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Biomedical subjects

Gilles Vergnaud

Publications and source records attributed to Gilles Vergnaud.

18 recordsLinked to original sources
PubMed2006

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.

Animals↗
PubMed2006

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing.

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). RESULTS: By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. CONCLUSION: Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.

Animals↗
PubMed2006

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci multiple locus variable-number tandem repeats analysis.

BACKGROUND: The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. RESULTS: Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. CONCLUSION: In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.

Automation↗
PubMed2006

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.

Animals↗
PubMed2005

Evaluation and selection of tandem repeat loci for Streptococcus pneumoniae MLVA strain typing.

BACKGROUND: Precise identification of bacterial pathogens at the strain level is essential for epidemiological purposes. In Streptococcus pneumoniae, the existence of 90 different serotypes makes the typing particularly difficult and requires the use of highly informative tools. Available methods are relatively expensive and cannot be used for large-scale or routine typing of any new isolate. We explore here the potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats), a method of growing importance in the field of molecular epidemiology, for genotyping of Streptococcus pneumoniae. RESULTS: Available genome sequences were searched for polymorphic tandem repeats. The loci identified were typed across a collection of 56 diverse isolates and including a group of serotype 1 isolates from Africa. Eventually a set of 16 VNTRs was proposed for MLVA-typing of S. pneumoniae. These robust markers were sufficient to discriminate 49 genotypes and to aggregate strains on the basis of the serotype and geographical origin, although some exceptions were found. Such exceptions may reflect serotype switching or horizontal transfer of genetic material. CONCLUSION: We describe a simple PCR-based MLVA genotyping scheme for S. pneumoniae which may prove to be a powerful complement to existing tools for epidemiological studies. Using this technique we uncovered a clonal population of strains, responsible for infections in Burkina Faso. We believe that the proposed MLVA typing scheme can become a standard for epidemiological studies of S. pneumoniae.

Bacterial Typing Techniques↗
PubMed2005

Failure of a short-term antibiotic therapy for human brucellosis using ciprofloxacin. A study on in vitro susceptibility of Brucella strains.

BACKGROUND: Human brucellosis is characterized byfocal complications, chronic courses, and therapeutic failures. METHODS: In a relapsed case of brucellosis after short-term antibiotic therapy using doxycycline and ciprofloxacin two Brucella strains were isolated, before and after treatment. In vitro susceptibilities of both isolates were determined by E tests including a great variety of antibiotics. In a killing rate experiment the bactericidal activities of doxycycline, streptomycin, rifampin and ciprofloxacin as single agents and in combinations were determined. RESULTS: Lowest MIC values were measured for doxycycline and ciprofloxacin. MICs did not change under therapy. Streptomycin alone exhibited the most effective killing within 6 h, whereas the other single agents did not show bactericidal activity. Doxycycline plus ciprofloxacin was the most active combination in vitro. CONCLUSION: Routine susceptibility testing of Brucellae is not obligatory as most of the 'traditional' anti-Brucella antibiotics are active in vitro and bactericidal efficacy may differ in vivo.

Anti-Infective Agents↗
PubMed2005

Minisatellite polymorphism as a tool to distinguish closely related Lactococcus lactis strains.

Genome plasticity is considered as a means for bacteria to adapt to their environment. Plasticity in tandem repeat sequences on bacterial genomes has been recently exploited to trace the epidemiology of pathogens. Here, we examine the utility of minisatellite (i.e., a repeat unit of six nucleotides or more) typing in non-pathogenic food bacteria of the species Lactococcus lactis. Thirty-four minisatellites identified on the sequenced L. lactis ssp. lactis strain IL1403 genome were first analyzed in 10 closely related ssp. lactis strains, as determined by randomly amplified polymorphic DNA (RAPD). The selected tandem repeats varied in length, percent identity between repeats, and locations. We showed that: (i) the greatest polymorphism was in orfs encoding exported proteins or in intergenic regions; (ii) two thirds of minisatellites were little- or non-variable, despite as much as 90% identity between tandem repeats; and (iii) dendrograms based on either RAPD or minisatellite analyses were similar. Seven minisatellites identified in this study are potentially useful for lactococcal typing. We then asked whether tandem repeats in L. lactis were stable upon very long-term (up to two years) storage. Despite large rearrangements previously reported in derivative strains, just one of 10 minisatellites tested underwent an alteration, suggesting that tandem repeat rearrangements probably occur during active DNA replication. We conclude that multiple locus minisatellite analysis can be a valuable tool to follow lactococcal strain diversity.

Lactococcus lactis↗
PubMed2005

A nose-only apparatus for airborne delivery of Mycobacterium tuberculosis to mice: calibration of biological parameters.

