the bacteria that can confront with immune system, and the complement system and can cause death in the cell. The aim of this study was to detect slyA, stn, sopB, Phop/Q and spvc genes in Salmonella typhimurium strains isolated from clinical samples by the multiplex PCR method and to determine antibiotic resistance patterns. Material and Methods: In this descriptive cross-sectional study, in 2016, 60 stool samples in order to identify Salmonella typhimurium from Alborz-Karaj Hospital were collected. After confirmation of the strains by using standard biochemical and microbiological tests, an antibiotic susceptibility test was performed on a Muller Hinton Agar medium and based on Clinical and Laboratory Standards Institute (CLSI) guidelines. Multiplex PCR assay was performed to detect virulence genes using specific primers. Results: The results of the antibiotic susceptibility test showed that all isolates were sensitive to imipenem, gentamicin and amikacin. Also, molecular findings showed that the prevalence rates of Phop/Q, slyA and stn genes were 100%, 98.3%, and 91.6%, respectively. While sopB and Spvc genes were not observed in isolates of Salmonella typhimurium. Conclusion: The results of this study indicate that the prevalence of virulence genes in clinical Salmonella typhimurium isolates can serve as an alarm for the prevalence of these genes to the other Salmonella serotypes.
Rowlands RE, Ristori CA, Ikuno AA, Barbosa ML, Jakabi M, Franco BD. Prevalence of drug resistance and virulence features in Salmonella spp. isolated from foods associated or not with salmonellosis in Brazil. Rev Inst Med Trop Sao Paulo. 2014; 56(6): 461-7.
Ktsoyan Z, Ghazaryan K, Manukyan G, Martirosyan A, Mnatsakanyan A, Arakelova K, et al. Inflammatory responses to Salmonella infections are serotype-specific. Int J Bacteriol. 2013; 2013: 168-79.
Amini K, Salehi TZ, Nikbakht G, Ranjbar R, Amini J, Ashrafganjooei SB. Molecular detection of invA and spv virulence genes in Salmonella enteritidis isolated from human and animals in Iran. Afr J Microbiol Res. 2010; 4(21): 2202-10.
Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O'brien SJ, et al. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis. 2010; 50(6): 882-9.
Ke B, Ran L, Wu S, Deng X, Ke C, Feng Z, et al. Survey of physician diagnostic and treatment practices for patients with acute diarrhea in Guangdong province, China. Foodborne Pathog Dis. 2012; 9(1): 47-53.
Mao X, Hu J, Liu X. Estimation on disease burden of foodborne non-typhoid salmonellosis in China using literature review method. Chinese J Disease Control Prevention. 2011; 15(7): 622-5.
Abdollahi A, Najafipour S, Kouhpayeh SA, Meshkibaf MH, Naghdi M. Salmonella enterica: Serotyping, Drug Resistance & Extended Spectrum of β-Lactamase (ESBLs). J Fasa Univ Med Sci. 2011; 1(1): 38-44.
Hur J, Choi YY, Park JH, Jeon BW, Lee HS, Kim AR, et al. Antimicrobial resistance, virulence-associated genes, and pulsed-field gel electrophoresis profiles of Salmonella enterica subsp. enterica serovar Typhimurium isolated from piglets with diarrhea in Korea. Can J Vet Res. 2011; 75(1): 49-56.
Glynn MK, Bopp C, Dewitt W, Dabney P, Mokhtar M, Angulo FJ. Emergence of Multidrug-Resistant Salmonella enterica Serotype Typhimurium DT104 Infections in the United States. N Engl J Med. 1998; 338(19): 1333-9.
Yang SJ, Park KY, Kim SH, No KM, Besser TE, Yoo HS, et al. Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: comparison of phenotypic and genotypic resistance characterization. Vet Microbiol.2002; 86(4): 295-301.
Wayne PA. Clinical and Laboratory Standards Institute: Performance standards for antimicrobial susceptibility testing: 20th informational supplement. CLSI document M100-S20. 2010.
