Subtyping β -lactamase-producing Escherichia coli strains isolated from patients with UTI by MLVA and PFGE methods

Document Type : Original Article


1 Department of Microbiology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran

2 Department of Microbiology, Research Center of Reference Health Laboratory, Ministry of Health and Medical Education, Tehran, Iran

3 Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran


Objective(s): Strain subtyping is an important epidemiological tool to trace contamination, determine clonal relationships between different strains, and the cause of outbreaks. Current subtyping methods, however, yield less than optimal subtype discrimination. Pulsed-field gel electrophoresis is the gold standard method for Escherichia coli and Multiple-Locus Variable-number tandem repeat Analysis is a rapid PCR-based method. The purpose of this study was to evaluate MLVA and PFGE methods for subtyping β -lactamase-producing E. coli strains isolated from urinary tract infections.
Materials and Methods: Overall, 230 E. coli isolates from patients with urinary tract infections were examined for antimicrobial susceptibility testing.   10-loci and 7-loci MLVA and PFGE methods were used for molecular typing of β -lactamase-producing E. coli 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 MLVA, respectively.  The discriminatory power of PFGE showed a value of 0.87. The discordance between the methods was high.
Conclusion: Our study showed that PFGE is more discriminatory than MVLA.  MLVA is a PCR- based method and can generate unmistakable data, in contrast to PFGE. Optimization of polymorphic VNTR is essential to improve the discriminatory power of MLVA based on geographical region.


