Multiple-locus variable number tandem repeat analysis of Neisseria gonorrhoeae isolates in Russia

Abstract

In the present study, a multiple-locus variable number tandem repeat analysis (MLVA) was used to assess the molecular epidemiology of Neisseria gonorrhoeae clinical isolates originating from different regions of Russia. MLVA, based on seven loci, was performed on 218 isolates that were previously tested for susceptibility to penicillin, tetracycline and ciprofloxacin and for the presence of certain genetic determinants of drug resistance. In total, 83 MLVA types were identified, indicating that MLVA is a highly discriminatory technique with a Hunter–Gaston discriminatory index of 0.963 (95% CI, 0.950–0.977). MLVA type 16 was shown to be the most prevalent type and is undoubtedly associated with a multidrug resistant phenotype. The spread of MLVA type 16 from Moscow to Irkutsk suggests that this type has a highly successful transmission rate. Hierarchical cluster analysis of the MLVA profiles classified 208 isolates (95%) into six large groups (containing more than 10 isolates). Clusters differed in geographical characteristics and susceptibility profiles. MLVA cluster A comprised in total 34 isolates and was unambiguously associated with multidrug resistance. Most isolates in cluster A carried mutations in penA, ponA, rpsJ, mtrR, gyrA, and parC genes. MLVA cluster D was associated with resistance to penicillin and with mutations in ponA and rpsJ genes and the presence of plasmid-borne bla_(TEM-1) gene. MLVA clusters B, C and E were associated with susceptibility to ciprofloxacin and had a lack of mutations in ponA, rpsJ, gyrA, and parC genes. We conclude that MLVA will be a useful tool for N. gonorrhoeae epidemiological studies.

Introduction

Neisseria gonorrhoeae is a causative agent of the sexually transmitted disease, gonorrhea, which is characterized by purulent inflammation of the mucous membranes of the genitourinary system. The emergence and spread of multidrug-resistant N. gonorrhoeae strains and the absence of an effective vaccine are major problems worldwide.

Although the estimated incidence of gonorrhea in Russia has decreased from 229.25 cases per 100,000 inhabitants in 1993 to 38.35 cases in 2011, the rate remains higher in Russia than in the Netherlands (21.49 cases), Hungary (13.74 cases), Sweden (10.09 cases) or other countries of European Union (World Health Organization, 2011). Antimicrobial resistance of N. gonorrhoeae across Russia is exceedingly high, as in most countries of the European Union; however, between 2005 and 2008, all Russian isolates were susceptible to ceftriaxone (Kubanova et al., 2010). Continuous local, national and international surveillance of N. gonorrhoeae infection is crucial for public health purposes.

Molecular typing is an important tool for the study of the epidemiology and population structure of N. gonorrhoeae. Many different typing methods have been developed and applied to characterize gonococcal isolates (Li and Dillon, 1995, Van Looveren et al., 1999, O’Rourke et al., 1995, Poh and Lau, 1993, Trees et al., 2000, Unemo and Dillon, 2011). In recent years, the most frequently used genotyping methods included: sequencing of the porB gene (Hobbs et al., 1999), multilocus sequence typing (MLST) (Viscidi and Demma, 2003) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) (Martin et al., 2004). These methods are based on DNA sequence analysis and are highly discriminatory and reproducible, and results can be objectively compared between laboratories using global online databases (http://www.ng-mast.net and http://www.pubmlst.org/neisseria).

Previously, multilocus variable number tandem repeat (VNTR) analysis (MLVA) was developed and applied successfully to the typing of many bacterial species, including Neisseria meningitidis (Liao et al., 2006, Schouls, 2006, Yazdankhah et al., 2005). In addition, Heymans et al. have recently developed and evaluated a MLVA method with five VNTR loci for genotyping of N. gonorrhoeae clinical isolates (Heymans et al., 2011, Heymans et al., 2012a, Heymans et al., 2012b).

In this study, we applied the MLVA method described previously (Kushnir et al., 2012) to analyze a collection of 218 N. gonorrhoeae isolates from Russia to provide new information about the molecular epidemiology and population structure of this important pathogen.