Original Article

Multilocus Variable-Number Tandem-Repeat Analysis for Genotyping of Escherichia coli Strains Isolated from Hospital Wastewate


Background: Escherichia coli is one of the most frequent causes of many common bacterial infections. As a potential reservoir, hospital wastewater is considered for the dissemination of bacterial pathogens such as E. coli. Therefore, research on hospital waste’s bacteria by low-cost, rapid and easy molecular typing methods such as multilocus variable-number tandem-repeat analysis (MLVA) can be helpful for the study of epidemics.

Methods: E. coli strains were isolated from hospital wastewater sources in Tehran, Iran, over a 24-month sampling period (Jun 2014- Jun 2016) and identified by standard bacteriological methods. The diversity of repeated sequences of seven variable-number tandem-repeat (VNTR) loci was studied by MLVA method base on polymerase chain reaction (PCR).

Results: Overall, 80 E. coli isolates were discriminated into 51 different genotypes. Analysis of the MLVA profiles using a minimum spanning tree (MST) algorithm showed two clonal complexes with 71 isolates and only nine isolates were stayed out of clonal complexes in the form of a singleton. High genotypic diversity was seen among E. coli strains isolated from hospital wastewaters; however, a large number of isolates showed a close genetic relationship.

Conclusion: MLVA showed to be a rapid, inexpensive and useful tool for the analysis of the phylogenetic relationships between E. coli strains under the study.

