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International audience; We sought to determine the relative value of conventional molecular methods and whole-genome sequencing (WGS) for subtyping Salmonella enterica serovar Enteritidis recovered from 2000 to 2015 in Tunisia and to investigate the genetic diversity of this serotype. A total of 175 Salmonella Enteritidis isolates were recovered from human, animal, and foodborne outbreak samples. Pulsed-field gel electrophoresis (PFGE), multiple locus variable-number tandem repeat analysis (MLVA), and wholegenome sequencing were performed. Eight pulsotypes were detected for all isolates with PFGE (DI = 0.518). Forty-five Salmonella Enteritidis isolates were selected for the MLVA and WGS techniques. Eighteen MLVA profiles were identified and classified into two major clusters (DI = 0.889). Core genome multilocus typing (cgMLST) analysis revealed 16 profiles (DI = 0.785). Whole-genome analysis indicated 660 single-nucleotide polymorphism (SNP) divergences dividing these isolates into 43 haplotypes (DI = 0.997). The phylogenetic tree supported the classification of Salmonella Enteritidis isolates into two distinct lineages subdivided into five clades and seven subclades. Pairwise SNP differences between the isolates ranged between 302 and 350. We observed about 311 SNP differences between the two foodborne outbreaks, while only less or equal to 4 SNP differences within each outbreak. SNP-based WGS typing showed an excellent discriminatory power comparing with the conventional methods such as PFGE and MLVA. Besides, we demonstrate the added value of WGS as a complementary subtyping method to discriminate outbreak from non-outbreak isolates belonging to common subtypes. It is important to continue the survey of Salmonella Enteritidis lineages in Tunisia using WGS.

International audience; Duplex DNA naturally folds into a right-handed double helix in physiological conditions. Some sequences of unusual base composition may nevertheless form alternative structures, as was shown for many repeated sequences in vitro. However, evidence for the formation of noncanonical structures in living cells is difficult to gather. It mainly relies on genetic assays demonstrating their function in vivo or through genetic instability reflecting particular properties of such structures. Efforts were made to reveal their existence directly in a living cell, mainly by generating antibodies specific to secondary structures or using chemical ligands selected for their affinity to these structures. Among secondary structure-forming DNAs are G-quadruplexes, human fragile sites containing minisatellites, AT-rich regions, inverted repeats able to form cruciform structures, hairpin-forming CAG/CTG triplet repeats, and triple helices formed by homopurine-homopyrimidine GAA/TTC trinucleotide repeats. Many of these alternative structures are involved in human pathologies, such as neurological or developmental disorders, as in the case of trinucleotide repeats, or cancers triggered by translocations linked to fragile sites. This review will discuss and highlight evidence supporting the formation of alternative DNA structures in vivo and will emphasize the role of the mismatch repair machinery in binding mispaired DNA duplexes, triggering genetic instability.

International audience; Bartonella genus includes an increasing number of species and subspecies, especially among wild felids, the positioning of which, with regards to the zoonotic species Bartonella henselae, is important to determine.The aim of this study was to test the ability of a molecular typing technique to distinguish between various Bartonella isolates obtained from four different species of free-ranging and captive wild felids and to identify key profiles or markers allowing differentiating them from each other and/or from B. henselae or B. koehlerae.A molecular typing technique for B. henselae based on the polymorphism of variable number tandem repeat units (VNTR) called MLVA (Multiple Locus VNTR Analysis) was applied to 24 Bartonella isolates from free-ranging or captive wild felids, 19 of which were obtained from California and five from three countries in Southern Africa, and compared with 49 B. henselae isolates from cats, dog or humans from the United States including the human ATCC (American Type Culture Collection) reference strain, B. henselae Houston 1.MLVA allowed distinguishing Bartonella isolates from wild felids from either B. henselae or B. koehlerae. We confirmed infection of semi-captive cheetahs with an isolate similar to a Californian bobcat isolate. MLVA also confirmed the unique profile of a free-ranging cheetah isolate from Namibia.Specific profiles were observed making MVLA a useful identification/classification tool of these wild felid isolates and suggesting that they are highly adapted to a specific feline reservoir. Finally, circulation of B. henselae isolates between domestic cats, wild felids and humans is likely occurring, based on the close allelic profiles of some isolates.

