Molecular characterization of Brucella species from Zimbabwe.

Brucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world.

Genotyping of French Bacillus anthracis strains based on 31-loci multi locus VNTR analysis: epidemiology, marker evaluation, and update of the internet genotype database.

BACKGROUND: Bacillus anthracis is known to have low genetic variability. In spite of this lack of diversity, multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) and single nucleotide polymorphisms (SNPs) including the canonical SNPs assay (canSNP) have proved to be highly effective to differentiate strains. Five different MLVA schemes based on a collection of 31 VNTR loci (MLVA8, MLVA15, MLVA20, MLVA25 and MLVA31) with increased resolving power have been described. RESULTS: MLVA31 was applied to characterize the French National Reference Laboratory collection. The total collection of 130 strains is resolved in 35 genotypes. The 119 veterinary and environmental strains collection in France were resolved into 26 genotypes belonging to three canSNP lineages and four MLVA clonal complexes (CCs) with particular geographical clustering. A subset of seven loci (MLVA7) is proposed to constitute a first line assay. The loci are compatible with moderate resolution equipment such as agarose gel electrophoresis and show a good congruence value with MLVA31. The associated MLVA and SNP data was imported together with published genotyping data by taking advantage of major enhancements to the MLVAbank software and web site. CONCLUSIONS: The present report provides a wide coverage of the genetic diversity of naturally occurring B. anthracis strains in France as can be revealed by MLVA. The data obtained suggests that once such coverage is achieved, it becomes possible to devise optimized first-line MLVA assays comprising a sufficiently low number of loci to be typed either in one multiplex PCR or on agarose gels. Such a selection of seven loci is proposed here, and future similar investigations in additional countries will indicate to which extend the same selection can be used worldwide as a common minimum set. It is hoped that this approach will contribute to an efficient and low-cost routine surveillance of important pathogens for biosecurity such as B. anthracis.

Yersinia pseudotuberculosis ST42 (O:1) Strain Misidentified as Yersinia pestis by Mass Spectrometry Analysis.

We report here the draft sequence of strain CEB14_0017, alias HIAD_DUP, recovered from a human patient and initially identified as Yersinia pestis by mass spectrometry analysis. Genotyping based on tandem repeat polymorphism assigned the strain to Yersinia pseudotuberculosis sequence type 42 (ST42). The total assembly length is 4,894,739 bp.

MLVA16 typing of Portuguese human and animal Brucella melitensis and Brucella abortus isolates.

To investigate the epidemiological relationship of isolates from different Portuguese geographical regions and to assess the diversity among isolates, the MLVA16(Orsay) assay (panels 1, 2A and 2B) was performed with a collection of 126 Brucella melitensis (46 human and 80 animal isolates) and 157 B. abortus field isolates, seven vaccine strains and the representative reference strains of each species. The MLVA16(Orsay) showed a similar high discriminatory power (HGDI 0.972 and 0.902) for both species but panel 1 and 2A markers displayed higher diversity (HGDI 0.693) in B. abortus compared to B. melitensis isolates (HGDI 0.342). The B. melitensis population belong to the "Americas" (17%) and "East Mediterranean" (83%) groups. No isolate belonged to the "West Mediterranean" group. Eighty-five percent of the human isolates (39 in 46) fit in the "East-Mediterranean" group where a single lineage known as MLVA11 genotype 116 is responsible for the vast majority of Brucella infections in humans. B. abortus isolates formed a consistent group with bv1 and bv3 isolates in different clusters. Four MLVA11 genotypes were observed for the first time in isolates from S. Jorge and Terceira islands from Azores. From the collection of isolates analysed in this study we conclude that MLVA16(Orsay) provided a clear view of Brucella spp. population, confirming epidemiological linkage in outbreak investigations. In particular, it suggests recent and ongoing colonisation of Portugal with one B. melitensis lineage usually associated with East Mediterranean countries.

Yersinia pestis lineages in Mongolia.

BACKGROUND: Whole genome sequencing allowed the development of a number of high resolution sequence based typing tools for Yersinia (Y.) pestis. The application of these methods on isolates from most known foci worldwide and in particular from China and the Former Soviet Union has dramatically improved our understanding of the population structure of this species. In the current view, Y. pestis including the non or moderate human pathogen Y. pestis subspecies microtus emerged from Yersinia pseudotuberculosis about 2,600 to 28,600 years ago in central Asia. The majority of central Asia natural foci have been investigated. However these investigations included only few strains from Mongolia. METHODOLOGY/PRINCIPAL FINDINGS: Clustered Regularly Interspaced Short Prokaryotic Repeats (CRISPR) analysis and Multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) with 25 loci was performed on 100 Y. pestis strains, isolated from 37 sampling areas in Mongolia. The resulting data were compared with previously published data from more than 500 plague strains, 130 of which had also been previously genotyped by single nucleotide polymorphism (SNP) analysis. The comparison revealed six main clusters including the three microtus biovars Ulegeica, Altaica, and Xilingolensis. The largest cluster comprises 78 isolates, with unique and new genotypes seen so far in Mongolia only. Typing of selected isolates by key SNPs was used to robustly assign the corresponding clusters to previously defined SNP branches. CONCLUSIONS/SIGNIFICANCE: We show that Mongolia hosts the most recent microtus clade (Ulegeica). Interestingly no representatives of the ancestral Y. pestis subspecies pestis nodes previously identified in North-western China were identified in this study. This observation suggests that the subsequent evolution steps within Y. pestis pestis did not occur in Mongolia. Rather, Mongolia was most likely re-colonized by more recent clades coming back from China contemporary of the black death pandemic, or more recently in the past 600 years.