Guerra B, Laconcha I, Soto SM, González-Hevia MÁ, Mendoza MC. Molecular characterisation of emergent multiresistant Salmonella enterica serotype [4, 5, 12: i:−] organisms causing human salmonellosis. FEMS Microbiol Lett.2000; 190(2): 341-7.
Switt AIM, den Bakker HC, Cummings CA, Rodriguez-Rivera LD, Govoni G, Raneiri ML, et al. Identification and characterization of novel Salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission. PLoS One. 2012; 7(7): e41247.
Arjmand-Asl M, Amini K. Molecular identification of virulence genes in clinical Salmonella typhimurium strains using the multiplex-PCR method and their antibiotic resistance profile. Feyz. 2016; 20(4): 376-82. [In Persian]
Aminia K, Nazarib Z, Mokhtaric A. Molecular detection of inv A and spv virulence genes in Salmonella typhimurium isolated from human and animals in Iran. Sci J Microbiol. 2015; 4(7) 49-57.
Eshraghi S, Dalall MM, Fardsanei F, Salehi TZ, Ranjbar R, Nikmanesh B, et al. Salmonella enteritidis and antibiotic resistance patterns: a study on 1950 children with diarrhea. Tehran Univ Med J.2010; 67(12).
Chu C, Chiu CH, Wu WY, Chu CH, Liu TP, Ou JT. Large drug resistance virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis. Antimicrob Agents Chemother. 2001; 45(8): 2299-303.
Herrero A, Mendoza M, Threlfall EJ, Rodicio M. Detection of Salmonella enterica serovar Typhimurium with pUO-StVR2-like virulence-resistance hybrid plasmids in the United Kingdom. Eur J Clin Microbiol Infect Dis.2009; 28(9): 1087-93.
Madadgar O, Salehi TZ, Tadjbakhsh H, Mahzounieh M, Feizabadi M. Genomic and phenotypic evaluation of Salmonella typhimurium and Salmonella enteritidis in Iran. Comp Clin Pathol. 2008; 17(4): 229-35.
Soto SM, Rodríguez I, Rodicio MR, Vila J, Mendoza MC. Detection of virulence determinants in clinical strains of Salmonella enterica serovar Enteritidis and mapping on macrorestriction profiles. J Med Microbiol. 2006; 55(4): 365-73.
Pan TM, Liu YJ. Identification of Salmonella enteritidis isolates by polymerase chain reaction and multiplex polymerase chain reaction. J Microbiol Immunol Infect.2002; 35(3): 147-51.
_||_Rowlands RE, Ristori CA, Ikuno AA, Barbosa ML, Jakabi M, Franco BD. Prevalence of drug resistance and virulence features in Salmonella spp. isolated from foods associated or not with salmonellosis in Brazil. Rev Inst Med Trop Sao Paulo. 2014; 56(6): 461-7.
Ktsoyan Z, Ghazaryan K, Manukyan G, Martirosyan A, Mnatsakanyan A, Arakelova K, et al. Inflammatory responses to Salmonella infections are serotype-specific. Int J Bacteriol. 2013; 2013: 168-79.
Amini K, Salehi TZ, Nikbakht G, Ranjbar R, Amini J, Ashrafganjooei SB. Molecular detection of invA and spv virulence genes in Salmonella enteritidis isolated from human and animals in Iran. Afr J Microbiol Res. 2010; 4(21): 2202-10.
Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O'brien SJ, et al. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis. 2010; 50(6): 882-9.
Ke B, Ran L, Wu S, Deng X, Ke C, Feng Z, et al. Survey of physician diagnostic and treatment practices for patients with acute diarrhea in Guangdong province, China. Foodborne Pathog Dis. 2012; 9(1): 47-53.
Mao X, Hu J, Liu X. Estimation on disease burden of foodborne non-typhoid salmonellosis in China using literature review method. Chinese J Disease Control Prevention. 2011; 15(7): 622-5.