Tuberculosis among foreign-born patients is a key indicator of country-level epidemiological profiles and, of an increasing concern in Europe given the more intensified migratory waves of refugees. Since Portugal presents a lower immigrant-associated TB incidence rate when compared to other European countries, we sought to characterize the epidemiology and transmission dynamics among the foreign-born population coming from Portuguese-speaking countries that are associated with higher TB incidences. In the present study we analyzed 133 Mycobacterium tuberculosis isolates obtained from foreign-born individuals over a three-year period in Lisbon, Portugal, using molecular epidemiological methods such as spoligotyping and 24-loci MIRU-VNTR. Moreover, all strains were subjected to drug susceptibility testing. The genetic profiles obtained suggest that strain importation from Portuguese speaking countries plays a less important role in TB epidemiology but instead argue in favor of a high degree of penetrance of Portuguese endemic strains to the migrant population, including multidrug resistant strains, which is particularly relevant to active screening programs. Financial support was provided by the European Society of Clinical Microbiology and Infectious Diseases, for which we would like to would like to acknowledge the Study Group for Mycobacterial Infections;JP was supported by a post doc fellowship [SFRH/BPD/95406/2013] from FCT. The phylogenetic analysis work at Nalin Rastogi's lab was supported by a FEDER grant financed by the European Union and Guadeloupe Region (Programme Opérationnel FEDER-Guadeloupe-Conseil Régional 2014-2020, Grant number 2015-FED-192).MV is thankful to [GHTM UID/Multi/04413/2013] from FCT and toprojects “ForDILAB-TB” and “A implementação de um novo método de identificação rápida do complexo M. tuberculosis nos Laboratórios de Referência da Tuberculose de Maputo e Beira” from Fundação Calouste Gulbenkian and the Community of the Portuguese Speaking Countries (CPLP), as well as toour colleagues Ana Tavares and Sonia Dias for their valuable contribution with the epidemiological and health-care services provided to migrants in Portugal related aspects. CS was supported by FCT [SFRH/BD/73579/2010]. info:eu-repo/semantics/publishedVersion

Background Combining different molecular typing methods for Mycobacterium tuberculosis complex (MTBC) can be a powerful tool for molecular epidemiology-based investigation of TB. However, the current standard method that provides high discriminatory power for such a combination, mycobacterial interspersed repetitive units-variable numbers of tandem repeats typing (MIRU-VNTR), is laborious, time-consuming and often too costly for many resource-limited laboratories. We aimed to evaluate a reduced set of loci for MIRU-VNTR typing in combination with spoligotyping and SNP-typing for routine molecular epidemiology of TB. Method Spoligotyping and SNP-typing, in combination with the 15 loci MIRU-VNTR typing, were first used to type clinical MTBC isolates (n = 158) from Madagascar. A step by step reduction of MIRU-VNTR loci number was then performed according to the Hunter and Gaston Discriminatory Index (HGDI) and to the Principal component analysis (PCA) correlation with the spoligotype profiles to evaluate the discrimination power inside the generated spoligotype clusters. The 15 MIRU-VNTR was used as reference and SNP-typing was used to determine the main MTBC lineages. Results Of the 158 clinical isolates studied, the SNP-typing classified 23 into Lineage 1 (14.6%), 31 into Lineage 2 (19.6%), 23 into Lineage 3 (14.6%) and 81 into Lineage 4 strains (51.3%). 37 different spoligotypes profiles were obtained, 15 of which were unique and 20 in clusters. 15-loci MIRU-VNTR typing revealed 144 different genotypes: 132 isolates had a unique MIRU-VNTR profile and 27 isolates were grouped into 12 clusters. After a stepwise reduction of the MIRU-VNTR loci number within each main spoligotype families, three different sets composed of 5 customised MIRU-VNTR loci had a similar discrimination level to the reference 15 loci MIRU-VNTR in lineage 1, lineage 2 and lineage 3. For lineage 4, a set of 4 and 3 MIRU-VNTR loci were proposed to subtype the Harleem and LAM spoligotype families, respectively. For the T spoligotype family, a set of 5 MIRU-VNTR loci was proposed. Conclusion According to the lineages and the spoligotype families, the number of MIRU-VNTR loci can be reduced to get an optimal classification of MTBC. These customized sets of MIRU-VNTR loci reduce workload and save resources while maintaining optimal discriminatory power.

