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Tuberculosis (TB) remains a major health problem within the Community of Portuguese Language Speaking Countries (CPLP). Despite the marked variation in TB incidence across its member-states and continued human migratory flux between countries, a considerable gap in the knowledge on the Mycobacterium tuberculosis population structure and strain circulation between the countries still exists. To address this, we have assembled and analysed the largest CPLP M. tuberculosis molecular and drug susceptibility dataset, comprised by a total of 1447 clinical isolates, including 423 multidrug-resistant isolates, from five CPLP countries. The data herein presented reinforces Latin American and Mediterranean (LAM) strains as the hallmark of M. tuberculosis populational structure in the CPLP coupled with country-specific differential prevalence of minor clades. Moreover, using high-resolution typing by 24-loci MIRU-VNTR, six cross-border genetic clusters were detected, thus supporting recent clonal expansion across the Lusophone space. To make this data available to the scientific community and public health authorities we developed CPLP-TB (available at http://cplp-tb.ff.ulisboa.pt), an online database coupled with web-based tools for exploratory data analysis. As a public health tool, it is expected to contribute to improved knowledge on the M. tuberculosis population structure and strain circulation within the CPLP, thus supporting the risk assessment of strain-specific trends., Highlights • The Community of Portuguese Speaking Countries (CPLP) occupies a vast geographical area. • Three CPLP countries are shortlisted in the WHO's list of Top 30 high-burden countries. • Common Mycobacterium tuberculosis population structure denote historical strain flow. • Cross-border clusters suggest recent intercontinental tuberculosis transmission. • CPLP-TB: a novel strain database and framework for collaborative studies and strain tracing.

In order to provide a global overview of genotypic, epidemiologic, demographic, phylogeographical, and drug resistance characteristics related to the prevailing tuberculosis (TB) epidemic, we hereby report an update of the 6th version of the international genotyping database SITVIT2. We also make all the available information accessible through a dedicated website (available at http://www.pasteur-guadeloupe.fr:8081/SITVIT2). Thanks to the public release of SITVIT2 which is currently the largest international multimarker genotyping database with a compilation of 111,635 clinical isolates from 169 countries of patient origin (131 countries of isolation, representing 1032 cities), our major aim is to highlight macro- and micro-geographical cleavages and phylogeographical specificities of circulating Mycobacterium tuberculosis complex (MTBC) clones worldwide. For this purpose, we retained strains typed by the most commonly used PCR-based methodology for TB genotyping, i.e., spoligotyping based on the polymorphism of the direct repeat (DR) locus, 5-loci Exact Tandem Repeats (ETRs), and MIRU-VNTR minisatellites used in 12-, 15-, or 24-loci formats. We describe the SITVIT2 database and integrated online applications that permit to interrogate the database using easy drop-down menus to draw maps, graphics and tables versus a long list of parameters and variables available for individual clinical isolates (year and place of isolation, origin, sex, and age of patient, drug-resistance, etc.). Available tools further allow to generate phylogenetical snapshot of circulating strains as Lineage-specific WebLogos, as well as minimum spanning trees of their genotypes in conjunction with their geographical distribution, drug-resistance, demographic, and epidemiologic characteristics instantaneously; whereas online statistical analyses let a user to pinpoint phylogeographical specificities of circulating MTBC lineages and conclude on actual demographic trends. Available associated information on gender (n = 18,944), age (n = 16,968), drug resistance (n = 19,606), and HIV serology (n = 2673), allowed to draw some important conclusions on TB geo-epidemiology; e.g. a positive correlation exists between certain Mycobacterium tuberculosis lineages (such as CAS and Beijing) and drug resistance (p-value.001), while other lineages (such as LAM, X, and BOV) are more frequently associated with HIV-positive serology (p-value.001). Besides, availability of information on the year of isolation of strains (range 1759-2012), also allowed to make tentative correlations between drug resistance information and lineages - portraying probable evolution trends over time and space. To conclude, the present approach of geographical mapping of predominant clinical isolates of tubercle bacilli causing the bulk of the disease both at country and regional level in conjunction with epidemiologic and demographic characteristics allows to shed new light on TB geo-epidemiology in relation with the continued waves of peopling and human migration.

Buenos Aires is an overpopulated port city historically inhabited by people of European descent. Together with its broader metropolitan area, the city exhibits medium tuberculosis rates, and receives migrants, mainly from tuberculosis highly endemic areas of Argentina and neighboring countries. This work was aimed to gain insight into the Mycobacterium tuberculosis population structure in two suburban districts of Buenos Aires which are illustrative of the overall situation of tuberculosis in Argentina. The Lineage 4 Euro-American accounted for >99% of the 816 isolates analyzed (one per patient). Frequencies of spoligotype families were T 35.9%, LAM 33.2%, Haarlem 19.5%, S 3.2%, X 1.5%, Ural 0.7%, BOV 0.2%, Beijing 0.2%, and Cameroon 0.2%. Unknown signatures accounted for 5.3% isolates. Of 55 spoligotypes not matching any extant shared international type (SIT) in SITVIT database, 22 fitted into 15 newly-issued SITs. Certain autochthonous South American genotypes were found to be actively evolving. LAM3, which is wild type for RDrio, was the predominant LAM subfamily in both districts and the RDrio signature was rare among autochthonous, newly created, SITs and orphan patterns. Two genotypes that are rarely observed in neighboring countries ̶ SIT2/H2 and SIT159/T1 Tuscany ̶ were conspicuously represented in Argentina. The infrequent Beijing patterns belonged to Peruvian patients. We conclude that the genotype diversity observed reflects the influence of the Hispanic colonization and more recent immigration waves from Mediterranean and neighboring countries. Unlike in Brazil, the RDrio type does not play a major role in the tuberculosis epidemic in Buenos Aires. Fil: Monteserin, Johana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Paul, Roxana Elizabeth. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Gravina, Elida. Hospital Interzonal General de Agudos “Dr. Diego Paroissien”; Argentina Fil: Reniero, Ana. Hospital Central de San Isidro; Argentina Fil: Hernandez, Teresa. Centro de Zooantroponosis de la Matanza; Argentina Fil: Mazzeo, Eduardo. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Togneri, Ana Maria. Hospital Interzonal de Agudos Evita; Argentina Fil: Simboli, Norberto. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: López, Beatriz. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina Fil: Couvin, David. Institut Pasteur de Guadeloupe; Francia Fil: Rastogi, Nalin. Institut Pasteur de Guadeloupe; Francia Fil: Ritacco, Gloria Viviana. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentina

