تایپینگ مولکولی اشریشیاکلی تولید کننده بتالاکتاماز: مقایسهMLVA و PFGE
محورهای موضوعی : میکروب شناسی مولکولی
علیرضا دولت یار دهخوارقانی
1
,
محمد رهبر
2
,
ستاره حقیقت
3
,
مرجان رهنمای فرزامی
4
1 - گروه میکروب شناسی، دانشکده فناوری های نوین، واحد علوم پزشکی تهران دانشگاه آزاد اسلامی
2 - ریاست بخش میکروب شناسی
3 - عضو هیئت علمی دانشگاه آزاد اسلامی واحد تهران پزشکی-دانشکده فناوری های نوین-گروه میکروب شناسی
4 - ریاست آزمایشگاه رفرانس ایران
کلید واژه: تنوع ژنتیکی, اشریشیا کلی, تولید کننده بتالاکتاماز, ویروتایپینگ,
چکیده مقاله :
سابقه و هدف: اشریشیا کلی تولید کننده بتالاکتاماز مهمترین عامل ایجاد کننده عفونت‌های دستگاه ادراری در جامعه و بیمارستان‌ها است. روش‌های تعیین تایپ سویه‌ها مانند MLVA و PFGE معمول‌ترین ابزار اپیدمیولوژیک نه تنها برای تشخیص انتقال متقاطع پاتوژن‌های بیمارستانی بلکه برای تعیین منبع عفونت می‌باشند.مواد و روشها: این پژوهش به به منظور ارزیابی قدرت تمیز روش‌های MLVA و PFGE انجام شد. در مجموع 230 جدایه اشریشیا کلی بدست آمده از بیماران مبتلا به عفونت دستگاه ادراری از نظر حساسیت ضد میکروبی آزمایش و به منظور مقایسه MLVA و PFGE برای تایپ مولکولی جدایه‌ها مورد بررسی قرار گرفتند.یافته ها: از 230 جدایه، 130 جدایه اشریشیا کلی تولید کننده بتالاکتاماز (56/5%) در این مطالعه یافت شد. شاخص تنوع لوکوس‌های VNTR به ترتیب برای روش 7 و 10 لوکوسی 0/48 و 0/54 تعیین شدند. قدرت تمیز PFGE 0/87 محاسبه گردید.نتیجه گیری: نتایج نشان داد که قدرت تمیز PFGE بیشتر از MVLA است. MLVA روشی مبتنی بر PCR است و برخلاف PFGE، داده‌های غیرقابل اشتباه تولید می‌کند. بهینه سازی VNTR پلی مورفیک برای بهبود قدرت تمیز MLVA در هر منطقه جغرافیایی ضروری است.
Background & Objectives: β -lactamase -producing E. coli is the most important agent causing urinary tract infections both in community and hospitals. Strain typing including MLVA and PFGE are the most common epidemiologically tools not only for detecting the cross transmission of nosocomial pathogens but also for determining the source of infection.Materials and methods: The present study was carried of for comparison of discriminatory power of MLVA and PFGE methods. A Total of 230 isolates of E. coli from patients with urinary tract infections were examined for identifying and antimicrobial susceptibility testing MLVA and PFGE methods were used for molecular typing of all isolates.Results: Out of 230 isolates, 130 ( 56.5%) β -lactamase -producing E. coli isolates were found in this study. The diversity indices of the VNTR loci showed an average diversity of 0.48 and 0.54 for 7-loci and 10-loci respectively. The discriminatory power of PFGE showed a value of 0.87. Conclusion: Our study showed that PFGE is more discriminatory than MVLA. MLVA is a PCR- based method and generates unmistakable data, in contrast to PFGE. Optimization of polymorphic VNTR is essential to improve the discriminatory power of MLVA basis on geographical region.
costs. Am. J. Med. 2002; 113: a5-13.
2. Bergeron RC, Prussing C, Boerlin P P, Daignault D, Dutil L,Reid-Smith RJ, Zhanel
GG,Manges AR. Chicken as Reservoir for Extraintestinal Pathogenic Escherichia coli in
Humans, Canada; Emerg. Infect. Dis. 2013; 18: 415-421.
3. Al-Badr A, Al-Shaikh G. Recurrent Urinary Tract Infections Management in Women: A
review. Sultan Qaboos Univ Med J. 2013; 13:359‐367.
4. Rodríguez-Baño J, Navarro MD, Romero L, Luis Martínez-Martínez L, Muniain MA,Perea EJ,
Pérez-Cano R, Pascual A. Epidemiology and clinical features of infections caused by
extended-spectrum beta-lactamase-producing Escherichia coli in nonhospitalized patients. J
Clin Microbiol. 2004; 42:1089‐1094.
5. Hyytiä-Trees EK, Cooper K, Ribot EM, et al.. Recent developments and future prospects in
subtyping of foodborne bacterial pathogens.; Future Microbiol. 2007; 2: 175-185.
