non tb mycobacterium :: Article CreatorNon-tuberculous Mycobacteria
The author reported a case of mycobacterial infection, which was diagnosed after a lengthy process.
However, the case is actually not one of tuberculosis in the stricter sense: the confirmed species—Mycobacterium avium intracellulare complex—belongs to the group of non-tuberculous mycobacteria. These are often found in the environment and, in cultures, are possibly only contaminants. However, in this case the diagnosis was firmly established following the criteria of the American Thoracic Society (ATS) – the specimens came from otherwise sterile body fluids (1).
Microscopy using different staining methods, as mentioned in the article, is less relevant than cultures and PCR for confirming a diagnosis, since microscopy has lower sensitivity and, in contrast to the other methods, cannot differentiate between species (2). It therefore needs to be emphasized that the diagnostic mistake in the case report consisted mainly in not doing culture and PCR.
Non-tuberculous mycobacteria—such as the described species—display sensitivities to anti-mycobacterial chemotherapy different from Mycobacterium tuberculosis complex. After resistance testing, recommended initial treatment should therefore consist of a combination of clarithromycin, ethambutol, and, if required, rifabutin. By contrast to the treatment described in the article, however, isoniazid is not the treatment of choice (3).DOI: 10.3238/arztebl.2010.0147b
Dr. Med. Norbert HeinrichPD Dr. Med. Michael HoelscherAbteilung für Infektions- und TropenmedizinKlinikum der LMULeopoldstr. 580802 München, Germanyheinrich@lrz.Uni-muenchen.De
PD Dr. Rer. Nat. Elvira RichterForschungszentrum BorstelNationales Referenzzentrum fürMykobakterienParkallee 1823845 Borstel, Germany
"Achilles Heel" Of Drug-Resistant TB Identified Using CRISPR Interference
Drug-resistant tuberculosis (TB) is a major contributor to global antimicrobial resistance with around half a million people falling ill with drug-resistant strains annually. Mycobacterium tuberculosis (M. Tuberculosis), the primary agent of TB, can usually be treated with a four-drug four-month regimen often involving the front-line antibiotic isoniazid. The emergence of resistance to these antibiotics necessitates novel antibiotic and drug combinations.
In a study published in Nature Communications, researchers have used a genetic platform to identify biological pathways in a drug-resistant strain of M. Tuberculosis that are highly sensitive to inhibition.
This technology enabled the researchers to find the pathogen's "Achilles Heel" and identify drugs that target these weaknesses to rapidly kill drug-resistant strains. The researchers believe this technology could be applied to other drug-resistant pathogens to help treat drug-resistant infections and prevent the emergence of new drug-resistant strains.
Whole genome screening of drug-resistant TB
While drug resistance can help a strain of bacteria gain the upper hand against antibiotics, it can be a double-edged sword for the pathogen. Drug-resistant strains must rely on specific cellular pathways that become more important for growth to mitigate the fitness costs associated with drug resistance. As a result, these pathways are more vulnerable to inhibition compared to a drug-sensitive parent.
"Prior attempts to identify vulnerabilities, including our own, have largely relied on the use of existing antibiotics that target only a very small subset of biological pathways," Dr. Matthew McNeil, senior research fellow at the University of Otago and senior author, told Technology Networks. "Whilst these studies have been useful, we hypothesized that they weren't providing a complete picture of potential vulnerabilities in drug-resistant M. Tuberculosis."
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In the current study, researchers used whole genome CRISPR interference (WG-CRISPRi) screening, transcriptomics and metabolomics to generate a genome-wide map of cellular vulnerabilities in an isoniazid-resistant strain of M. Tuberculosis.
"By combining all the outputs [WG-CRISPRi, transcriptomics, metabolomics] we get a really good idea of how this resistant strain of M. Tuberculosis was trying to adapt to the fitness cost imposed on it by the initial mutation. What was really important is that each approach provided unique insights, that we simply would have missed if we were using one approach," stated McNeil.
Using WG-CRISPRi, the researchers were able to quantifiably measure vulnerabilities associated with target inhibition at a genome-wide level. Importantly the approach enabled them to assess both non-essential and essential genes.
What is CRISPR interference (CRISPRi)?
Similar to CRISPR knockout experiments, CRISPR interference (CRISPRi) uses guide RNA sequences to direct a Cas9 protein to target a specific sequence. The major difference is that CRISPRi uses a catalytically inactive Cas9 (dCas9) protein that is unable to cut DNA and instead binds to the target sequence to block transcription at that site. CRISPRi lacks the toxicity caused by DNA double-strand breaks, allowing for the silencing of noncoding RNAs and the discovery of noncoding regulatory regions.
"CRISPR interference is a great technology. One of the big benefits is that not only can you determine if a gene is non-essential or essential, but it describes essentiality as a variable," said McNeil.
"Some genes can be highly essential in which they are really important for growth so only need a small level of inhibition, or some pathways can be weakly essential and require a high level of inhibition or need to be inhibited for a long time to have an effect. By comparing how essential a gene is across different backgrounds we can identify changes in genetic vulnerability."
From the screening data, the researchers identified vulnerabilities in respiration, ribosome biogenesis and nucleotide and amino acid metabolism in the isoniazid-resistant strain of M. Tuberculosis.
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"At a clinical level, we observed that respiration, particularly the ATP synthase operon was more vulnerable to inhibition by both CRISPRi and the inhibitor bedaquiline that targets this enzyme in our isoniazid-resistant strains and clinically relevant genotypes," explained McNeil.
These findings help explain the efficacy of the six-month all-oral BPaL (bedaquiline, pretomanid and linezolid) regimen that is currently recommended by the World Health Organization to treat drug-resistant TB. McNeil hypothesized the efficiency of this treatment is likely to be "partly driven by bedaquiline inadvertently targeting these collateral drug susceptibilities in drug-resistant M. Tuberculosis."
By defining the genes and cellular pathways that are more vulnerable to inhibition in isoniazid-resistant cells, the researchers hope this will bring about the development of novel drugs for tackling drug-resistant TB infections.
Beyond TB, the researchers believe the genomics platform utilized in this study could be applied to other resistant bacteria to identify novel drug targets. "Our next steps are to apply this approach to other drug-resistant strains of M. Tuberculosis, to hopefully identify some shared and unique genetic vulnerabilities across genetically diverse resistant strains," said McNeil.
"Whilst the specific platform is optimized for use in Mycobacteria, in principle, this experimental approach can be applied to other drug-resistant pathogens."
Reference: Wang X, Jowsey WJ, Cheung CY, et al. Whole genome CRISPRi screening identifies druggable vulnerabilities in an isoniazid resistant strain of Mycobacterium tuberculosis. Nat Commun. 2024;15(1):9791. Doi: 10.1038/s41467-024-54072-w
About the interviewee:
Headshot of Dr. Mathew McNeil, senior research fellow at the University of Otago. Credit: Sharon Bennett.
Dr. Matthew McNeil is a senior research fellow at the University of Otago. He holds a PhD from the University of Otago. His current research focuses on the development of novel treatment strategies to combat Mycobacterium tuberculosis. McNeil's research utilizes a combination of molecular biology, microbiology, antimicrobial susceptibility testing, biochemical assays, next-generation sequencing and metabolomics.
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