The work done at Institut Pasteur de la Guadeloupe was supported by a FEDER grant, financed by the European Union and Guadeloupe Region (Programme Op?rationnel FEDER-Guadeloupe-Conseil R?gional 2014?2020, Grant number 2015-FED-192). The ECC and IPF received support from Capes and FAPESPA respectively. Universidade Federal do Rio de Janeiro. Instituto de Microbiologia. Rio de Janeiro, RJ, Brazil. Institut Pasteur de Guadeloupe. Unit? de la Tuberculose et des Mycobact?ries. WHO Supranational TB Reference Laboratory. Abymes, Guadeloupe, France. Institut Pasteur de Guadeloupe. Unit? de la Tuberculose et des Mycobact?ries. WHO Supranational TB Reference Laboratory. Abymes, Guadeloupe, France. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade do Estado do Par?. Programa de P?s-gradua??o em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brazil. Funda??o Oswaldo Cruz. Laborat?rio de Biologia Molecular Aplicada as Micobact?rias. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Laborat?rio de Biologia Molecular Aplicada as Micobact?rias. Rio de Janeiro, RJ, Brazil. Funda??o Oswaldo Cruz. Laborat?rio de Biologia Molecular Aplicada as Micobact?rias. Rio de Janeiro, RJ, Brazil / Institute of Tropical Medicine. Mycobacterium Unit. Antwerp, Belgium. Universidade Federal do Par?. Instituto de Ci?ncias Biol?gicas. Laborat?rio de Polimorfismo de DNA. Bel?m, PA, Brazil. Universidade Federal do Par?. N?cleo de Medicina Tropical. Bel?m, PA, Brazil. Univ. Paris-Sud. Institut de Biologie Int?grative de la Cellule. Univ. Paris-Saclay, France. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil. Universidade Federal do Rio de Janeiro. Instituto de Microbiologia. Rio de Janeiro, RJ, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Ananindeua, PA, Brasil / Universidade do Estado do Par?. Programa de P?s-gradua??o em Biologia Parasit?ria na Amaz?nia. Bel?m, PA, Brazil. There is only scarce information available on genotypic diversity of the Mycobacterium tuberculosis complex (MTBC) clinical isolates circulating in the Northern part of Brazil, a relatively neglected region regarding research on tuberculosis. We therefore characterized 980 MTBC clinical isolates from the state of Par?, by spoligotyping and data was compared with patterns from around the world, besides analyzing drug susceptibility, and collecting sociodemographic data. We also performed 24 loci MIRU-VNTR typing to evaluate phylogenetic inferences among the East-African-India (EAI) lineage strains. The Geographic Information System analyses were performed to generate a descriptive visualization of MTBC strain distribution in the region. A total of 249 different spoligopatterns primarily belonging to evolutionary recent Euro-American lineages, as well as Central-Asian, Manu and ancestral EAI lineages, were identified, in addition to strains with reportedly unknown lineage signatures. The most frequent lineages were Latin American Mediterranean, T and Haarlem. Interestingly, EAI lineage strains were found in a higher proportion in a significantly higher proportion in comparison with previous studies from South America. Regarding EAI lineage, the absence of spacers 4?9 and 23?24 co-related to 24 loci MIRU-VNTRs may suggest a close evolutionary relationship between such strains in Par? and those prevalent in Mozambique, which might have contributed to the genetic diversity of MTBC strains in this region.

This study was carried out to characterize Mycobacterium tuberculosis population in Ghatampur, Kanpur, North India, by spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTRs) typing. A total of 335 isolates were genotyped by spoligotyping and Central Asian (CAS) sub-lineage was the most prevalent, comprising 59.1% of all isolates. Other lineages were: East-African Indian (EAI) (19.10%), T (5.07%), Beijing (3.28%), Manu (2.98%), X (2.68%), S (0.89%), H3 (0.59%), Ural (0.59%), LAM 9 (0.29%) and unknown (5.37%). This data was compared with 8444 clinical isolates from other parts of India and neighboring countries. Thanks to interrogation of the SITVIT2 database, which shows that China is unique in having a predominance of Beijing lineage; Iran in having an almost equal proportion of Ural and CAS lineages; while the rest of the Middle-East and Indian subcontinent shows a gradient of CAS lineage predominating in the north of tropic of cancer, and the ancestral EAI lineage in South India and South-East Asia. Additionally, 12 loci MIRU-VNTR typing efficiently discriminated 13 spoligotype-defined clusters into 92 patterns; 53 isolates showed >70% homology. It was observed that Beijing lineage strains were more frequently associated with MDR strains (p-value = 0.001). A multi-step application of combination of spoligotyping and MIRU-VNTR typing for analyzing the molecular epidemiology of TB may provide a better means of fingerprinting and studying transmission dynamics.

International audience; Emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) isolates is a major public health problem that threatens progress made in tuberculosis (TB) care and control worldwide. In Colombia, the prevalence of MDR tuberculosis (MDR-TB) has increased slowly but steadily since 2001. However, the population structure of the MDR-TB strains circulating in Colombia is sparsely known. In this work, 203 MDR isolates isolated in 2012-2013 were collected, and characterized by spoligotyping, followed by 24-loci MIRU-VNTR (data available for 190 isolates). The most prevalent genotypes corresponded to SIT42/LAM9 (12.81%), SIT62/H1 (10.34%), and SIT190/Beijing (10.34%). A fine analysis showed that although the MDR strains came from 29 of the 33 departments of Colombia, the distribution of these main lineages was not at random and depended on the city of isolation (p-value

Mutations in rpsL, rrs, and gidB are well linked to streptomycin (STR) resistance, some of which are suggested to be potentially associated with Mycobacterium tuberculosis genotypic lineages in certain geographic regions. In this study, we aimed to investigate the mutation characteristics of streptomycin resistance and the relationship between the polymorphism of drug-resistant genes and the lineage of M. tuberculosis isolates in Sichuan, China. A total of 227 M. tuberculosis clinical isolates, including 180 STR-resistant and 47 pan-susceptible isolates, were analyzed for presence of mutations in the rpsL, rrs and gidB loci. Mutation K43R in rpsL was strongly associated with high-level streptomycin resistance (P < 0.01), while mutations in rrs and gidB potentially contributed to low-level resistance (P < 0.05). No general association was exhibited between STR resistance and Beijing genotype, however, in STR-resistant strains, Beijing genotype was significantly correlated with high-level STR resistance, as well as the rpsL mutation K43R (P < 0.01), indicating that Beijing genotype has an evolutionary advantage under streptomycin pressure. Notably, in all isolates of Beijing genotype, a dual mutation E92D (a276c) and A205A (a615g) in gidB was detected, suggesting a highly significant association between this dual mutation and Beijing genotype.

International audience; We argue in favor of a concerted and coordinated response to stop tuberculosis (TB) by monitoring global TB spread, drug-resistance surveillance and populations at risk using available molecular and web tools to identify circulating clones of Mycobacterium tuberculosis complex (MTBC). We took specific example of the Beijing lineage associated with worldwide emergence of both multiple, and extensively drug resistant (MDR/XDR)-TB. The study dataset (n=10,850 isolates, 92 countries of patient origin) was extracted from our multimarker SITVIT2 database on MTBC genotyping (n=111,635 isolates, 169 countries of patient origin). Epidemiological and demographic information in conjunction with spoligotyping (n=10,850), MIRU-VNTR minisatellites (n=2896), and drug resistance (n=2846) data was mapped at macro-geographical (United Nations subregions) and country level, followed by statistical, bioinformatical, and phylogenetical analysis. The global male/female sex ratio was 1.96, the highest being 4.93 in Russia vs. range of 0.8-1.13 observed in Central America, Caribbean, Eastern Africa and Northern Europe (p < 0.0001). The major patient age-group was 21-40 yrs worldwide except Japan (with majority of patients >60 yrs). Younger patients were more common in South America, South Asia, and Western Africa since 25-33% of TB cases due to Beijing genotype occurred in the age group 0-20 yrs. A continuous progression in the proportion of MDR and XDR strains is visible worldwide since 2003 and 2009 respectively. Pansusceptible TB mainly concerned older patients >60 yrs (44%) whereas Drug resistant, MDR and XDR-TB concerned patients preferentially aged 21-40 yrs (between 52 and 58%). Although the proportion of SIT1 pattern vs. other patterns was very high (93%); the proportion of MDR was highest for an emerging genotype SIT190 (p < 0.0001). Lastly, proportion of pansusceptible strains was highest in Japan, while MDR/XDR strains were most common in Russia and Northern Europe. We underline remarkable macro/micro-geographical cleavages in phylogenetic and epidemiologic diversity of Beijing genotype, with phylogeographical specificity of certain genotypes.

