Andreas Roth

Publications of Andreas Roth

• Analytical performance of the Roche LightCycler® Mycobacterium Detection Kit for the diagnosis of clinically important mycobacterial species.

  Authors: Shaheed V Omar, Andreas Roth, Nazir A Ismail, Linda Erasmus, Marthie Ehlers, Marleen Kock, Nuraan Paulse, Halima M Said, Anwar A Hoosen, Udo Reischl

  PloS one. 01/2011; 6(9):e24789.

  The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed.The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility. Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay's reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent. The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.

• Early bactericidal activity of moxifloxacin in treatment of pulmonary tuberculosis: a prospective, randomized study.

  Authors: Mathias W R Pletz, Andres De Roux, Andreas Roth, Karl-Heinz Neumann, Harald Mauch, Hartmut Lode

  Antimicrobial agents and chemotherapy. 04/2004; 48(3):780-2.

  Moxifloxacin is the most active fluoroquinolone against Mycobacterium tuberculosis in vitro. However, data about the efficacy in patients are not available. We enrolled 17 patients with tuberculosisMoxifloxacin is the most active fluoroquinolone against Mycobacterium tuberculosis in vitro. However, data about the efficacy in patients are not available. We enrolled 17 patients with tuberculosis in a prospective, randomized study. After 5 days of monotherapy with either moxifloxacin or isoniazid, we detected significant decreases in mean CFU per milliliter in sputum in both groups. The calculated early bactericidal activities for isoniazid and moxifloxacin were 0.209 and 0.273 log(10) CFU per ml of sputum per day, respectively. According to the data from our study, moxifloxacin exhibits an early bactericidal activity that is comparable to that of isoniazid.

• RIDOM: comprehensive and public sequence database for identification of Mycobacterium species.

  Authors: Dag Harmsen, Stefan Dostal, Andreas Roth, Stefan Niemann, Jörg Rothgänger, Michael Sammeth, Jürgen Albert, Matthias Frosch, Elvira Richter

  BMC infectious diseases. 12/2003; 3:26.

  BACKGROUND: Molecular identification of Mycobacterium species has two primary advantages when compared to phenotypic identification: rapid turn-around time and improved accuracy. The informationBACKGROUND: Molecular identification of Mycobacterium species has two primary advantages when compared to phenotypic identification: rapid turn-around time and improved accuracy. The information content of the 5' end of the 16S ribosomal RNA gene (16S rDNA) is sufficient for identification of most bacterial species. However, reliable sequence-based identification is hampered by many faulty and some missing sequence entries in publicly accessible databases. METHODS: In order to establish an improved 16S rDNA sequence database for the identification of clinical and environmental isolates, we sequenced both strands of the 5' end of 16S rDNA (Escherichia coli positions 54 to 510) from 199 mycobacterial culture collection isolates. All validly described species (n = 89; up to March 21, 2000) and nearly all published sequevar variants were included. If the 16S rDNA sequences were not discriminatory, the internal transcribed spacer (ITS) region sequences (n = 84) were also determined. RESULTS: Using 5'-16S rDNA sequencing a total of 64 different mycobacterial species (71.9%) could be identified. With the additional input of the ITS sequence, a further 16 species or subspecies could be differentiated. Only Mycobacterium tuberculosis complex species, M. marinum/M. ulcerans and the M. avium subspecies could not be differentiated using 5'-16S rDNA or ITS sequencing. A total of 77 culture collection strain sequences, exhibiting an overlap of at least 80% and identical by strain number to the isolates used in this study, were found in the GenBank. Comparing these with our sequences revealed that an average of 4.31 nucleotide differences (SD +/- 0.57) were present. CONCLUSIONS: The data from this analysis show that it is possible to differentiate most mycobacterial species by sequence analysis of partial 16S rDNA. The high-quality sequences reported here, together with ancillary information (e.g., taxonomic, medical), are available in a public database, which is currently being expanded in the RIDOM project http://www.ridom-rdna.de), for similarity searches.

• Evaluation of RIDOM, MicroSeq, and Genbank services in the molecular identification of Nocardia species.

  Authors: Alexander Mellmann, Joann L Cloud, Sebastian Andrees, Kym Blackwood, Karen C Carroll, Amin Kabani, Andreas Roth, Dag Harmsen

  International journal of medical microbiology : IJMM. 11/2003; 293(5):359-70.

