Claudia Ritter

Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis

Treatment of multidrug-resistant Mycobacterium tuberculosis is a challenge. This letter describes the emergence of resistance to new therapies, bedaquiline and delamanid.

Quantitative Drug Susceptibility Testing of Mycobacterium tuberculosis by Use of MGIT 960 and EpiCenter Instrumentation

ABSTRACT Since numbers of drug-resistant Mycobacterium tuberculosis strains are on the rise, the simple classification into “susceptible” and “resistant” strains based on susceptibility testing at “critical concentrations” has to be reconsidered. While future studies have to address the correlation of phenotypic resistance levels and treatment outcomes, a prerequisite for corresponding investigations is the ability to exactly determine levels of quantitative drug resistance in clinical M. tuberculosis isolates. Here we have established the conditions for quantitative drug susceptibility testing for first- and second-line agents using MGIT 960 instrumentation and EpiCenter software equipped with the TB eXiST module. In-depth comparative analysis of a range of well-characterized susceptible and resistant clinical isolates has allowed us to propose conditions for testing and to develop criteria for interpretation.

Septicemia Caused by Tick-borne Bacterial Pathogen Candidatus Neoehrlichia mikurensis

We have repeatedly detected Candidatus Neoehrlichia mikurensis, a bacterium first described in Rattus norvegicus rats and Ixodes ovatus ticks in Japan in 2004 in the blood of a 61-year-old man with signs of septicemia by 16S rRNA and groEL gene PCR. After 6 weeks of therapy with doxycycline and rifampin, the patient recovered.

Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study

OBJECTIVES Treatment outcome of MDR-TB is critically dependent on the proper use of second-line drugs as per the result of in vitro drug susceptibility testing (DST). We aimed to establish a standardized DST procedure based on quantitative determination of drug resistance and compared the results with those of genotypes associated with drug resistance. METHODS The protocol, based on MGIT 960 and the TB eXiST software, was evaluated in nine European reference laboratories. Resistance detection at a screening drug concentration was followed by determination of resistance levels and estimation of the resistance proportion. Mutations in 14 gene regions were investigated using established techniques. RESULTS A total of 139 Mycobacterium tuberculosis isolates from patients with MDR-TB and resistance beyond MDR-TB were tested for 13 antituberculous drugs: isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide, streptomycin, para-aminosalicylic acid, ethionamide, amikacin, capreomycin, ofloxacin, moxifloxacin and linezolid. Concordance between phenotypic and genotypic resistance was >80%, except for ethambutol. Time to results was short (median 10 days). High-level resistance, which precludes the therapeutic use of an antituberculous drug, was observed in 49% of the isolates. The finding of a low or intermediate resistance level in 16% and 35% of the isolates, respectively, may help in designing an efficient personalized regimen for the treatment of MDR-TB patients. CONCLUSIONS The automated DST procedure permits accurate and rapid quantitative resistance profiling of first- and second-line antituberculous drugs. Prospective validation is warranted to determine the impact on patient care.

Acquisition of clarithromycin resistance mutations in the 23S rRNA gene of Mycobacterium abscessus in the presence of inducible erm(41)

OBJECTIVES Antibiotic therapy of pulmonary Mycobacterium abscessus infection is based on a combination treatment including clarithromycin. Recent data demonstrated that M. abscessus may carry a chromosomal, inducible erm gene coding for the ribosomal methylase Erm(41). The purpose of this study was to investigate whether in patients with chronic M. abscessus infection undergoing clarithromycin therapy, M. abscessus acquires clarithromycin resistance mutations in the rrl gene in addition to the presence of an inducible Erm(41) methylase. METHODS We determined clarithromycin MICs, erm(41) and rrl sequences for 29 clinical M. abscessus subsp. abscessus isolates of five different patients. The isolates were obtained between 2007 and 2011 covering a longitudinal observation period of 2-4 years for the individual patients. RESULTS In three out of five patients with an initial rrl wild-type isolate, follow-up isolates demonstrated acquisition of resistance mutations in the rrl gene in addition to the presence of an inducible Erm methylase. CONCLUSIONS Our results show that in M. abscessus, clarithromycin resistance mutations in the 23S rRNA peptidyltransferase region provide an additional selective advantage independent of a functional erm(41) gene.

Lack of antimicrobial bactericidal activity in Mycobacterium abscessus

ABSTRACT Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organisms natural resistance toward most clinically available antimicrobials. We investigated the bactericidal activity of antibiotics commonly administered in M. abscessus infections in order to better understand the poor therapeutic outcome. Time-kill curves were generated for clinical M. abscessus isolates, Mycobacterium smegmatis, and Escherichia coli by using antibiotics commonly categorized as bactericidal (amikacin and moxifloxacin) or bacteriostatic (tigecycline and linezolid). In addition, the impact of aminoglycoside-modifying enzymes on the mode of action of substrate and nonsubstrate aminoglycosides was studied by using M. smegmatis as a model organism. While amikacin and moxifloxacin were bactericidal against E. coli, none of the tested compounds showed bactericidal activity against M. abscessus. Further mechanistic investigations of the mode of action of aminoglycosides in M. smegmatis revealed that the bactericidal activity of tobramycin and gentamicin was restored by disruption of the chromosomal aac(2′) gene in the mycobacterial genome. The lack of bactericidal antibiotics in currently recommended treatment regimens provides a reasonable explanation for the poor therapeutic outcome in M. abscessus infection. Our findings suggest that chromosomally encoded drug-modifying enzymes play an important role in the lack of aminoglycoside bactericidal activity against rapidly growing mycobacteria.