International audience; Mycobacterium tuberculosis genetic structure, and evolutionary history have been studied for years by several genotyping approaches, but delineation of a few sublineages remains controversial and needs better characterization. This is particularly the case of T group within lineage 4 (L4) which was first described using spoligotyping to pool together a number of strains with ill-defined signatures. Although T strains were not traditionally considered as a real phylogenetic group, they did contain a few phylogenetically meaningful sublineages as shown using SNPs. We therefore decided to investigate if this observation could be corroborated using other robust genetic markers. We consequently made a first assessment of genetic structure using 24-loci MIRU-VNTRs data extracted from the SITVIT2 database (n = 607 clinical isolates collected in Russia, Albania, Turkey, Iraq, Brazil and China). Combining Minimum Spanning Trees and Bayesian population structure analyses (using STRUCTURE and TESS softwares), we distinctly identified eight tentative phylogenetic groups (T1-T8) with a remarkable correlation with geographical origin. We further compared the present structure observed with other L4 sublineages (n = 416 clinical isolates belonging to LAM, Haarlem, X, S sublineages), and showed that 5 out of 8 T groups seemed phylogeographi-cally well-defined as opposed to the remaining 3 groups that partially mixed with other L4 isolates. These results provide with novel evidence about phylogeographically specificity of a proportion of ill-defined T group of M. tuberculosis. The genetic structure observed will now be further validated on an enlarged worldwide dataset using Whole Genome Sequenc-ing (WGS).

International audience; The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

International audience; From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1-18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19-48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans.

The Beijing genotype of Mycobacterium tuberculosis has been shown in many settings to be hypervirulent and associated with multi-drug resistance. Its presently global and rapid dissemination makes it an important issue of public health. Here, I present a significantly enlarged update of the MIRU-VNTR global database of the M. tuberculosis Beijing genotype (11 loci). I further attempted to link the observed mycobacterial diversity with relevant events of the known human history. Large water masses have been the most efficient and drastic generators of the genetic divergence between human populations. The same situation appears true also for M. tuberculosis, which general diversity pattern amazingly resembles that of its human host. At the same time, less expected affinities observed between distant populations of M. tuberculosis may reflect hidden patterns of human migrations or yet unknown epidemiological links between distant regions.

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.

International audience; BackgroundSerotype 1 was the most prevalent pneumococcal meningitis serotype encountered in Niger over the period 2003–2011 (pre-vaccination era), accounting for 45.3% of infections.MethodsMultiple locus VNTR analysis (MLVA) was used to create a genotypic snapshot of a representative subset of the pneumococcal population of serotype 1.ResultsMLVA using 16 markers revealed a homogeneous genetic background of pneumococci serotype 1 from Niger, which clustered with few serotype 1 pneumococci from some African countries, while other African countries displayed different clonal complexes. DNA from Niger and from other African countries were different from pneumococci serotype 1 from European countries.ConclusionsMLVA-typing revealed a low genetic diversity among pneumococci serotype 1 from meningitis cases in Niger in the pre-vaccination era.

Background Leptospirosis is the most common bacterial zoonoses and has been identified as an important emerging global public health problem in Southeast Asia. Rodents are important reservoirs for human leptospirosis, but epidemiological data is lacking. Methodology/Principal Findings We sampled rodents living in different habitats from seven localities distributed across Southeast Asia (Thailand, Lao PDR and Cambodia), between 2009 to 2010. Human isolates were also obtained from localities close to where rodents were sampled. The prevalence of Leptospira infection was assessed by real-time PCR using DNA extracted from rodent kidneys, targeting the lipL32 gene. Sequencing rrs and secY genes, and Multi Locus Variable-number Tandem Repeat (VNTR) analyses were performed on DNA extracted from rat kidneys for Leptospira isolates molecular typing. Four species were detected in rodents, L. borgpetersenii (56% of positive samples), L. interrogans (36%), L. kirschneri (3%) and L. weilli (2%), which were identical to human isolates. Mean prevalence in rodents was approximately 7%, and largely varied across localities and habitats, but not between rodent species. The two most abundant Leptospira species displayed different habitat requirements: L. interrogans was linked to humid habitats (rice fields and forests) while L. borgpetersenii was abundant in both humid and dry habitats (non-floodable lands). Conclusion/Significance L. interrogans and L. borgpetersenii species are widely distributed amongst rodent populations, and strain typing confirmed rodents as reservoirs for human leptospirosis. Differences in habitat requirements for L. interrogans and L. borgpetersenii supported differential transmission modes. In Southeast Asia, human infection risk is not only restricted to activities taking place in wetlands and rice fields as is commonly accepted, but should also include tasks such as forestry work, as well as the hunting and preparation of rodents for consumption, which deserve more attention in future epidemiological studies., Author Summary Leptospirosis is the most prevalent bacterial zoonosis worldwide. Rodents are believed to be the main reservoirs of Leptospira, yet little epidemiological research has been conducted on rodents from Southeast Asia. Previous studies suggest that activities which place humans in microenvironments shared by rodents increase the probability of contracting leptospirosis. We therefore investigated the circulation of leptospiral species and strains in rodent communities and human populations in seven localities scattered throughout Southeast Asia; in Thailand, Lao PDR and Cambodia. Molecular typing assays were used to characterize leptospiral species and strains in both rodents and humans, which demonstrated common strains between humans and rodents. Additionally, we observed that the two most abundant leptospiral species; L. borgpetersenii and L. interrogans, have different habitat requirements, which supposes different modes of transmission. Lastly, in Southeast Asia, the risk of leptospiral transmission to humans is not solely limited to wetlands and rice paddy fields, but is also linked to forested areas, and activities such as the hunting and/or preparation of rodents for consumption.