In this study we used spoligotyping and 15-loci MIRU-VNTRs for a finer characterization of Mycobacterium tuberculosis strains isolated from patients residing in Guyana (n = 74) and Suriname (n = 80). The mean age of the patients was 38.5 years (36.5 and 40.2 years for Guyana vs. Suriname), with a male-to-female sex-ratio of 2.25 for Guyana vs. 4.27 in Suriname. Spoligotyping and 15-loci MIRU-VNTRs led to a total of 41 and 65 different patterns respectively, with an overall clustering rate of 83.8% vs. 68.8%. Combined spoligotyping and VNTR analysis led to the detection of 18 clusters of 2–41 isolates, with an overall clustering of 67.5% and a recent “n − 1” transmission rate of 55.8%. Importantly, Guyana was characterized by a significantly higher percentage of clustered isolates than Suriname (79.7% vs. 56.3%; p = 0.0019). Three big spoligo/MIRU (SIT/MIT) clusters containing >10 isolates each were shared between the 2 countries, and concerned: T1 sublineage cluster 53/861 (n = 41, 37 in Guyana vs. 4 in Suriname); EAI6-BGD1 sublineage cluster 1340/860 (n = 16, 3 in Guyana vs. 13 in Suriname); and T1 sublineage cluster 131/146 (n = 11, 6 in Guyana vs. 5 in Suriname); as well as 2 smaller clusters of 2 and 3 isolates respectively. However, the relative phylogeographical specificities of strains in circulation as well as a lack of drug-resistance observed among strains from Suriname suggested that trans-border transmission of drug-resistant isolates occurred less frequently than thought. Tracing and interrupting transmission channels of a specific clone (SIT53/15-MIT861) should become a priority in Guyana, not only because it is by far most abundant but also because it accounts for almost half of the drug resistant isolates (n = 8/17, 47.1%) in our study, and clustered 5/12 (41.7%) MDR isolates.

International audience; We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world.

In the present study, Mycobacterium tuberculosis complex (MTBC) clinical isolates from culture-positive TB patients in Morocco were studied by spoligotyping and 12-loci MIRU-VNTR typing methods to characterize prevalent genotypes (n = 219 isolates from 208 patients). Spoligotyping resulted in 39 unique patterns and 167 strains in 30 clusters (2-50 strains per cluster). Comparison with international database showed that 29 of 39 unique patterns matched existing shared spoligotype international types (SITs). Nine shared types containing 10 strains were newly created (SIT 2891 to SIT 2899); this led to the description of 69 SITs with 206 strains and two orphan patterns. The most prevalent spoligotype was SIT42 (LAM; n = 50 or 24% of isolates). The repartition of strains according to major MTBC clades was as follows LAM (46.1%)> Haarlem (26%) >ill-defined T superfamily (22.6%) and S clade (0.96%). On the other hand, Beijing, CAS (Central Asian) and EAI (East-African Indian) strains were absent in this setting. Subsequent 12-Loci MIRU typing resulted in a total of 25 SIT/MIT clusters (n = 66 isolates, 2-6 isolates per cluster), with a resulting recent transmission rate of 22.3%. The MIRU-VNTR patterns corresponded to 69 MITs for 138 strains and 46 orphan patterns. The most frequent patterns were MIT43 (n = 8), MIT9 (n = 7) and MIT42 (n = 7). HGDI analysis of the 12 MIRU loci showed that loci 10, 23 and 40 were highly discriminative in our setting. The results also underlined the usefulness of spoligotyping and MIRU-VNTR to detect mixed infections among certain of our TB patients. Globally, the results obtained showed that TB is almost exclusively transmitted in Morocco through evolutionary-modern MTBC lineages belonging to principal genetic groups 2/3 strains (Haarlem, LAM, T), with a high level of biodiversity seen by MIRU typing. This study provides with a 1st global snapshot of MTBC population structure in Morocco, and validates the potential use of spoligotyping in conjunction with minisatellites for future investigations in Morocco that should in future ideally include optimized 15- or 24-loci MIRU-VNTRs.

summary Kyrgyzstan is a post-Soviet country in Central Asia marked with high incidence and mortality rates of tuberculosis (TB). The present study provided first assessment of Mycobacterium tuberculosis population structure and drug-resistance in civilian population here. The collection included 103 M. tuberculosis DNA samples subjected to the analysis of rifampin and isoniazid resistance mutations and spoligotyping. The major spoligotype-defined families were Beijing (n ¼ 62), T (n ¼ 14), LAM (n ¼ 9), Ural-2 (n ¼ 6) and Ural1( n ¼ 3). Genotypically, 20 isolates were RIF-resistant, 28 were INH-resistant, 17 were multidrugresistant. Drug resistant isolates were more prevalent among Beijing than non-Beijing groups (P ¼ 0.03). The predominance of the mainly “Russian” spoligotypes among the non-Beijing strains (LAMRUS and Ural-1) in this study along with previously demonstrated prevalence of the Russia-specific subtype of the Beijing family in Kyrgyz prison (Mokrousov et al., 2009) suggest that the current population structure of M. tuberculosis in Kyrgyzstan has been mainly formed within the course of the 20th century when the country was a part of the Russian Empire and Soviet Union. On the other hand, a prevalence of the Asia-specific Ural-2 type in the oldest age group (68e85 years old; P < 0.0001) may present a heritage of the more distant historical events. In summary, we suggest: (i) a clear shift of the local M. tuberculosis population structure during the last 100 years and (ii) a critical impact of the Beijing genotype on the current situation with drug resistant TB in Kyrgyzstan.

Tuberculosis (TB) continues to be a major health problem in India, and there is very little information about the prevalent genotypes of tubercle bacilli that cause TB in India, especially in Kerala. Our aim was to study the different circulating strains of Mycobacterium tuberculosis (MTB) that are prevalent in Kerala, India. We analyzed 168 MTB isolates from as many pulmonary TB patients using IS6110-RFLP, spoligotyping and MIRU-VNTRs. The results of IS6110-RFLP revealed that majority of isolates had null copy (10.89%) or single copy (44.87%) of IS6110 insertion. Low copy (

Genotypic classification in Mycobacterium tuberculosis has greatly contributed to the comprehension of phylogenetic and population genetic relationships. It is, therefore, necessary to verify the robustness of the genetic markers for phylogenetic classification. In this study, we report some examples of homoplasy for two molecular markers, the IS6110 insertion at the NTF region, and a single nucleotide polymorphism (SNP) at locus 909166, through genotyping of 1054 Beijing family strains. Our data revealed that a small fraction of strains traditionally classified into modern sublineages by IS6110 insertion at NTF actually belong to an ancient sublineage. We also proved that the robustness of branches in the evolutionary tree established using the putative homoplasious SNP 909166 is relatively low. Our findings highlight the importance of validating genetic markers used to establish phylogeny, evolution, and phenotypic characteristics.

Resume Introduction Les migrants residant en Guadeloupe sont originaires pour la plupart d’entre eux des iles de la Caraibe, dont certaines presentent une forte incidence de la tuberculose. L’objectif de notre etude etait de definir les caracteristiques epidemiologiques de la tuberculose affectant les sujets migrants et natifs en Guadeloupe. Methodes et patients Dans cette etude retrospective et observationnelle, nous avons recense 128 cas de tuberculose survenus en Guadeloupe, du 1er juillet 2006 au 30 juin 2011. Resultats L’incidence de la tuberculose parmi les migrants en Guadeloupe etait sept fois plus elevee que celle des sujets natifs en 2010 (33,4 vs 5,5 nouveaux cas/100 000 habitants). Par rapport aux patients natifs, la tuberculose du sujet migrant se caracterisait par des patients plus jeunes (42 vs 55 ans), et, une proportion importante de co-infection par le virus de l’immunodeficience humaine (VIH) (47 vs 14 %, p Conclusions La population migrante en Guadeloupe constitue dans cette etude un groupe a risque de tuberculose qu’il faudra considerer comme une cible prioritaire des actions de lutte antituberculeuse.