6. M. A. Bhat, S. Sageerabanoo, R. Kowsalya, and G. Sarkar. The occurrence of CTX-M3 type
extended spectrum beta lactamases among Escherichia coli causing urinary tract infections in a
tertiary care hospital in puducherry. J Clin Diagn Res. 2012; 6.7 : 1203-1206.
7. Bhattacharjee A, Sen M R, Prakash P, Gaur A, Anupurba S. Increased prevalence of extended
spectrum β lactamase producers in neonatal septicaemic cases at a tertiary referral hospital.
Indian J Med Microbiol. 2008; 26:356-60.
8. Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization,
epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001;
14:933–951.
9. Paterson DL, Hujer KM, Hujer AM, et al.. Extended-spectrum beta-lactamases in Klebsiella
pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type beta-lactamases. Antimicrob Agents Chemother. 2003; 47
(11):3554–3560.
10. Foley SL, Lynne AM, Nayak R. Molecular typing methodologies for microbial source
tracking and epidemiological investigations of Gram-negative bacterial foodborne pathogens.
Infect. Genet. Evol. 2009; 9: 430-440.
11. Lindstedt BA, Brandal LT, Aas L, Vardund T, Kapperud G. Study of polymorphic
variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic
Escherichia coli and Shigella isolates for use in a genotyping assay. J Microbiol Methods.
2007; 69: 197-205.
12. Løbersli I, Haugum K, Lindstedt BA. Rapid and high resolution genotyping of all
Escherichia coli serotypes using 10 genomic repeat-containing loc. J Microbiol Methods.
2012; 88: 134-139.
13. www.Pulsnet International.com
14. Helldal L, Karami N, Florén K, Welinder-Olsson C, Moore ERB, Åhrén C.. Shift of CTX-M
genotypes has determined the increased prevalence of extendedspectrum
β-lactamase-producing Escherichia coli in south-western Sweden. Clin. Microbiol. Infect.
2013; 19: E87-90.
15. Nicolas-Chanoine MH, Bertrand X, Madec JY. Escherichia coli st131, an intriguing clonal
group. Clin. Microbiol. Rev. 2014; 27: 543-574.
16. Cantón R, Novais A, Valverde A, Machado E, Peixe L, Baquero F, et al.; 2008; Prevalence
and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe. Clin.
Microbiol. Infect. 14: 144-153.
17. Karami N, Helldal L, Welinder-Olsson C, Åhrén C, Moore ERB. Sub-typing of
extended-spectrum-β-lactamase-producing isolates from a nosocomial outbreak: Application
of a 10-loci generic Escherichia coli multi-locus variable number tandem repeat analysis.
PLOS One. 2013; 8: e83030.
18. Naseer U, Olsson-Liljequist BE, Woodford N, Dhanji H, Cantón R, Sundsfjord. Multi-locus
variable number of tandem repeat analysis for rapid and accurate typing of virulent multidrug
resistant Escherichia coli clones. PLOS One. 2012; 7: e41232.
19. Ribot, E. M., M. A. Fair, R. Gautom, D. N. Cameron, S. B andHunter, B. Swaminathan, and
T. J. Barrett. Standardization of pulsedfield gel electrophoresis protocols for the subtyping of
Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis.
2006; 3: 59-67.
20. P.Wayne. Performance standards for antimicrobial susceptibility. CLSI Document M100-S29,
Clinical and Laboratory Standards Institute. 2019.
21. Kim J, Hyeon JY, Lee E, Lee D, Kim YJ, Kim S. Molecular epidemiological analysis of five
outbreaks associated with Salmonella enterica serovar Enteritidis between 2008 and 2010 on
Jeju Island, Republic of Korea. Foodborne Pathog Dis. 2014; 11: 38-42.
22. Boxrud D, Pederson-Gulrud K, Wotton J, Medus C, Lyszkowicz E, Besser J. Comparison of
multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and
phage typing for subtype analysis of Salmonella enterica serotype Enteritidis. J. Clin.
Microbiol. 2007; 45: 536-543.
23. Cho S, Boxrud DJ, Bartkus JM, Whittam TS, Saeed M. Multiple-locus variable-number
tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources
using a single multiplex PCR. FEMS Microbiol. Lett. 2007; 275 (1): 16-23.
_||_
costs. Am. J. Med. 2002; 113: a5-13.
2. Bergeron RC, Prussing C, Boerlin P P, Daignault D, Dutil L,Reid-Smith RJ, Zhanel
GG,Manges AR. Chicken as Reservoir for Extraintestinal Pathogenic Escherichia coli in
Humans, Canada; Emerg. Infect. Dis. 2013; 18: 415-421.
3. Al-Badr A, Al-Shaikh G. Recurrent Urinary Tract Infections Management in Women: A
review. Sultan Qaboos Univ Med J. 2013; 13:359‐367.
