Thomas Bodmer

Multilocus Sequence Analysis and rpoB Sequencing of Mycobacterium abscessus (Sensu Lato) Strains

ABSTRACT Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536T, M. massiliense CIP 108297T, and M. bolletii CIP 108541T) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

Clinical Implications of Mycobacterium kansasii Species Heterogeneity: Swiss National Survey

ABSTRACT Several subtypes of Mycobacterium kansasii have been described, but their respective pathogenic roles are not clear. This study investigated the distribution of subtypes and the pathogenicity of M. kansasii strains (n = 191) isolated in Switzerland between 1991 and 1997. Demographic, clinical, and microbiological information was recorded from clinical files. Patients were classified as having an infection according to the criteria of the American Thoracic Society. Subtypes were defined by PCR-restriction enzyme analysis of the hsp65 gene. Subtype 1 comprised 67% of the isolates (n = 128), while subtypes 2 and 3 comprised 21% (n = 40) and 8% (n = 15), respectively. Other subtypes (subtypes 4 and 6 and a new subtype, 7) were recovered from only 4% of patients (n = 8). M. kansasii subtype 1 was considered pathogenic in 81% of patients, while M. kansasii subtype 2 was considered pathogenic in 67% of patients and other subtypes were considered pathogenic in 6% of patients. The majority of patients with M. kansasii subtype 2 were immunocompromised due to the use of corticosteroids (21% of patients) or coinfection with HIV (62.5% of patients). Subtyping M. kansasii may improve clinical management by distinguishing pathogenic from nonpathogenic subtypes.

Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis.

ABSTRACT Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter −15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter −15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.

“Pseudo-Beijing”: Evidence for Convergent Evolution in the Direct Repeat Region of Mycobacterium tuberculosis

Background Mycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as “Beijing” has repeatedly been associated with drug resistance and has been emerging in some parts of the world. “Beijing” strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC) strains exhibiting the typical “Beijing” spoligotyping pattern. Methods and Findings MTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS) rather than to Lineage 2 (also known as East-Asian lineage). Sequencing of the RD207 in one of these strains showed that the deletion responsible for this “Pseudo-Beijing” spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical “Beijing” strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR) region of MTBC. Conclusions We provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of “Beijing” strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes.

Development and Application of Real-Time PCR Assay for Quantification of Mycobacterium ulcerans DNA

ABSTRACT Buruli ulcer, an infection caused by Mycobacterium ulcerans, is, after tuberculosis and leprosy, the third most common mycobacterial disease. The mode of transmission of M. ulcerans is not exactly known, but since Buruli ulcer often occurs in focalized swampy areas, it is assumed that there is a reservoir of the pathogen in stagnant water. Buruli ulcer usually starts as a painless nodule and can lead to massive destruction of skin, subcutaneous tissue, and eventually muscle and bone. Currently the only recommended treatment is wide surgical excision. In this report we describe the development of a real-time PCR method for the quantification of M. ulcerans DNA (IS2404 TaqMan). The highly specific assay is based on the detection of the M. ulcerans specific insertion sequence IS2404. The IS2404 TaqMan assay turned out to be about 10 times more sensitive than the available conventional PCR-based diagnostic test. It is demonstrated that the IS2404 TaqMan assay is suitable for the quantitative assessment of the dissemination of the mycobacteria in Buruli ulcer lesions. Prototype results obtained with excised tissue from a patient with a late preulcerative Buruli ulcer lesion reconfirmed earlier histopathological findings indicating that tissue damage occurs far beyond the regions in which large numbers of mycobacteria are detectable. The IS2404 TaqMan assay should be a useful tool for both diagnosis and research into the pathology and mode of transmission of this still inadequately investigated mycobacterial disease.

Use of the Immunodominant 18-Kilodalton Small Heat Shock Protein as a Serological Marker for Exposure to Mycobacterium ulcerans

ABSTRACT While it is well established that proximity to wetlands is a risk factor for contracting Buruli ulcer, it is not clear what proportion of a population living in an area where the etiologic agent, Mycobacterium ulcerans, is endemic is actually exposed to this disease. Immunological cross-reactivity among mycobacterial species complicates the development of a specific serological test. Among immunodominant proteins recognized by a panel of anti-M. ulcerans monoclonal antibodies, the M. ulcerans homologue of the M. leprae 18-kDa small heat shock protein (shsp) was identified. Since this shsp has no homologues in M. bovis and M. tuberculosis, we evaluated its use as a target antigen for a serological test. Anti-18-kDa shsp antibodies were frequently found in the sera of Buruli ulcer patients and of healthy household contacts but rarely found in controls from regions where the infection is not endemic. The results indicate that only a small proportion of M. ulcerans-infected individuals contract the clinical disease.