Among various genotyping methods to study Mycobacterium tuberculosis complex (MTC) genotypic polymorphism, spoligotyping and mycobacterial interspersed repetitive units-variable number of DNA tandem repeats (MIRU-VNTRs) have recently gained international approval as robust, fast, and reproducible typing methods generating data in a portable format. Spoligotyping constituted the backbone of a publicly available database SpolDB4 released in 2006; nonetheless this method possesses a low discriminatory power when used alone and should be ideally used in conjunction with a second typing method such as MIRU-VNTRs for high-resolution epidemiological studies. We hereby describe a publicly available international database named SITVITWEB which incorporates such multimarker data allowing to have a global vision of MTC genetic diversity worldwide based on 62,582 clinical isolates corresponding to 153 countries of patient origin (105 countries of isolation). We report a total of 7105 spoligotype patterns (corresponding to 58,180 clinical isolates) - grouped into 2740 shared-types or spoligotype international types (SIT) containing 53,816 clinical isolates and 4364 orphan patterns. Interestingly, only 7% of the MTC isolates worldwide were orphans whereas more than half of SITed isolates (n=27,059) were restricted to only 24 most prevalent SITs. The database also contains a total of 2379 MIRU patterns (from 8161 clinical isolates) from 87 countries of patient origin (35 countries of isolation); these were grouped in 847 shared-types or MIRU international types (MIT) containing 6626 isolates and 1533 orphan patterns. Lastly, data on 5-locus exact tandem repeats (ETRs) were available on 4626 isolates from 59 countries of patient origin (22 countries of isolation); a total of 458 different VNTR patterns were observed - split into 245 shared-types or VNTR International Types (VIT) containing 4413 isolates) and 213 orphan patterns. Datamining of SITVITWEB further allowed to update rules defining MTC genotypic lineages as well to have a new insight into MTC population structure and worldwide distribution at country, sub-regional and continental levels. At evolutionary level, the data compiled may be useful to distinguish the occasional convergent evolution of genotypes versus specific evolution of sublineages essentially influenced by adaptation to the host. This database is publicly available at: http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE.

International audience; China ranks second next to India among 22 high-burden countries despite decades' effort on tuberculosis (TB) control. The Sichuan province today contains the second-largest number of TB cases among Chinese provinces, where the prevalence of drug-resistant TB, especially MDR-TB, is much higher than the average level in eastern China. In this study, the population structure and the transmission characteristics of drug-resistant TB in Sichuan province were studied by spoligotyping and 24-locus Mycobacterial interspersed repetitive units-variable number tandem DNA repeats (MIRU-VNTR), applied to a total of 306 clinical isolates. Spoligotyping-based analysis showed that Beijing family represented 69.28% of all isolates and constituted the largest group (66.24%) of MDR-TB in Sichuan. The remaining isolates, accounting for 33.76% of MDR isolates, belonged to the ill-defined T family, Manu2, H3, LAM9, and other minor unassigned clades. The discriminatory power evaluated for spoligotyping was poor (HGI=0.595), but high for 24-locus MIRU-VNTRs (HGI=0.999). The number of the most discriminatory loci (h>0.6) was 12, including locus 424, 802, 960, 1644, 1955, 2163b, 2996, 3007, 3192, 3690, 4348 and 4052. It was concluded that 24-locus MIRU-VNTRs could be a more discriminatory tool for differentiating clinical isolates from Sichuan region. The small clustering size obtained from the current population structure analysis suggested that the high prevalence of drug-resistant TB in this region might be attributed partially to the acquired resistance due to inappropriate drug use rather than active transmission of drug-resistant TB (primary resistance).

We report on a fine molecular and phylogenetical characterization of circulating Mycobacterium tuberculosis strains isolated from patients during a 1-year period in Trinidad and Tobago (TT). The spoligotyping data coupled to minisatellite typing and available epidemiological data showed that a single major clone of "evolutionary modern" tubercle bacilli (SIT566) was responsible for more than half of the tuberculosis (TB) cases. It preferentially infected younger age groups (mean 39.1 years versus 47.7 years for other genotypes, p0.0005), and was overrepresented in Port-of-Spain (1 out of 3 patients). A comparison of genotyping results to data gathered for 6 Caribbean countries (n=2653 clinical isolates) showed that the overall lineage distribution in TT was completely different from its neighbors, e.g., TT was the only country harboring a unique sublineage of the Latin AmericanMediterranean (LAM) family, designated LAM-10CAM with phylogeographical specificity for Cameroon and neighboring countries in West Africa; interestingly 3/4 of the patients within this group in TT were African descendants. Similarly, strains belonging to East African Indian (EAI) lineage with phylogeographical specificity for the Indian subcontinent, were found in TT (13% of all strains), but were absent among the neighboring countries. Although the predominant SIT566 was not yet detected elsewhere in the Caribbean, available information underlined that this genotype was already present in the United States as imported cases of disease among TT-born patients. Characterization of SIT566 strains using 12-, 15- and 24-loci MIRU typing, and comparison of results to international databases showed that these isolates were characterized by a common 12-loci MIRU pattern 224315153324 corresponding to MIRU International Type-MIT633 in 21/25 strains tested, as well as its 4 variants; an orphan pattern , MIT27-, MIT117-, and MIT1158-. Extended 24-loci MIRU typing led to a predominant pattern 224315153324323483334323 in a total of 16/21 MIT633 isolates, as well as identification of 3 supplemental patterns. Comparison of 24-loci MIRU data with the international database MIRU-VNTRplus showed the unique nature of the patterns obtained in TT. Further analysis using the Levenshtein algorithm showed that the first 2 closest matches with the SIT566/MIT633 clone belonged to the X lineage strains in MIRU-VNTRplus. This observation corroborates our preliminary spoligotyping-based analysis using minimum spanning and neighbor-joining trees, which suggested a phylogenetical relatedness of the SIT566 clone with SIT119, which represents X1 lineage prototype in SpolDB4 database. We hypothesize that the predominant SIT566 clone might have evolved from a pool of X lineage M. tuberculosis strains with phylogeographical affinity for Anglo-Saxon descendants.

The most prevalent strain of Mycobacterium tuberculosis in Madrid, Spain (strain 5) was recovered from 45 cases between 1997 and 2004 and showed a highly homogeneous genetic composition. This strain was not exclusive to Spain, and its spoligotyping signature (ST20) was found in entries from different countries in the SITVIT1 database. Patients infected with strain 5 were more frequently positive for human immunodeficiency virus and autochthonous, and had been in prison more frequently, but strain 5 did not show increased infectivity in an in-vitro model of infection.

