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Multispacer Sequence Typing for Mycobacterium tuberculosis Genotyping  [PDF]
Zoheira Djelouadji, Catherine Arnold, Saheer Gharbia, Didier Raoult, Michel Drancourt
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002433
Abstract: Background Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis. Methods and Findings In M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing <8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts ≤ 42 cycles in internal transcribed spacer-real time PCR. Conclusions These results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTub?erculosis/mst).
Rapid detection of laboratory cross-contamination with Mycobacterium tuberculosis using multispacer sequence typing
Zoheira Djelouadji, Jean Orehek, Michel Drancourt
BMC Microbiology , 2009, DOI: 10.1186/1471-2180-9-47
Abstract: MST analysis indicated a unique and common sequence profile between a strain isolated from a patient with proven pulmonary tuberculosis and a strain isolated from a patient diagnosed with lung carcinoma. Using this approach, we were able to provide a clear demonstration of laboratory cross-contamination within just four working days. Further epidemiological investigations revealed that the two isolates were processed for culture on the same day.The application of MST has been demonstrated to serve as a rapid and efficient method to investigate cases of possible cross-contamination with M. tuberculosis.The isolation of Mycobacterium tuberculosis complex organisms from clinical specimens collected from suspected patients serves as the gold standard for the proper diagnosis of tuberculosis in the laboratory [1]. However, false-positive cultures have been reported that result from the cross-contamination of specimens via a contaminated bronchoscope [2,3] or, more often, by laboratory cross-contamination [4]. The latter situation has been reported at a frequency ranging from 0.1% to 3% of M. tuberculosis [1,4-8]. Laboratory cross-contamination should be suspected when M. tuberculosis is cultured from a smear-negative specimen processed in the same batch as a culture from a smear-positive specimen. The factors that increase the likelihood of cross-contamination include instances when only one of several specimens from the same patient is culture-positive and instances when the clinician is considering a diagnosis other than tuberculosis, which the clinician believes to be more likely based on clinical observations [8]. Such false-positives resulting from cross-contaminated specimens are disadvantageous since, besides resulting in a misdiagnosis, they result in unnecessary treatment and delay further diagnostic investigations in an effort to derive a definitive and correct diagnosis [9]. Finally, these false-positive cultures lead to an overestimation of the incidence and
Multispacer Sequence Typing Relapsing Fever Borreliae in Africa  [PDF]
Elbir Haitham,Gregory Gimenez,Cheikh Sokhna,Kassahun Desalegn Bilcha,Jemal Ali,Stephen C. Barker,Sally J. Cutler,Didier Raoult,Michel Drancourt
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001652
Abstract: Background In Africa, relapsing fevers are neglected arthropod-borne infections caused by closely related Borrelia species. They cause mild to deadly undifferentiated fever particularly severe in pregnant women. Lack of a tool to genotype these Borrelia organisms limits knowledge regarding their reservoirs and their epidemiology. Methodology/Principal Findings Genome sequence analysis of Borrelia crocidurae, Borrelia duttonii and Borrelia recurrentis yielded 5 intergenic spacers scattered between 10 chromosomal genes that were incorporated into a multispacer sequence typing (MST) approach. Sequencing these spacers directly from human blood specimens previously found to be infected by B. recurrentis (30 specimens), B. duttonii (17 specimens) and B. crocidurae (13 specimens) resolved these 60 strains and the 3 type strains into 13 species-specific spacer types in the presence of negative controls. B. crocidurae comprised of 8 spacer types, B. duttonii of 3 spacer types and B. recurrentis of 2 spacer types. Conclusions/Significance Phylogenetic analyses of MST data suggested that B. duttonii, B. crocidurae and B. recurrentis are variants of a unique ancestral Borrelia species. MST proved to be a suitable approach for identifying and genotyping relapsing fever borreliae in Africa. It could be applied to both vectors and clinical specimens.