Leptospirosis is a worldwide zoonosis which is responsible for the typical form of Weil's disease. The epidemiological surveillance of the Leptospira species agent is important for host prevalence control. Although the genotyping methods have progressed, the identification of some serovars remains ambiguous. We investigated the multispacer sequence typing (MST) method for genotyping strains belonging to the species Leptospira interrogans , which is the main agent of leptospirosis worldwide. A total of 33 DNA samples isolated from the reference strains of L. interrogans serogroups Icterohaemorrhagiae, Australis, Canicola, and Grippotyphosa, which are the most prevalent serogroups in France, were analyzed by both the variable-number tandem-repeat (VNTR) and MST methods. An MST database has been constructed from the DNA of these reference strains to define the MST profiles. The MST profiles corroborated with the VNTR results. Moreover, the MST analysis allowed the identification at the serovar level or potentially to the isolate level for strains belonging to L. interrogans serovar Icterohaemorrhagiae, which then results in a higher resolution than VNTR (Hunter-Gaston index of 0.94 versus 0.68). Regarding L. interrogans serogroups Australis, Canicola, and Grippotyphosa, the MST and VNTR methods similarly identified the genotype. The MST method enabled the acquisition of simple and robust results that were based on the nucleotide sequences. The MST identified clinical isolates in correlation with the reference serovar profiles, thus permitting an epidemiological surveillance of circulating L. interrogans strains, especially for the Icterohaemorrhagiae serogroup, which includes the most prevalent strains of public health interest.

Leprosy, a chronic human disease with potentially debilitating neurological consequences, results from infection with Mycobacterium leprae . This unculturable pathogen has undergone extensive reductive evolution, with half of its genome now occupied by pseudogenes. Using comparative genomics, we demonstrated that all extant cases of leprosy are attributable to a single clone whose dissemination worldwide can be retraced from analysis of very rare single-nucleotide polymorphisms. The disease seems to have originated in Eastern Africa or the Near East and spread with successive human migrations. Europeans or North Africans introduced leprosy into West Africa and the Americas within the past 500 years.

International audience; OBJECTIVES: To investigate the resistance mechanisms of β-lactam-resistant Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients in France. METHODS: Two-hundred-and-four P. aeruginosa CF isolates were collected in 10 French university hospitals in 2007. Their susceptibility to 14 antibiotics and their resistance mechanisms to β-lactams were investigated. Their β-lactamase contents were characterized by isoelectric focusing, PCR and enzymatic assays. Expression levels of efflux pumps and the intrinsic β-lactamase AmpC were quantified by reverse transcription real-time quantitative PCR. Genotyping was performed using multiple-locus variable number of tandem repeats analysis (MLVA). The oprD genes were sequenced and compared with those of reference P. aeruginosa strains. To assess deficient OprD production, western blotting experiments were carried out on outer membrane preparations. RESULTS: MLVA typing discriminated 131 genotypes and 47 clusters. One-hundred-and-twenty-four isolates (60.8%) displayed a susceptible phenotype to β-lactams according to EUCAST breakpoints. In the 80 remaining isolates, resistance to β-lactams resulted from derepression of intrinsic cephalosporinase AmpC (61.3%) and/or acquisition of secondary β-lactamases (13.8%). Efflux pumps were up-regulated in 88.8% of isolates and porin OprD was lost in 53.8% of isolates due to frameshifting or nonsense mutations in the oprD gene. CONCLUSIONS: β-Lactam resistance rates are quite high in CF strains of P. aeruginosa isolated in France and not really different from those reported for nosocomial strains. Development of β-lactam resistance is correlated with patient age. It results from intrinsic mechanisms sequentially accumulated by bacteria isolated from patients who have undergone repeated courses of chemotherapy.

Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.

Background Plague was introduced to Madagascar in 1898 and continues to be a significant human health problem. It exists mainly in the central highlands, but in the 1990s was reintroduced to the port city of Mahajanga, where it caused extensive human outbreaks. Despite its prevalence, the phylogeography and molecular epidemiology of Y. pestis in Madagascar has been difficult to study due to the great genetic similarity among isolates. We examine island-wide geographic-genetic patterns based upon whole-genome discovery of SNPs, SNP genotyping and hypervariable variable-number tandem repeat (VNTR) loci to gain insight into the maintenance and spread of Y. pestis in Madagascar. Methodology/Principal Findings We analyzed a set of 262 Malagasy isolates using a set of 56 SNPs and a 43-locus multi-locus VNTR analysis (MLVA) system. We then analyzed the geographic distribution of the subclades and identified patterns related to the maintenance and spread of plague in Madagascar. We find relatively high levels of VNTR diversity in addition to several SNP differences. We identify two major groups, Groups I and II, which are subsequently divided into 11 and 4 subclades, respectively. Y. pestis appears to be maintained in several geographically separate subpopulations. There is also evidence for multiple long distance transfers of Y. pestis, likely human mediated. Such transfers have resulted in the reintroduction and establishment of plague in the port city of Mahajanga, where there is evidence for multiple transfers both from and to the central highlands. Conclusions/Significance The maintenance and spread of Y. pestis in Madagascar is a dynamic and highly active process that relies on the natural cycle between the primary host, the black rat, and its flea vectors as well as human activity., Author Summary Plague, caused by the bacterium Yersinia pestis, has been a problem in Madagascar since it was introduced in 1898. It mainly affects the central highlands, but also has caused several large outbreaks in the port city of Mahajanga, after it was reintroduced there in the 1990s. Despite its prevalence, the genetic diversity and related geographic distribution of different genetic groups of Y. pestis in Madagascar has been difficult to study due to the great genetic similarity among isolates. We subtyped a set of Malagasy isolates and identified two major genetic groups that were subsequently divided into 11 and 4 subgroups, respectively. Y. pestis appears to be maintained in several geographically separate subpopulations. There is also evidence for multiple long distance transfers of Y. pestis, likely human mediated. Such transfers have resulted in the reintroduction and establishment of plague in the port city of Mahajanga where there is evidence for multiple transfers both from and to the central highlands. The maintenance and spread of Y. pestis in Madagascar is a dynamic and highly active process that relies on the natural cycle between the primary host, the black rat, and its flea vectors as well as human activity.

Background Staphylococcus aureus infection in patients with cystic fibrosis (CF) is frequent and may be due to colonization by a few pathogenic lineages. Systematic genotyping of all isolates, methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) is necessary to identify such lineages and follow their evolution in patients. Multiple-locus variable-number tandem repeat analysis (MLVA/VNTR) was used to survey S. aureus clinical isolates in a French paediatric CF centre. Results During a 30 months period, 108 patients, aged 2 to 21 years, regularly followed up at the centre, provided sputum for culture. From 79 patients, a total of 278 isolates were genotyped by MLVA, resolving into 110 genotypes and 19 clonal complexes (CC) composed of similar or closely related isolates. 71% of the strains were distributed into four main CCs, in term of number of isolates and number of genotypes. Spa (Staphylococcus protein A) typing was performed on representative samples, showing an excellent concordance with MLVA. In 17 patients, strains from two to four different CCs were recovered over time. On six occasions, S. aureus isolates with the same genotype were shared by 2 different patients and they belonged to one of the four main clusters. Methicillin-resistance was observed in 60% of the isolates, 90% of which belonged to the main clonal complexes CC8, CC45 and CC5. In 5 patients, methicillin-resistance of S. aureus isolates was not associated with the mecA gene: for four patients, it was due to overproduction of β-lactamase, leading to BOR-SA (borderline S. aureus) isolates, while a strain showing probably a new modified penicillin-binding capacity (MOD-SA) was observed from one patient. Conclusion Systematic genotyping of S. aureus isolates recovered from sputum of CF children allows a thorough analysis of the strains responsible for sporadic as well as chronic colonization and the follow up of their evolution over time. We show here that more than 70% of these strains belong to 4 major CCs. MSSA as well as MRSA, BOR-SA and MOD-SA isolates can persist over several years, despite antibiotic treatments.

