Iranian Journal of Microbiology 2018. 10(1):30-36.

MLVA typing of Haemophilus influenzae isolated from two Iranian university hospitals
Faranak Nejati, Abolfazl Fateh, Seyed Ali Nojoumi, Mohammad Rahbar, Ava Behrouzi, Farzam Vaziri, Seyed Davar Siadat


Background and Objectives: Different serotypes of Haemophilus influenzae is now divided into 2 divisions: encapsulated and unencapsulated. Multiple locus variable number tandem repeat analysis (MLVA) includes such specifications as the extra power of separation, ease of data interpretation, and epidemiological data accordance, which have made it an appropriate molecular device for good typing and phylogenetic analysis of bacterial pathogens.

Materials and Methods: In this research, cultured samples were studied and strains identified through biochemical tests were recognized. Moreover, DNA was extracted and studied qualitatively and quantitatively. Four pairs of specialized primers related to H. influenzae variable number tandem repeats (VNTR) and preparation of PCR were designed according to the regulated program. Also, electrophoresis of PCR products was performed. Finally, the interpretation of electrophoresis gel was done with respect to the observable bands showing the presence or absence of the required sequence in the samples related to every primer.

Results: This study was the first MLVA typing of the unencapsulated H. influenzae in Iran. In this research, the VNTR sequences were tested in 30 strains without the unencapsulated H. influenzae. Among 30 mentioned strains, for which MLVA profile was obtained in this research, 25 different MLVA types were observed. Likewise, there was no repetition in VNTR sequences resulting from PCR in few H. influenzae. In all these cases, the number of repetitions in MLVA profile was determined as 0, except for one of the primers in 4 strains, which was 16%. However, this did not occur for the other VNTRs.

Conclusion: The highest diversity of the repeats was for VNTR5 (7 types), followed by VNTR6 with 6 types of repeats, and VNTR12-1 and VNTR12-2 with 3 different types.


Haemophilus influenzae, VNTR, MLVA

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Erwin AL, Nelson KL, Mhlanga-Mutangadura T, Bojthuis PJ, Geelhood JL, Morlin G, et al. Characterization of genetic and phenotypic diversity of invasive nontypeable Haemophilus influenzae. Infect Immun 2005;73: 5853-5863.

Novotny LA, Partida-Sánchez S, Munson RS, Bakaltez LO. Differential uptake and processing of a Haemophilus influenzae P5-derived immunogen by chinchilla dendritic cells. Infect Immun 2008;76: 967-977.

Ryan K, Ray CG. Medical Microbiology 2004; McGraw Hill.

Choi J, Cox AD, Li J, McCready W, Ulanova M. Activation of innate immune responses by Haemophilus influenzae lipooligosaccharide. Clin Vaccine Immunol 2014;21: 769-776.

Malani PN. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. JAMA 2010;304(18): 2067-2071.

Singh B, Brant M, Kilian M, Hallstrom B, Riesbeck K. Protein E of Haemophilus influenzae is a ubiquitous highly conserved adhesin. J Infect Dis 2010;201: 414-419.

van Ketel R de, De Wever B, Van AlphenL. Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification. J Med Microbiol 1990;33: 271-276.

Hassan-King M., Adegbola R, Baldeh I, Mulholland K, Omosigho Ch, Oparaugo A, et al. A polymerase chain reaction for the diagnosis of Haemophilus influenzae type b disease in children and its evaluation during a vaccine trial. Pediatr Infect Dis J 1998;17: 309-312.

Davis GS, Petel M, Hammond J, Zhang L, Dawid S, Marrs CF, et al. Prevalence, distribution, and sequence diversity of hmwA among commensal and otitis media non-typeable Haemophilus influenzae. Infect Genet Evol 2014;28: 223-232.

Schouls LM, van der Ende A, van de Pol I, Schot C, Spanjaard L, Vauterin P, et al. Increase in genetic diversity of Haemophilus influenzae serotype b (Hib) strains after introduction of Hib vaccination in The Netherlands. J Clin Microbiol 2005;43:2741-2749.

Harrison OB, Brueggemann AB, Vaugant DA, van der Ende A, Frosch M, Gray S, et al. Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review. Microbiology 2011;157(Pt 8):2181-2195.

Chang C-H, Chang Y-C, Underwood A, Chiou C-S, Kao C-Y. VNTRDB: a bacterial variable number tandem repeat locus database. Nucleic Acids Res 2007;35(Database issue):D416-21.

Techaruvichit P, Takahashi H, Vesaratvhavest M, Keeratipibul S, Kuda T, Kimura B. Development of multiple-locus variable-number tandem-repeat analysis for molecular subtyping of Campylobacter jejuni by using capillary electrophoresis. Appl Environ Microbiol 2015;81(16): 5318-5325.