Abdollahi A, Najafipour S, Kouhpayeh SA, Meshkibaf MH, Naghdi M. Salmonella enterica: Serotyping, Drug Resistance & Extended Spectrum of β-Lactamase (ESBLs). J Fasa Univ Med Sci. 2011; 1(1): 38-44.
Hur J, Choi YY, Park JH, Jeon BW, Lee HS, Kim AR, et al. Antimicrobial resistance, virulence-associated genes, and pulsed-field gel electrophoresis profiles of Salmonella enterica subsp. enterica serovar Typhimurium isolated from piglets with diarrhea in Korea. Can J Vet Res. 2011; 75(1): 49-56.
Glynn MK, Bopp C, Dewitt W, Dabney P, Mokhtar M, Angulo FJ. Emergence of Multidrug-Resistant Salmonella enterica Serotype Typhimurium DT104 Infections in the United States. N Engl J Med. 1998; 338(19): 1333-9.
Yang SJ, Park KY, Kim SH, No KM, Besser TE, Yoo HS, et al. Antimicrobial resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from animals in Korea: comparison of phenotypic and genotypic resistance characterization. Vet Microbiol.2002; 86(4): 295-301.
Wayne PA. Clinical and Laboratory Standards Institute: Performance standards for antimicrobial susceptibility testing: 20th informational supplement. CLSI document M100-S20. 2010.
Guerra B, Laconcha I, Soto SM, González-Hevia MÁ, Mendoza MC. Molecular characterisation of emergent multiresistant Salmonella enterica serotype [4, 5, 12: i:−] organisms causing human salmonellosis. FEMS Microbiol Lett.2000; 190(2): 341-7.
Switt AIM, den Bakker HC, Cummings CA, Rodriguez-Rivera LD, Govoni G, Raneiri ML, et al. Identification and characterization of novel Salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission. PLoS One. 2012; 7(7): e41247.
Arjmand-Asl M, Amini K. Molecular identification of virulence genes in clinical Salmonella typhimurium strains using the multiplex-PCR method and their antibiotic resistance profile. Feyz. 2016; 20(4): 376-82. [In Persian]
Aminia K, Nazarib Z, Mokhtaric A. Molecular detection of inv A and spv virulence genes in Salmonella typhimurium isolated from human and animals in Iran. Sci J Microbiol. 2015; 4(7) 49-57.
Eshraghi S, Dalall MM, Fardsanei F, Salehi TZ, Ranjbar R, Nikmanesh B, et al. Salmonella enteritidis and antibiotic resistance patterns: a study on 1950 children with diarrhea. Tehran Univ Med J.2010; 67(12).
Chu C, Chiu CH, Wu WY, Chu CH, Liu TP, Ou JT. Large drug resistance virulence plasmids of clinical isolates of Salmonella enterica serovar Choleraesuis. Antimicrob Agents Chemother. 2001; 45(8): 2299-303.
Herrero A, Mendoza M, Threlfall EJ, Rodicio M. Detection of Salmonella enterica serovar Typhimurium with pUO-StVR2-like virulence-resistance hybrid plasmids in the United Kingdom. Eur J Clin Microbiol Infect Dis.2009; 28(9): 1087-93.
Madadgar O, Salehi TZ, Tadjbakhsh H, Mahzounieh M, Feizabadi M. Genomic and phenotypic evaluation of Salmonella typhimurium and Salmonella enteritidis in Iran. Comp Clin Pathol. 2008; 17(4): 229-35.
Soto SM, Rodríguez I, Rodicio MR, Vila J, Mendoza MC. Detection of virulence determinants in clinical strains of Salmonella enterica serovar Enteritidis and mapping on macrorestriction profiles. J Med Microbiol. 2006; 55(4): 365-73.
Pan TM, Liu YJ. Identification of Salmonella enteritidis isolates by polymerase chain reaction and multiplex polymerase chain reaction. J Microbiol Immunol Infect.2002; 35(3): 147-51.