The Genome Sequence of ‘Mycobacterium massiliense’ Strain CIP 108297 Suggests the Independent Taxonomic Status of the Mycobacterium abscessus Complex at the Subspecies Level  [PDF]
Yong-Joon Cho, Hana Yi, Jongsik Chun, Sang-Nae Cho, Charles L. Daley, Won-Jung Koh, Sung Jae Shin
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0081560
Abstract: Members of the Mycobacterium abscessus complex are rapidly growing mycobacteria that are emerging as human pathogens. The M. abscessus complex was previously composed of three species, namely M. abscessus sensu stricto, ‘M. massiliense’, and ‘M. bolletii’. In 2011, ‘M. massiliense’ and ‘M. bolletii’ were united and reclassified as a single subspecies within M. abscessus: M. abscessus subsp. bolletii. However, the placement of ‘M. massiliense’ within the boundary of M. abscessus subsp. bolletii remains highly controversial with regard to clinical aspects. In this study, we revisited the taxonomic status of members of the M. abscessus complex based on comparative analysis of the whole-genome sequences of 53 strains. The genome sequence of the previous type strain of ‘Mycobacterium massiliense’ (CIP 108297) was determined using next-generation sequencing. The genome tree based on average nucleotide identity (ANI) values supported the differentiation of ‘M. bolletii’ and ‘M. massiliense’ at the subspecies level. The genome tree also clearly illustrated that ‘M. bolletii’ and ‘M. massiliense’ form a distinct phylogenetic clade within the radiation of the M. abscessus complex. The genomic distances observed in this study suggest that the current M. abscessus subsp. bolletii taxon should be divided into two subspecies, M. abscessus subsp. massiliense subsp. nov. and M. abscessus subsp. bolletii, to correspondingly accommodate the previously known ‘M. massiliense’ and ‘M. bolletii’ strains.
Molecular Longitudinal Tracking of Mycobacterium abscessus spp. during Chronic Infection of the Human Lung  [PDF]
Kaj M. Kreutzfeldt, Paul R. McAdam, Pauline Claxton, Anne Holmes, A. Louise Seagar, Ian F. Laurenson, J. Ross Fitzgerald
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0063237
Abstract: The Mycobacterium abscessus complex is an emerging cause of chronic pulmonary infection in patients with underlying lung disease. The M. abscessus complex is regarded as an environmental pathogen but its molecular adaptation to the human lung during long-term infection is poorly understood. Here we carried out a longitudinal molecular epidemiological analysis of 178 M. abscessus spp. isolates obtained from 10 cystic fibrosis (CF) and 2 non CF patients over a 13 year period. Multi-locus sequence and molecular typing analysis revealed that 11 of 12 patients were persistently colonized with the same genotype during the course of the infection while replacement of a M. abscessus sensu stricto strain with a Mycobacterium massiliense strain was observed for a single patient. Of note, several patients including a pair of siblings were colonized with closely-related strains consistent with intra-familial transmission or a common infection reservoir. In general, a switch from smooth to rough colony morphology was observed during the course of long-term infection, which in some cases correlated with an increasing severity of clinical symptoms. To examine evolution during long-term infection of the CF lung we compared the genome sequences of 6 sequential isolates of Mycobacterium bolletii obtained from a single patient over an 11 year period, revealing a heterogeneous clonal infecting population with mutations in regulators controlling the expression of virulence factors and complex lipids. Taken together, these data provide new insights into the epidemiology of M. abscessus spp. during long-term infection of the CF lung, and the molecular transition from saprophytic organism to human pathogen.
Conditional Gene Expression in Mycobacterium abscessus  [PDF]
Mélanie Cortes, Anil Kumar Singh, Jean-Marc Reyrat, Jean-Louis Gaillard, Xavier Nassif, Jean-Louis Herrmann
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0029306
Abstract: Mycobacterium abscessus is an emerging human pathogen responsible for lung infections, skin and soft-tissue infections and disseminated infections in immunocompromised patients. It may exist either as a smooth (S) or rough (R) morphotype, the latter being associated with increased pathogenicity in various models. Genetic tools for homologous recombination and conditional gene expression are desperately needed to allow the study of M. abscessus virulence. However, descriptions of knock-out (KO) mutants in M. abscessus are rare, with only one KO mutant from an S strain described so far. Moreover, of the three major tools developed for homologous recombination in mycobacteria, only the one based on expression of phage recombinases is working. Several conditional gene expression tools have recently been engineered for Mycobacterium tuberculosis and Mycobacterium smegmatis, but none have been tested yet in M. abscessus. Based on previous experience with genetic tools allowing homologous recombination and their failure in M. abscessus, we evaluated the potential interest of a conditional gene expression approach using a system derived from the two repressors system, TetR/PipOFF. After several steps necessary to adapt TetR/PipOFF for M. abscessus, we have shown the efficiency of this system for conditional expression of an essential mycobacterial gene, fadD32. Inhibition of fadD32 was demonstrated for both the S and R isotypes, with marginally better efficiency for the R isotype. Conditional gene expression using the dedicated TetR/PipOFF system vectors developed here is effective in S and R M. abscessus, and may constitute an interesting approach for future genetic studies in this pathogen.