4. Rodríguez-Baño J, Navarro MD, Romero L, Luis Martínez-Martínez L, Muniain MA,Perea EJ,
Pérez-Cano R, Pascual A. Epidemiology and clinical features of infections caused by
extended-spectrum beta-lactamase-producing Escherichia coli in nonhospitalized patients. J
Clin Microbiol. 2004; 42:1089‐1094.
5. Hyytiä-Trees EK, Cooper K, Ribot EM, et al.. Recent developments and future prospects in
subtyping of foodborne bacterial pathogens.; Future Microbiol. 2007; 2: 175-185.
6. M. A. Bhat, S. Sageerabanoo, R. Kowsalya, and G. Sarkar. The occurrence of CTX-M3 type
extended spectrum beta lactamases among Escherichia coli causing urinary tract infections in a
tertiary care hospital in puducherry. J Clin Diagn Res. 2012; 6.7 : 1203-1206.
7. Bhattacharjee A, Sen M R, Prakash P, Gaur A, Anupurba S. Increased prevalence of extended
spectrum β lactamase producers in neonatal septicaemic cases at a tertiary referral hospital.
Indian J Med Microbiol. 2008; 26:356-60.
8. Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization,
epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001;
14:933–951.
9. Paterson DL, Hujer KM, Hujer AM, et al.. Extended-spectrum beta-lactamases in Klebsiella
pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type beta-lactamases. Antimicrob Agents Chemother. 2003; 47
(11):3554–3560.
10. Foley SL, Lynne AM, Nayak R. Molecular typing methodologies for microbial source
tracking and epidemiological investigations of Gram-negative bacterial foodborne pathogens.
Infect. Genet. Evol. 2009; 9: 430-440.
11. Lindstedt BA, Brandal LT, Aas L, Vardund T, Kapperud G. Study of polymorphic
variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic
Escherichia coli and Shigella isolates for use in a genotyping assay. J Microbiol Methods.
2007; 69: 197-205.
12. Løbersli I, Haugum K, Lindstedt BA. Rapid and high resolution genotyping of all
Escherichia coli serotypes using 10 genomic repeat-containing loc. J Microbiol Methods.
2012; 88: 134-139.
13. www.Pulsnet International.com
14. Helldal L, Karami N, Florén K, Welinder-Olsson C, Moore ERB, Åhrén C.. Shift of CTX-M
genotypes has determined the increased prevalence of extendedspectrum
β-lactamase-producing Escherichia coli in south-western Sweden. Clin. Microbiol. Infect.
2013; 19: E87-90.
15. Nicolas-Chanoine MH, Bertrand X, Madec JY. Escherichia coli st131, an intriguing clonal
group. Clin. Microbiol. Rev. 2014; 27: 543-574.
16. Cantón R, Novais A, Valverde A, Machado E, Peixe L, Baquero F, et al.; 2008; Prevalence
and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe. Clin.
Microbiol. Infect. 14: 144-153.
17. Karami N, Helldal L, Welinder-Olsson C, Åhrén C, Moore ERB. Sub-typing of
extended-spectrum-β-lactamase-producing isolates from a nosocomial outbreak: Application
of a 10-loci generic Escherichia coli multi-locus variable number tandem repeat analysis.
PLOS One. 2013; 8: e83030.
18. Naseer U, Olsson-Liljequist BE, Woodford N, Dhanji H, Cantón R, Sundsfjord. Multi-locus
variable number of tandem repeat analysis for rapid and accurate typing of virulent multidrug
resistant Escherichia coli clones. PLOS One. 2012; 7: e41232.
19. Ribot, E. M., M. A. Fair, R. Gautom, D. N. Cameron, S. B andHunter, B. Swaminathan, and
T. J. Barrett. Standardization of pulsedfield gel electrophoresis protocols for the subtyping of
Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis.
2006; 3: 59-67.
20. P.Wayne. Performance standards for antimicrobial susceptibility. CLSI Document M100-S29,
Clinical and Laboratory Standards Institute. 2019.
21. Kim J, Hyeon JY, Lee E, Lee D, Kim YJ, Kim S. Molecular epidemiological analysis of five
outbreaks associated with Salmonella enterica serovar Enteritidis between 2008 and 2010 on
Jeju Island, Republic of Korea. Foodborne Pathog Dis. 2014; 11: 38-42.
22. Boxrud D, Pederson-Gulrud K, Wotton J, Medus C, Lyszkowicz E, Besser J. Comparison of
multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and
phage typing for subtype analysis of Salmonella enterica serotype Enteritidis. J. Clin.
Microbiol. 2007; 45: 536-543.
23. Cho S, Boxrud DJ, Bartkus JM, Whittam TS, Saeed M. Multiple-locus variable-number
tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources
using a single multiplex PCR. FEMS Microbiol. Lett. 2007; 275 (1): 16-23.