1. Córdoba G, Holm A, Hansen F, Hammerum AM, Bjerrum L. Prevalence of antimicrobial resistant Escherichia coli from patients with suspected urinary tract infection in primary care, Denmark. BMC infectious diseases 2017; 17:670-676.
2. Bergeron CR, Prussing C, Boerlin P, Daignault D, Dutil L, Reid-Smith RJ, et al. Chicken as reservoir for extraintestinal pathogenic Escherichia coli in humans, Canada. Emerging infectious diseases. 2012; 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, Martínez-Martínez L, Muniain MA, Perea EJ, et al. 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, Gerner-Smidt P. Recent developments and future prospects in subtyping of foodborne bacterial pathogens. Future Microbiol 2007; 2:175-185.
6. Malini A. Bhat SS, Kowsalya R., Gautam 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;6:1203-1206.
7. Bhattacharjee A SMR, 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; 26:356-360.
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, Yeiser B, Bonomo MD, Rice LB, 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; 47:3554-3560.
10. Lee K, Izumiya H, Iyoda S, Ohnishi M. Effective surveillance using multilocus variable-number tandem-repeat analysis and whole-genome sequencing for enterohemorrhagic Escherichia coli O157. Appl Environ Microbiol 2019; 85: e00728-19
11. 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.
12. Brisse S, Brehony C, Conceição T, Cubero M, Glasner C, Le Gouil M, et al. Microbial molecular markers and epidemiological surveillance in the era of high throughput sequencing: an update from the IMMEM-10 conference. Res  Microbiol. 2014; 165:140-153.
13. Karama M, Mainga AO, Cenci-Goga BT, Malahlela M, El-Ashram S, Kalake A. Molecular profiling and antimicrobial resistance of Shiga toxin-producing Escherichia coli O26, O45, O103, O121, O145 and O157 isolates from cattle on cow-calf operations in South Africa. Scientific Reports 2019;9:11930.
14. Neoh HM, Tan XE, Sapri HF, Tan TL. Pulsed-field gel electrophoresis (PFGE): A review of the “gold standard” for bacteria typing and current alternatives. Infect Genet Evol 2019; 74:103935.
15. Smith AM. Review of molecular subtyping methodologies used to investigate outbreaks due to multidrug-resistant enteric bacterial pathogens in sub-Saharan Africa.  Afr J lab Med 2019;8:760-783.
16. Memariani M, Najar-Peerayeh S, Zahraei Salehi T. Multi locus vntR (MLVA) typing and detection of the OI-122 pathogenicity island in typical and atypical enteropathogenic escherichia coli isolated from children with acute diarrhea. Arch Clin Infect Dis 2019;14:e65855.
17. Nadon CA, Trees E, Ng LK, Møller Nielsen E, Reimer A, Maxwell N, et al. Development and application of MLVA methods as a tool for inter-laboratory surveillance. Euro Surveill 2013;18:20565.
18. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. CLSI supplement M100. S29 ed2019. 33-40 p.
19. 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.
20. Løbersli I, Haugum K, Lindstedt BA. Rapid and high resolution genotyping of all Escherichia coli serotypes using 10 genomic repeat-containing loci.  J Microbial Methods 2012; 88:134-139.
21. International P. Standard operating procedure for pulsenet PFGE of Escherichia coli O157:H7, Escherichia coli non-O157 (STEC), Salmonella serotypes, Shigella sonnei and Shigella flexeneri 2013 [Available from:
22. Babenko D. Comparison of four genotyping methods for Pseudomonas aeruginosa: In silico study. Georgian Med News 2016:98-103.
23. Estaji M, Tabasi M, Sadeghpour Heravi F, Kheirvari Khezerloo J, Radmanesh A, Raheb J, et al. Genotypic identification of Pseudomonas aeruginosa strains isolated from patients with urinary tract infection. Comp Immunol Microbiol Infect Dis 2019;65:23-28.
24. Farajzadeh-Sheikh A, Savari M, Ahmadi K, Hosseini Nave H, Shahin M, Afzali M. Distribution of genes encoding virulence factors and the genetic diversity of enteroinvasive Escherichia coli (EIEC) Isolates from patients with diarrhea in Ahvaz, Iran. Infect Drug Resist 2020; 13:119-127.
25. Babenko D, Turmuhambetova A, Sandle T, Pestrea SA, Moraru D, Chesca A. In silico comparison of different types of MLVA with PFGE based on Pseudomonas aeruginosa genomes. Acta Medica Mediterranea 2017;33 :607-612.
26. Rumore JL, Tschetter L, Nadon C. The impact of multilocus variable-number tandem-repeat analysis on pulseNet Canada Escherichia coli O157:H7 laboratory surveillance and outbreak support, 2008-2012. Foodborne Pathog Dis 2016;13:255-261.
27. Sobral D, Mariani-Kurkdjian P, Bingen E, Vu-Thien H, Hormigos K, Lebeau B, et al. A  new highly discriminatory multiplex capillary-based MLVA assay as a tool for the epidemiological survey of Pseudomonas aeruginosa in cystic fibrosis patients. Eur J Clin Microbiol Infect Dis. 2012; 31:2247-56.