PubMedID: 17462962 To highlight the transmission rate and major phylogenetic clades of drug-resistant Mycobacterium tuberculosis isolates, a total of 200 drug-resistant strains isolated in four different regions of Turkey (Marmara n = 81; Mediterranean n = 39; Aegean n = 42; East Anatolia n = 38), were typed by spoligotyping and IS6110-restriction fragment length polymorphism (RFLP). The major spoligotyping-defined shared-types (STs) and corresponding lineages were, ST 41 (22.5%, LAM7-TUR), ST53 (19.5%, ill-defined T super-family), ST 50 (6.5%, Haarlem 3), ST 1261 (4.5%, LAM7-TUR), ST 47 (3.5%, Haarlem 1), as well as two STs that belonged to undefined clades (ST 284, 3%, and ST 2067, 2.5%). The global distribution of major M. tuberculosis lineages among drug-resistant strains was as follows: T super-family (29%), Latin-American & Mediterranean (33.5%), Haarlem (14%), and the S lineage (3%). A high number of strains (n = 29, 14.5%) showed patterns that did not fall within major clades described so far. A combination of spoligotyping and IS6110-RFLP fingerprinting methods resulted in a final clustering rate of 38.5% and a recent transmission rate of 25.5%. Our results underline the highly diverse nature of drug-resistant tuberculosis in our study population, as well as its ongoing transmission with lineages that are specific to these regions, the most predominant being the LAM7-TUR lineage which shows an enhanced phylogeographical specificity for Turkey. © 2007 Elsevier B.V. All rights reserved. T.Z. received a Ph.D. fellowship awarded by European Union and the Regional Council of Guadeloupe and the International Network of the Pasteur Institutes. The international database project coordinated by Institut Pasteur de Guadeloupe has benefited by an active feedback from various research groups worldwide and currently financed through the European Union and the Regional Council of Guadeloupe, which is greatly acknowledged.

Spoligotyping was performed on 540 Mycobacterium tuberculosis isolates in order to evaluate the genetic biodiversity of tubercle bacilli in India. One hundred and forty seven patterns were unique and 393 were grouped in 48 clusters. Comparison with an international spoligotype database showed that the most predominant clades among tuberculosis (TB) isolates were Central Asian (CAS) and East-African Indian (EAI) with shared-types (ST) ST26 and ST11 alone being responsible for 34% of all TB cases. Twenty one (3.8%) isolates belonged to the Beijing genotype. Marked variations were observed among circulating strains, STs belonging to CAS family predominated in the North, whereas the EAI family was more common in the Southern India. TB in India is predominantly caused by strains belonging to the principal genetic group 1 (PGG1), suggesting that most of the TB burden in India may be traced to ancestral clones of the tubercle bacilli. This study gives an insight into the global M. tuberculosis genetic biodiversity in India, the predominant spoligotypes and their impact on disease transmission.

Despite well-developed tuberculosis (TB) control policies in Madagascar, the incidence of TB remains high and is estimated at about 100 new cases per 100 000 inhabitants. This paper describes genetic characteristics of TB bacilli in Madagascar. Using an international spoligotyping database, SpolDB4, we also attempted to identify the origin of strains circulating in Madagascar. DNA polymorphism of 333 Mycobacterium tuberculosis complex isolates was assessed. A total of 301 isolates belonging to 60 spoligotyping-defined clusters were found, whereas 32 isolates harbored orphan patterns. By comparison with the international database, we identified a new genetic group of closely genetically related M. tuberculosis strains which we suggested to be specific from Madagascar. Most of them belonging to the East-African-Indian (EAI) superfamily of strains that are responsible for 14% of total TB cases (shared types ST1514-1525). These strains are closely related to the most prevalent shared type ST109, whose distribution is mainly confined to Madagascar. The observed distribution of genotypes shows that principal genetic group 1 strains (EAI, Beijing, CAS, Afri, “Manu”) is high (35.4%) suggesting an ancient evolutionary history of tuberculosis in Madagascar, in relation to the origin of peopling and the demographic history.

Tuberculosis remains a major health problem in India, with 2 million new cases and 421,000 deaths each year. In this paper, we describe the spoligotyping results of 216 Mycobacterium tuberculosis culture isolates from patients with pulmonary tuberculosis in Mumbai, India. As spoligotyping data from India have rarely been described until now, and as there is limited information on the major circulating clades of M. tuberculosis, the data obtained were also compared to an international spoligotype database (SpolDB4) that contained patterns from 22,546 isolates from more than 100 countries. Eighty-four (39%) of the isolates were definitively marked as orphan strains, indicating the paucity of such data from India. The remaining 132 isolates clustered among 59 shared types; among these, 42 shared types were already present in the database, 17 were newly created, and 5 of them were specifically reported from Mumbai. A total of 9 major types in this study clustered 32% of the isolates. At the phylogenetic level, 30% of the isolates belonged to the Central Asian families CAS1 and CAS2, of the major genetic group (MGG) 1, 29% to MGG 2 and 3 families (spacers 33-36 missing) and 17% to the ancestral East African Indian (EAI) family. Finally, nearly 10% of the isolates belonged to the W-Beijing family in a broad sense, also in the MGG 1 group. In conclusion, historic clones of the MGG 1 group of M. tuberculosis are responsible for roughly 60% of all tuberculosis cases in Mumbai. Together with the fact that organisms presumably of European descent (such as the Haarlem family) were only rarely found, our observations suggest that tuberculosis in Mumbai, India is essentially caused by historical clones of tubercle bacilli undergoing active circulation due to uncontrolled demography, high prevalence of the disease, and a paucity of resources.

Resume La tuberculose est une maladie infectieuse et contagieuse en pleine recrudescence et dont la surveillance epidemiologique a ete renforcee par des techniques de biologie moleculaire. Dans ce contexte, nous nous sommes particulierement interesses aux cas de tuberculose de la region Antilles-Guyane sur une periode de deux ans (1997 et 1998) par l’analyse de leurs genotypes. Nos resultats ont ete confrontes a une region metropolitaine (l’Aquitaine) equivalente demographiquement avec des liens privilegies vis-a-vis de notre region et beneficiant d’un reseau de surveillance pour la tuberculose comparable au notre. Ainsi, nous avons employe la methode PCR appelee spoligotypage, utilisee en premiere intention afin d’optimiser la methode IS6110–RFLP appliquee depuis plusieurs annees mais demeurant contraignante. Ainsi 105 souches des Antilles-Guyane et 172 souches d’Aquitaine ont ete typees par spoligotypage et par la methode de reference IS6110–RFLP. Les resultats du premier groupage par spoligotypage ont fait l’objet d’une analyse comparative par les empreintes genomiques obtenues par IS6110–RFLP. Ainsi le taux de transmission recente a ete estime a 34,3 % dans la region Antilles-Guyane et a 10,5 % en Aquitaine lui conferant un degre de polymorphisme important. Grâce au differents profils obtenus par spoligotypage, nous avons pu etudier leur repartition dans les trois differentes regions et mettre en evidence des types qui leurs sont communs comme les types 53, 50 et 42, des types plus localises comme les types 33 et 14 retrouves respectivement en Aquitaine et aux Antilles ainsi que des types endemiques comme le 76 retrouve uniquement en Guyane. De ce fait, nous avons pu emettre des hypotheses quant a l’evolution des souches au cours du temps.