Background Expressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut). Results A catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher. Conclusion We have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.

Background Mental deficiency has been linked to abnormalities in cortical neuronal network connectivity and plasticity. These mechanisms are in part under the control of two interacting signalling pathways, the serotonergic and the brain-derived neurotrophic (BDNF) pathways. The aim of the current paper is to determine whether particular alleles or genotypes of two crucial genes of these systems, the serotonin transporter gene (SLC6A4) and the brain-derived neurotrophic factor gene (BDNF), are associated with mental deficiency (MD). Methods We analyzed four functional polymorphisms (rs25531, 5-HTTLPR, VNTR, rs3813034) of the SLC6A4 gene and one functional polymorphism (Val66 Met) of the BDNF gene in 98 patients with non-syndromic mental deficiency (NS-MD) and in an ethnically matched control population of 251 individuals. Results We found no significant differences in allele and genotype frequencies in the five polymorphisms studied in the SLC6A4 and BDNF genes of NS-MD patients versus control patients. While the comparison of the patterns of linkage disequilibrium (D') in the control and NS-MD populations revealed a degree of variability it did not, however, reach significance. No significant differences in frequencies of haplotypes and genotypes for VNTR/rs3813034 and rs25531/5-HTTLPR were observed. Conclusion Altogether, results from the present study do not support a role for any of the five functional polymorphisms of SLC6A4 and BDNF genes in the aetiology of NS-RM. Moreover, they suggest no epistatic interaction in NS-MD between polymorphisms in BDNF and SLC6A4. However, we suggest that further studies on these two pathways in NS-MD remain necessary.

Epidemics of Vibrio parahaemolyticus in Chile have occurred since 1998. Direct genome restriction enzyme analysis (DGREA) using conventional gel electrophoresis permitted discrimination of different V. parahaemolyticus isolates obtained from these outbreaks and showed that this species consists of a highly diverse population. A multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) approach was developed and applied to 22 clinical and 91 environmental V. parahaemolyticus isolates from Chile to understand their clonal structures. To this end, an advanced molecular technique was developed by applying multiplex PCR, fluorescent primers, and capillary electrophoresis, resulting in a high-resolution and high-throughput (HRHT) genotyping method. The genomic basis of this HRHT method was eight VNTR loci described previously by Kimura et al. (J. Microbiol. Methods 72:313-320, 2008) and two new loci which were identified by a detailed molecular study of 24 potential VNTR loci on both chromosomes. The isolates of V. parahaemolyticus belonging to the same DGREA pattern were distinguishable by the size variations in the indicative 10 VNTRs. This assay showed that these 10 VNTR loci were useful for distinguishing isolates of V. parahaemolyticus that had different DGREA patterns and also isolates that belong to the same group. Isolates that differed in their DGREA patterns showed polymorphism in their VNTR profiles. A total of 81 isolates was associated with 59 MLVA groups, providing fine-scale differentiation, even among very closely related isolates. The developed approach enables rapid and high-resolution analysis of V. parahaemolyticus with pandemic potential and provides a new surveillance tool for food-borne pathogens.