Mycobacterium tuberculosis is the main cause of tuberculosis and is still a public health concern worldwide. This mycobacterium is transmitted through aerosols from human beings suffering from pulmonary tuberculosis to susceptible persons. To study this natural route of infection, we designed a new nose-only aerosol apparatus--system of aerosolisation of microorganisms (SAM)--in a carefully designed biohazard facility. For safety reasons, Mycobacterium smegmatis was first used to calibrate several parameters, such as inoculum density, atmospheric conditions (i.e. hygrometry) and particle size distribution. We present evidence that our apparatus is totally adapted to airborne delivery; the particle size of generated aerosol ranges from 1 to 7 microm, which is ideal for an infection by inhalation. We found that 99% of generated particles (<7 microm) could be retained by the respiratory tract, and among these particles, 62-79% (<3.3 microm) were able to reach pulmonary compartments. The next step was to simultaneously challenge 48 mice with M. tuberculosis in a highly reproducible way. We showed that a moderate dose (4 log10 colony-forming units (CFU) per mice) of M. tuberculosis was capable of causing progressive lung pathology and death in mice 30 days post-aerosolisation. Therefore, our apparatus, once calibrated, is easy to handle, safe, and can be used with any pathogen, which is spread by aerosol.

Aerosols↗
PubMed2005

Comparison of minisatellite polymorphisms in the Bacillus cereus complex: a simple assay for large-scale screening and identification of strains most closely related to Bacillus anthracis.

Polymorphism of five tandem repeats that are monomorphic in Bacillus anthracis was investigated in 230 isolates of the B. cereus group and in 5 sequenced B. cereus genomes in search for markers allowing identification of B. cereus and B. thuringiensis strains most closely related to B. anthracis. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from B. anthracis and was also proposed to be closest to B. anthracis by MLVA analysis. However, this strain remains separated from B. anthracis by a number of significant genetic events observed in B. anthracis, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of B. cereus and B. thuringiensis strains closely related to B. anthracis. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the B. cereus complex, with modest equipment and at a low cost, to eventually fill the gap with B. anthracis and better understand the origin and making of this dangerous pathogen.

Animals↗
PubMed2004

Identification of polymorphic tandem repeats by direct comparison of genome sequence from different bacterial strains: a web-based resource.

BACKGROUND: Polymorphic tandem repeat typing is a new generic technology which has been proved to be very efficient for bacterial pathogens such as B. anthracis, M. tuberculosis, P. aeruginosa, L. pneumophila, Y. pestis. The previously developed tandem repeats database takes advantage of the release of genome sequence data for a growing number of bacteria to facilitate the identification of tandem repeats. The development of an assay then requires the evaluation of tandem repeat polymorphism on well-selected sets of isolates. In the case of major human pathogens, such as S. aureus, more than one strain is being sequenced, so that tandem repeats most likely to be polymorphic can now be selected in silico based on genome sequence comparison. RESULTS: In addition to the previously described general Tandem Repeats Database, we have developed a tool to automatically identify tandem repeats of a different length in the genome sequence of two (or more) closely related bacterial strains. Genome comparisons are pre-computed. The results of the comparisons are parsed in a database, which can be conveniently queried over the internet according to criteria of practical value, including repeat unit length, predicted size difference, etc. Comparisons are available for 16 bacterial species, and the orthopox viruses, including the variola virus and three of its close neighbors. CONCLUSIONS: We are presenting an internet-based resource to help develop and perform tandem repeats based bacterial strain typing. The tools accessible at http://minisatellites.u-psud.fr now comprise four parts. The Tandem Repeats Database enables the identification of tandem repeats across entire genomes. The Strain Comparison Page identifies tandem repeats differing between different genome sequences from the same species. The "Blast in the Tandem Repeats Database" facilitates the search for a known tandem repeat and the prediction of amplification product sizes. The "Bacterial Genotyping Page" is a service for strain identification at the subspecies level.

Bacteria↗
PubMed2004

Variable number of tandem repeats in Salmonella enterica subsp. enterica for typing purposes.

The genomic sequences of Salmonella enterica subsp. enterica strains CT18, Ty2 (serovar Typhi), and LT2 (serovar Typhimurium) were analyzed for potential variable number tandem repeats (VNTRs). A multiple-locus VNTR analysis (MLVA) of 99 strains of S. enterica supsp. enterica based on 10 VNTRs distinguished 52 genotypes and placed them into four groups. All strains tested were independent human isolates from France and did not reflect isolates from outbreak episodes. Of these 10 VNTRs, 7 showed variability within serovar Typhi, whereas 1 showed variability within serovar Typhimurium. Four VNTRs showed high Nei's diversity indices (DIs) of 0.81 to 0.87 within serovar Typhi (n = 27). Additionally, three of these more variable VNTRs showed DIs of 0.18 to 0.58 within serovar Paratyphi A (n = 10). The VNTR polymorphic site within multidrug-resistant (MDR) serovar Typhimurium isolates (n = 39; resistance to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline) showed a DI of 0.81. Cluster analysis not only identified three genetically distinct groups consistent with the present serovar classification of salmonellae (serovars Typhi, Paratyphi A, and Typhimurium) but also discriminated 25 subtypes (93%) within serovar Typhi isolates. The analysis discriminated only eight subtypes within serovar Typhimurium isolates resistant to ampicillin, chloramphenicol, spectinomycin, sulfonamides, and tetracycline, possibly reflecting the emergence in the mid-1990s of the DT104 phage type, which often displays such an MDR spectrum. Coupled with the ongoing improvements in automated procedures offered by capillary electrophoresis, use of these markers is proposed in further investigations of the potential of MLVA in outbreaks of salmonellosis, especially outbreaks of typhoid fever.