Tracking a Tuberculosis Outbreak Over 21 Years: Strain-Specific Single-Nucleotide Polymorphism Typing Combined With Targeted Whole-Genome Sequencing

BACKGROUND Whole-genome sequencing (WGS) is increasingly used in molecular-epidemiological investigations of bacterial pathogens, despite cost- and time-intensive analyses. We combined strain-specific single-nucleotide polymorphism (SNP) typing and targeted WGS to investigate a tuberculosis cluster spanning 21 years in Bern, Switzerland. METHODS On the basis of genome sequences of 3 historical outbreak Mycobacterium tuberculosis isolates, we developed a strain-specific SNP-typing assay to identify further cases. We screened 1642 patient isolates and performed WGS on all identified cluster isolates. We extracted SNPs to construct genomic networks. Clinical and social data were retrospectively collected. RESULTS We identified 68 patients associated with the outbreak strain. Most received a tuberculosis diagnosis in 1991-1995, but cases were observed until 2011. Two thirds were homeless and/or substance abusers. Targeted WGS revealed 133 variable SNP positions among outbreak isolates. Genomic network analyses suggested a single origin of the outbreak, with subsequent division into 3 subclusters. Isolates from patients with confirmed epidemiological links differed by 0-11 SNPs. CONCLUSIONS Strain-specific SNP genotyping allowed rapid and inexpensive identification of M. tuberculosis outbreak isolates in a population-based strain collection. Subsequent targeted WGS provided detailed insights into transmission dynamics. This combined approach could be applied to track bacterial pathogens in real time and at high resolution.

Evaluation of Decontamination Methods and Growth Media for Primary Isolation of Mycobacterium ulcerans from Surgical Specimens

ABSTRACT We evaluated four decontamination methods and one nondecontamination procedure in combination with four egg-based media for the primary isolation of Mycobacterium ulcerans from tissue specimens. With mycobacterial recovery and contamination rates of 75.6 and 2.4%, respectively, the combination of the oxalic acid decontamination method with Lowenstein-Jensen medium supplemented with glycerol yielded the best results.

Multicenter Evaluation of the MB/BACT System for Susceptibility Testing of Mycobacterium tuberculosis

ABSTRACT The reliability of the MB/BACT system for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide, rifampin, isoniazid, streptomycin, and ethambutol was compared to the BACTEC 460TB system. The proportion method was used to resolve discrepant results by an independent arbiter. Two interpretative methods were used, with an undiluted control (direct control) and a diluted control (10−1 control). As no significant difference was observed between the two controls, the method with the direct control was adopted as the most accurate one. One hundred sixty-six strains were tested, with an overall agreement of 98.3%. After resolution of the 18 discrepant results by the proportion method, the sensitivity and specificity of the MB/BACT system were 100% for rifampin, isoniazid, and pyrazinamide. For ethambutol, sensitivity was 92.3% at the critical concentration and 33% at the high concentration, and specificity was 100% at both concentrations. For streptomycin, sensitivity was 100% at the critical concentration and 80% at the high concentration, and specificity was 98.6% at the critical concentration and 100% at the high concentration. The rifampin, isoniazid, streptomycin, and ethambutol susceptibility test results were obtained in 6.6 days with the MB/BACT versus 5 days with the BACTEC 460TB. The pyrazinamide susceptibility test results were obtained in 7.8 days with the MB/BACT, versus 6.7 days with the BACTEC 460TB. These data demonstrate that the fully automated MB/BACT system is a very reliable method for rapid susceptibility testing of M. tuberculosis against rifampin, isoniazid, and pyrazinamide. Sensitivity results have to be improved for ethambutol and streptomycin, especially at the high concentration.

HIV Infection Disrupts the Sympatric Host–Pathogen Relationship in Human Tuberculosis

The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV–infected and HIV–negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV–infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host–pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21–infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5–19.1, p<0.0001). This association remained when adjusting for frequent travelling, contact with foreigners, age, sex, and country of birth (adjusted OR 5.6, 95% CI 1.5–20.8, p = 0.01). Moreover, it became stronger with greater immunosuppression as defined by CD4 T-cell depletion and was not the result of increased social mixing in HIV–infected patients. Our observation was replicated in a second independent panel of 440 M. tuberculosis strains collected during a population-based study in the Canton of Bern between 1991 and 2011. In summary, these findings support a model for TB in which the stable relationship between the human host and its locally adapted M. tuberculosis is disrupted by HIV infection.