Background French Guiana has the highest tuberculosis (TB) burden among all French departments, with a strong increase in the TB incidence over the last few years. It is now uncertain how best to explain this incidence. The objective of this study was to compare three different methods evaluating the extent of recent TB transmission in French Guiana. Methods We conducted a population-based molecular epidemiology study of tuberculosis in French Guiana based on culture-positive TB strains (1996 to 2003, n = 344) to define molecular relatedness between isolates, i.e. potential transmission events. Phylogenetic relationships were inferred by comparing two methods: a "cluster-graph" method based on spoligotyping results, and a minimum spanning tree method based on both spoligotyping and variable number of tandem DNA repeats (VNTR). Furthermore, three indices attempting to reflect the extent of recent TB transmission (RTIn, RTIn-1 and TMI) were compared. Results Molecular analyses showed a total amount of 120 different spoligotyping patterns and 273 clinical isolates (79.4%) that were grouped in 49 clusters. The comparison of spoligotypes from French Guiana with an international spoligotype database (SpolDB4) showed that the majority of isolates belonged to major clades of M. tuberculosis (Haarlem, 22.6%; Latin American-Mediterranean, 23.3%; and T, 32.6%). Indices designed to quantify transmission of tuberculosis gave the following values: RTIn = 0.794, RTIn-1 = 0.651, and TMI = 0.146. Conclusion Our data showed a high number of Mycobacterium tuberculosis clusters, suggesting a high level of recent TB transmission, nonetheless an estimation of transmission rate taking into account cluster size and mutation rate of genetic markers showed a low ongoing transmission rate (14.6%). Our results indicate an endemic mode of TB transmission in French Guiana, with both resurgence of old spatially restricted genotypes, and a significant importation of new TB genotypes by migration of TB infected persons from neighgouring high-incidence countries.

A multiple-locus variable-number tandem-repeat analysis (MLVA) using six microsatellite markers was assessed in 127 Candida glabrata isolates. Thirty-seven different genotypes, stable both in vitro and in vivo, were observed. The highest discriminatory power ( D = 0.902) was reached by using only four markers. MLVA seems to be relevant for C. glabrata typing.

The utility of a genotypic typing assay for Legionella pneumophila was investigated. A multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) scheme using PCR and agarose gel electrophoresis is proposed based on eight minisatellite markers. Panels of well-characterized strains were examined in a multicenter analysis to validate the assay and to compare its performance to that of other genotyping assays. Excellent typeability, reproducibility, stability, and epidemiological concordance were observed. The MLVA type or profile is composed of a string of allele numbers, corresponding to the number of repeats at each VNTR locus, separated by commas, in a predetermined order. A database containing information from 99 L. pneumophila serogroup 1 strains and four strains of other serogroups and their MLVA profiles, which can be queried online, is available from http://bacterial-genotyping.igmors.u-psud.fr/ .

The influence of changes in population structures of modern humans and human pathogens is likely reciprocal. In my opinion, a quantitative approach to study this co-influence in a historical perspective requires, in particular, adequate estimators of genetic distances that are well developed for human but not yet for microbial populations. Here, I propose a simple measure of genetic distance between geographic populations within a microbial species based on the observed difference in the frequencies of its genotypes. Further, I apply the proposed method to principal components analysis of Mycobacterium tuberculosis, and interpret the geographic distribution of its VNTR haplotypes in the light of human historical and recent migrations. The proposed approach may be helpful for a quantitative understanding of human-microbial interactions that constitute an integral part of the global history.

International audience; CONTEXT: Polymorphism at the insulin gene (INS) variable number of tandem repeat (VNTR) shows variable associations with childhood body mass index (BMI) in different populations. OBJECTIVE: The objective of this study was to observe INS VNTR associations with body composition and insulin secretion in children. DESIGN: The study was designed as a prospective birth cohort study. PARTICIPANTS: A total of 947 children genotyped for the INS VNTR participated. MAIN OUTCOME MEASURES: Main outcome measures were whole body dual x-ray emission absorptiometry at 9 yr to estimate height-corrected fat mass index (FMI), truncal FMI, and fat-free mass, and insulin secretion after oral glucose at 8 yr. RESULTS: Homozygous III/III children had higher BMI (P = 0.020), FMI (P = 0.015), and truncal FMI (P = 0.022) at 9 yr than class I bearers, but no difference in fat-free mass (P = 0.23). Gain in weight sd score between birth and 3 yr was associated positively with BMI, FMI, and truncal FMI in class I bearers, but not in III/III children (p-interaction with genotype = 0.009-0.066). INS VNTR genotype was not associated overall with insulin secretion at 8 yr (P = 0.64), but class I bearers showed a stronger positive correlation between insulin secretion and BMI at 8 yr (regression coefficient +/- se, 0.26 +/- 0.05; P < 0.0001) than III/III children (-0.10 +/- 0.07; P = 0.48) (p-interaction = 0.003). CONCLUSION: We clarified that the overall association between INS VNTR class III/III genotype and larger BMI in this population relates to fat mass, but not fat-free mass. In contrast, among the subgroup of children who showed rapid infancy weight gain, class I bearers tended to have larger BMI and fat mass than III/III children. This genetic interaction could relate to insulin secretion, which, in class I bearers, increased more rapidly with overweight and obesity.

