Dataset Information

Differential detection of tuberculous and non-tuberculous mycobacteria by qPCR in lavage fluids of tuberculosis-suspicious white rhinoceros.

ABSTRACT: Tuberculosis (TB) occurs in a wide range of mammalian species and thus poses a health risk to humans living or working in close proximity with TB infected animals. Despite a high incidence of M. bovis infections in domestic or wildlife species tuberculosis infections in rhinoceros have so far been very limited. Over the past 53 years, tuberculosis of the respiratory tract has been confirmed in just 22 rhinoceros, most of those infected not by M. bovis but M. tuberculosis. However, because of the zoonotic risk TB testing is recommended or becomes even mandatory in endangered species. The dilemma in rhinoceros and many other wildlife species; non-validated tests are highly inconsistent in their ability to identify TB infection. Current lack of TB diagnostics may result in TB positive rhinoceros living with the infection, transmitting it to those around them or in euthanasia of animals found unconfirmed at necropsy. This is an unacceptable diagnostic status considering that some species are critically endangered and therefore should not be euthanized in order to confirm suspicion of disease. To overcome this shortcoming we used bronchoscopy to detect mycobacteria in respiratory fluids of TB suspicious rhinoceros. Fluids from seven, TB suspicious white rhinoceros were harvested during 21 bronchoscopies. Our new approach: In addition to bacterial culture a dual quantitative PCR system tested for the general presence of DNA from NTM and more specifically for DNA from MTC. Both, bacterial culture and qPCR were negative for MTC in respiratory fluids of all rhinoceros (7/7). At the same time, respiratory fluids from six rhinoceros tested positive for the presence of NTM or other closely related bacteria (6/7). M. tuberculosis was found only once in an oesophageal aspirate. The high incidence of mycobacterial DNA in the respiratory tract suggests that white rhinoceros, as strict grazers, are immensely exposed to environmental bacteria of this genus. Presence of NTM in the respiratory or intestinal system could possibly cause false positive results in intradermal tests. A wider use of bronchoalveolar lavage is warranted to further elucidate immunologic response to NTM and exposure to, incidence and prevalence of MTC infections in rhinoceros.

SUBMITTER: Hermes R

PROVIDER: S-EPMC6261570 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Differential detection of tuberculous and non-tuberculous mycobacteria by qPCR in lavage fluids of tuberculosis-suspicious white rhinoceros.

Hermes Robert R Saragusty Joseph J Moser Irmgard I Barth Stefanie A SA Holtze Susanne S Lecu Alexis A Cracknell Jonathan J Williams Duncan D Göritz Frank F Hildebrandt Thomas Bernd TB

PloS one 20181128 11

Tuberculosis (TB) occurs in a wide range of mammalian species and thus poses a health risk to humans living or working in close proximity with TB infected animals. Despite a high incidence of M. bovis infections in domestic or wildlife species tuberculosis infections in rhinoceros have so far been very limited. Over the past 53 years, tuberculosis of the respiratory tract has been confirmed in just 22 rhinoceros, most of those infected not by M. bovis but M. tuberculosis. However, because of the ...[more]

PMID: 30485372

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Project description:Reactive multi-target 'fragment drugs' represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small 'dirty' drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM pathogens. These hits will now be evaluated in animal models of mycobacterial infection to determine whether any of them can be moved forward as a new antimycobacterial fragment drug candidate.

Project description:Four species of non-tuberculous mycobacteria (NTM) rated as biosafety level 1 or 2 (BSL-1/BSL-2) organisms and showing higher genomic similarity with Mycobacterium tuberculosis (Mtb) than previous comparator species Mycobacterium kansasii and Mycobacterium marinum were subjected to genomic and phenotypic characterization. These species named Mycobacterium decipiens, Mycobacterium lacus, Mycobacterium riyadhense, and Mycobacterium shinjukuense might represent "missing links" between low-virulent mycobacterial opportunists and the highly virulent obligate pathogen Mtb. We confirmed that M. decipiens is the closest NTM species to Mtb currently known and found that it has an optimal growth temperature of 32°C-35°C and not 37°C. M. decipiens showed resistance to rifampicin, isoniazid, and ethambutol, whereas M. lacus and M. riyadhense showed resistance to isoniazid and ethambutol. M. shinjukuense was sensitive to all three first-line TB drugs, and all four species were sensitive to bedaquiline, a third-generation anti-TB drug. Our results suggest these four NTM may be useful models for the identification and study of new anti-TB molecules, facilitated by their culture under non-BSL-3 conditions as compared to Mtb. M. riyadhense was the most virulent of the four species in cellular and mouse infection models. M. decipiens also multiplied in THP-1 cells at 35°C but was growth impaired at 37°C. Genomic comparisons showed that the espACD locus, essential for the secretion of ESX-1 proteins in Mtb, was present only in M. decipiens, which was able to secrete ESAT-6 and CFP-10, whereas secretion of these antigens varied in the other species, making the four species interesting examples for studying ESX-1 secretion mechanisms.IMPORTANCEIn this work, we investigated recently identified opportunistic mycobacterial pathogens that are genomically more closely related to Mycobacterium tuberculosis (Mtb) than previously used comparator species Mycobacterium kansasii and Mycobacterium marinum. We confirmed that Mycobacterium decipiens is the currently closest known species to the tubercle bacilli, represented by Mycobacterium canettii and Mtb strains. Surprisingly, the reference strain of Mycobacterium riyadhense (DSM 45176), which was purchased as a biosafety level 1 (BSL-1)-rated organism, was the most virulent of the four species in the tested cellular and mouse infection models, suggesting that a BSL-2 rating might be more appropriate for this strain than the current BSL-1 rating. Our work establishes the four NTM species as interesting study models to obtain new insights into the evolutionary mechanisms and phenotypic particularities of mycobacterial pathogens that likely have also impacted the evolution of the key pathogen Mtb.

Dataset Information

Differential detection of tuberculous and non-tuberculous mycobacteria by qPCR in lavage fluids of tuberculosis-suspicious white rhinoceros.

Publications

Differential detection of tuberculous and non-tuberculous mycobacteria by qPCR in lavage fluids of tuberculosis-suspicious white rhinoceros.

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