  The molecular identification of Nocardia species, when compared to phenotypic identification, has two primary advantages: rapid turn-around time and improved accuracy. The information content in theThe molecular identification of Nocardia species, when compared to phenotypic identification, has two primary advantages: rapid turn-around time and improved accuracy. The information content in the 5'-end of the 16S ribosomal RNA gene is sufficient for identification of most bacterial species. An evaluation was performed to demonstrate the quality of results provided by two specialized databases (RIDOM and MicroSeq 500 versions 1.1 and 1.4.3, library version 500-0125, respectively) and the more general GenBank database. In addition, these results were compared with phenotypic identifications. Partial 5'-16S rDNA sequences from 64 culture collection strains (DSM, CIP, JCM, and ATCC) were derived, in duplicate, independently in two laboratories. Furthermore, the sequences and the conventional identification results of 91 clinical Nocardia isolates were determined. With the exception of N. soli and N. cummidelens, all Nocardia type strains were distinguishable using 5'-16S rDNA sequencing. Assuming a normal distribution for the pairwise distances of all unique Nocardia sequences and choosing a reporting criterion of > or = 99.12% similarity for a "distinct species", a statistical error probability of 1.0% can be calculated. When the various databases were searched with the clinical isolate sequences RIDOM gave a perfect match in 71.4% of cases whereas MicroSeq yielded a perfect match in only 26.4%. The GenBank service gave a 100% similarity in 59.3% but in 70.4% of these cases the results obtained were not exclusive for a single Nocardia species. Conventional methods gave a correct identification in 59 cases, although most recent taxonomic changes were not taken into account. The RIDOM service (http://www.ridom-rdna.de/) is in the process of making available a comprehensive and high-quality database for bacterial identification purposes and provides excellent results for the majority of Nocardia isolates.

• Phylogeny of the genus Nocardia based on reassessed 16S rRNA gene sequences reveals underspeciation and division of strains classified as Nocardia asteroides into three established species and two unnamed taxons.

  Authors: Andreas Roth, Sebastian Andrees, Reiner M Kroppenstedt, Dag Harmsen, Harald Mauch

  Journal of clinical microbiology. 03/2003; 41(2):851-6.

  Conventional identification of Nocardia in the routine laboratory remains problematic due to a paucity of reliable phenotypic tests and due to the yet-unresolved taxonomy of strains classified asConventional identification of Nocardia in the routine laboratory remains problematic due to a paucity of reliable phenotypic tests and due to the yet-unresolved taxonomy of strains classified as belonging to the species Nocardia asteroides, which comprises the type strain and isolates with drug pattern types II and VI. The 16S rRNA gene of 74 representative strains of the genus Nocardia, encompassing 25 established species, was sequenced in order to provide a molecular basis for accurate species identification and with the aim of reassessing the phylogeny of taxons assigned to the species N. asteroides. The result of this phylogenetic analysis confirms that the interspecies heterogeneity of closely related nocardial species can be considerably low (a sequence divergence of only 0.5% was found between N. paucivorans and N. brevicatena). We observed a sequence microheterogeneity (sequence divergence of fewer than five bases) in 8 of 11 species of which more than one strain in the species was studied. At least 10 taxons were found that merit description as new species. Strains previously classified as N. asteroides fell into five distinct phylogenetic groups: the type strain cluster (N. asteroides sensu strictu), N. abscessus, N. cyriacigeorgica, and two clusters closely related to N. carnea or N. flavorosea. The strains within the latter two groups probably represent new species, pending further genetic and phenotypic evaluation. Restricted phenotypic data revealed that N. abscessus, N. cyriacigeorgica, and the two Nocardia species taxons are equivalent to drug patterns I, VI, and II, respectively. In the future, these data will help in finding species-specific markers after adoption of a more precise nomenclature for isolates closely related to N. asteroides and unravel confusing phenotypic data obtained in the past for unresolved groups of strains that definitely belong to separate taxons from a phylogenetic point of view.

• Differentiation between Mycobacterium farcinogenes and Mycobacterium senegalense strains based on 16S-23S ribosomal DNA internal transcribed spacer sequences.

  Authors: Mohamed E. Hamid, Andreas Roth, Olfert Landt, Reiner M Kroppenstedt, Michael Goodfellow, Harald Mauch

  Journal of clinical microbiology. 03/2002; 40(2):707-11.

  16S ribosomal DNA (rDNA) and 16S-23S internal transcribed spacer rDNA sequence analyses were performed on Mycobacterium farcinogenes and M. senegalense strains and 26 strains of other rapidly growing16S ribosomal DNA (rDNA) and 16S-23S internal transcribed spacer rDNA sequence analyses were performed on Mycobacterium farcinogenes and M. senegalense strains and 26 strains of other rapidly growing mycobacteria to investigate the phylogenetic structure of bovine farcy mycobacteria within the M. fortuitum complex. M. farcinogenes and M. senegalense were indistinguishable in their 5"-end 16S rDNA but showed both considerable interspecies spacer sequence divergence and a high level of intraspecies sequence stability. A rapid detection assay using PCR and hybridization with species-specific probes was developed. The assay was specific among 46 species other than M. farcinogenes and M. senegalense and correctly identified all M. farcinogenes and M. senegalense strains. PCR- and 16S-23S rDNA sequence-based detection will be a valuable approach for diagnosis of the causal agents of African bovine farcy in cattle.