The recent introduction of molecular methods has gained increased acceptance as a powerful tool for epidemiology and phylogeny of tuberculosis (TB). In this investigation, the efficiency of molecular typing using mycobacterial interspersed repetitive units (MIRUs) was assessed on a set of 116 Mycobacterium tuberculosis complex clinical isolates from 11 different geographic origins. The results obtained were compared with spoligotyping and variable number of tandem DNA repeats (VNTRs) typing data. Eighty-nine different MIRU profiles were obtained on the sample studied. Spoligotyping- or VNTR-defined clusters were split into subclusters by MIRU typing. Conversely, almost all of the clinical isolates clustered by MIRUs were shown to belong to spoligotyping-based defined clusters. The calculation of the discriminative power by the Hunter-Gaston index (HGI) for VNTR, spoligotyping and MIRU typing gave the values of, respectively, 0.959, 0.965 and 0.988, showing the high discriminative power of the MIRUs. The allelic diversity of the sample was calculated for each of the MIRU-VNTR loci; five MIRU loci (MIRU nos. 10, 23, 26, 31 and 40) were "highly discriminant", four (MIRU nos. 4, 16, 24 and 39) were "moderately discriminant", and three (MIRU nos. 2, 20 and 27) were "poorly discriminant". Among the three complementary VNTRs (exact tandem repeats ETR-A, ETR-B and ETR-C), ETR-A was the most discriminant locus. A combined numerical analysis of spoligotyping, VNTR and MIRU typing results partly corroborated a recently hypothesized evolutionary scenario for the M. tuberculosis complex. M. canettii would be the first branch to have diverged from a common M. tuberculosis complex ancestor. The East-African Indian (EAI) clade could be the first family to have diverged thereafter. A third branching separated a M. africanum-M. bovis clade, followed by a node separating Beijing versus non-Beijing M. tuberculosis. The Beijing clade was distinct from the Central Asian 1 (CAS1) family. Among non-Beijing strains, branches such as the Latin-American and Mediterranean (LAM), X and Haarlem clades diverged later. In conclusion, the results obtained show the congruence between clades defined by spoligotyping, and MIRU-VNTR, and underline the potential of these methods for M. tuberculosis phylogeny reconstruction. We also conclude that MIRU typing is a very promising method that may be used in a "two PCR-based" genotyping strategy, in conjunction to conventional epidemiological investigations.

Resume Les nouvelles methodes de typage moleculaire completent efficacement les enquetes epidemiologiques classiques autour des cas de tuberculose, permettant d’envisager une approche globale du controle de certaines maladies infectieuses. Dans ce travail, nous avons etudie la diversite genetique des souches de bacilles tuberculeux isoles au cours de l’annee 1998 dans l’agglomeration angevine (260 000 habitants repartis en 29 communes), afin d’identifier des eventuelles grappes de bacilles identiques, suggestives de cas de transmission recente. Nous avons employe une strategie d’association de 3 methodes de typage par PCR (typage par oligonucleotides des espaceurs du locus DR ou spoligotyping, nombre variable des repetitions ADN en tandem ou VNTR, et PCR d’elements repetitifs doubles ou DRE-PCR). Une fois finalises, les fichiers de resultats de spoligotyping et de VNTR ont ete introduits dans des bases de donnees internationales creees et regulierement mises a jour a l’Institut Pasteur de Guadeloupe, et qui comptent actuellement environ 10 000 profils de spoligotypes et 500 profils de VNTR, representatifs d’une soixantaine de pays. Il apparait que la tuberculose de transmission recente est rare dans la region angevine et que la reactivation de cas contractes par le passe semble constituer la premiere cause de tuberculose dans cette region. Dans l’ensemble, les souches presentes dans cette region appartiennent majoritairement a des familles conservees de souches de bacilles tuberculeux typiquement rencontrees en Europe. Quelques cas de tuberculose d’origine asiatique et de rares cas specifiques d’epidemies decrites dans le monde sont egalement mis en evidence.

In this investigation, 94 clinical isolates of Mycobacterium tuberculosis obtained over a 3-year period (1996-1998) from French Guiana were characterized by spoligotyping and IS6110-RFLP and the patterns obtained were compared with genotypes representing the worldwide diversity in an international spoligotyping database (n = 4269) and a IS6110-RFLP database (n = 4189). All the clustered isolates giving < or = 6 copies of IS6110 were further typed using the double-repetitive element (DRE)-PCR. The results obtained underlined the highly diverse nature of the M. tuberculosis population in French Guiana with potential links to neighboring countries within the Americas. It may be hypothesized that the genetic heterogeneity of tubercle bacilli in French Guiana is linked to the high number of imported cases of tuberculosis, that may account for as high as 68% of all tuberculosis cases. Although an epidemiological investigation based on direct interrogation of patients was not performed, available medical records suggested that the clustering of isolates was mostly linked to the following risk factors: pulmonary tuberculosis, smear-positive samples, foreign-born nationals and/or immigrants, and a high rate of HIV-TB coinfection. Thus the persisting foci of endemic disease and increased active transmission due to high population flux and HIV coinfection may be largely responsible for the relatively high incidence of tuberculosis in French Guiana.

A sudden upsurge in the isolation of Mycobacterium simiae from terminally ill AIDS patients was recently reported on the Caribbean island of Guadeloupe. Identification of these M. simiae isolates was achieved using biochemical tests and further confirmed by PCR-restriction analysis (PRA) of a 439-bp fragment of hsp 65. A novel PRA profile III (three Bst EII fragments of 240/125/80 bp and four Hae III fragments of 145/125/40/25 bp) was observed in four blood isolates from two patients. The 16 S rRNA gene sequencing of the hypervariable A region confirmed that all the pattern III isolates were indeed M. simiae species, and the hsp 65 sequencing confirmed the existence of a new hsp 65 allele in these caribbean isolates. A hsp 65 sequence-based phylogenetic tree was also created for 39 species including M. simiae and related mycobacterial species as well as other rapid and slow growing mycobacteria, and may serve as an useful tool for identification of mycobacteria to species level.

The interaction of various pathogenic (Mycobacterium tuberculosis, M. avium, M. kansasii, M. xenopi), and non-pathogenic mycobacteria (M. smegmatis, M. phlei) with human macrophages at the level of macrophage cytokine expression (TNFalpha, IL1, IL6 and GM-CSF) was investigated. Both for TNFalpha and GM-CSF, the lowest levels were obtained with pathogenic mycobacterial species, whereas about 2-8 times higher levels were observed for non-pathogenic species. Contrary to the above, the differences for IL6 and IL1 were not marked, although IL6 appeared to be more elevated for non-pathogenic species. Heat-killed bacteria induced a lower level of the cytokines for all the three cytokines assayed (TNFalpha, IL6 and IL1), except for M. tuberculosis for whom a significantly higher proportion of TNFalpha was induced by killed bacilli. The RT-PCR experiments performed on M. avium (as a low inducer of the cytokines) and M. smegmatis (as a high inducer of the cytokines) showed that the differences observed among pathogenic vs non-pathogenic strains were also reflected at the transcriptional level for TNFalpha and to a lesser extent for IL6, but not for IL1. This investigation underlined important differences existing between the pathogenic and non-pathogenic species, particularly as regards TNFalpha and GM-CSF.

Mycobacterium tuberculosis clinical isolates (113 isolates from 78 patients) were typed using IS6110-RFLP, DR-RFLP, DR-based spoligotyping and direct repetitive element PCR (DRE-PCR). The similarities among isolates were compared for each individual method. The individual matrix distance files for each method were summed and averaged, and the resulting unique distance file was analysed by the UPGMA (unweighted pair group method with arithmetic averages). Combined numerical analysis with 3 genetic markers (IS6110-RFLP, DR-RFLP and spoligotyping) was performed for all 78 clinical isolates, whereas analysis with 4 genetic markers (with the addition of DRE-PCR) was performed on the 10 main clusters described. When compared to molecular analysis based on individual markers, the molecular description based on multiple genetic markers enabled comparison of the results obtained by individual methods and the obtaining of a more accurate view of strain identity and clusters comparison. The resulting cumulative dendrogram was more accurate for studying the population structure of M. tuberculosis and may be a good tool for elucidating intraspecies genetic microevolution.