Intraspecies biodiversity of the genetically homologous species Brucella microti.

Brucellosis is one of the major bacterial zoonoses worldwide. In the past decade, an increasing number of atypical Brucella strains and species have been described. Brucella microti in particular has attracted attention, because this species not only infects mammalian hosts but also persists in soil. An environmental reservoir may pose a new public health risk, leading to the reemergence of brucellosis. In a polyphasic approach, comprising conventional microbiological techniques and extensive biochemical and molecular techniques, all currently available Brucella microti strains were characterized. While differing in their natural habitats and host preferences, B. microti isolates were found to possess identical 16S rRNA, recA, omp2a, and omp2b gene sequences and identical multilocus sequence analysis (MLSA) profiles at 21 different genomic loci. Only highly variable microsatellite markers of multiple-locus variable-number tandem repeat (VNTR) analysis comprising 16 loci (MLVA-16) showed intraspecies discriminatory power. In contrast, biotyping demonstrated striking differences within the genetically homologous species. The majority of the mammalian isolates agglutinated only with monospecific anti-M serum, whereas soil isolates agglutinated with anti-A, anti-M, and anti-R sera. Bacteria isolated from animal sources were lysed by phages F1, F25, Tb, BK2, Iz, and Wb, whereas soil isolates usually were not. Rough strains of environmental origin were lysed only by phage R/C. B. microti exhibited high metabolic activities similar to those of closely related soil organisms, such as Ochrobactrum spp. Each strain was tested with 93 different substrates and showed an individual metabolic profile. In summary, the adaptation of Brucella microti to a specific habitat or host seems to be a matter of gene regulation rather than a matter of gene configuration.

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

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.

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.

BACKGROUND: Since 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. RESULTS: 294 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. CONCLUSION: The 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.

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.

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.

Assessment of genetic stability of Brucella melitensis Rev 1 vaccine strain by multiple-locus variable-number tandem repeat analysis.

The assessment of the genetic stability is one of the essential elements to guarantee the biological quality of live anti-bacteria vaccines. Live attenuated Brucella melitensis Rev 1 is the most effective vaccine against brucellosis in small ruminants. Thirty-six B. melitensis Rev 1 vaccine strains isolated from human or animal sources from different geographic regions, from different commercial batches or laboratory collections were typed by the multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) recently described for Brucella spp. Our results demonstrated that B. melitensis Rev 1 group as assayed by MLVA is genetically very homogeneous. We believe that MLVA methodology could be an essential assay to guarantee the quality and stability of live anti-bacterial vaccines being produced worldwide and can be included as in vitro control.

Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay.

BACKGROUND: The classification of Brucella into species and biovars relies on phenotypic characteristics and sometimes raises difficulties in the interpretation of the results due to an absence of standardization of the typing reagents. In addition, the resolution of this biotyping is moderate and requires the manipulation of the living agent. More efficient DNA-based methods are needed, and this work explores the suitability of multiple locus variable number tandem repeats analysis (MLVA) for both typing and species identification. RESULTS: Eighty tandem repeat loci predicted to be polymorphic by genome sequence analysis of three available Brucella genome sequences were tested for polymorphism by genotyping 21 Brucella strains (18 reference strains representing the six 'classical' species and all biovars as well as 3 marine mammal strains currently recognized as members of two new species). The MLVA data efficiently cluster the strains as expected according to their species and biovar. For practical use, a subset of 15 loci preserving this clustering was selected and applied to the typing of 236 isolates. Using this MLVA-15 assay, the clusters generated correspond to the classical biotyping scheme of Brucella spp. The 15 markers have been divided into two groups, one comprising 8 user-friendly minisatellite markers with a good species identification capability (panel 1) and another complementary group of 7 microsatellite markers with higher discriminatory power (panel 2). CONCLUSION: The MLVA-15 assay can be applied to large collections of Brucella strains with automated or manual procedures, and can be proposed as a complement, or even a substitute, of classical biotyping methods. This is facilitated by the fact that MLVA is based on non-infectious material (DNA) whereas the biotyping procedure itself requires the manipulation of the living agent. The data produced can be queried on a dedicated MLVA web service site.

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".

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.

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

BACKGROUND: Currently 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. RESULTS: A 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. CONCLUSIONS: Tandem-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.