28. Helldal L, Karami N, Florén K, Welinder-Olsson C, Moore ER, Åhrén C. Shift of CTX-M genotypes has determined the increased prevalence of extended-spectrum β-lactamase-producing Escherichia coli in south-western Sweden. Clin Microbiol Infect 2013;19:E87-90.
29. Nicolas-Chanoine MH, Bertrand X, Madec JY. Escherichia coli ST131, an intriguing clonal group. Clin Microbiol Rev 2014;27:543-574.
30. Cantón R, Novais A, Valverde A, Machado E, Peixe L, Baquero F, et al. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe. Clin Microbiol Infect 2008;14 Suppl 1:144-153.
31. Karami N HL, 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.
32. Naseer U, Olsson-Liljequist BE, Woodford N, Dhanji H, Cantón R, Sundsfjord A, et al. 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.
33. Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, et al. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet.  Foodborne Pathog Dis 2006;3:59-67.
34. Raimondi S, Righini L, Candeliere F, Musmeci E, Bonvicini F, Gentilomi G, et al. Antibiotic resistance, virulence factors, phenotyping, and genotyping of Escherichia coli isolated from the feces of healthy subjects.  Microorganisms 2019;7:251.
35. Caméléna F, Birgy A, Smail Y, Courroux C, Mariani-Kurkdjian P, Le Hello S, et al. Rapid and simple universal Escherichia coli genotyping method based on multiple-locus variable-number tandem-repeat analysis using single-tube multiplex PCR and standard gel electrophoresis. Appl Environ Microbiol 2019;85:e02812-2818.
36. Dudek B, Książczyk M, Krzyżewska E, Rogala K, Kuczkowski M, Woźniak-Biel A, et al. Comparison of the phylogenetic analysis of PFGE profiles and the characteristic of virulence genes in clinical and reptile associated Salmonella strains. BMC Vet Res 2019; 15:312.
37. Gupta G, Tak V, Mathur P. Detection of AmpC β lactamases in Gram-negative bacteria. J Lab Physicians 2014;6:1-6.
38. Dolatyar Dehkharghani A, Haghighat S; Rahnamaye Farzami M, Rahbar M. The mechanism of resistance in AmpC-producing Escherichia coli isolated from urinary tract infections.  Arch Med Lab Sci 2020; 6:1-9.
39. Kim J, Hyeon J-Y, Lee E, Lee D, Kim Y-J, Kim Y-J, et al. Molecular epidemiological analysis of five outbreaks associated with Salmonella enterica serovar Enteritidis between 2008 and 2010 on Jeju Island, Republic of Korea. Food borne Pathog Dis. 2014; 11:38-42.
40. Boxrud D, Pederson-Gulrud K, Wotton J, Medus C, Lyszkowicz E, Besser J, et al. 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.
41. Bustamante AVS, Mariel A. Multiple-locus variable-number of tandem-repeats analysis (MLVA) as subtyping technique for  foodborne pathogens. Handbook of Food Bioengineering. Chapter 14.2018; 10:423-442.
42. Omid F, Reza R, Sahar HJ, Bahareh A. Multilocus variable-number tandem-repeat Analysis for genotyping of Escherichia coli strains isolated from hospital wastewater. Iran J Public Health 2020;49:2409-2417.
43. Onteniente L, Brisse S, Tassios PT, Vergnaud G. Evaluation of the polymorphisms associated with tandem repeats for Pseudomonas aeruginosa strain typing.  J Clin Microbiol 2003;41:4991-4997.
44. Nakamura H, Iguchi A, Maehara T, Fujiwara K, Fujiwara A, Ogasawara J. Comparison of three molecular subtyping methods among O157 and non-O157 shiga toxin-producing Escherichia coli isolates from Japanese cattle.  Jpn J Infect Dis 2018;71:45-50.
45. Terletskiy VP, Novikova OB, Tyshchenko VI, Shinkarenko LA. In-silico  selection  of  enzymes  for  strain genotyping  of bacteria  belonging   to  Campylobacter  Genus   isolated from birds  J Agric Environ 2019;3: 23649.
46. Ferrari RG, Panzenhagen PHN, Conte-Junior CA. Phenotypic and Genotypic eligible methods for Salmonella typhimurium source tracking. Front Microbiol 2017;8:2587.
47. Najar-Peerayeh S, Navidinia M, Fallah F, Bakhshi B, Alebouyeh M. Evaluation of clonal relatedness among different sources of Escherichia coli isolates in Iranian children with urinary tract infection (UTI) and age-matched healthy people. Biomed Res 2019;30:229-237.  
48. Zheng J, Keys CE, Zhao S, Ahmed R, Meng J, Brown EW.   Simultaneous analysis of multiple enzymes increases accuracy of pulsed-field gel electrophoresis in assigning genetic relationships among homogeneous Salmonella strains. J Clin Microbiol 2011;49:85 -94.
49. Tang S, Orsi RH, Luo H, Ge C, Zhang G, Baker RC, et al. Assessment and comparison of molecular subtyping and characterization methods for Salmonella. Front Microbiol 2019;10:1591.
50. 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;275:16-23.
51. Ziebell K, Chui L, King R, Johnson S, Boerlin P, Johnson RP. Subtyping of Canadian isolates of Salmonella enteritidis using multiple locus variable number tandem repeat analysis (MLVA) alone and in combination with pulsed-field gel electrophoresis (PFGE) and phage typing. J Microbiol Methods 2017; 139:29-36.