Philippe Le Flèche

High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing

BackgroundCurrently available reference methods for the molecular epidemiology of the Mycobacterium tuberculosis complex either lack sensitivity or are still too tedious and slow for routine application. Recently, tandem repeat typing has emerged as a potential alternative. This report contributes to the development of tandem repeat typing for M. tuberculosis by summarising the existing data, developing additional markers, and setting up a freely accessible, fast, and easy to use, internet-based service for strain identification.ResultsA collection of 21 VNTRs incorporating 13 previously described loci and 8 newly evaluated markers was used to genotype 90 strains from the M. tuberculosis complex (M. tuberculosis (64 strains), M. bovis (9 strains including 4 BCG representatives), M. africanum (17 strains)). Eighty-four different genotypes are defined. Clustering analysis shows that the M. africanum strains fall into three main groups, one of which is closer to the M. tuberculosis strains, and an other one is closer to the M. bovis strains. The resulting data has been made freely accessible over the internet http://bacterial-genotyping.igmors.u-psud.fr/bnserver to allow direct strain identification queries.ConclusionsTandem-repeat typing is a PCR-based assay which may prove to be a powerful complement to the existing epidemiological tools for the M. tuberculosis complex. The number of markers to type depends on the identification precision which is required, so that identification can be achieved quickly at low cost in terms of consumables, technical expertise and equipment.

High Genetic Diversity Revealed by Variable-Number Tandem Repeat Genotyping and Analysis of hsp65 Gene Polymorphism in a Large Collection of “Mycobacterium canettii” Strains Indicates that the M. tuberculosis Complex Is a Recently Emerged Clone of “M. canettii”

ABSTRACT We have analyzed, using complementary molecular methods, the diversity of 43 strains of “Mycobacterium canettii” originating from the Republic of Djibouti, on the Horn of Africa, from 1998 to 2003. Genotyping by multiple-locus variable-number tandem repeat analysis shows that all the strains belong to a single but very distant group when compared to strains of the Mycobacterium tuberculosis complex (MTBC). Thirty-one strains cluster into one large group with little variability and five strains form another group, whereas the other seven are more diverged. In total, 14 genotypes are observed. The DR locus analysis reveals additional variability, some strains being devoid of a direct repeat locus and others having unique spacers. The hsp65 gene polymorphism was investigated by restriction enzyme analysis and sequencing of PCR amplicons. Four new single nucleotide polymorphisms were discovered. One strain was characterized by three nucleotide changes in 441 bp, creating new restriction enzyme polymorphisms. As no sequence variability was found for hsp65 in the whole MTBC, and as a single point mutation separates M. tuberculosis from the closest “M. canettii” strains, this diversity within “M. canettii” subspecies strongly suggests that it is the most probable source species of the MTBC rather than just another branch of the MTBC.

MLVA-16 typing of 295 marine mammal Brucella isolates from different animal and geographic origins identifies 7 major groups within Brucella ceti and Brucella pinnipedialis

BackgroundSince 1994, Brucella strains have been isolated from a wide range of marine mammals. They are currently recognized as two new Brucella species, B. pinnipedialis for the pinniped isolates and B. ceti for the cetacean isolates in agreement with host preference and specific phenotypic and molecular markers. In order to investigate the genetic relationships within the marine mammal Brucella isolates and with reference to terrestrial mammal Brucella isolates, we applied in this study the Multiple Loci VNTR (Variable Number of Tandem Repeats) Analysis (MLVA) approach. A previously published assay comprising 16 loci (MLVA-16) that has been shown to be highly relevant and efficient for typing and clustering Brucella strains from animal and human origin was used.Results294 marine mammal Brucella strains collected in European waters from 173 animals and a human isolate from New Zealand presumably from marine origin were investigated by MLVA-16. Marine mammal Brucella isolates were shown to be different from the recognized terrestrial mammal Brucella species and biovars and corresponded to 3 major related groups, one specific of the B. ceti strains, one of the B. pinnipedialis strains and the last composed of the human isolate. In the B. ceti group, 3 subclusters were identified, distinguishing a cluster of dolphin, minke whale and porpoise isolates and two clusters mostly composed of dolphin isolates. These results were in accordance with published analyses using other phenotypic or molecular approaches, or different panels of VNTR loci. The B. pinnipedialis group could be similarly subdivided in 3 subclusters, one composed exclusively of isolates from hooded seals (Cystophora cristata) and the two others comprising other seal species isolates.ConclusionThe clustering analysis of a large collection of marine mammal Brucella isolates from European waters significantly strengthens the current view of the population structure of these two species, and their relative position with respect to the rest of the Brucella genus. MLVA-16 is confirmed as being a rapid, highly discriminatory and reproducible method to classify Brucella strains including the marine mammal isolates. The Brucella2009 MLVA-16 genotyping database available at http://mlva.u-psud.fr/ is providing a detailed coverage of all 9 currently recognized Brucella species.

Isolation of Brucella microti from Mandibular Lymph Nodes of Red Foxes, Vulpes vulpes, in Lower Austria

From the mandibular lymph nodes of wild red foxes (Vulpes vulpes) hunted in the region of Gmünd, Lower Austria, two gram-negative, oxidase- and urease-positive, coccoid rod-shaped bacteria (strains 257 and 284) were isolated. Cells were fast growing, nonmotile, and agglutinated with monospecific anti-Brucella (M) serum. Both strains were biochemically identified as Ochrobactrum anthropi by using the API 20NE test. However, sequencing of the 16S rRNA and recA genes clearly identified strains 257 and 284 as Brucella spp. Further molecular analysis by omp2a/b gene sequencing, multilocus sequence typing and multilocus variable number tandem repeats analysis revealed Brucella microti, a recently described Brucella species that has originally been isolated from diseased common voles (Microtus arvalis) in South Moravia, Czech Republic in 2000. Our findings demonstrate that B. microti is prevalent in a larger geographic area covering the region of South Moravia and parts of Lower Austria. Foxes could have become infected by ingestion of infected common voles.

