Evaluation of Brucella MLVA typing for human brucellosis

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Abstract

Human brucellosis is still the most common bacterial zoonosis worldwide. Neither well-known molecular tools nor the classical biotyping methods are satisfactory for subtyping of Brucella spp. Loci containing Variable Number of Tandem Repeats (VNTRs) have recently proved their usefulness in typing strains from animal origin despite the high genetic homogeneity within the genus Brucella (DNA–DNA homology > 90%).

The aim of this study was to evaluate MLVA (Multiple Locus VNTR Analysis) for diagnostic and epidemiological use in human brucellosis. One hundred and twenty-eight B. melitensis isolates of all three biovars were typed using eight minisatellite (panel 1) and eight microsatellite (panel 2) markers. One hundred and ten different genotypes were identified. The MLVA clustering pattern correlated with the geographic origin of the strains. Brucella strains isolated from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. Fuchsin sensitive B. melitensis strains were found in closely related clusters giving evidence for an association between VNTRs and some phenotypic characteristics. However, the validity of biovars established by classical microbiological methods could not be confirmed by MLVA clustering. The original data can be queried on the genotyping web page at http://bacterial-genotyping.igmors.u-psud.fr. The MLVA assay is rapid, highly discriminatory, and reproducible within human Brucella isolates. MLVA can significantly contribute to epidemiological trace-back analysis of Brucella infections and may advance surveillance and control of human brucellosis.

Introduction

Human brucellosis is still the most common zoonotic disease worldwide with 500,000 new cases reported annually (Pappas et al., 2006). The disease causing agent Brucella (B.) is directly or indirectly transmitted from its animal reservoir to man. Four out of six nomen species are pathogenic for humans, i.e. B. abortus (transmitted from cattle and other Bovidae), B. melitensis (from sheep and goats), B. suis (mainly from pigs) and rarely B. canis (from dogs) (Godfroid et al., 2005). Single cases of human infections caused by potentially new species tentatively called ‘B. maris’ have been described. Brucella infections in domestic animals lead to substantial economic losses due to infertility and miscarriage. Human brucellosis is a flu-like disease without specific signs or characteristic symptoms. The mortality of human disease is low but the involvement of multiple organs may be debilitating for a long time.

Brucella spp. were among the first biological agents weaponized in the 1950s because of their high contagiosity and due to the enormous economic impact of human and animal brucellosis (Christopher et al., 2005). Effective human vaccines are not available and countermeasures therefore have to rely on early diagnosis, adequate antibiotic treatment and the control of animal husbandry and products.

The classical microbiological identification of Brucella strains is based on phenotypic characteristics, i.e. agglutination with monospecific sera, CO2 requirement, H2S production, urea hydrolysis, inhibition of growth by basic fuchsin and thionin, and phage typing (Alton et al., 1988). These criteria will allow the differentiation of species and define biovars. However, in various geographical regions specific species or biovars may predominate, e.g. B. melitensis in the Mediterranean Basin. In these areas, current biotyping methods cannot be used effectively for trace-back investigations.

Various methods have been established for molecular subtyping of Brucella strains (Al Dahouk et al., 2003), e.g. polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of different genetic loci (Al Dahouk et al., 2005c), enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR), repetitive intergenic palindromic sequence-PCR (REP-PCR) (Mercier et al., 1996, Tscherneva et al., 1996), random amplified polymorphic DNA-PCR (RAPD-PCR) (Tscherneva et al., 2000), arbitrary primed-PCR (AP-PCR) (Fekete et al., 1992), amplified fragment length polymorphism (AFLP) (Whatmore et al., 2005) or single nucleotide polymorphism (SNP) (Marianelli et al., 2006). However, most of these tests lack reproducibility, show a limited capability to differentiate single strains, and are not appropriate for routine typing. Because of the genetic homogeneity within the genus, subtyping of Brucella isolates remains a challenge.