High-level resistance to isoniazid and ethionamide in multidrug-resistant Mycobacterium tuberculosis of the Lisboa family is associated with inhA double mutations

OBJECTIVES The purpose of this study was to determine the levels of isoniazid and ethionamide resistance and to identify associated mutations in endemic multidrug-resistant (MDR) strains of Mycobacterium tuberculosis from the Lisbon metropolitan area, Portugal. METHODS Seventeen clinical MDR tuberculosis (TB) strains were characterized by standard and semi-quantitative drug susceptibility testing to assess the level of isoniazid and ethionamide resistance. The genes katG, inhA, ethA and ndh were screened for mutations. All strains were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) analysis. RESULTS All strains showed high-level resistance to both isoniazid (>1 mg/L) and ethionamide (>25 mg/L). MIRU-VNTR typing revealed the presence of two main clusters, Lisboa3 and Q1, in 16/17 strains, all of which showed the C-15T mutation in the promoter region of the inhA gene. The 16 strains belong to the Latino-American-Mediterranean (LAM) genotype and the other strain belongs to the Beijing genotype. Sequencing of the inhA open reading frame revealed that the 16 strains also had mutations in the structural region of the gene, leading to the S94A substitution in 9 strains and the I194T substitution in 7 strains. CONCLUSIONS The results reveal that the presence of a mutation in the inhA regulatory region together with a mutation in the inhA coding region can lead to the development of high-level isoniazid resistance and cross-resistance to ethionamide among the MDR-TB strains circulating in Lisbon. This mutational pattern also hints to a possible involvement of strain-specific factors that could be a feature of the Portuguese MDR-TB strains where the LAM family is the major circulating genotype.

Evaluation of Cobas TaqMan MTB for direct detection of the Mycobacterium tuberculosis complex in comparison with Cobas Amplicor MTB.

ABSTRACT The Roche Cobas Amplicor MTB assay, recently replaced by the Roche Cobas TaqMan MTB assay, was one of the first commercially available assays for detection of the Mycobacterium tuberculosis complex based on nucleic acid amplification. We reported previously on the limited specificity of the Cobas Amplicor MTB assay, in particular for positive samples with an optical density at 660 nm (OD660) of <2.0. Using a selected set of respiratory samples, which were scored as false positive by the Cobas Amplicor test, we demonstrate here that the specificity of the Cobas TaqMan assay is significantly improved. In addition, our study of a set of 133 clinical samples revealed that the Cobas TaqMan MTB assay showed significantly less PCR inhibition than the Cobas Amplicor test. An overall concordance of 98.2% was observed between the two assays. In a subsequent prospective study, we evaluated the performance of the Roche Cobas TaqMan MTB assay on 1,143 clinical specimens, including respiratory (n = 838) and nonrespiratory (n = 305) specimens. Using culture as the gold standard, we found a sensitivity of 88.4% and a specificity of 98.8% for the 838 respiratory specimens, compared to a sensitivity of 63.6% and a specificity of 94.6% for the 305 nonrespiratory specimens. We conclude that the Cobas TaqMan MTB assay is a significantly improved tool for the direct detection of M. tuberculosis DNA in clinical specimens.

Evaluation of the AID TB Resistance Line Probe Assay for Rapid Detection of Genetic Alterations Associated with Drug Resistance in Mycobacterium tuberculosis Strains

ABSTRACT The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected resistance mutations in clinical M. tuberculosis isolates from areas with low and high levels of endemicity (Switzerland, n = 104; South Africa, n = 52) and in selected Mycobacterium bovis BCG 1721 mutant strains (n = 5) with 100% accuracy. Subsequently, the line probe assay was shown to be capable of rapid genetic assessment of drug resistance in MGIT broth cultures, the results of which were in 100% agreement with those of DNA sequencing and phenotypic drug susceptibility testing. Finally, the line probe assay was assessed for direct screening of smear-positive clinical specimens. Screening of 98 clinical specimens demonstrated that the test gave interpretable results for >95% of them. Antibiotic resistance mutations detected in the clinical samples were confirmed by DNA sequencing. We conclude that the AID TB Resistance line probe assay is an accurate tool for the rapid detection of resistance mutations in cultured isolates and in smear-positive clinical specimens.

Mycobacterium algericum sp. nov., a novel rapidly growing species related to the Mycobacterium terrae complex and associated with goat lung lesions.

A previously undescribed, rapid-growing, non-chromogenic Mycobacterium isolate from a goat lung lesion in Algeria is reported. Biochemical and molecular tools were used for its complete description and showed its affiliation to the Mycobacterium terrae complex. 16S rRNA, rpoB and hsp65 gene sequences were unique. Phylogenetic analyses showed a close relationship with M. terrae sensu stricto and Mycobacterium senuense. Culture and biochemical characteristics were generally similar to those of M. terrae and M. senuense. However, in contrast to M. terrae and M. senuense, the isolate was positive for urease production and had faster growth. The mycolic acid profile was distinct from those of M. terrae and M. senuense, thus further supporting the new taxonomic position of the isolate. We propose the name Mycobacterium algericum sp. nov. for this novel species. The type strain is TBE 500028/10(T) ( = Bejaia(T) = CIP 110121(T) = DSM 45454(T)).