1. Moges F, Endris M, Belyhun Y, Worku W (2014). Isolation and characterization of multiple drug resistance bacterial pathogens from waste water in hospital and non-hospital environments, Northwest Ethiopia. BMC Res Notes, 7:215.
2. McLellan SL (2004). Genetic diversity of Escherichia coli isolated from urban rivers and beach water. Appl Environ Microbiol, 70:4658-4665.
3. Yang C, Lin M, Liao P, et al (2009). Comparison of antimicrobial resistance patterns between clinical and sewage isolates in a regional hospital in Taiwan. Lett Appl Microbiol, 48:560-565.
4. Ranjbar R, Masoudimanesh M, Dehkordi FS, et al (2017). Shiga (Vero)-toxin producing Escherichia coli isolated from the hospital foods; virulence factors, o-serogroups and antimicrobial resistance properties. Antimicrob Resist Infect Control, 6:4.
5. Anvarinejad M, Farshad S, Ranjbar R, et al (2012). Genotypic analysis of E. coli strains isolated from patients with cystitis and pyelonephritis. Iran Red Crescent Med J, 14(7):408-16.
6. Timmons C, Trees E, Ribot EM, et al (2016). Multiple-locus variable-number tandem repeat analysis for strain discrimination of non-O157 Shiga toxin-producing Escherichia coli. J Microbiol Methods, 125:70-80.
7. Trabulsi LR, Keller R, Gomes TAT (2002). 10.321/eid0805.Typical and Atypical Enteropathogenic Escherichia coli. Emerg Infect Dis, 8(5):508-13.
8. Giuffrè M, Geraci DM, Bonura C, et al (2016). The increasing challenge of multidrug-resistant Gram-negative bacilli: results of a 5-year active surveillance program in a neonatal intensive care unit. Medicine (Baltimore), 95(10):e3016.
9. Sabaté M, Prats G, Moreno E, et al (2008). Virulence and antimicrobial resistance profiles among Escherichia coli strains isolated from human and animal wastewater. Res Microbiol, 159:288-293.
10. Momtaz H, Karimian A, Madani M, et al (2013). Uropathogenic Escherichia coli in Iran: serogroup distributions, virulence factors and antimicrobial resistance properties. Ann Clin Microbiol Antimicrob, 12:8.
11. Miquel S, Peyretaillade E, Claret L, et al (2010). Complete genome sequence of Crohn's disease-associated adherent-invasive E. coli strain LF82. PLoS One, 5:e12714.
12. Sadeghifard N, Ranjbar R, Zaeimi J, et al (2010). Antimicrobial susceptibility, plasmid profiles, and RAPD-PCR typing of Acinetobacter bacteria. Asian biomedicine, 4(6):901-911.
13. Ranjbar R, Memariani M, Memariani H (2015). Diversity of variable number tandem repeat loci in Shigella species isolated from pediatric patients. Int J Mol Cell Med, 4(3):174-81.
14. Filliol-Toutain I, Chiou CS, Mammina C, et al. (2011). Global distribution of Shigella sonneiclones. Emerg Infect Dis, 17: 1910-1912.
15. Malachowa N, Sabat A, Gniadkowski M, et al (2005). Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates. J Clin Microbiol, 43:3095-3100.
16. Behzadi P, Najafi A, Behzadi E, et al (2016). Microarray long oligo probe designing for Escherichia coli: An in-silico DNA marker extraction. Cent European J Urol, 69(1): 105-11.
17. Association APH, Association AWW, Federation WPC, Federation WE (1915). Standard methods for the examination of water and wastewater. ed. American Public Health Association.
18. Memariani M, Peerayeh SN, Salehi TZ, Mostafavi SKS (2015). Occurrence of SHV, TEM and CTX-M β-lactamase genes among enteropathogenic Escherichia coli strains isolated from children with diarrhea. Jundishapur J Microbiol, 8(4):e15620.
19. Gorgé O, Lopez S, Hilaire V, et al (2008). Selection and validation of a multilocus variable-number tandem-repeat analysis panel for typing Shigella spp. J Clin Microbiol, 46:1026-1036.
20. Weniger T, Krawczyk J, Supply P, et al (2010). MIRU-VNTR plus: a web tool for polyphasic genotyping of Mycobacterium tuberculosis complex bacteria. Nucleic Acids Res, 38(Web Server issue):W326-31.
21. Ranjbar R, Memariani M (2015). Multilocus variable-number tandem-repeat analysis for genotyping of Shigella sonnei strains isolated from pediatric patients. Gastroenterol Hepatol Bed Bench, 8(3):225-32.
22. Yang Y, Wang Y, Poulsen E, et al (2017). Genotyping Brucella canis isolates using a highly discriminatory multilocus variable-number tandem-repeat analysis (MLVA) assay. Sci Rep, 7(1):1067.
23. Menshawy A, Perez-Sancho M, Garcia-Seco T, et al (2014). Assessment of genetic diversity of zoonotic Brucella spp. recovered from livestock in Egypt using multiple locus VNTR analysis. BioMed Res Int, 2014: 353876.
24. Wang H, Diao B, Cui Z, Yan M, Kan B (2016). Genotyping of Salmonella typhi using 8-loci multi locus VNTR analysis. Gut Pathog, 8:14.
25. Ghaderi R, Tadayon K, Avagyan S, et al (2013). The population structure of Salmonella enterica Enteritidis in Iran analyzed by multiple-locus variable-number tandem repeat analysis. Trop Anim Health Prod, 45:889-894.
26. Santos AS, Tilburg JJ, Botelho A, et al (2012). Genotypic diversity of clinical Coxiella burnetii isolates from Portugal based on MST and MLVA typing. Int J Med Microbiol, 302:253-256.
27. Swirski A, Pearl D, Williams M, et al (2014). Spatial epidemiology of Escherichia coli O157: H7 in dairy cattle in relation to night roosts of Sturnus vulgaris (European starling) in Ohio, USA (2007–2009). Zoonoses Public Health, 61:427-435.
28. Byrne L, Elson R, Dallman TJ, et al (2014). Evaluating the use of multilocus variable number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 as a routine public health tool in England. PLoS One, 9:e85901.
29. Perry N, Cheasty T, Dallman T, et al (2013). Application of multilocus variable number tandem repeat analysis to monitor Verocytotoxin‐producing Escherichia coli O157 phage type 8 in England and Wales: emergence of a profile associated with a national outbreak. J Appl Microbiol, 115:1052-1058.
30. Dyet K, Robertson I, Turbitt E, Carter P (2011). Characterization of Escherichia coli O157: H7 in New Zealand using multiple-locus variable-number tandem-repeat analysis. Epidemiol Infect, 139:464-471.
31. Nave HH, Mansouri S, Moghadam MT, Moradi M (2016). Virulence Gene Profile and Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) of Enteroinvasive Escherichia coli (EIEC) Isolates From Patients With Diarrhea in Kerman, Iran. Jundishapur J Microbiol, 9(6):e33529.
32. Nadon C, Trees E, Ng L, et al (2013). Development and application of MLVA methods as a tool for inter-laboratory surveillance. Euro Surveill, 18:20565.
33. Sekse C, Sunde M, Lindstedt B, et al (2011). Potentially human pathogenic Escherichia coli O26 in Norwegian sheep flocks. Appl Environ Microbiol, 77(14):4949-58.
IssueVol 49 No 12 (2020) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijph.v49i12.4829
Escherichia coli Multilocus variable number tandem repeat analysis Hospital wastewater Genotyping Phylogenetic relationships

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How to Cite
FARAHANI O, RANJBAR R, HONARMAND JAHROMY S, ARABZADEH B. Multilocus Variable-Number Tandem-Repeat Analysis for Genotyping of Escherichia coli Strains Isolated from Hospital Wastewate. Iran J Public Health. 2020;49(12):2409-2417.