CONTEXT: Both adverse intrauterine events and genetic background have been suggested to promote insulin resistance in subjects born small for gestational age (SGA). Among candidate genes that potentially influence both fetal growth and glucose metabolism is insulin. The potential effect of the insulin gene VNTR (INS) on birth weight has been controversial so far. OBJECTIVE: The present association study aimed at testing for the contribution of the INS VNTR locus on birth weight and on the metabolic profile of young adults born SGA (mean age, 22 yr). Two groups of subjects were selected on birth data: SGA (birth weight < 10th percentile; n = 735), and appropriate for gestational age (AGA; birth weight between 25th and 75th percentiles; n = 886). All subjects were genotyped for rs689 A/T single nucleotide polymorphism, in complete linkage disequilibrium with the INS VNTR classes I and III, respectively. RESULTS: Class I INS frequencies were similar in the two groups (70% in AGA; 72% in SGA; P = 0.42). There was significant effect on mean birth weight in neither SGA (P = 0.99) nor AGA (P = 0.18). Although the INS VNTR locus did not associate with anomalies of insulin resistance indices in the AGA group, in the SGA group, INS VNTR class III allele was associated with higher insulin resistance (quantitative insulin sensitivity check index = 0.38 vs. 0.39; P = 0.05). Furthermore, there was evidence of an interaction between the SGA/AGA status and INS VNTR locus on insulin resistance indices (P = 0.01) in a multivariate analysis. CONCLUSION: The INS VNTR locus does not associate in a major way with SGA in the French population. However, our data support an interaction between severe fetal growth restriction and INS VNTR locus, which were associated with insulin resistance in young adults born SGA.

International audience; Marfan syndrome (MFS) is an autosomal dominant connective-tissue disorder characterized by skeletal, ocular and cardiovascular defects of highly variable expressivity. The diagnosis relies solely on clinical criteria requiring anomalies in at least two systems. By excluding the chromosome 15 disease locus, fibrillin 1 (FBN1), in a large French family with typical cardiovascular and skeletal anomalies, we raised the issue of genetic heterogeneity in MFS and the implication of a second locus (MFS2). Linkage analyses, performed in this family, have localized MFS2 to a region of 9 centiMorgans between D3S1293 and D3S1283, at 3p24.2–p25. In this region, the highest lod score was found with D3S2336, of 4.89 (θ=0.05). By LINKMAP analyses, the most probable position for the second locus in MFS was at D3S2335.

Background This paper describes an efficient in silico method for detecting tandem gene arrays (TGAs) in fully sequenced and compact genomes such as those of prokaryotes or unicellular eukaryotes. The originality of this method lies in the search of protein sequence similarities in the vicinity of each coding sequence, which allows the prediction of tandem duplicated gene copies independently of their functionality. Results Applied to nine hemiascomycete yeast genomes, this method predicts that 2% of the genes are involved in TGAs and gene relics are present in 11% of TGAs. The frequency of TGAs with degenerated gene copies means that a significant fraction of tandem duplicated genes follows the birth-and-death model of evolution. A comparison of sequence identity distributions between sets of homologous gene pairs shows that the different copies of tandem arrayed paralogs are less divergent than copies of dispersed paralogs in yeast genomes. It suggests that paralogs included in tandem structures are more recent or more subject to the gene conversion mechanism than other paralogs. Conclusion The method reported here is a useful computational tool to provide a database of TGAs composed of functional or nonfunctional gene copies. Such a database has obvious applications in the fields of structural and comparative genomics. Notably, a detailed study of the TGA catalog will make it possible to tackle the fundamental questions of the origin and evolution of tandem gene clusters.

Comparative genomics analysis of the Tamil Nadu strain of Mycobacterium leprae has uncovered several polymorphic sites with potential as epidemiological tools. In this study we compared the stability of two different markers of genomic biodiversity of M. leprae in several biopsy samples isolated from the same leprosy patient. The first type comprises five different variable-number tandem repeats (VNTR), while the second is composed of three single nucleotide polymorphisms (SNP). Contrasting results were obtained, since no variation was seen in the SNP profiles of M. leprae from 42 patients from 7 different locations in Mali whereas the VNTR profiles varied considerably. Furthermore, since variation in the VNTR pattern was seen not only between different isolates of M. leprae but also between biopsy samples from the same patient, these VNTR may be too dynamic for use as epidemiological markers for leprosy.