• Novel Diagnostic Algorithm for Identification of Mycobacteria Using Genus-Specific Amplification of the 16S-23S rRNA Gene Spacer and Restriction Endonucleases

  Authors: Andreas Roth, Udo Reischl, Anna Streubel, Ludmila Naumann, Reiner M Kroppenstedt, Marion Habicht, Marga Fischer, Harald Mauch

  A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA geneA novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.

• Mycobacterium heckeshornense sp. nov., a New Pathogenic Slowly Growing Mycobacterium sp. Causing Cavitary Lung Disease in an Immunocompetent Patient

  Authors: Andreas Roth, Udo Reischl, Nicolas Schönfeld, Ludmila Naumann, Stefan Emler, Marga Fischer, Harald Mauch, Robert Loddenkemper, Reiner M Kroppenstedt

  A pathogenic scotochromogenic Mycobacterium xenopi-like organism was isolated from the lung of an immunocompetent young woman. This pathogen caused severe bilateral cavitary lung disease, making twoA pathogenic scotochromogenic Mycobacterium xenopi-like organism was isolated from the lung of an immunocompetent young woman. This pathogen caused severe bilateral cavitary lung disease, making two surgical interventions necessary after years of chronic disease. This case prompted us to characterize this mycobacterium by a polyphasic taxonomic approach. The isolate contained chemotaxonomic markers which were typical for the genus Mycobacterium, i.e., the meso isomer of 2,6-diaminopimelic acid, arabinose, and galactose as diagnostic whole-cell sugars, MK-9(H2) as the principal isoprenoid quinone, a mycolic acid pattern of α-mycolates, ketomycolates, and wax ester mycolates, unbranched saturated and unsaturated fatty acids plus a significant amount of tuberculostearic acid, and small amounts of a C20:0 secondary alcohol. On the basis of its unique 16S rRNA and 16S-23S spacer gene sequences, we propose that the isolate should be assigned to a new species, Mycobacterium heckeshornense. This novel species is phylogenetically closely related to M. xenopi. The type strain of M. heckeshornense is strain S369 (DSM 44428T). The GenBank accession number of the 16S rRNA gene of M. heckeshornense is AF174290.

• Differentiation of Phylogenetically Related Slowly Growing Mycobacteria Based on 16S-23S rRNA Gene Internal Transcribed Spacer Sequences

  Authors: Andreas Roth, Marga Fischer, Mohamed E. Hamid, Sabine Michalke, Wolfgang Ludwig, Harald Mauch

  Interspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they areInterspecific polymorphisms of the 16S rRNA gene (rDNA) are widely used for species identification of mycobacteria. 16S rDNA sequences, however, do not vary greatly within a species, and they are either indistinguishable in some species, for example, in Mycobacterium kansasii and M. gastri, or highly similar, for example, in M. malmoense and M. szulgai. We determined 16S-23S rDNA internal transcribed spacer (ITS) sequences of 60 strains in the genus Mycobacterium representing 13 species (M. avium, M. conspicuum, M. gastri, M. genavense, M. kansasii, M. malmoense, M. marinum, M. shimoidei, M. simiae, M. szulgai, M. triplex, M. ulcerans, and M. xenopi). An alignment of these sequences together with additional sequences available in the EMBL database (for M. intracellulare, M. phlei, M. smegmatis, and M. tuberculosis) was established according to primary- and secondary-structure similarities. Comparative sequence analysis applying different treeing methods grouped the strains into species-specific clusters with low sequence divergence between strains belonging to the same species (0 to 2%). The ITS-based tree topology only partially correlated to that based on 16S rDNA, but the main branching orders were preserved, notably, the division of fast-growing from slowly growing mycobacteria, separate branching for M. simiae, M. genavense, and M. triplex, and distinct branches for M. xenopi and M. shimoidei. Comparisons of M. gastri with M. kansasii and M. malmoense with M. szulgai revealed ITS sequence similarities of 93 and 88%, respectively. M. marinum and M. ulcerans possessed identical ITS sequences. Our results show that ITS sequencing represents a supplement to 16S rRNA gene sequences for the differentiation of closely related species. Slowly growing mycobacteria show a high sequence variation in the ITS; this variation has the potential to be used for the development of probes as a rapid approach to mycobacterial identification.