Antimycobacterial drugs acting efficiently against Mycobacterium avium complex have in common low MICs and MBC/MIC ratios. The recently reported clinical efficacy of some of the newer drugs is also clearly linked to their pharmacokinetic properties such as higher serum level and/or intracellular concentrations and half-life. In the present investigation, comparative postantibiotic effects (PAEs) of amikacin, rifampin, sparfloxacin, clofazimine and clarithromycin were investigated. Bacteria were exposed to MIC, MIC x 4 and MIC x 8 concentrations of each drug for 2 h, the drug was removed by centrifugation and cells were thoroughly washed and resuspended in drug-free medium. Growth was compared to control organisms which underwent a similar treatment (but without drugs) and PAEs were assessed using the equation "T-C", where T equals the time required for colony counts to increase by 1 log10 in test samples after antibiotic exposure and C equals the time for 1 log10 growth in control. Our results underlined two distinct patterns concerning PAE: pattern I included drugs for which PAE (in hours) was dose-dependent and varied (for MIC, MIC x 4 and MIC x 8 concentrations) for amikacin (10.3 +/- 1.7, 14.7 +/- 1.9 and 17.7 +/- 4.1), rifampin (28.0 +/- 7.6, 62.0 +/- 18.5 and 71.0 +/- 3.2) and clarithromycin (2.6 +/- 1.0, 15.0 +/- 4.0 and 22.0 +/- 4.0), whereas pattern II included drugs with a stable PAE, relatively independent of the drug concentrations: sparfloxacin (11.0 +/- 2.5, 12.3 +/- 6.4 and 13.0 +/- 2.1) and clofazimine (26.0 +/- 2.8, 28.8 +/- 2.5 and 27.3 +/- 1.3). These results may be useful for guidance in scheduling of drug administration in M. avium-infected AIDS patients overburdened with too many drugs given for various opportunistic infections.

Summary A total of 33 clinical isolates of the Mycobacterium avium complex from 25 patients, identified by means of biochemical and cultural characteristics, the Accuprobe system and DT1/DT6 PCR, were further analysed using novel insertion elements IS 1245 and IS 1311 in a French Caribbean setting. PvuII-cleaved DNA and non-radioactive Southern hybridization and detection systems were used for fingerprinting with both IS elements. The data confirmed the specificity of the two probes for M. avium in our setting and highlighted a significant proportion of M. intracellulare-infected patients in this region. Two distinct groups composed of 2–3 bands and 6–27 bands were found among M. avium isolates, and were composed of the same isolates both with IS 1245 and IS 1311. The computer analysis of polymorphic banding patterns identified two prevalent genotypes: one contained 4 isolates from 3 patients while a second 2-banded cluster was composed of 6 isolates from 4 patients; all the patients were from the same hospital in Guadeloupe. A single isolate from Martinique was falsely included in the 2-banded cluster initially upon IS 1245 fingerprinting, but could be discriminated from other isolates on the basis of IS 1311 fingerprinting of PvuII-cleaved DNA. These results were also confirmed upon IS 1245 fingerprinting of Pstl-digested DNA, as well as DT6 fingerprinting. A single case of polyclonal infection was also discovered in a patient at a 75-day interval. This is the first study comparing the two IS elements and constitutes a first description of dissseminated M. avium complex disease from the Caribbean. We conclude that both elements possess a similar discriminatory potential for M. avium isolates. Coupled with computer analysis, this methodology would appear to be particularly suitable for larger epidemiological studies.

As our understanding of the mycobacterium avium complex (MAC) progresses, it is clear that the ability of this organism to survive and modulate immune responses in the host is related to the architecture of the cell envelope and the constituents contained therein. Because these constituents are among the initial factors contributing to host responsiveness, it is important to identify specific cell envelope components and characterize their ability to modulate immune responses in the infected host. In addition, it is apparent that cell envelope architecture is instrumental in confirming drug resistance, particularly after M. avium has been phagocytosed and multiplies intracellularly

The in vitro activity of 13 drugs against 552 clinical isolates of atypical mycobacteria representing 12 species was performed in 7H11 agar medium at the National Reference Laboratory for Mycobacteria, using the 1% proportion method. All the species tested were resistant to isoniazid and pyrazinamide. In general, clofazimine and D-cycloserine showed the widest spectrum of activity except in the case of Mycobactrium fortuitum and M. chelonei which were resistant to both drugs, and the M. szulgai and M. terrae complex which was resistant to D-cycloserine. The next broad-spectrum drug was ethionamide, followed by ansamycin, rifampin, capreomycin, kanamycin, streptomycin and ethambutol. Among the fluoroquinolones, both ciprofloxacin and ofloxacin were active against M. xenopi, M. gordonae and M. fortuitum whereas M. kansasii and M. gastri were sensitive to ofloxacin only. When the species were listed in respect of the number of drugs to which they were susceptible (less than 10% of resistant strains), they were classified as follows; 7/13 drugs for M. kansasii, M. gastri and M. xenopi; 6/13 for M. gordonae; 5/13 for M. marinum; 3/13 for M. szulgai; 2/13 for M. fortuitum; 1/13 for the M. avium, M. scrofulaceum, M. simiae, and M. terrae complex, and none of the 13 in the case of M. chelonei. These results are discussed in relation to the multiple drug resistance of atypical mycobacteria. We conclude that the critical concentrations of drugs established for M. tuberculosis are not appropriate for atypical mycobacteria.

A radiometric method using the "Bactec 460-TB" apparatus now enables rapid obtaining of drug-susceptibility data and differentiation of clinical mycobacterial specimens into those belonging to the Mycobacterium tuberculosis complex (comprised of M. tuberculosis, M. bovis, M. bovis BCG and M. africanum) and non-tuberculous mycobacteria, using a specific inhibitor (p-nitro-alpha-acetyl-amino-beta-hydroxypropiophenone). In the present work, further differentiation of the M. tuberculosis complex into 4 individual species was achieved using the susceptibility of strains to 1.25 micrograms/ml of thiophene-2-carboxylic acid hydrazide, 40 micrograms/ml of D-cycloserine, and 100 micrograms/ml of pyrazinamide.

The J-774 macrophage-like cell line has been established as a model for intracellular multiplication of pathogenic mycobacteria, permitting assessment of the intracellular bactericidal action of the macrophages after addition of both the drugs and immunomodulators. In this study, the action of immunomodulators was investigated. Significant morphological changes were demonstrated under the optical and scanning electron microscope (SEM), and the degree of macrophage activation was also measured by acid phosphatase (AcPase) cytochemistry, release of free oxygen radicals and by their ability to hinder the intracellular multiplication of virulent strains of Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av). For This purpose, the macrophages were left to multiply during 3 days in the presence of 50 U/ml of recombinant murine gamma-interferon (INF), 4 micrograms/ml of 1,25 dihydroxyvitamin D3 (D3) and 50 micrograms/ml of lipopeptide RP-56142 (RP) added separately or in various possible combinations, and these "activated" cells were then challenged with viable bacteria. Parallel controls included bacterial multiplication in nonactivated macrophages and also extracellularly but under the same experimental conditions as in the macrophage experiments. Transmission electron microscopy (TEM) using the AcPase marker to localize phagosome-lysosome fusion (PLF) in infected cells was also performed. Although all the immunomodulators used significantly changed the morphology of treated cells and increased the % of AcPase-positive cells, none had any effect on the release of oxygen radicals. On the other hand, guinea-pig alveolar macrophages which served as a parallel positive control, were activated by INF and D3 (but not RP) to release superoxide anions. Our data suggest that differential killing mechanisms for intracellular M.tb and M.av may exist. The results obtained also showed that established mycobactericidal mechanisms of the host could not solely account for the antimycobacterial effects observed. Consequently, mechanisms not yet revealed may account for some of the antimycobacterial effects observed.

