| 1. |
- Idh, Jonna, et al.
(författare)
-
Susceptibility of Clinical Strains of Mycobacterium tuberculosis to Reactive Nitrogen Species in Activated Macrophages
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: Nitric oxide (NO) is produced in macrophages by the inducible NO synthase (iNOS) upon activation by pro-inflammatory cytokines. NO has been shown to be essential for the control of Mycobacterium tuberculosis infection in murine models whereas its importance in man is not as clear. There is a lack of studies regarding the susceptibility to reactive nitrogen species (RNS) in clinical strains of M. tuberculosis and the relation to first-line drug resistance, such as to isoniazid (INH). The aim of this study was to explore susceptibility to RNS and intracellular survival of clinical strains of M. tuberculosis, with or without INH resistance. Method: Seven clinical strains of M. tuberculosis were transformed with the pSMT1-plasmid encoding Vibrio harveyi luciferase. Survival was analysed by luminometry following exposure to the NO donor DETA/NO or peroxynitrite (SIN-1). Intracellular killing was studied in murine macrophages (RAW 264.7) activated with interferon gamma (IFN-γ) and lipopolysaccharide (LPS). Results: There was a significant effect on growth control of M. tuberculosis strains upon macrophage activation, which showed variability among clinical isolates. In the cell-free system, all strains showed a dose-dependent susceptibility to DETA/NO and SIN-1, and clinical strains were in general more resistant than H37Rv to DETA/NO. INH-resistant strains with an inhA mutation were significantly more tolerant to DETA/NO than inhA wild type. Conclusion: Reactive nitrogen species inhibited growth of clinical M. tuberculosis isolates both in an intra- and extracellular model with significant difference between strains. Increased tolerance to NO was associated with isoniazid resistance mediated by inhA.
|
|
| 2. |
- Juréen, Pontus
(författare)
-
Molecular characterisation of antibiotic resistance in Mycobacterium tuberculosis
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To counteract the emergence of multi-drug resistant tuberculosis (MDR-TB) a number of approaches have been proposed, amongst which are the sound usage of the present anti-TB drugs and the development of prompt and specific tools for the diagnosis of resistance. To examine the role of drugs in the treatment of resistant TB, we investigated the general crossresistance between kanamycin and amikacin, which has been reported in numerous studies. Forty clinical isolates were shown to be low-to intermediate resistant to kanamycin but fully sensitive to amikacin. Sequencing of the 16S rRNA gene, rrs, revealed no specific genotype correlating to this. We could however confirm the previously reported correlation between mutations at position 1400 and dually highly resistant strains. Since cross-resistance is not present in all strains, caution should thus be taken when extrapolating the results of susceptibility testing between the closely related drugs kanamycin and amikacin. A rapid detection of MDR-TB infections makes it possible to promptly change to a more effective treatment of the patient, which could shorten the patient's period of infectiousness and thus reduce the risk for additional new cases. We evaluated the usefulness of the commercially available hybridisation Line Probe Assay (INNO-LiPATM Rif.TB). We also developed an assay based on Pyrosequencing technology, for the identification of rpoB mutations, and thus the rapid detection of rifampicin resistance. Both methods detected mutations in all rifampicin resistant strains. Among the susceptible strains, the Pyrosequencing assay detected additional mutations whereas these could not be discriminated by LiPA. Since the M. tuberculosis Beijing family has been associated with major outbreaks and MDR-TB, we investigated whether rifampicin resistance-levels and rpoB mutations could be strain dependent. We studied 189 in vitro generated rifampicin resistant mutants, of which approximately half were of the Beijing family. There was no general difference in resistance-level or mutations between the two subsets of mutants. The two most common mutations found, irrespective of strain origin, were the Ser531Leu and His526Tyr, which reflect what is found among clinical isolates. Thus, the predominance of Beijing strains in terms of resistance and high prevalence in certain geographical areas are likely due to other factors than mutations in the rpoB gene. Lastly, we investigated the use of a sequencing assay directed to the pncA gene and sequences in its proximity, for the detection of pyrazinamide resistance. The phenotypic drug-susceptibility tests for the first-line agent pyrazinamide are cumbersome to use and known to be difficult to reproduce. Not only would a sequencing assay circumvent the obstacles associated with the phenotypic methods but also provide a shorter turnaround time. We identified mutations in all but one resistant strain. Among the susceptible strains, only a few mutations were found, and these were silent mutations. Thus, pncA sequencing seems to offer an attractive alternative to phenotypic tests. In the present studies, we have shown that DNA-based methods directed to rpoB and pncA correlate well to the phenotypic methods for the detection of rifampicin and pyrazinamide resistance. A hybridisation-based method would be of second choice as there is a risk for false positive readings. Thus, we recommend the use of sequencing assays that will detect and directly define mutations. Unfortunately, for aminoglycosidic resistance there is yet no clear correlation between mutations and resistance, and only highly resistant strains could be identified using our sequencing assay. More importantly however, in contrast to what has earlier been described, we showed that cross-resistance between amikacin and kanamycin is not a general rule. Therefore we propose that, if these two drugs are being considered for treatment, drug-susceptibility testing on both drugs should be conducted simultaneously. These studies show that molecular techniques not only offer tools for a rapid detection of drug resistance, but also increase our understanding of how resistance is developed.
|
|
| 3. |
- Larsson, Marie C, et al.
(författare)
-
A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability
- 2014
-
Ingår i: Journal of Microbiological Methods. - : Elsevier BV. - 0167-7012 .- 1872-8359. ; 106, s. 146-150
-
Tidskriftsartikel (refereegranskat)abstract
- The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5 days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4 mg/L, 8 mg/L and 2 mg/L, respectively) compared to extracellularly (MIC90: 0.5 mg/L for AMI and EMB; 0.25 mg/L for LEV). The reverse was the case for INH (IC: 0.064 mg/L vs EC: 0.25 mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs. (C) 2014 Elsevier B.V. All rights reserved.
|
|
| 4. |
- Merker, Matthias, et al.