Alleles↗
PubMed2004

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.

Bacterial Proteins↗
PubMed2003

Predicting human minisatellite polymorphism.

We seek to define sequence-based predictive criteria to identify polymorphic and hypermutable minisatellites in the human genome. Polymorphism of a representative pool of minisatellites, selected from human chromosomes 21 and 22, was experimentally measured by PCR typing in a population of unrelated individuals. Two predictive approaches were tested. One uses simple repeat characteristics (e.g., unit length, copy number, nucleotide bias) and a more complex measure, termed HistoryR, based on the presence of variant motifs in the tandem array. We find that HistoryR and percentage of GC are strongly correlated with polymorphism and, as predictive criteria, reduce by half the number of repeats to type while enriching the proportion with heterozygosity >/=0.5, from a background level of 43% to 59%. The second approach uses length differences between minisatellites in the two releases of the human genome sequence (from the public consortium and Celera). As a predictor, this similarly enriches the number of polymorphic minisatellites, but fails to identify an unexpectedly large number of these. Finally, typing of the highly polymorphic minisatellites in large families identified one new hypermutable minisatellite, located in a predicted coding sequence. This may represent the first coding human hypermutable minisatellite.

Chromosomes, Human, Pair 21↗
PubMed2003

DNA-DNA hybridization study of Burkholderia species using genomic DNA macro-array analysis coupled to reverse genome probing.

The present study was aimed at simplifying procedures to delineate species and identify isolates based on DNA-DNA reassociation. DNA macro-arrays harbouring genomic DNA of reference strains of several Burkholderia species were produced. Labelled genomic DNA, hybridized to such an array, allowed multiple relative pairwise comparisons. Based on the relative DNA-DNA relatedness values, a complete data matrix was constructed and the ability of the method to discriminate strains belonging to different species was assessed. This simple approach led successfully to the discrimination of Burkholderia mallei from Burkholderia pseudomallei, but also discriminated Burkholderia cepacia genomovars I and III, Burkholderia multivorans, Burkholderia pyrrocinia, Burkholderia stabilis and Burkholderia vietnamiensis. Present data showed a sufficient degree of congruence with previous DNA-DNA reassociation techniques. As part of a polyphasic taxonomic scheme, this straightforward approach is proposed to improve species definition, especially for application in the rapid screening necessary for large numbers of clinical or environmental isolates.

Bacterial Typing Techniques↗
PubMed2003

Evaluation of the polymorphisms associated with tandem repeats for Pseudomonas aeruginosa strain typing.

We report on the development of a scheme for the typing of Pseudomonas aeruginosa, multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA). We first evaluated the polymorphisms of 201 tandem repeat loci selected from more than 3,000 such sequences present in strain PAO1 with a test collection of 12 genotypically distinct clinical strains. Seven VNTR loci which can be easily scored with the technology used here were identified and used to genotype a collection of 89 clinical isolates that had previously been classified into 46 ribotypes, including 2 widespread ribotypes. Seventy-one different MLVA genotypes could be distinguished. With only two exceptions, strains with identical ribotypes were grouped together upon cluster analysis of the MLVA data. The 27 isolates with the most frequent ribotype were divided into 14 MLVA types, and the 18 isolates with the second most frequent ribotype were divided into 15 MLVA types. Analysis of a subset of 17 strains belonging to the major ribotype by pulsed-field gel electrophoresis with the enzyme SpeI distinguished seven types, identical to the number of MLVA types in this subset. Our data show that MLVA typing of P. aeruginosa based on the first set of loci has a high discriminatory power. Because MLVA is highly reproducible and easily portable among laboratories, it represents a very promising tool for the molecular surveillance of P. aeruginosa. A free, online strain identification service based on the genotyping data produced herein has been developed.

Base Sequence↗
PubMed2002

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.

DNA, Bacterial↗
PubMed2002

Segmental polymorphisms in the proterminal regions of a subset of human chromosomes.

The subtelomeric domains of chromosomes are probably the most rapidly evolving structures of the human genome. The highly variable distribution of large duplicated subtelomeric segments has indicated that frequent exchanges between nonhomologous chromosomes may have been taking place during recent genome evolution. We have studied the extent and variability of such duplications using in situ hybridization techniques and a set of well-defined subtelomeric cosmid probes that identify discrete regions within the subtelomeric domain. In addition to reciprocal translocation and illegitimate recombination events that could explain the observed mosaic pattern of subtelomeric regions, it is likely that homology-based recombination mechanisms have also contributed to the spread of distal subtelomeric sequences among particular groups of nonhomologous chromosome arms. The frequency and distribution of large-scale subtelomeric polymorphisms may have direct implications for the design of chromosome-specific probes that are aimed at the identification of cryptic subtelomeric deletions. Furthermore, our results indicate that the relevance of some of the telomere closures proposed within the present Human Genome Sequence draft are restricted to specific allelic variants of unknown frequencies.

Black People↗