Liu Y, Shi X, Li Y, Chen Q, Jiang M, Li W, et al. The evaluation and application of multilocus variable number tandem repeat analysis (MLVA) for the molecular epidemiological study of Salmonella enterica subsp. enterica serovar Enteritidis infection. Ann Clin Microbiol Antimicrob 2016;15: 4.

Rumore JL, Tschetter L, Nadon C. The Impact of Multilocus variable-number tandem-repeat analysis on pulse net Canada Escherichia coli O157: H7 laboratory surveillance and outbreak support, 2008-2012. Foodborne Pathog Dis 2016;13: 255-261.

Chiou C-S. Multilocus variable-number tandem repeat analysis as a molecular tool for subtyping and phylogenetic analysis of bacterial pathogens. Expert Rev Mol Diagn 2010;10: 5-7.

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.

Gherardi G, Creti R, Pompilio A, Di Bonaventura G. An overview of various typing methods for clinical epidemiology of the emerging pathogen Stenotrophomonas maltophilia. Diagn Microbiol Infect Dis 2015;81: 219-226.

Zhou K, AertsenA, Michiels CW. The role of variable DNA tandem repeats in bacterial adaptation. FEMS Microbiol Rev 2014;38: 119-141.

Keim P, Van Ert MN, Pearson T, Vogler AJ, Huynh LY, Wagner DM. Anthrax molecular epidemiology and forensics: using the appropriate marker for different evolutionary scales. Infect Genet Evol 2004;4: 205-213.

Chiou C-S, Watanabe H, Wang Y-W, Wang W-L, Terajima J, Thong K-L, et al. Utility of multilocus variable-number tandem-repeat analysis as a molecular tool for phylogenetic analysis of Shigella sonnei. J Clin Microbiol 2009;47: 1149-1154.

Van Belkum A. Tracing isolates of bacterial species by multilocus variable number of tandem repeat analysis (MLVA). FEMS Immunol Med Microbiol 2007;49: 22-27.

Bruun T, Sørensen G, Forshell LP, Jensen T, Nygård K, Kapperud G, et al. An outbreak of salmonella typhimurium infections in Denmark, Norway and Sweden, 2008. Euro Surveill 2009;14(10). pii: 19147.

Currie BJ, Haslem A, Pearson T, Hornstra H, Leadem B, Mayo M, et al. Identification of melioidosis outbreak by multilocus variable number tandem repeat analysis. Emerg Infect Dis 2009;15: 169-174.

Cooley M, Carychao D, Crawford-miksza L, Jay MT, Myers C, et al. Incidence and tracking of Escherichia coli O157: H7 in a major produce production region in California. PloS one 2007;2(11): e1159.

Nygard K, Lindstedt BA, Wahl W, Jensvoll L, Kjelso C, Molbak K, et al. Outbreak of Salmonella Typhimurium infection traced to imported cured sausage using MLVA-subtyping. Euro Surveill 2007;12(3):E070315.5.

Ahrén IL, Janson H, Forsgren A, Riesbeck K. Protein D expression promotes the adherence and internalization of non-typeable Haemophilus influenzae into human monocytic cells. Microb Pathog 2001;31: 151-158.

Khodashahri SB, Siadat SD, Rahbar M, Abdollahpour-Alitappeh M, Vaziri F, Rahnamaye-Farzami M, et al. Genotyping of Haemophilus influenzae type b strains and their incidence in the clinical samples isolated from Iranian patients. Iran J Microbiol 2015;7:136-143.

Ueyama T, Kurono Y, Shirabe K, Takeshita M, Mogi G. High incidence of Haemophilus influenzae in nasopharyngeal secretions and middle ear effusions as detected by PCR. J Clin Microbiol 1995;33: 1835-1838.

Hood DW, Deadman ME, Jennings MP, Bisercic M, Fleischmann RD, Venter JC, et al. DNA repeats identify novel virulence genes in Haemophilus influenzae. Proc Natl Acad Sci U S A 1996;93: 11121-11125.

Van Belkum A, Schere S, Van Leeuwen W, Willemse D, Van Alphen L, Verbrugh H. Variable number of tandem repeats in clinical strains of Haemophilus influenzae. Infect Immun 1997;65: 5017-5027.

Renders N, Licciardello L, Ijsseldijk C, Sijmons M, van Alphen L, Verbrugh H, et al. Variable numbers of tandem repeat loci in genetically homogeneous Haemophilus influenzae strains alter during persistent colonisation of cystic fibrosis patients. FEMS Microbiol Lett 1999;173: 95-102.

Van Belkum A, Melchers WJ, Ijsseldjik C, Nohlmans L, Verbrugh H, Meis JF. Outbreak of amoxicillin-resistant Haemophilus influenzae type b: variable number of tandem repeats as novel molecular markers. J Clin Microbiol 1997;35: 1517-1520.


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