(författare)
-
Phylogenetically informative mutations in genes implicated in antibiotic resistance in Mycobacterium tuberculosis complex
- 2020
-
Ingår i: Genome Medicine. - : BMC. - 1756-994X. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). Methods We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. Results Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). Conclusions Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.
|
|
| 5. |
- Niward, Katarina, et al.
(författare)
-
Susceptibility testing breakpoints for Mycobacterium tuberculosis categorize isolates with resistance mutations in gyrA as susceptible to fluoroquinolones : implications for MDR-TB treatment and the definition of XDR-TB
- 2016
-
Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 71:2, s. 333-338
-
Tidskriftsartikel (refereegranskat)abstract
- Fluoroquinolones (FQs) are important in the treatment of MDR-TB and in the definition of XDR-TB. Our objective was to investigate how discrepancies in the phenotypic and genotypic methods for antimicrobial susceptibility testing could affect the interpretation of antimicrobial susceptibility test results. We analysed MICs of ofloxacin and levofloxacin in Middlebrook 7H10 broth (7H10) as well as sequencing of the quinolone resistance-determining region of the gyrA gene and the MTBDRsl assay in 75 resistant isolates, including MDR and XDR strains of Mycobacterium tuberculosis. Among 75 resistant isolates, 27 had mutations associated with FQ resistance. Among isolates with resistance mutations in gyrA, 26% (seven of 27) were susceptible to levofloxacin and ofloxacin by phenotypic testing at 1 mg/L and 2 mg/L. The most common mutation was in codon 94 and these isolates had significantly increased MICs of levofloxacin (2-8 mg/L) compared with isolates with mutations in codon 90 (0.25-2 mg/L, PaEuroS < aEuroS0.05). The sensitivity and specificity for the MTBDRsl assay compared with gyrA sequencing were 96% and 98%, respectively. Current critical concentrations may classify up to 26% of isolates with gyrA mutations as susceptible to FQs due to a close relationship between susceptible and resistant populations. These results should be considered while improving clinical breakpoints for M. tuberculosis and may have an impact on the definition of XDR-TB.
|
|
| 6. |
- Schon, Thomas, et al.
(författare)
-
Evaluation of wild-type MIC distributions as a tool for determination of clinical breakpoints for Mycobacterium tuberculosis
- 2009
-
Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 64:4, s. 786-793
-
Tidskriftsartikel (refereegranskat)abstract
- Objectives: The aim of this study was to establish wild-type MIC distributions of first-line drugs for Mycobacterium tuberculosis, as well as to explore the usefulness of such distributions when setting clinical breakpoints. Methods: We determined the MICs of rifampicin, isonlazid and ethambutol for M. tuberculosis using a Middlebrook 7H10 dilution method for 90 consecutive clinical isolates, 8 resistant strains and 16 isolates from the WHO proficiency test panel. M. tuberculosis H37Rv was used for quality control and susceptibility results using 7H10 were compared with the results obtained with BACTEC460. Results: The agreement with BACTEC460 was very high for isonlazid (99.1%) and rifampicin (99.1%) but lower for ethambutol (94.7%). Intra- and inter-assay variation was below one MIC dilution. The MIC distributions for isoniazid and rifampicin provided a clear separation between susceptible and resistant strains. Regarding ethambutol, the current breakpoint for 7H10 (5 mg/L) is close to the wild-type and all strains (n=6) showing a disagreement between BACTEC460 and 7H10 were distributed very close to the breakpoint (MIC 4-8 mg/L). This problematic relation was confirmed by investigating isolates from the WHO panel with an agreement <95% (64%-88% among 26 laboratories, n=4) for which the MICs were 4-8 mg/L . Conclusions: Utilizing the wild-type MIC distribution was found to be as useful in M. tuberculosis as in other bacteria when setting clinical breakpoints. We suggest that the present clinical breakpoints should be re-evaluated, taking into account wild-type MIC distributions and available pharmacokinetic data.
|
|
| 7. |
- Thelaus, Johanna, et al.
(författare)
-
Network Experiences from a Cross-Sector Biosafety Level-3 Laboratory Collaboration : A Swedish Forum for Biopreparedness Diagnostics
- 2017
-
Ingår i: Health Security. - : Mary Ann Liebert. - 2326-5094 .- 2326-5108. ; 15:4, s. 384-391
-
Tidskriftsartikel (refereegranskat)abstract
- The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration among the 4 agencies with responsibility for the laboratory diagnostics of high-consequence pathogens, covering animal health and feed safety, food safety, public health and biodefense, and security. The aim of the network is to strengthen capabilities and capacities for diagnostics at the national biosafety level-3 (BSL-3) laboratories to improve Sweden's biopreparedness, in line with recommendations from the EU and WHO. Since forming in 2007, the FBD network has contributed to the harmonization of diagnostic methods, equipment, quality assurance protocols, and biosafety practices among the national BSL-3 laboratories. Lessons learned from the network include: (1) conducting joint projects with activities such as method development and validation, ring trials, exercises, and audits has helped to build trust and improve communication among participating agencies; (2) rotating the presidency of the network steering committee has fostered trust and commitment from all agencies involved; and (3) planning for the implementation of project outcomes is important to maintain gained competencies in the agencies over time. Contacts have now been established with national agencies of the other Nordic countries, with an aim to expanding the collaboration, broadening the network, finding synergies in new areas, strengthening the ability to share resources, and consolidating long-term financing in the context of harmonized European biopreparedness.
|
|