Evaluation of a Multilocus Variable-Number Tandem-Repeat Analysis Scheme for Typing Human Brucella Isolates in a Region of Brucellosis Endemicity

ABSTRACT Brucellosis remains an important anthropozoonosis worldwide. Brucella species are genetically homogeneous, and thus, the typing of Brucella species for epidemiological purposes by conventional molecular typing methods has remained elusive. Although many methods could segregate isolates into the phylogenetically recognized taxa, limited within-species genetic diversity has been identified. Recently, multilocus variable-number tandem-repeat analysis (MLVA) was found to have a high degree of resolution when it was applied to collections of Brucella isolates from geographically widespread locations, and an assay comprising 16 such loci (MLVA-16) was proposed. This scheme includes eight minisatellite loci (panel 1) and eight microsatellites (panel 2, which is subdivided into panels 2A and 2B). The utility of MLVA-16 for the subtyping of human Brucella isolates from geographically restricted regions needs to be further evaluated, and genotyping databases with worldwide coverage must be progressively established. In the present study, MLVA-16 was applied to the typing of 42 human Brucella isolates obtained from 41 patients recovered from 2002 to 2006 at a tertiary-care center in Lebanon. All isolates were identified as Brucella melitensis by MLVA-16 and were found to be closely related to B. melitensis isolates from neighboring countries in the Middle East when their genotypes were queried against those in the web-based Brucella2007 MLVA database (http://mlva.u-psud.fr/ ). Panel 2B, which comprised the most variable loci, displayed a very high discriminatory power, while panels 1 and 2A showed limited diversity. The most frequent genotype comprised seven isolates obtained over 7 weeks in 2002, demonstrating an outbreak from a common source. Two isolates obtained from one patient 5 months apart comprised another genotype, indicating relapsing disease. These findings confirm that MLVA-16 has a good discriminatory power for species determination, typing of B. melitensis isolates, and inferring their geographical origin. Abbreviated panel 2B could be used as a short-term epidemiological tool in a small region of endemicity.

Comparison of multiple-locus variable-number tandem-repeat analysis with other PCR-based methods for typing Brucella suis isolates.

ABSTRACT Multiple-locus variable-number tandem-repeat analysis (MLVA), multiplex PCR, and PCR-restriction fragment length polymorphism analysis were compared for typing Brucella suis isolates. A perfect concordance was obtained among these molecular assays. However, MLVA was the only method to demonstrate brucellosis outbreaks and to confirm that wildlife is a reservoir for zoonotic brucellosis.

Imported brucellosis in Denmark: Molecular identification and multiple-locus variable number tandem repeat analysis (MLVA) genotyping of the bacteria

Abstract A polymerase chain reaction was used to identify Brucella species isolated from humans in Denmark. Consecutive analysis of referred bacteria and re-examination of historical isolates identified all as Brucella melitensis. Multiple-locus variable number tandem repeat analysis (MLVA) placed the isolates in the previously defined ‘East Mediterranean’ B. melitensis group.

Evaluation of a Multilocus Variable Number Tandem Repeat Analysis Scheme for Typing Human Brucella Isolates in an Endemic Country

ABSTRACT Brucellosis remains an important anthropozoonosis worldwide. Brucella species are genetically homogeneous, and thus, the typing of Brucella species for epidemiological purposes by conventional molecular typing methods has remained elusive. Although many methods could segregate isolates into the phylogenetically recognized taxa, limited within-species genetic diversity has been identified. Recently, multilocus variable-number tandem-repeat analysis (MLVA) was found to have a high degree of resolution when it was applied to collections of Brucella isolates from geographically widespread locations, and an assay comprising 16 such loci (MLVA-16) was proposed. This scheme includes eight minisatellite loci (panel 1) and eight microsatellites (panel 2, which is subdivided into panels 2A and 2B). The utility of MLVA-16 for the subtyping of human Brucella isolates from geographically restricted regions needs to be further evaluated, and genotyping databases with worldwide coverage must be progressively established. In the present study, MLVA-16 was applied to the typing of 42 human Brucella isolates obtained from 41 patients recovered from 2002 to 2006 at a tertiary-care center in Lebanon. All isolates were identified as Brucella melitensis by MLVA-16 and were found to be closely related to B. melitensis isolates from neighboring countries in the Middle East when their genotypes were queried against those in the web-based Brucella2007 MLVA database (http://mlva.u-psud.fr/ ). Panel 2B, which comprised the most variable loci, displayed a very high discriminatory power, while panels 1 and 2A showed limited diversity. The most frequent genotype comprised seven isolates obtained over 7 weeks in 2002, demonstrating an outbreak from a common source. Two isolates obtained from one patient 5 months apart comprised another genotype, indicating relapsing disease. These findings confirm that MLVA-16 has a good discriminatory power for species determination, typing of B. melitensis isolates, and inferring their geographical origin. Abbreviated panel 2B could be used as a short-term epidemiological tool in a small region of endemicity.