Among new fluoroquinolone derivatives, the N-cyclopropylanalogs Ciprofloxacin (CIPRO) and ofloxacin (OFLO) have recently been found to have a high in vitro activity against a variety of mycobacteria (2). In this study, we have investigated their action against intracellularly growing Mycobacterium tuberculosis. For this purpose, the J-774 macrophage cell line was infected with the H37Rv type strain of M. tuberculosis, and drugs (at their serum level concentrations obtainable in healthy individuals (= 5 micrograms/ml; 8) were added after 2 days of intracellular growth of the bacteria. The bacterial growth during the experimentation was followed both electron microscopically and also by lysing the macrophages at various time intervals, and enumerating the bacterial viable counts after plating the lysate on appropriate media. Both drugs at the concentrations used did not affect macrophage viability (as assessed by trypan blue staining), and were highly bactericidal against the virulent tubercle bacilli multiplying in our J-774 macrophage model.

Mycobacterium leprae extracted and purified from experimentally infected armadillo was coated with rabbit sera raised against the total antigens of the following species of mycobacteria: M. leprae, M. avium, M. bovis BCG, and M. fallax. In addition, the bacteria were also coated either with serum from a lepromatous (LL), or a tuberculoid (TT) leprosy patient. The effectiveness of surface coating was verified by electron microscopy, with the aid of gold immunolabelling. The coated bacilli were phagocytized by mice bone marrow-derived macrophages, and the phagosome-lysosome fusions (PLF) were assessed during phagocytosis using acid-phosphatase (AcPase) cytochemistry. As compared to control preparations (like-wise treated with non-immune serum), significant but partial reversion of PLF inhibition was observed in all cases except when bacteria had been incubated with M. fallax antiserum (rapidly growing, non-pathogenic species). The results obtained suggest that some of the antimycobacterial antibodies may offer partial protection to the host during early events of infection by reverting the usual pattern of inhibition of PLF in infected macrophages.

Cell-free extracts of various strains belonging to Mycobacterium paratuberculosis (Ptb) and "wood-pigeon" (WP) mycobacteria were compared by polyacrylamide gel electrophoresis and the various protein bands obtained were tested for peroxidase enzyme activity. One strain of Mycobacterium avium served as a control. Bacterial extracts were also analysed by crossed immunoelectrophoresis (CRIEP) and fused rocket immunoelectrophoresis (FRIEP) using antisera raised in rabbit against M. paratuberculosis and WP mycobacteria. The immunoprecipitates obtained both in CRIEP and FRIEP plates were subsequently stained for selective peroxidase enzyme staining. Our results showed that, although Ptb and WP mycobacteria shared common peroxidase isoenzymes and antigens, they also had specific immunoprecipitates showing the differences between the two groups of bacteria.

International audience; 9th forum in microbiology

The potential of p-nitrobenzoic acid (PNB) and p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) for discriminating the Mycobacterium tuberculosis (M.tb) complex from other mycobacteria was evaluated in cases of clinical isolates. For this purpose, 500 micrograms/ml of PNB and 5 or 10 micrograms/ml of NAP were incorporated in 7H11 agar medium, and appropriate dilutions corresponding to about 10(6) viable units were then plated on PNB- or NAP-containing media. Results were reported as growth or no growth as compared to a parallel control. Our data reconfirmed the potential of PNB to discriminate M.tb complex bacteria. This study also showed for the first time that the NAP test (used until now in 7H12a broth only) can also be successfully applied using 7H11 agar. Also, preliminary data with 5 micrograms/ml of NAP on a limited number of strains suggested that it may discriminate the M.tb complex bacilli from M. xenopi and M. gastri better than PNB in 7H11 agar.

Summary The antibiotic colistin (polymyxin E) inhibited the lytic cycle of the mycobacteriophage D 29 in the tubercle bacilli, but not the D 29 adsorption. The protein and nucleic acid synthesis in D 29 -infected bacteria were not affected significantly. The inhibitory activity was reversed by washing off the antibiotic, and by addition of Ca ++ , but not in media made iso-osmotic by addition of NaCl or sucrose. Transmission electron microscopy revealed an asymmetric to symmetric transition in the staining profile of the cytoplasmic membrane. Though no mature phage particles were ever observed in colistin-treated, D 29 -infected tubercle bacilli, loosely arranged aggregates resembling phage proheads were occasionally found. Judging from the above data, it was concluded that colistin inhibited D 29 lytic cycle by causing molecular displacements in the inner leaflet of the cytoplasmic membrane, and consequently, the binding sites for D 29 structural proteins were not available.

After phagocytosis by bone-marrow macrophages, Mycobacterium avium was surrounded by a thick electron-transparent zone (ETZ). The use of various fixation and embedding procedures showed that ETZ did not seem to be an artifactual structure. A quantitative assessment of ETZ frequency was performed at different times after infection of macrophages with SmD and SmT colony variants of M. avium. For SmT-variant-infected macrophages, a higher percentage of ETZ+ bacilli paralleled a higher percentage of intact bacilli than was the case for SmD-infected macrophages. Macrophages were also infected with bacteria killed with UV or gamma rays, H2O2, heat or glutaraldehyde. About 50% of bacilli killed with any of these treatments were found ETZ+ instead of 80-85% with live bacteria. Unlike live bacilli, for which the percentage of ETZ frequency remained stable throughout incubation time, ETZ frequency for killed bacilli decreased with time. ETZ assessment performed on M. tuberculosis H37 Rv for comparison showed that, despite a very low ETZ frequency (8-15%), the percentage of intact bacteria was identical to that observed with M. avium. In contrast, three rapidly growing non-pathogenic species (M. smegmatis, M. phlei and M. fallax) presented a low ETZ frequency after phagocytosis and were rapidly degraded. The process of ETZ formation and its role in bacterial survival are discussed.

The extracellular and intracellular activities of 4 antituberculous drugs (rifampicin, streptomycin, ansamycin and kanamycin) and 7 beta-lactam antibiotics (penicillin G, amoxycillin, carbenicillin, ticarcillin, ampicillin, cloxacillin and imipenem) were determined against the bacteria of the Mycobacterium avium complex (MAC). The extracellular activities were determined against 14 strains by the broth-dilution method, whereas the intracellular activities were determined against type strain ATCC 15769 multiplying actively inside murine J-774 macrophages. Six of the beta-lactam drugs (except the beta-lactamase-stable drug imipenem) were also used in association with the beta-lactamase inhibitor clavulanic acid. Our results showed that the screening of MAC strains against beta-lactams may be useful, and that correlation of usual in vitro minimal inhibitory concentrations with the intracellular activities of the same drugs against pathogenic mycobacteria, whenever feasible, would be the method of choice to designate the therapeutic protocols against these intracellular pathogens.

Minimal inhibitory concentrations and minimal bactericidal concentrations of colistin (polymyxin E) were determined for the type strains of fifteen mycobacterial species. Colistin was found to be active against pathogenic species Mycobacterium xenopi, M. intracellulare, M. tuberculosis, M. fortuitum and also against the rapidly growing, non-pathogenic species M. phlei and M. smegmatis. The discriminatory potential of susceptibility to colistin as a test was investigated on 25 strains of the M. fortuitum/M. chelonei complex, and also on 11 strains of the M. avium/M. intracellulare complex. The experimental data indicated the potential of colistin susceptibility testing for discriminating M. fortuitum from M. chelonei.