Variable number tandem repeats (VNTRs) which are commonly used for DNA fingerprinting in forensic applications also exist in bacterial genomes and seem to be highly discriminatory markers, even when the pathogens investigated belong to monomorphic species e.g. Yersinia pestis, Bacillus anthracis, Francisella tularensis or Brucella (Johansson et al., 2004, Pourcel et al., 2004, Lindstedt, 2005, Le Flèche et al., 2001, Le Flèche et al., 2006, Vergnaud and Pourcel, 2006, Whatmore et al., 2006). Tandem repeats are composed of perfect or imperfect copies of an elementary unit and various alleles can be observed in different bacterial strains within a species. Tandem repeats are classified in satellites (megabases of DNA) present in many eukaryotic genomes, minisatellites (spanning hundreds of basepairs with a repeat unit size of at least 9 bp), and microsatellites (spanning a few tens of nucleotides with a repeat unit size up to 8 bp) (Vergnaud and Denoeud, 2000). Fingerprints resulting from analysis of multiple loci can be highly discriminating or even unique.

Recently, the genomes of B. melitensis 16M (DelVecchio et al., 2002), B. suis 1330 (Paulsen et al., 2002) and B. abortus strain 9–941 (Halling et al., 2005) have been sequenced. DNA sequence variability required for subtyping or epidemiological trace-back of Brucella strains can now be deduced by direct comparison using the appropriate bioinformatic tools (Denoeud and Vergnaud, 2004, Le Flèche et al., 2006). Thus, 107 tandem repeat loci with a repeat unit of at least 5 bp and predicted to display size polymorphism were identified. Eighty of those were further evaluated in 21 reference strains including the six classical species and their biovars as well as 3 marine mammal strains. Each strain was characterized by the number of repeat units (alleles) at each locus generating a specific fingerprint. A subset of 15 loci which preserved the strain clustering and achieved a satisfying discrimination of the strains was consecutively selected. Finally, a large number of animal isolates was investigated for polymorphism. The proposed MLVA-15 assay comprised eight user-friendly minisatellite markers to identify Brucella species (panel 1) and seven microsatellite markers of higher discriminatory power (panel 2). However, different marker combinations can be selected from the available polymorphic loci (Le Flèche et al., 2006, Whatmore et al., 2006). The value of each MLVA set has to be determined in various scenarios before a standardized test will reach a consensus. The aim of our study was the evaluation of an MLVA assay for diagnostic and epidemiological purposes in human brucellosis.

Section snippets

Brucella strains

One hundred and twenty-eight B. melitensis strains isolated from humans were investigated (28 of biotype 1, 55 of biotype 2, 42 of biotype 3, and 3 rough strains). Previously published MLVA data of animal isolates and reference strains were used for comparison (Le Flèche et al., 2006). The reference strains represented the three biovars (bv) of B. melitensis, i.e. bv 1 (16M; ATCC 23456), bv 2 (63/9; ATCC 23457), and bv 3 (Ether; ATCC 23458). All Brucella isolates were classified using the

Results

The combination of alleles amplified from each isolate defined its genotype which is reminiscent of a genetic fingerprint. Variability occurred among the three biovars of B. melitensis and among isolates within each biovar. The different allele sizes found at each locus are shown in Table 2. The allele size is given in basepairs (PCR product) and as number of repeats.

Different strains may have different allele sizes and alleles with a high repeat copy number might be more prone to

Discussion

Because of the close relatedness of Brucella spp. the development of molecular assays efficiently distinguishing species, biovars or eventually single strains proved to be very difficult. The question may arise why these tools are needed in human brucellosis although species identification does not influence therapy. However, molecular epidemiology significantly contributes to the analysis and understanding of infections caused by pathogenic bacteria (van Belkum, 2003). In epidemiology,

Acknowledgements

We gratefully thank Cornelia Göllner for strain identification and Angelika Draeger and Csilla Lodri for DNA preparation. Work on typing of dangerous pathogens and the establishment of reference databases is supported by DGA grants (Délégation Générale pour l'Armement, PEA02–36–01). This project is part of the European biodefence project CEPA 13.14 coordinating work on dangerous pathogens on behalf of Sweden, Germany, France, Italy, The Netherlands, and Norway and of COST Action 845

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    Current address: Central Military Hospital of the Bundeswehr, Department of Internal Medicine, Rübenacherstr. 170, D-56072 Koblenz, Germany.

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    Current address: Friedrich Loeffler Institute, German Reference Center for Human and Animal Brucellosis, Naumburger Str. 96a, D-07743 Jena, Germany.

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