Diagnosis of endocarditis caused by Mycobacterium abscessus  [cached]
Al-Benwan Khalifa,Ahmad Suhail,Mokaddas Eiman,Johny Molly
Annals of Saudi Medicine , 2010,
Abstract: We report a fatal case of native valve endocarditis due to Mycobacterium abscessus in a hemodialysis patient. The diagnosis was based on culture isolation of acid-fast bacilli from peripheral blood and a permanent catheter tip, and their identification as M abscessus by a reverse hybridization-based assay and direct DNA sequencing of the 16S-23S internal transcribed spacer region. Rapid diagnosis and combination therapy are essential to minimize mortality due to this pathogen. Although combination therapy was started with clarithromycin and tigecycline, the patient refused to take clarithromycin due to severe abdominal pain. The patient became afebrile after therapy with tigecycline alone although bacteremia persisted. He was discharged against medical advice and readmitted three months later for persistent fever. His blood cultures again yielded M abscessus and a transesophageal echocardiogram showed two mobile vegetations. The patient was noncompliant with therapy and died due to cardiac arrest and multiorgan failure. This report shows that M abscessus should also be considered in the differential diagnosis of infective endocarditis in hemodialysis patients.
Non Mycobacterial Virulence Genes in the Genome of the Emerging Pathogen Mycobacterium abscessus  [PDF]
Fabienne Ripoll, Sophie Pasek, Chantal Schenowitz, Carole Dossat, Valérie Barbe, Martin Rottman, Edouard Macheras, Beate Heym, Jean-Louis Herrmann, Mamadou Daffé, Roland Brosch, Jean-Loup Risler, Jean-Louis Gaillard
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005660
Abstract: Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a “mycobacterial” gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the “non mycobacterial” factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients.
Genomics of glycopeptidolipid biosynthesis in Mycobacterium abscessus and M. chelonae
Fabienne Ripoll, Caroline Deshayes, Sophie Pasek, Fran?oise Laval, Jean-Luc Beretti, Franck Biet, Jean-Loup Risler, Mamadou Daffé, Gilles Etienne, Jean-Louis Gaillard, Jean-Marc Reyrat
BMC Genomics , 2007, DOI: 10.1186/1471-2164-8-114
Abstract: We have recently sequenced the complete genomes of two fast-growers causing human infections, Mycobacterium abscessus (CIP 104536T) and M. chelonae (CIP 104535T). We show here that these two species contain genes corresponding to all those of the M. smegmatis "GPL locus", with extensive conservation of the predicted protein sequences consistent with the production of GPL molecules indistinguishable by biochemical analysis. However, the GPL locus appears to be split into several parts in M. chelonae and M. abscessus. One large cluster (19 genes) comprises all genes involved in the synthesis of the tripeptide-aminoalcohol moiety, the glycosylation of the lipopeptide and methylation/acetylation modifications. We provide evidence that a duplicated acetyltransferase (atf1 and atf2) in M. abscessus and M. chelonae has evolved through specialization, being able to transfer one acetyl at once in a sequential manner. There is a second smaller and distant (M. chelonae, 900 kb; M. abscessus, 3 Mb) cluster of six genes involved in the synthesis of the fatty acyl moiety and its attachment to the tripeptide-aminoalcohol moiety. The other genes are scattered throughout the genome, including two genes encoding putative regulatory proteins.Although these three species produce identical GPL molecules, the organization of GPL genes differ between them, thus constituting species-specific signatures. An hypothesis is that the compact organization of the GPL locus in M. smegmatis represents the ancestral form and that evolution has scattered various pieces throughout the genome in M. abscessus and M. chelonae.Mycobacterium abscessus and M. chelonae are both species of rapidly growing mycobacteria (RGM) that have emerged as significant pathogens in humans during the last ten years: both species are major causes of skin and soft tissue infections following medical or surgical procedures [1]; M. abscessus also causes pulmonary infections and is increasingly recovered from patients with cyst
MabsBase: A Mycobacterium abscessus Genome and Annotation Database  [PDF]
Hamed Heydari, Wei Yee Wee, Naline Lokanathan, Ranjeev Hari, Aini Mohamed Yusoff, Ching Yew Beh, Amir Hessam Yazdi, Guat Jah Wong, Yun Fong Ngeow, Siew Woh Choo
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062443
Abstract: Summary Mycobacterium abscessus is a rapidly growing non-tuberculous mycobacterial species that has been associated with a wide spectrum of human infections. As the classification and biology of this organism is still not well understood, comparative genomic analysis on members of this species may provide further insights on their taxonomy, phylogeny, pathogenicity and other information that may contribute to better management of infections. The MabsBase described in this paper is a user-friendly database providing access to whole-genome sequences of newly discovered M. abscessus strains as well as resources for whole-genome annotations and computational predictions, to support the expanding scientific community interested in M. abscessus research. The MabsBase is freely available at http://mabscessus.um.edu.my.
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